Genetics in Dentistry

Introduction And Mendel’s Laws Of Inheritance

Definition Of Genetics

We all have observed that children of same parents, more often than not, resemble each other as well as resemble their parents. We also try to guess the proximity of this resemblance towards one of the two parents. It is quite interesting to observe that the resemblances are not just confined to their physical appearances (facial features, height, color of skin and hair, etc.), but are often perceptible in their mental attributes (intelligence, tastes, attitudes, etc.) also.

This is because the characteristics of parents are passed on to the children through the gametes furnished by each parent (sperm an dovum). The process of transmission of characters from one generation to the next (parents to children) is called inheritance or heredity.

The question that crops in our mind is about what are the substrates that actually determine the characters in an individual. The characters, in fact, are determined by certain factors called genes; the fundamental units of inheritance. For details about genes refer to Chapter 4. The genes determining specific characters in an individual are transmitted to them physically through gametes of the parents.

The individual in turn passes these trains onto its offsprings through his or her gametes. An individual is either short or tall in stature or with black or blond hair entirely due to the presence or absence of specific genes responsible for a particular character or trait. Since an individual receives genes from parents (through sperm and ovum), he or she inherits characters both from the father and the mother.

An important fact about gene transmission is that when they are transmitted from one generation to the next, the transmission of a trait is not random but it follows some discreet statistical laws depending upon the type of the character and of course, the behavior of the gene during gamete formation. Therefore the science of genetics can be defined as the study of genes and of the principles that govern the passage of genes from one generation to the next.

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Divisions Of Genetics

Human genetics can now be divided into several branches. Few important subdivisions of genetics are as under.

Molecular genetics: Includes the study of chemical structure of gene at molecular level. This branch also includes the study of function of gene and regulation of its activity.

Cytogenetic: Deals with the study of chromosomes. Cytogenetics provides the cytological explanation of different genetic principles.

Biochemical genetics: Concerns with the study of genes and their products, the enzymes, which control important stages of various metabolic processes. This branch deals with the inborn errors of metabolism.

Cancer genetics: The cell cycle is under genetic surveillance and control. The cycle progresses from one stage to the next through several stages called the checkpoints. The structure of all genes is scrutinized at these periodic intervals for allowing only the healthy genes to proceed to the next stage. Cancer genetics studies the abnormalities related to these checkpoints to find the reasons that cause cancer.

Immunogenetics: The immunological make-up of an individual is under strict control of certain genes. Immunogenetics deals with the genetics of production of different types of antibodies.

Developmental genetics: Deals with the genetic control of development of an embryo.

Population genetics: This branch deals with frequencies and distribution of genes in human population and the rates of their mutation.

Classification Of Genetic Diseases

Diverse genetic mechanisms are involved in different hereditary diseases. The cause of a genetic disorder may have its base in the abnormality of the structure of a single gene or multiple genes. Genetic diseases may be also due to a gross abnormality in the structure of an entire chromosome. Thus genetic diseases may be classified as under:

• Disorders due to mutation in single gene: Single gene mutations are responsible for these disorders and they follow laws of Mendelian inheritance. These disorders may be autosomal dominant, autosomal recessive or X-linked. Thousands of disorders can be categorized in this group. Some examples related to dentistry are given below.
  • Autosomal Dominant
    • Achondroplasia
    • Dentinogenesis imperfecta type 1
    • Amelogenesis imperfecta hypoplastic type 2 (AIH2)
    • Amelogenesis imperfecta hypocalcification type
    • Hypodontia
    • Osteogenesis imperfecta.
  • Autosomal Recessive
    • Cystic fibrosis
    • Amelogenesis imperfecta (local hypoplastic type)
    • Amelogenesis imperfecta (pigmented hypomaturation type)
    • Neonatal osseous displasia 1.
  • X-Linked Dominant
    • Amelogenesis imperfecta (Hypoplastic)
    • Vit. D resistant rickets.
  • X-Linked Recessive
    • Hemophilia
    • Ectodermal dysplasia type 4
    • Amelogenesis imperfecta hypomaturation type (AIH)
    • Chondrodysplasia punctata – 1
• Multifactorial disorders: Cumulative or additive effects of multiple genes are implicated in these disorders. The normal characters like height, color of skin, intellignece and physique are determined by the interaction of many genes. Common congenital malformations like cleft lip and palate and diseases like hypertension and diabetes mellitus ae multifactorial disorders. Some kind of oral conditions like dental caries, periodontitis and malocclusion have strong genetic susceptibility. These kinds of disorders are results of interplay between gene expression and environmental factors.
  The multifactorial disorders follow different pattern of inheritance as compared to single gene disorders.
• Disorders due to chromosomal abnormality: This group includes gross structural anomalies that give rise to alterations in the number of chromosomes (absence of a chromosome or presence of an extra chromosome), i.e., Trisomy 21 (Down’s syndrome) or Turner’s syndrome (XO). This class also includes disorders, which result due to abnormality in the structure of chromosomes such as deletions and translocations. The invention of banding and FISH (fluorescent in sity hybridization) techniques has helped to detect even minor abnormalities in chromosomes. Subtle or point chromosomal abnormalities are included in the single gene disorders.
• Somatic genetic disease: Cell divisions (mitosis in somatic and meiosis in germ cells) constantly occur during the life time of an individual. During each cell division there are chances that a change in the structure of a gene (gene mutation) may take place due to an error in DNA replication. It may also happen that at the end of a cell division (mitosis or meiosis) one of the daughter cells might receive an unequal number of chromosomal (due to erro in chromosomal separation). These kinds of mutation or mistakes in chromosomal distribution are accountable for numerous somatic and germ line diseases.

Genetics In Dentistry

It was first observed by the French biologist Maupertius (1689-1759) that the conditions like polydactyly and albinism were inherited in human beings. Likewise John Dalton (1766-1844) observed that color blindness and hemophilia were inherited diseases. However, human genetics was recognized as a science only after rediscovery of Mendel’s Laws of Inheritance in early 1900.

From the mid 20th century onwards, oral health care professionals had started realizing that many diseases related to the oral cavity were in fact inheritable. Information from the Human Genome Project (2001) and recent genetic researches has clearly indicated that many dieseases with the dental, oral and craniofacial manifestations have a genetic basis both in terms of heritability (disease running in families) as well as arising from structural mutation in a particular individual.

Tooth Agenesis

The etiology of tooth agenesis was largely unknown till the recent past. But today we know that the development of tooth is strictly under the control of many genes. Several mutations in the developmental genes could result into failure of tooth development. Familial tooth agenesis may be transmitted as an autosomal dominant, recessive or an X-linked condition.

Similarly, most cases of hypodontia exhibit polygenic inheritance pattern. Hypodontia is associated with syndromes like Down’s syndrome, ectodermal dysplasia and the Ellis-van Creveld syndroms. This demonstrates that the development of other organs and tissues of the body is closely related to the development of dentition and perhaps regulated by common genes.

Dental Caries

Certain microorganisms have been incriminated as the causal factors for two major diseases of oral cavity, i.e. dental caries and periodontal diseases. Recent research date have pointed that these conditions have a strong genetic predisposition. Different people have different susceptibility risk for developing periodontics.

Studies have shown that the increase vulnerability to severe adult periodontics is due to variation in the interlukin-1 (IL – 1) gene cluster that is situated on chromosome number 2.

Craniofacial Syndromes

The development of craniofacial region during the early stages of development is genetically determined in terms of migration of definite neural crest cell and through this to the expression of certain sequential homeobox genes. Epithelial-mesenchymal interaction during embryogenesis is regulated by growth factors and the retinoic acid super families.

Conditions like hemifacial microsomia and craniosynostosis have their origin in neural crest cell disorders. The nutation in fibroflast growth factor receptor genes are responsible for abnormal suture development and found to occur in Apert, Crouzon and Pfeiffer syndromes. Cleidocranial dysplasia is characterized by defects in the membranous bones of the cranial vault and clavicle.

The nutations responsible for this deformity have been found to occur in the core binding factor 1 gene (CBFAI). The gene responsible for a well known craniofacial abnormality the Treacher Collins syndrome is situated on the long arm of chromosome 5. Many craniofacial abnormalities are due to interaction between environmental and genetic factors.

Cleft Lip, Cleft Palate and Cancers

Among the common occurring malformations of the oral cavity Cleft lip and cleft palate are amongst the top in the list. These congenital malformations are inherited as multifactorial traits. The same is true for malocclusion.

The head and neck region are very common sites for carcinomas in general and oral cancer are the ones seen quite frequently. The dynamics of cancer involves changes in the genome that result in uncontrolled cellular proliferation and metastasis.

The growth factor and growth factor receptor genes regulate the proliferation of cells. Genes responsible for cancer are known as oncogenes. These genes function normally in regulating cellular activity. A mutation in these genes may trigger them to acquired oncogenic properties. Cell division is strictly under genetic control and each of the steps is under constant surveillance of cellular mechanisms.

Cell cycle checkpoints exist at appropriate transition points of the cell cycle. The activites at these checkpoints are executed by special proteins that are synthesized by specific genes like the p54 gene. Anomalies in these genes lead to abnormal cell division and subsequently to tumor formation. Structural integrity of the DNA us determined and checked at the checkpoints before allowing it to proceed to subsequent stages of cell division.

Mutations of the check point controlling genes and proteins cause several cancers. The tumor suppressor genes constitute another important cell cycle controlling element. These genes apply brakes to the events in a cell division in case of detection of an abnormality at any stage. These genes are constitutively active or in simple terms, active by default in all normal cells. A mutation causing abnormal activity of any tumor supressor gene may lead to cancer.

Researchers have also identified several tumor-forming genes that occur in normal cells but remain inactive by themselves. Such proto-oncogensea trigger unwarranted cell division if they are activated by any means or when any normally occurring inhibition acting on them is withdrawn .

It is hopeted that the near future will witness a lot of excfiting advances in: Use of primary teeth as source of stem cells, tissues engineering in dentistry, use of saliva as a diagnostic fluid in deteching genetic dental disorders and salivary gland gene transfer.

It is quite imperative that dental practitioners now will increasingly require knowledge of human genetics and the awareness of the applications of new molecular based diagnostic and therapeutic technologies. Thus a sound knowledge of genetics will definitely improve the ability of dentists to diagnose and treat patients suffering from inherited and genetically caused dental diseases.

Since recent past more and more diseases are being recognized as having something related to genes and genetics. This is perhaps due to interpretations based on our new and expanding knowledge at the molecular level and progress in modern diagnostic techniques.

On other hand this may also be due to the fact that owing to the overall improvement in hygiene and health care, the incidence of communicable diseases and nutritional deficiency has reduced thereby shifting our attention to diseases resulting from gene-related etiology. Genetic disorders are now considered significant causes for disease in all age groups.

Mendel And His Laws Of Inheritance

Johann Gregor Mendel was born in Austria on July 22, 1822. He had to face relentless difficulties in his childhood and youth due to poverty and ill health. It was to the credit of his young man that he remained steadfast in the face of all the adversities in the face of all the adversities for the pursuit of knowledge. It took him almost eight years to complete his initial experiments on pea plants.

Mendel published his reports in the proceedings of the Brunn Natural Science Society in 1866. Mendel’s work remained unappreciated and unnoticed till the turn of the century when the postulates of Mendel were rediscovered and revisited by three independently working scientists, Erich Von Tshermak, Hugo de Varies and Carl Correns in the beginning of the 20th century.

Mendel’s work did not get recognition during his lifetime. He passed away in 1844, much before his monumental work immortalized him as the ‘father of modern genetics’.

The Pea Plants Experiments

Mendel’s experiments were designed to find out the mechanisms responsible of inheritance of traits in the peas plants. His experiments basically involved two types of crosses. One between plants differing in a single pair of contrasting characters such as cross between a pure tall and a pure dwarf plant called the Monohybrid cross and subsequent crosses within the offsprings in each generation obtained from the monohybrid crosses.

The other type of experiment called the Dihybrid cross was carried out between plants differing in two pairs of contrasting characters; a cross, for example, done between plants having yellow and round seeds and plants having green and wrinkled seeds. The contrasting pairs of characters in the dihybrid cross were represented by the color and the texture of the seeds in the two different varieties of plants.

Explaining Certain Terms

Self-pollination: Polines of a flower pollinating the stigma of the same flower is called as self-pollination (self-fertilization).

Cross-pollination: Pollens of a flower pollinating different flower stigma is called cross-pollination. (cross-fertilization). The offspring which result from cross breeding between pure strains is called a hybrid.

Monohybrid cross: The cross between the plants or animals differing in single pair of contrasting characters is called monohybrid cross, e.g. cross between tall and dwarf plants.

Dihybrid cross: The cross between plants or animals differing in two pairs of contrasting characters is called dihybrid cross, e.g. plants with yellow and round seeds are crossed with plants having green and wrinkled seeds.

Monohybrid Crosses Experiments

Mendel crossed plants that differed only in a single pair of contrasting character or trait. He crossed between pure tall plants and pure dwarf plants. The character or trait in case of these plants was the height of the stem and the pair of contrast was the tallness and dwarfness of the respective plants. The purity was verified with repeated self-pollination where the tall plants always inbreeded tall and the dwarf plants yielded dwarf offsprings on repeated inbreeding for several generations.

And when these two varieties of plants (tall and short) were crossed, Mendel observed that:

• All the hybrid members of the first generation, called the First filial generation (F1), were tall plants.
• When the F1 population was allowed to self-pollinate, the palnts of the F1 generation gave rise to the Second filial generation (F2) with the following features.
  • The character of dwarfness that had disappeared in the F1 reappeared in F2.
  • 75% of the F2 plants were tall and 25% were dwarfs.

• After self-pollination the F2 generation gave the following results:
  • The dwarf plants of F2 when pollinated with dwarfs in the F2, always yielded dwarfs.
  • The tall plants when pollinated with the other tall plants within the F2, olny 1/3rd of plants always yielded tall offsprings.
  • Remaining 2/3rd of the tall plants yielded tall and dwarf plants in the ratio of 3:1.

Similar to the experiments in monohybrid crosses related to the height of the stem of plants, Mendel conducted experiments with other contrasting characters such as teh shape and color of the seeds and pods, etc. and remarkably got statistically comparable results.

Mendel was not only able to put forward the principles of heredity; he could also predict many of the outcomes of his experiments. He derived several conclusions related to the governance of hereditary traits that hold well till today.

• Inheritable characters are transferred with the help of factors through generations. These factors were later identified as genes.
• The heritable factors are transmitted through gametes (sperms and ova).
• The factors for each character or trait exist as a pair.
  Each of the factors (genes, as we know them today) is responsible for a trait and is located at identical positions on each chromosome of a particular pair of chromosomes (homologous chromosomes). The fixed position on a particular chromosome for a definite gene is called the locus (plural loci) for the gene.
  The same positions (loci) on two homologous chromosomes contain genes responsible for the same character. For example the loci representing the height of a plant may contain genes responsible for tallness (T) or dwarfness (t) in different combinations as (TT, Tt or tt). The alternative form of genes, e.g. ‘T’ and ‘t’ present at the same locus are called allelomorphs or alleles.
  The alleles define a particular character depending upon their dominance with respect to each other. Homolous chromosomes carrying identical alleles (same gene) are termed homozygous. A situation with different genes at the loci defining a particualr trait is called heterozygous.
• The members of the homologous pair of chromosomes separate from each other at the time of gametogenesis.
  Each of the gametes carries only a single chromosome out of the homologous pair. A single gamate will carry a single locus containing either a dominant or a recessive gene depending on the chromosomal constitution of the individual parent.

• Crossing between plants of pure variety differing in a single pair of contrasting character yields only the dominant character in first generation, whereas both the characters are expressed in second generation.
  Punnett squares are grids that are extensively used to compute the genetic constitution of an individual by entering the constitution of the gametes on the top and side squares of the grid. Analyzing the above shown cross that is same as the first cross in Mendel’s monohybrid cross experiment to yield the first filial generation in the Punnett square, looks like as below.

The genotype of an individual is defined as its genetic constitution for any particular trait. The term phenotype denotes the physical appearance for a particular trait. A gene is considered to be a dominant if it is able to express itself in the phenotype even when it is present in a heterozygous condition, e.g. the gene for tallness ‘T’ in (Tt). The gene for shortness ‘t’ is only expressed when it is present in a homozygous state (tt) in an individual and is called a recessive gene.

It is thus evident that all the progeny tall plants of the F1 generation (Tt) have different genotypes than the pure bred parent tall plants (TT) though they are same in their phenotypes.

Self-pollination of F1 plants can be analyzed in the F2 generation with the Punnett square.

Self-pollination in the F1 generation yielded the F2 progeny. The F2 progeny constituted two varieties of phenotype with the reappearance of short stature in the plants. The genotype of the tall plants showed two varieties; the homozygous tall the heterozygous tall plants. The concepts of dominance and recessive perspectives are also clear from the results in the grid squares.

Characters are transmitted from one generation to next following statistical laws. When the plants of F1 generation were self-pollinated both tall and short plants appeared int he ratio of 3:1. When the plants of F2 generation were self-pollinated the tall and short plants always appeared in the fixed ratio.

The results of the F3 generation in the Punnett square show the genotype and the phenotype of the individual progeny.

Dihybrid Crosses Experiments

For his dihybrid cross experiments Mendel selected two varieties of pea plants that differed in two pairs of contrasting characters. He selected, for example, pure variety of plants having yellow and rounded seeds and crossed them with another pure variety of plants having green and wrinkled seeds (dihybrid cross).

These crosses were conducted to study the inheritance of a pair of contrasting characters with relation oto the inheritance of the other coexisting pair of contrasting character in successive generations.

Mendel found that in F1 generation all plants were yellow with round seeds indicating that the yellow color and the round shape as were dominant over the green color and wrinkled shape that were recessive in nature.

On self-pollination, the F1 plants yielded the F2 progeny. These offsprings were of four different phenotypes in the ration 9:3:3:1 with 9 yellow and round, 3 yellow and wrinkled, 3 green and round and 1 green and wrinkled types of seeds.

It was thus observed that the two pairs of contrasting characters actually were transmitted independent of each other. The offsprings even demonstrated the new combination of characters in the form of yellow and wrinkled and green and round seeds in the F2 generation. The details of the experiments and their discussion are not discussed here.

Carl Correns, one of the rediscoverers of Mendel’s work in 1900, promoted the ideas of Mendel as the “laws of inheritance”. Following three concepts are recognized as Mendel’s Laws.

Mendel’s Laws

It was earlier believed that traits or characters of parents become blended, diluted and lost in the offsprings of subsequent generations. Mendel’s experiments have shown that these parental characters are determining by certain ‘factors’ (genes) and do not “mix” ir “contaminate each other” and expressin the progeny at a later stage. Mendel’s first law of inheritance was based on this evidence.

The Law of Uniformity

Plants with two contrating (one tall and the other short) characters when crossed, the characters do not blend. If any character is not expressed in the girst generation it may reappear without change in a subsequent generation.

The Law of Segregation

An individual possesses two factors (genes) for a particular character with each of these factors situated on one of the chromosomes of a homologous pair. At the time of formation of gametes each member of the pair of chromosome separate independently from one another so that each gamete carries only one chromosome of the pair and as such only one of the two factors (gene) responsible for the determination of a character.

In Mendel’s words, neither of the factors has “taken over anything from the other”. The genes of a pair are separated completely unaltered on a chromosome that migrates to a gamete during gametogenesis.

The Law of Independent Assortment

Members of different gene pairs (determining different sets of characters) that exist on the same chromosome, assort independent of each other during gametogenesis to migrate into a gamete. Because of such independent assortment new combinations between different sets of characters are produced in an offspring. For explanation refer to dihybrid cross.

The dihybird cross experiments yielded four different phenotypes in the F2 generation with yellow-round, yellow-wrinkled, green-round and green-wrinkled seeds. This implied that the genes responsible for yellow and green colors and round and wrinkled shapes of seeds separated out independently tha tresulted in four different phenotypes and 9 different genotypes in the F2 generation.

Summary

• Conclusions from hybridization experiments:
  • The factors responsible for inheritance of character are basically the genes.
  • The factors or genes for each character occur in pair.
  • These genes are transmitted from one generation to next through gametes.
  • Members of a pair of genes separate from each other at the time of gametogenesis so that each gamete carries only one gene.
  • Only one character (dominant) is expressed in first generation and both characters (dominant and recessive) are expressed in second generation when pure bred plants differing in pair of contrasting character is crossed.
  • Statistical laws are followed in transmission of characters.
  • Inheritance of one pair of factors is independent to other pair of factors in case of dihybrid cross experiments.
• Hybrid: Offspring of cross (mating) between two generically different organisms.
• Monohydrid Crosses: Cross (mating) between individuals or plants differing in a single pair of contrasting characters. Such cross yields monohybrids which are genetically heterozygous for the particular trait and factor.
• Dihybrid crosses: Cross (mating) between individuals or plants differing in two pairs of contrasting characters.
• Locus: The position of a gene on a chromosome is called locus.
• Allele: Alternative form of a gene present at any particular locus.
• Homologous: Chromosomes come in pairs in autosomes and as sex-chromosomes in the female. The members of the pair are identical to each other in their morphology. These chromosomes of a pair are called homologous.
• Homozygous: A condition of having same allele at a given loci on homologous pair of chromosomes.
• Heterozygous: A condition of having different alleles at a given loci on a homologous pair of chromosomes.
• Genotype: The genetic constitution or makeup of an individual.
• Phenotype: It is the physical, mental or biochemical manifestation of an individual in relation to a particular character resulting from the expression of associated genes. Phenotype may be influences by environmental factors.
• Dominant: Is a trait can express itself even in heterozygous state of a particualr gene (single dose) eg. tallenss.
• Recessive: It is a trait which is expressed only in homozygous condition (double dose) e.g. shortness of a gene.
• Mendel’s laws of inheritance:
  • The law of uniformity.
  • The law of segregation.
  • The law of independent assortment.

Techniques Used In Genetics

The understanding of the science of genetics has evolved along with the development of several molecular techniques. These techniques are based on basic principles of genetics and are applied to use genetic mechanisms for the benefit of humanity. Overviews of certain techniques are elicited in this chapter. Details of the techniques can be found in standard biotechnology textbooks.

Recombinant DNA Technology

In very basic terms recombination of DNA implies the insertion of fragments or more specifically, insertion of desired genes into certain host cells to utilize the inherent replication mechanisms of the host to produce multiple copies of the gene. This is nothing but cloning of the specific sequences of DNA and thus the process is also termed ‘genetic engineering’.

The specific gene to be cloned may be derived from sources such as another genome of an organism or artificially synthesized in the laboratory. Before the desired fragment of DNA is inserted into a suitable host cell, it is processed by separation from its source. This processed segment has two cut ends that integrate into the host genome and is called the recombinant DNA.

Idea of Recombination from the Nature

Interesting observations on the genetic behavior of bacteria and viruses have inspired the application of those mechanisms to evolve genetic techniques.

Bacteria and virus are called prokaryotes as they do not have cell nuclei. Eukaryotes are all the other organisms that possess a well-defined nuclear membrane. Bacterial DNA exists in the form of a looped thread-like chromosome or in the form of several smaller ring-shaped genetic material called plasmids. The enormously rapid replication rates of bacteria and virus make them favorites for becoming the host cells in recombination techniques.

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Plasmids have the unique property of easily entering a cell and promptly using the cellular mechanism for its replication. Scientists target plasmids for attaching the desired gene to transport them into the host cell. Thus the plasmids act as vectors of DNA.

Though the virus contain nuclear material inside their protein coat, it is mandatory for them to take the help of any other host cell for replication as they lack replicatory enzyme mechanisms of their own. The viruses usually infect bacteria as the host cell, replicate its components inside them, assemble and eventually rupture the bacteria to come out. Recombinant DNA is integrated into viral (bacteriophage) genome and then the virus acts as a vector for the integrated genome.

On the other hand certain enzymes evolve in the bacteria to fight such an invasion by viruses. One of them, the restriction enzymes is used as important tool by scientists in genetic engineering. Viral DNA segments can be fragment at desired sites with the enzyme. This enzyme is used extensively in cleaving required DNA segments from its source. Other enzymes like the ligases are used appropriately to anneal or join ends of DNA fragments.

Process of Recombinant DNA Technique

The sequential procedure of obtaining the desired fragment of DNA (gene) which is to be cloned, multiplication of the obtained gene in a suitable vector, combining the DNA fragment with that of the DNA of vector and transferring of the recombinant vector to the host organism comprise the process of the technique.

Production of the DNA Fragment

The cloning begins by cutting off the DNA at specific sites with enzymes like restriction endonucleases that recognize a set of short DNA sequences (4 to 8 base pair long). The enzymes are named as per their sources, e.g. Eco RI is from the organism E. coli and Hind III from Hemophilus influenzae, and number at about 300.

The endonucleases cleave both the strands of DNA but between specific pairs of bases. This cleaving produces either staggered ends or blunt ends at the interface of the cut ends of the DNA. These ends are called ‘sticky’ ends as these cut ends can unite with complementary sequences at any other cut end produced by the same enzyme on a DNA molecule.

Processing of the Vector

Vectors can be obtained from natural sources that can incorporate the desired DNA segment easily into its own genetic environment and transfer the integrated molecule into the host cell for independent and rapid replication. Plasmids, bacteriophages and cosmids are some of the common examples of vectors.

Plasmids, as already mentioned, consist of circular duplex of DNA and occur naturally in bacteria. Plasmids are obtained after disruption of bacteria and then cleaved by restriction enzymes. Restriction enzymes only act at specific sites on the plasmid.

Along with the desired DNA the plasmids also may contain genes expressing factors for antibiotic resistance and thus a particular strain of plasmid can be identified by detecting its resistance against a specific antibiotic.

Integration or Recombination of Desired DNA Fragment with the DNA of Vector

Same restriction enzymes are used to cleave out the desired DNA fragment from its source as well as to break the plasmid between specific bases. This type of breaks in the DNA fragment and in the plasmid’s DNA produces reciprocally complementary ends in both the ‘foreign’ DNA and the cleaved plasmid that combine easily.

As mentioned earlier, the ends of the cleaved DNA are termed ‘sticky’ as they easily combine with cut ends of the plasmid. The enzyme DNA ligase seals and secures the attached ends. The united DNA and the plasmid molecule are then called the recombinant DNA molecule.

Transfer of Recombinant Vector to Host Organism

After the recombination of the DNA fragment and the plasmid, this particle is introduced into the host cell by increasing the porosity of the cell membrane with the application of certain chemicals or high electric voltage across its membrane.

The recombinant molecule starts replication within the cell along with the nuclear material of the cell at each cycle of cell division. Eventually hundreds and thousands of copies of the desired DNA are produced with the help of the host cell machinery.

Screening of Recombinant Vectors

The host cell or bacteria does not accept all plasmids. This selectivity of acceptance creates two kinds of bacteria in the culture media-one type containing the DNA fragment whereas the other not containing it.

The plasmids contain certain genes along their genome that impart them resistance against certain antibiotics. In case the foreign DNA is inserted into the plasmid by cleaving a particular gene responsible for developing resistance for a particular antibiotic, such resistance would be lost in this plasmid though the same plasmid would maintain resistance against some other antibiotics, the genes for which remain intact.

This property of loss of resistance due to recombination is utilized for the detection of bacteria that have accepted the DNA fragments. Separate colonies of bacteria are exposed to the recombination. Bacteria from these colonies are cultured separately and representative bacteria from individual colonies are tested for susceptibility for different antibiotic.

The colony in the subculture showing susceptibility to a particular antibiotic specifies the cleavage in the plasmid and integration of the DNA fragment. These colonies from the master (primary) plate are picked and cultured separately and will contain only bacteria (host cells) with recombinant vectors (plasmids).

Screening of Clones with Specific DNA Sequence

The detection of the recombinant DNA integrated bacterium can be pursued with more refined techniques including nucleic acid hybridization. This method entails direct hybridization of labeled probes on to specific sequences on the recombinant molecule. The identified bacteria are isolated and culture to obtain the desired DNA segment.

The recombinant DNA molecules are thus generated and collected and this collection constitutes the DNA library.

Some Important Applications of Recombinant DNA Technology

Application of the recombinant DNA technology is widely accepted now as an important tool for several useful purposes such as:

• Preparation of chromosome maps and analysis of DNA sequences.
• Production of drugs like insulin, somatostatin, blood clotting factors, growth hormones, synthetic vaccines like antirabies, antimalarial, antihepatitis and cholera vaccines, interferon from genetically engineered E. coli to combat viral infections and monoclonal antibodies against certain organisms.
• Using in the diagnosis of genetic diseases and gene therapy.

Polymerase Chain Reaction

The amplification of DNA sequences (genes) described in the above sections is based on utilization of the host cellular mechanisms and thus is known as “in vivo” cell-based cloning. Genes can be cloned by non cellular “in vitro” techniques such as the polymerase chain reaction (PCR) that is done in machines.

Copies of DNA sequences can be produced in large amounts with PCR. The essential prerequisite for this technique is that we must know the sequences of the DNA of the either sides (flanking regions) of the desired segment to be cloned. This knowledge is mandatory for the formulation of the ‘primers’ (discussed later). Only a very small amount of DNA (even of a single cell) is needed to produce millions of copies of the DNA fragment.

Concept of PCR Technique

As shown in the figure below, DNA replication needs at least a single molecule of a double stranded DNA to begin with. The two strands are separated (denatured) with regulation of temperature. Enzymes, nucleotides and primers are then added to make-up a mixture in the PCR machine (thermo-cyclers). Once added, the nucleotides get arranged on each of the denatured DNA single strand. Thus the new complementary strand along with the old strand together forms the double helix.

The nucleo-tides are attached one by one to the primer CLEIC ACID PROBES to their 3’ends. The primers, as seen in the figure below, are attached one on each of the denatured starting molecule of the DNA at opposite ends. In the PCR technique, the primers (short nucleotide stretches) are added in the machine along with enzymes and the nucleotides. The primers are actually very short DNA sequences called deoxyoligo-nucleotides.

DNA polymerase as well as the four nucleotides are added to the cloning mixture. It is therefore essential to have an idea of the flanking sequences of the desired gene to be cloned in order to produce the primers. These primers essentially limit the stretch of the big DNA molecule to be replicated. The DNA molecule confined between the primers is acted upon by the artificial cloning machinery to replicate the trapped segment of the DNA.

The DNA fragments, the primers, the oligonucle-otides and DNA polymerase enzyme (the heat stable “Taq polymerase” derived from the Thermus aquaticus) are all incubated in the machine and the required temperature for amplification is regulated externally.

Each cycle of replication is repeated with fresh denaturation of the double helix and annealing of added nucleotides to the annealed primers. This results in the replication of a DNA segment in an exponential proportion. PCR thermal cyclers are automatic and need not be set again after each round of amplification. Modification of techniques can also produce mutations in DNA fragment, as desired.

PCR is a valuable technique used for detecting infectious agents like viruses, for prenatal investigations, tissue typing for transplantation, studying polymorphisms, evolution and several other applications.

Nucleic Acid Probes

Radiolabeled probes help to recognize complementary sequences in DNA or RNA molecule. This helps to identify and isolate the specific DNA sequences from an organism. Nucleic acid probes are small stretches of DNA that can be derived from various sources. These probes anneal to complementary sequences, if these complementary target sequences are present in the sample DNA.

These probes also help in the diagnosis of infectious diseases and identification of specific causal organisms. Forensic tests (DNA fingerprinting) are based on the same principle.

Detection of DNA Segments with Nucleic Acid Hybridization

The following steps are followed sequentially to identify DNA segments of interest from a given genomic population

• After the DNA molecules are extracted, they are digested with application of restriction enzyme so that they are cleaved into multiple segments of different sizes.
• The DNA sample is run in electrophoresis where the fragments are arranged according to their sizes along the gel.
• Bands appear on the gel at specific intervals depending on the molecular weights of the fragments.
• These bands are stained and visualized directly in the gel.
• These bands can be isolated for analyzing their DNA sequences. A particular gene (DNA segment) can be identified within those bands with the help of radio tagged molecular probes that bind to definite denatured strand.

A particular segment in a band in the gel can be identified by hybridization with molecular probes. This process requires transferring of the band from the gel to a nitrocellulose paper. This transferring technique is called ‘blotting’. Several types of blotting are enumerated according to the involvement of different molecules.

Blotting of DNA bands on nitrocellulose paper- Southern blotting.

Blotting of mRNA bands on nitrocellulose- Northern blotting.

Blotting of protein on nitrocellulose membrane- Western blotting.

Southern Blotting

Southern blotting is one of the most used techniques in genetics. DNA is extracted from a source and then digested using specific restriction enzymes that cut the DNA strand into several smaller segments at sites specific to the enzymes. This sample is then run in a gel electrophoresis equipment. The bands thus formed on the gel are then denatured with alkali.

This gel is now placed between a buffer saturated paper and a sheet of nitrocellulose membrane. The movement of the buffer from the paper to the nitrocellulose membrane passes through the gel and carries the denatured single strands of DNA from the gel to the membrane (blotting). The transferred DNA is now fixed on the membrane by heating it at 80°C for 2 to 3 hours.

The membrane can now be subjected to exposure to radio-labeled probes that hybridize to specific sequences in the DNA. After proper washing of the membrane, an autoradiograph of hybridized DNA may be taken on an X-ray film for the presence of the desired strand of molecule in any given band on the membrane.

The appearance of a band at a particular level will happen only when a DNA segment of the particular length is present in the sample after the application of restriction enzyme. Similarly, attachment of specific probes will depend upon the presence of the particular DNA segment complementary to the probe.

Bands at a particular level denote the presence of similar fragments that can be compared with a given reference. Bands may be of different thickness at the same levels of reference and across the length of the gel.

DNA Sequence Of Gene Or A DNA Segment

As an extension to the step of DNA isolation and segment identification, one may proceed to determine the sequencing of nucleotides on the DNA molecule (DNA sequencing). Several methods are utilized as tools for sequencing and are designed on the basis of different principles of genetics.

The most commonly used technique has been the dideoxy chain termination method. The more recent automatic sequencers apply a variant of this method.

Dideoxy Chain Termination Method (enzymatic)

This is one of the earliest processes employed in the late 70’s to determine DNA sequences. Denatured single stranded DNA fragments that are to be sequenced are taken in four different reaction tubes as the first step. All the tubes contain several identical copies of fragments of DNA molecules to be sequenced.

Radioactively labeled four different deoxy- nucleotides, enzyme DNA polymerase I and oligonucleotide primers all are added to each of the four tubes. The deoxynucleotides are molecules that anneal against complementary nucleotides on the denatured DNA strand and maintain the elongation of the new strand that is being synthesized.

Each tube also receives one of the four dideoxynucleotides and as such each tube has a different dideoxynucleotide. The dideoxynucleotide molecules are different from the deoxynucleotide molecules as the former lack in a hydroxyl group in one of their carbon atoms. The attachment of a dideoxynucleotide to the growing strand immediately stops the chain elongation.

This termination of chain elongation is randomly affected. This means that the termination of chain elongation depends on the ‘chance’ of attachment of the particular type of a dideoxynucleotide to its complementary nucleotide in the template strand. Therefore, in a given tube we can find millions of chains of different lengths terminated randomly on the event of attachment with the specific dideoxynucleotide in the tube .

Once (in a given tube) the particular dideoxy- nucleotides are incorporated in the chain with stopping of chain elongation, the fragments are taken out from the tube and run in electrophoresis. Terminated chains in all the four tubes are run in electrophoresis equipment on four adjoining lanes, each lane denoting chain terminations due to attachment of four different dideoxynucleotides. These attachments are random, giving rise to chains of different lengths that arrange according to their lengths in the gel.

From this information we can identify the particular terminal dideoxynucleotide that stops chain elongation and hence identify the complementary terminal nucleotide in that chain. In this way all the terminal nucleotides in all the chain fragments in all the four lanes can be computed in a sequence yielding precise sequencing of a DNA molecule. Autoradiographic methods are applied in detection of the radiolabeled dideoxynucleotides in the electrophoretic bands.

Automatic Sequencers

Modern day automatic sequencing machines are computerized and highly accurate and rapid. Different fluorescent dyes are attached to the oligonucleotide primer in each of the four reaction tubes. The resultant gel mixture is then electrophoresed in a single gel tube instead of four.

A fluorescence detector measures the color from the gel tube and automatically records sequences. This method is of course a further modification of the dideoxy process. The credit for rapidity with which the human genome project was conducted goes to these automatic sequencers.

DNA Fingerprinting

In all humans the genome comprises of the coding as well as noncoding regions. In the noncoding DNA regions the sequences are very repetitive and are called tandemly repeated DNA sequences. The collection of these repetitive sequences imparts unique identities to individuals. The pattern of occurrence, length and number of these repeats are unique and specific for each individual. The concept of DNA fingerprinting is based on the above principle.

DNA fingerprint is an important tool for identification of individuals, settlement of disputed paternity, criminal investigations and related purposes. DNA fingerprints in identical (mono- zygotic) twins are exactly the same.

The DNA Fingerprinting Technique

The technique begins with obtaining DNA from a source that may be as varied as the body fluids, cells or sequestrated dead tissues often several years old (DNA is usually a very stable molecule). Obtained DNA is cleaved into smaller fragments with the help of endonuclease enzymes.

The action of the endonuclease enzyme differs in individuals as the enzyme cuts individual genomes at different places due to the presence different patterns of the tandem repeats sequences in different individuals. These ununiform cuts in the genome give rise to DNA fragments of different lengths in individuals. The fragments of DNA are subsequently separated by agarose gel electrophoresis. Southern blotting is then applied to transfer the bands on to nitrocellulose.

As mentioned earlier, variability in individual genomes occurs specially in the noncoding regions in the forms of several polymorphisms. These polymorphisms exist as short (2-3 bp), inherited tandem sequence repeats like CACACA……that run repeatedly at several locations (loci) in the genome. The sequences are called microsatellites which occur throughout the genome at microsatellite loci. The numbers of such tandem sequences running at each locus may vary from 4 to 40 in different individuals.

This variation in the number of nucleotide runs at these loci is called Variable Number of Tandem Repeats (VNTR) or a hypervariable satellite sequence. The numbers of these variations are different in individuals and when several of these loci are taken into account, the diversity between individuals is enormous and the chance of two random individuals sharing the same genetic pattern is one in a billion.

Primers that bracket important loci with VNTRS have been developed. These bracketed variable sequences can be amplified with the help of PCR. A set of 5 to 10 such VNTR loci are chosen and amplified for the complete comparison between individuals. The band patterns of these variable sequences produced after electrophoresis are akin to the molecular fingerprint of an individual.

As discussed earlier, the presence of these variable sequences also alter the sites of action of restriction enzymes. Action of these enzymes on different individual genomes would cut the DNA at different places in different individuals. This polymorphism is called the Restriction Fragment Length Polymorphism (RFLP).

RFLP in the DNA produce bands at different levels (according to the different lengths of DNA segments) in the electrophoretic gel. This property of variance is used to detect the molecular finger- printing of a person or used to detect associations of a type of RLFP with a disease.

In the given example, a sample from the site of crime (e.g. a strand of hair from the victim’s nails) is amplified by PCR. A set of 3 variable loci are selected and amplified from the sample. Since each of the loci is represented in two homologous chromosomes, 6 bands are obtained after the amplification. Samples are collected from the suspects and the same loci are amplified and banded.

Identification of the culprit (individual 2 in the example) is accurate using this technique when the overall pattern of its bands matches significantly with that with the sample. This approach can be used in the testing of paternity. In cases of paternity disputes matching of at least half of the bands usually settles the issue.

Human Genome Project

A monumental project was started in 1991 in the USA involving 16 laboratories, 1,100 biologists, computer scientists and technicians from countries like USA, UK, France, Japan and others. The design behind the human genome project (HGP) was to sequence the entire human genome with a view to provideing an extensive understanding of DNA sequences, the organization, function and evolution of the human genome, to map both normal and anomalous disease- specific genes, give boost to functional and comparative genomics and bioinformatics.

Constant upgradation of techniques helped to complete this public funded project in February 2001; well before the stipulated time of 15 years. Two research facilities the Celera Genomic Corporation and the HGP working on the project released their data simultaneously to the world.

The project yielded several important observations about the human genome. About 3.2 billion base pairs constitute the human genome with approximately 30,000 genes in contrast to the earlier concept of presence of greater number of genes in the genome. This finding, supplemented with further studies in proteomics, has forced us to reconsider the validity of the “one gene-one function (enzyme)” concept in favor of a better explanation for protein synthesis. The genes comprise only about 5% of the genomic bulk. Rest of the genome is made up of noncoding DNA (junk DNA) containing segments of repetitive DNA.

Human DNA can be dated (DNA dating) with the analysis of such repeats and family trees can be assembled that explain the source, the point of first occurrence and subsequent evolution and dispersion of any segment of the genome in an individual, farly, clan or an entire race.

It is interesting to observe that several bacterial genes have been introduced by nature directly into the human genome without undergoing the grind of evolution.

Though smeared with its share of controversies, the HGP was a historical and colossal endeavor in Though smeared with its share of controversies, science that has brought forth vast data and understanding that would eventually benefit the mankind, with its proper utilization.

Stem Cell Research

Stem cells are those cells that are capable of self- renewal (can divide to produce cells with same properties) and also are able to differentiate into specific types (lineages) of cells. Thus stem cells are basically pluripotent and multipotent (if not totipotent) progenitor cells that not only divide to produce cells of the same nature but given the appropriate environment, they differentiate into other tissues as well.

It is evident that potency of cells diminishes as an individual grows from a zygote towards an adult. Therefore, it is understood that early embryos are the best sources of stem cells (embryonic stem cells). Stem cells can also be harvested from the umbilical cord at a later stage of life (cord blood stem cells).

In adults the bone marrow is a good source of stem cells (mesenchymal stem cells). It is to be noted that the potency of the stem cells depends on its source. After collection of the stem cell rich tissue, the stem cells are separated by various cell sorting techniques using sophisticated equipment as per the requirements. The stem cells bear several cell markers on their cell walls that give them specific identities.

Cells are preserved at very low temperatures (cryopreservation). When desired the cells can be thawed back to normal conditions. Cells can be then cultured for multiplication or put in an environment that leads the cells to differentiate into required progenitor cells. These cells are then injected into damaged tissues where they are thought to replicate and repair the damaged organs.

Embryonic stem cells are good models to study the effects of bacterial toxins, drugs, etc. One can preserve his/her own cord blood stem cells (banking) in case of a need to him (autologous transplantation) or others (allogenic transplantation) in the future. An important aspect of stem cell therapy is that it does not need stringent compatibility match for allogenic transplantations.

Prenatal Diagnosis, Techniques And Genetic Counselling

The term “Prenatal diagnosis” is used to define the process of detecting a disease or the risk of occurrence of a disease in humans. These tests are done well before the birth of the fetus. This approach of detecting severe debilitating or fatal diseases proves to be very helpful in pregnancies with a high risk for any such genetic disease. This gives an opportunity to the couple to decide the fate of the affected pregnancy before it reaches to a very advanced stage.

Prenatal detection of dental diseases is not practiced. Prenatal diagnosis of congenital and other genetic disorders is common in cases of diseases that are incompatible to life or diseases that cause severe physical and mental disability. Detection of dental disorders may be a matter of correlation or coincidence occurring as a part of these systemic diseases.

Prenatal detection of diseases is advised in cases of high-risk pregnancies with the history of previous incidence of the disease in the family.

Indications for Prenatal Diagnosis and Techniques

Couples having a history of any genetic disorder or undiagnosed physical abnormality in the family are ideal candidates for prenatal diagnosis of the fetus. History of a neural tube defect in the family or any genetic syndrome in a previous child, pregnant women of 35 years or above calls for a prenatal diagnostic test in the unborn child. In case of suspicion of an X-linked recessive disorder, mothers should be screened for their carrier status.

Few of the prenatal diagnostic techniques are summarized below:

Amniocentesis

Amniocentesis entails the process of collection of fetal cells for chromosomal analysis. The technique is preferably applied between 14-16 weeks of gestation. 10 to 20 ml of amniotic fluid is tapped through the abdominal wall of the mother and the cells that are collected are used for karyotyping (Refer Chapter 3).

Neural tube defects can be diagnosed in the prenatal state by estimation of a-fetoprotein levels in the amniotic fluid. Its level is raised in the amniotic fluid as a result of leakage from the open neural tube defects. The procedure of amniocentesis carries only 1% risk of abortion.

Chorionic Villus Sampling

Chorionic villi samples are aspirated with a catheter introduced through the cervix under strict asepsis and ultrasound guidance. Analysis of the cells is possible even without culturing them as the cells in these tissues grow rapidly. The biopsy of the tissue is used for biochemical assay or DNA analysis to detect any genetic disorder.

Chorionic villus sampling is preferably done during 10 to 11 weeks of pregnancy, i.e. a few weeks earlier than the designated period for an amniocentesis (14-16 weeks). The risk of abortion is enhanced due to the small gestation size and manipulation of the vital villus structures. It is rated at 2 to 3% and is higher than that with amniocentesis. The procedure may be associated with limb anomalies if carried out prior to 9 weeks of gestation.

Ultrasonography

It is a safe method of prenatal diagnosis both for the fetus and the mother. It is routinely performed at 12 weeks of pregnancy for detection of multiple pregnancies, fetal malformations, fetal age determination and classification of the type of placenta. Nuchal translucency (NT), exomphalos, rocker- bottom foot, etc. are some of the features related to certain chromosomal abnormalities detected with ultrasonography.

Serum Screening in the Mother and Blood Sampling in the Fetus

Detection of certain elements in maternal blood sample may act as clues for a probable disease in the offspring. The presence of “alpha-feto-protein” (AFP) in maternal serum at 16 weeks gestation indicates towards a neural tube defect (anencephaly or spina bifida).

Fetal blood is drawn under ultrasound guidance from one of the umbilical vessels by putting a transabdominal percutaneous needle into the mother’s abdomen. The procedure is often referred to as “cordocentesis”. The indication of this procedure is to use the collected fetal blood to arrive at a prenatal diagnosis of blood disorders and for chromosomal analysis. A high risk of abortion (about 10%) is associated with the procedure.

Genetic Counselling

At the existing levels of medical understanding and therapeutics the scope of curing genetically incurred diseases is quite remote. Interventions in terms of replacement of defective genes or their products by genetic engineering are not commonly practiced. Therefore, the knowledge of the principles of genetics can be used to detect the risk of incidence of some of the genetic diseases. This information, then, can be used to prevent their occurrences or reduce the severity of their outcome.

The services of a counselor should be sought by patients distressed with genetic disorders to help them attenuate their sufferings or couples, who are at risk of having a genetically abnormal child, to consider specific detection and treatment measures. The counselor should provide realistic and. appropriate suggestions about the disease.

Diagnosis Of Genetic Disease

It is vital that the patient and his relatives should be informed about the correct diagnosis of the disease as well as the mode of its inheritance. The risks for occurrence of the disease are calculated on the basis of the understanding of laws of Mendelian inheritance. The prognosis and availability of treatment, if any, should also be positively discussed. Arriving at a diagnosis may involve 3 pertinent steps.

• Family history
  One should make a detailed pedigree chart to analyze the mode of inheritance of the trait or disease.
• Examination of patient
  A careful clinical examination of patient will help to reach a correct diagnosis.
• Laboratory investigations
  This may include biochemical investigations, chromosomal analysis and molecular studies. Antenatal diagnostic interventions may be sought in high-risk pregnancies, e.g. amniocentesis, chorionic villi sampling, imaging, etc. that would aid correct diagnosis.

Management Of Genetic Disease

Parents as well as individuals should be clearly told about the diagnosis and the risk of occurrence or recurrence (as applicable) involved in case they decide to continue an affected pregnancy or to have children in future. A counselor should also tell them about all the options available for the management of any genetic disease.

As most of the genetic disorders are incurable, one should try to prevent or limit the disabilities of the disorder. Couples left with no other alternatives of having a normal child, should be recommend to think for an adoption. Subsequent to a positive prenatal detection and all explanations given by the counselor, the decision regarding the fate of the pregnancy has to be made only by the couple involved.

Gene Therapy In Dentistry

We have seen already in the preceding sections of this book that diseases are caused by defective gene structure and function. Several strategies have been adopted to treat genetic diseases caused by such defects. These strategies are either direct or indirect interventions directed to correct those defects. Indirect methods apparently try to treat the ‘results’ of the disease whereas direct interventions try to mend the ’cause’ (genetic defects) of these diseases.

Attempts have been made to cure genetic diseases with the correction or replacement of defective genes by molecular tools of genetic engineering. Several methods that have been adopted to treat genetic diseases at different levels have been discussed in the subsequent sections of this chapter.

Common Strategies To Treat Genetic Diseases

Prenatal Treatment

The scope of treating genetic diseases in fetuses inside the uterus is absolutely negligible till date. Positive prenatal detection of any genetic disease mostly results in its spontaneous or voluntary termination due to the absence of the option of a permanent cure at the genetic level. There is of course some hope that prenatal treatment for few diseases may evolve in the near future.

Read and Learn More Genetics in Dentistry Notes

A few disorders like congenital adrenal hyperplasia (CAH) and severe combined immunodeficiency can be treated inside the uterus (in-utero) to some extent. Low doses of dexamethasone are given throughout the pregnancy on detection of congenital adrenal hyperplasia (CAH). The later disorder may be corrected by transfusion of stem cells that give rise to immune precursor cells. In both the cases the intervention or the therapeutic correction is aimed at the level of the gene products and not at the level of the genes.

It is hoped that gene therapy may become a possibility in near future to treat a genetic disease at the level of the genes. This treatment can be extended not only to living patients but to fetuses detected with the disease in-utero. It is hoped that stem cell transplantations in-utero may treat many serious early onset genetic diseases. In-utero gene therapy has successfully treated cystic fibrosis in mouse.

Treatments at the molecular level include transplantation of stem cells (containing normal genes) at the site of defects or comprise replacement of the defective genes from affected cells within the body. Somatic cells can also be taken out of the body, injected with the normal replacement of genes and then put back inside the body.

Postnatal Treatment

Cure for most of the genetic disorders is not available due to our incomplete understanding of links between defective genes and their products. The therapies directed towards genetic disorders today mainly depend upon supplementation of deficient gene products (enzyme, protein, etc.) from extraneous sources.

Compounding our limited knowledge of the dynamics of genetic disorders is the difficulty to deliver gene products into the cell for intracellular metabolism. Replenishment of secretory products of cells into the extracellular milieu is comparatively a better strategy to combat a genetically mediated deficiency. On the other hand corrections have been attempted in the abnormal genes themselves.

Genes in the germ-line as well as somatic cells in affected tissues have been manipulated in order to integrate normally expressing elements in them. Though no single and foolproof therapy exists in treating genetic diseases, strategies as described below have evolved to minimize the disabilities rising thereof.

The following section discusses the strategies that have been conceived and are being worked upon for developing therapies for treating genetic disorders including dental diseases.

Supplementing a Gene Product

Genetic disorders resulting in the deficiency or reduced effectivity of a gene product (enzyme or protein) can be treated with supplementation of the product from outside. Recombinant DNA technology has proven to be a boon in this regard as it can yield large amounts of polypeptides that can be introduced in affected individuals.

Treating with Drugs

Drugs with varied pharmacotherapeutic effects intervene and allay symptoms in a few of the genetic dis- orders of metabolism. Cholestyramine helps to reduce cholesterol levels in familial hypercholesterolemia as does the chelating agent penicillamine in Wilson’s disease (in defective copper metabolism).

Transplantation or Removal of Tissue

Several approaches of reconstructive surgeries (autologous or allograft bone replacement) are usually tried in the patients who suffer from a major loss of alveolar bone loss. It is a priority to preserve the affected tooth and/or restoration of the diseased tooth is favored instead of sacrificing the tooth. The results of conventional therapeutic modalities in treating genetic diseases are mostly unpredictable at best.

Activation of body’s own reconstructive mechanisms can be targeted with gene therapy in such cases to hasten recovery. Manufacturing of tissues like bone has been attempted from within the body rather than from without.

Stem Cell Transplantation

Stem cell transplantation seems to be a viable option in the near future as a strategy directed for treating genetic diseases. Patients suffering from certain genetic disorders involving the blood cells can be injected with precursor stem cells that differentiate into the required population of matured blood cells. Compatibility matching between the donor and the recipient is mandatory except for administration of stem cells derived from fetal umbilical cord or bone marrow derived mesenchymal stem cells.

Concept Of Gene Therapy And Its Applications

Gene therapy is based on intricate principles of genetic engineering that involves correction of defective genes or their replacement with normally functioning genes in cells. Gene therapy may be of two kinds, Germ line gene therapy and Somatic cell gene therapy. Germ line gene therapy involves genetic manipulation of the defective gamete producing cells so that a normal gamete is produced and a corrected haploid complement of chromosome is transferred to the future generations.

This kind of gene therapy is of course associated with its own moral and ethical issues. Somatic cell gene therapy on the other hand the change of a given somatic environment of an inditargets only particular tissues or organs resulting invidual. This kind of therapy is universally accepted.

Treatments with protein delivery systems have been tried for sometime now. Supplementation with growth factor enhancement can be useful in replenishing bone loses in the alveoli of the mandible. These factors increase the turnover of bone production. The effect of externally introduced growth factors is extremely short-lived with the factors getting dissolved or being broken down by proteolysis. Gene therapy can be adopted as an alternative option for sustaining the delivery of such factors for a prolonged period.

The transfer of genes can be achieved in two ways. In one procedure the desired gene and the vector (within which resides the injected gene) is introduced directly into the area of interest or indirectly through the intravenous route. The vectors are taken up by the target cells.

This direct application of target gene is called the in-vivo process. On the other hand genes may be introduced into cells after being taken out of the body (biopsy) in the laboratory, with the help of inoculating vector viruses. These cells are further cultured (multiplied) and then put back into the host. This process is termed as the ex-vivo method of gene transfer.

Gene Therapy Involves the Following Steps

Identification of the Defective Gene

Several molecular techniques are used to detect defective genes (structural genes, promoter genes, etc.) that are responsible for causing disease. Identification of such genes can be done both in somatic as well as in the germ line cells.

Cloning of Normal Healthy Gene

Cloning or duplication of DNA sequences involves copying of structural genes, promoter regions and other segments of DNA that regulate the expression of that gene. The desired gene is generally cloned or copied inside a vector. The vector is capable of penetrating and depositing the foreign or corrected gene into a target cell. Once inside the cell, a structural gene may take the help of promoters that are already present in the cell for its activation.

Identification of Target Cell Tissue/Organ

Target cells or tissues are the ones that are affected by the functioning of the abnormal gene or genes, e.g. alveoli of the mandible that suffer from bone loss. Cells are taken out from the organ/tissue, genetically manipulated and then they are introduced back into the blood stream. The engineered cells ‘home’ at the target regions to resume normal function. Corrected genes may also be introduced directly at a desired location inside the organism.

The Method of Insertion of a Normal Functional Gene in the Host DNA

A physical and chemical method of gene transfer includes microinjection of DNA into the cells by electroporation (permeability of the cell membrane is increased by application of electric current), Calcium- phosphate precipitation where endocytosis of the DNA element is facilitated by precipitating it with calcium-phosphate.

Cationic liposome mediated gene transfer is another technique in which synthetic cationic lipid vesicles encapsulating DNA particles fuse with specific cell membranes and release DNA into the cell. The methods of physical gene transfer techniques have evolved with time and graduated from the most basic direct injection of the DNA (micro- seeding technique), usage of electrically charged aqueous liposomes (bags of lipid associated DNA) that pass through the cell wall, to the more sophisticated processes of gene delivery by the macromolecular conjugate method where a negatively charged target DNA molecule is attached to an oppositely charged chemical substance or antibodies that bind to certain receptors on the cell wall with subsequent endocytosis of the DNA construct to the interior of the cell.

More advanced physical methods include the transfer of genes with the gene activated matrices (GAMS) where naked DNA fragments are carried on polymer matrix tools for gene delivery as they are safer than the viral sponges. Non-viral or physical methods are attractive methods. DNA of relatively large size also can be delivered with the physical technique. The drawback of the physical method is that it is not as efficient as the viral methods because of its complicated designing and application. Though repeated application of gene transfer is possible with the physical technique but the effect of transfer is short-lived.

The most common method used in gene therapy is the viral vector method for gene transfer. Adeno- viruses and retroviruses are the most used vectors. Adenoviruses are DNA viruses and do not integrate their DNA into the host genome. The disadvantage with this vector is that the introduced gene may be unstable. The inserted gene is activated outside the host genome.

Retroviruses are RNA viruses that integrate into the host DNA. The inserted stretch of viral RNA uses the cellular machinery of the host to synthesize selected proteins from the viral genome but multiplication of the entire virus particle is not allowed by deliberately silencing certain regions of the incorporated viral genome. This prevents propagation of the virus themselves.

Though their uses have not been widely reported, lentivirus and herpes simplex virus are some of the other example of viruses used for this purpose. Adeno-associated viruses are also gaining acceptance for their selective benefits for this technique. All said and done, the transferred gene would only function normally when the coding regions for the gene as well as their regulatory elements are present in the host and more so, when they are correctly aligned. The designing of a perfect genomic architecture is the biggest challenge for the scientists.

The selection of types of promoters that influence the expression of introduced genes is an important aspect of gene transfer. The promoters are responsible for persistent, stable and elevated levels of gene expression. Erroneous selection of viral promoters has shown unregulated expression of undesired mammalian host genes. Promoters are varied in function and as such are put to trials before tagging with specific transferrable genes. The application of tissue-specific promoters is gaining popularity as they allow the genes to be expressed only in specified tissues.

Characteristics of Different Viruses used in Gene Transfer

Viral methods are actually nature’s own mode of gene transfer. Scientists have adopted this technique for delivery of genes to the target cells. Though an efficient technique, viral transfers of genes have their own safety concerns. The criteria for selection of a definite type of viral vector depend upon the tissue target, the duration for which the expression of the transferred gene is desired and the size of the concerned gene to be transferred.

Viruses have different characteristics in terms of their replication. Retroviruses infect only dividing cells whereas adenoviruses and adeno- associated viruses infect both dividing and non- dividing cells. Retroviruses can attach into desired region of the host cell DNA leading to a prolonged and stable expression of the gene. The disadvantage with retroviruses is that its application may cause mutations in the host genome by integrating the gene at ‘risky’ regions in the genome.

Adenoviruses on the other hand introduce DNA into the host cell where these DNA remain independent (called Episomes) and do not integrate into the host genome. Thus with each cell division the number of cells that contain the introduced DNA is reduced.

This results in the period of expression of the introduced gene being reduced. However, adenoviruses can be generated in huge numbers and as such the viruses can be introduced in large numbers directly to the desired tissue (in-vivo). Adeno-associated viruses integrate desired genes to sites in the genome that are not ‘risky’ in terms of mutagenesis.

Retroviruses are preferred and used for an ex-vivo type of gene transfer where cells like the blood or bone marrow cells are briefly taken out of the body and infected with the virus. These cells are then reintroduced into the body. The size of the introduced gene is a limiting factor in developing a fully functional vector. Adenoviruses are the tiniest of viruses and can accommodate a foreign DNA that is only a fraction of the size of its own DNA.

An important step before introduction of the vector virus into the host cell is to render it completely harmless and incapable of self replication within the host cell to cause damage or disease. Viruses are rendered deficient in replication by means of deleting certain elements from their genomes that are involved in replication. These viruses can be manipulated to grow only in laboratory settings and not in any settings outside the laboratory.

Ribozymes are certain types of RNA molecules that can act like an enzyme to cleave and destroy mRNA transcripts of cancer producing genes. Experimentally designed ribozymes directed against transcripts of the E6 and E7 genes of the oral cancer producing Human Papilloma Viruses (type 16 and 18) have been shown to cut and destroy the mRNA of those E6 and E7 proteins that cause defects in the cell growth regulation and produce tumors, especially oral cancers.

The DNA encoding such ribozymes can be introduced inside replication-deficient viral vectors and then these vectors could be used to transfer the gene into the oral mucosal cells to stop E6 and E7 translation and prevent unregulated cellular proliferation.

As discussed in the previous chapter as well as in appropriate segments in the book, the advancement in molecular biology has enabled us to understand the nuances of the development of human structure and the importance of several molecules that work in tandem and with immaculate precision to bring forth flawless and wonderful functional structures. The concept of molecular dentistry is fast gaining its due acceptance as research is progressing toward a detailed understanding of dental diseases and their management.

The human genome project, transcriptomics, proteomics and related developments have revolutionized the discipline of basic sciences. Clinical research is facilitating the application of the ideas of basic science to the benefit of the patients. The oral health professional community, of late, has emphasized their commitment to the need of improving standards of oral health care, education and training about research innovations, discoveries and their clinical applications like never before.

The capacity to design and fabricate tissues and organs has been achieved with interdisciplinary research involving material scientists and biologists, and is no longer a distant dream. Revelation of the regulations of molecular biology has enabled scientists to design models that simulate or mimic biological system.

‘Biomimetics’ is a new concept that uses genetics and stem cell biology methods to engineer biomimetic cartilage, bone, muscle and nerve tissues that have been applied to tackle clinical problems. Such an approach can be applied through molecular dentistry to improve soft and hard tissue engineering and towards regeneration of tooth and salivary glands.

It is to the credit of scientific advancement that it has also transformed imaging procedures. Starting from the application of simple dental X-rays, to the use of magnetic resonance imaging (MRI), 360 degrees craniofacial-oral-dental imaging, computer-assisted tomography, ultrasound imagining, digital radiography and innovations such as biomarker reporter molecule detection, usage of these modalities have changed dimensions of medical intervention.

Recent advancement in molecular genetics has not only aided to the diagnostic confirmation of a disease but also have pinpointed to the etiology of a disorder. Molecular techniques have identified the disease causing events or molecules to ultimate perfection and these tools have also given precise insights to the genetic maps and mechanisms dynamically involved in producing the disease.

Genetic as well as environmental factors affect tooth agenesis. Hypodontic individuals may show the characteristic in isolation or as a feature along with other traits of a syndrome. In both the cases though, hypodontia is determined genetically. Non-syndromic hypodontia involve the Msx1, Pax9, and Axin2 genes.

A few important genes associated with early embryonic development like the Shh, Pitx2, Irf6, and p63 have been implicated in several syndromes that induce dental agenesis. Molecular therapies and bioengineering methods can be used along with dental implants and other conventional treatment modalities for treating tooth agenesis.

The interaction between the genetic and environ- mental factors is complex and thus it is more difficult to implicate a single factor in the development of a dental disorder. Yet we can simplify to understand that there are conditions that are simple and result from single gene defects. The more complex conditions result from the interaction between a set of defective genes and environmental influences.

It is also imperative that one understands the mechanisms and events that shape the development of the craniofacial complex. These events are guided under strict molecular control. Details of each of the genetically defined dental anomalies are available in the book. In the subsequent section of the chapter we would preview the potential application of molecular treatment in dental disorders.

Genetic conditions may be simple (single gene regulated) and complex (multiple gene and environmentally regulated) situations. Genetics and molecular events related to single and multiple gene disorders are discussed in the appropriate sections of the book.

Gene theraphy was tried for the first time on a child suffering from ADA deficiency. Absence of the Gene therapy was tried for the first time on a child adenosine deaminase (ADA) enzyme results in inactivation of the white blood cells leading to incapacitation of the immune system. WBC’s culled from the boy were allowed to mix with viral vectors containing normal ADA genes.

The normal genes got transferred into the white blood cells through the vectors. These WBC’s were cultured further and transfused back in large numbers into the patient. Though the patient required repeated transfusions of the same kind at repeated increasing intervals, this effort paved the way for others improved techniques to follow.

Applications Of Gene Therapy In Dentistry

Use in Bone Repair

In-vivo gene transfer technology is utilized with adenovirus acting as vectors to carry the BMP genes to the diseased area. This recombinant adenovirus (Ad- BMP) population is directly injected to the site of the bony lesion. Lesions of periodontal diseases or surgical wounds can be healed and osseous defects can be treated with new bone replacement.

After being delivered, the genes encoding for bone morphogenetic protein-7 (BMP-7) in the virus tend to upregulate the bone forming mechanisms in the local diseased area and heal large wounds around dental implants in the supporting bones. BMP-7 belongs to the family of cartilage and bone producing gene family.

A mixture of the BMP gene and Adeniovirus has been successfully introduced into target cells at the defect. When inside the host cell, BMP-7 genes are seen to be guided near the host genome by the virus to precise locations where they are required to be present. The host cell stimulates the expression of the BMP that peaks in about ten days. The expression gradually tapers with time as the target gene does not get integrated into the target cell genome and do not get multiplied or replicated at the time of cell division. Thus the effect of the gene is temporary and to the advantage of the treatment.

Ex-vivo methods are also used to transfer BMP 2 and BMP 7 to the target cells. These genes are introduced into cultured keratinocytes outside the body and then introduced to the desired affected areas. The genes help to repair bones, ligaments and the cementum. New bone and blood vessels can also be formed from stem cells that are induced to express bone morphogenetic proteins.

Use in Salivary Glands

Gene transfer has successfully been tried in the salivary glands both with the in-vivo and ex-vivo models. The salivary glands are vulnerable to radiations applied to treat cancers of the head and neck regions. These structures also commonly get affected irreversibly with several autoimmune diseases (Sjögren’s syndrome, etc.). Repair in salivary glands has been achieved by inserting the gene that encodes the water channel protein aquaporin-1 or AQP-1 into the ductal cells of the gland.

This results in the nonsecretory cells of the ducts of the glands being converted into secretory cells thereby restoring the function of the gland. In another example of gene therapy, a definite gene for example, the one responsible for synthesis of the polypeptide histatin is delivered into the cells in the gland resulting in increased levels of its production in the saliva. Histatin being a natural anticandidal polypeptide is postulated to be effective in preventing or treating resistant oral candidal infection. Oral candidiasis is common in AIDS and also occurs secondary to dental implants.

Though the salivary glands are exocrine glands, they can be manipulated to act as an endocrine gland by gene transfer. Genes encoding hormones like the growth hormone can be introduced into the salivary gland. The new endocrine secretions from these glands are carried from the acini directly into the blood and serum.

Application of immunomodulatory properties of stem cells can be utilized to combat autoimmune dis- eases. Specific and local activation of certain genes also can act as mediators of immunomodulation and can prove to be good methods for restricting autoimmune diseases related to salivary glands found commonly in dental practice.

Use in Pain Management

The management of pain involves the participation of maximal resources in dental as well as medical practice. As it is well-established that the intrinsic mechanisms in the body to combat pain depends upon the expression of the endogenous opioids and their receptors, gene therapy has emerged as a promising tool for the management of pain at different levels.

Managing or eliminating pain is a major part of dental practice. The use of viral vector mediated gene transfer is being experimented as the technique to achieve expression of specific genes in the host cell.

The genes enhance the expression of endorphins and enkephalins and simultaneously upregulating the expression of the μ, delta and kappa receptors. This activation of opioid systems at the levels of the peripheral and the central nervous system causes delayed conduction of nociception with induction of analgesia.

Use in Periodontal Diseases

The introduction of the Porphyromonas gingivalis (P. gingivalis) fimbrial gene into the salivary glands through plasmids has been tried successfully with adenovirus recombination. This experiment has resulted in two outcomes. The DNA delivered directly into the salivary glands of the mice has lead to the production of immunoglobulins like IgA, IgG in the saliva as well as antibodies IgG in the serum.

The salivary antibodies are able to reduce plaque formation by neutralizing the plaque forming organism P.gingivalis. Researchers have also identified and isolated the fimbrillin gene. The fimbrillin protein is one of the surface proteins of the organism P.gingivalis.

Recombination and transfer of the fimbrillin gene through adenovirus vectors into salivary glands is expected to secrete the protein fimbrillin locally around the gland and in the saliva. The availability of fimbrillin in the saliva would attach to the pellicle elements and thus prevent the harmful P. gingivalis to attach to the pellicle and form plaque.

Periodontal diseases can be controlled by pre- venting the process of tight microbial attachment to the infecting surfaces. The degree of virulence of a pathogen depends to a great extent to the levels of attachment of the pathogen to a surface. Adherence is brought about by the expression of “tight adherence genes” as found in a certain strain of Actinobacillus. Localized and destructive periodontitis results from Actinobacillus.

The strength of adherence adds to the degree of pathogenicity of organisms. A strategy has been evolved that uses application of artificially mutated strains of the organism deficient in the ‘Tight adherence’ gene. These strains when introduced with the virulent strains of organism colonize with them. This cocolonization of the mutated with the virulent strain causes limitation in the extent of pathogenic colonization of the organism. The spread of periodontitis can be prevented with the help of application of this model of genetic engineering.

Similar to the strategy applied to expedite bone growth, osseous defects in the periodontal region can be addressed by the application of in-vivo or ex-vitro gene transfer of BMP 7 and BMP 9 genes with the help adenovirus vectors into affected regions in the oral cavity. BMP 2 can expedite the formation of blood vessels.

Stem cells with specifically activated genes may also differentiate into osseous tissue on application into the defects.

Hard and soft tissue regeneration is distinctly related to the growth factor called the Platelet Derived Growth Factor (PDGF). This factor is a potent sub- stance and has profound action on cellular proliferation. In situations of tissue injury the interactions between the receptors for this molecule and the PDGF is disrupted that limits the activity of the growth factor. Investigators have tried to transfer the PDGF gene through an adenovirus to the injured areas in order to enhance cell signaling and proliferation.

Use in Keratinocytes

Keratinocytes are preferentially used as targets for the study and therapeutic application of gene therapy. This is due to the fact that keratinocytes being epidermal cells are easily accessible. Culture models for keratinocytes are well-founded techniques. The technique of gene transfer as well as their subsequent therapeutic application and monitoring are simpler in keratinocytes.

Researchers have used the ex-vivo method to transfer genes into cultured keratinocytes with retroviral vectors. These viruses insert the foreign gene permanently into the keratinocyte genome. The keratinocytes are then cultured easily in sheets and are applied for treatment in specified areas.

This technique can be used as gene product delivery systems in the oral mucosa and elsewhere. As keratinocytes are well-designed to deliver proteins, epithelial sheets have already been experimentally made that deliver proteins like apolipoprotein E.

Use in Cancers of the Head and Neck

As mentioned elsewhere in this book the role played by the p53 molecule in detecting structural DNA damage is of immense importance. This system of surveillance identifies defective DNA and stops the progress of the cell cycle and instructs either a DNA repair or cellular apoptosis. Efforts are being made to develop adenoviruses that when introduced into the system replicate and destroy only those abnormal cells that contain a mutated p53 gene.

Normal cells remain unaffected and repopulate the tissue. Such a therapy can boost the outcome of treatment in cancers if they are tried along with the conventional modes of cancer therapy. The genomes of these viruses are manipulated in such a way that their propagating machinery is activated only in conditions where it detects an abnormality in the host p53 molecule.

As discussed earlier, the application of the ribozymes to inactivate the Human Papilloma Virus (HPV 16 and HPV 18) proteins E6/E7 that mediate cancerous growth in the oral cavity has led scientists to create recombinants using the DNA coding for those ribozymes from the protein mRNA.

This strategy is under development and investigators hope that its application would not only halt progression of a primary tumor but also help to scavenge dysplastic cells not yet turned malignant.

Use in Growing New Teeth

Though quite futuristic in outlook, the idea of growing teeth in the laboratory and transplantation to edentulous patients has been worked upon for some time now. This feat of bioengineering would create teeth almost with the composition similar to normal teeth but without nerves or blood vessels.

This effort would involve the identification and activation of several genes that are associated with synthesis of over more than 25 proteins constituting dental tissues. The discovery of the role of the master gene PAX 9 will help to understand the sequence of gene activation critical for fabrication of tooth in time to come.

Dental tissues or dissociated dental cells have been used for at least sometime now for tooth engineering purposes as a part of recombination experiment. Recently, of course, certain type of stem cells and types of non-dental cells have been applied in tooth bio- engineering. These cells range from mesenchymal stem cells, bone marrow stromal cells to dental pulp stem cells.

In 2009, researchers at the Akita University in Japan have reported a novel epithelial-mesenchymal interaction experiment. The report explains an attempt of tooth regeneration by recombination of intact dental epithelium with a transformed, continuous dental mesenchymal cell line (see Suggested Readings) called the odontoblast-lineage cells (OLC).

Interestingly, these cell lines were grown on three dimensional, Use in Periodontal Vaccination in-vitro organ culture constructs and also transplanted beneath the renal capsule in mice as an in-vivo experiment. The OLC seem to have shown induction of dental development in both the in-vivo and the in-vitro models.

Other Modalities of Bone Repair with Gene Therapy

The introduction of Bone sialoprotein (BSP) in areas deficient in osseous tissue can trigger alveolar and periodontal bone proliferation. BSP is expressed in the event of bone repair and regeneration. This gene controls cell differentiation. It has also been found that the BSP is under the control of the mastergene Cbfa. Bone sialoprotein is non-collagenous in nature and one of the chief constituents of bone.

The application of the new NTF-hydrogel technology is based upon the delivery of a nonviral gene mixed with a hyaluronic acid-derived, non-immuno- genic gel at the site of an osseous defect. This technique can be used as an adjuvant to conventional therapies.

This method does not invoke any immune reaction and helps in bone regeneration by inducing the resident cells at the neighboring sites of the wound to add new bone to fill the defect.

Vascular endothelial growth factor (VEGF) delivery into rat mandibular condyles involving in-vivo technique have proven to be of help in cases of craniofacial deformities. This growth factor when delivered using adeno-associated virus (rAAV), have shown subsequent increases in certain osteogenetic and chondrogenetic markers accompanied by increase in the size of the mandibular condyle (See Suggested Readings).

Delivery of antiapoptotic genes like the Bcl2 gene to the site of tissue injury could be effective in recovery. This process involves much more localized delivery of the gene. The gene is actually processed with the gene activated matrix (GAM) technology (as done with NTF-Hydrogen) prior to its application.

These “DNA devices,” are the latest concepts in fabrication of special dental implants. Implantable products are made biocompatible by coating them with polymers capable of incorporating intact DNA molecules. The delivery of specific genes at the required sites creates implants with site-specific gene delivery.

Use in Periodontal Vaccination

The immunization of the salivary glands with non- virulent DNA encoding P. gingivalis and its fimbrial protein using plasmids and adenovirus has been discussed in the preceding paragraphs. Vectors like the Streptococcus gordonii have successfully been tested in animal models against the organisms like the P. gingivalis that cause periodontitis.

It has been observed that inoculation of hemagglutinin in a certain variety of rats increases the levels of IgG antibodies as well as enhances the production of interleukins as an immune response. The availability of these immune mediating factors induces protection against attack of P. gingivalis. Since hemagglutinin has been identified as one of the virulence factors of P. gingivalis, the production of antibodies against hemagglutinin provides such a protection.

Genetic Approach to Biofilm Antibiotic Resistance

It is interesting to note that certain microorganisms become resistant manifolds to antibiotics as they start living in microbial colonies attached to surfaces. This phenomenon is called biofilm formation. The reasons for the development of such resistance are not well understood and may be attributed to the activation of definite genes like the ndyB, which is related to the synthesis of the enzyme glycosyl transferase.

Glycosyl transferase is further linked to the production of periplasmic glucans that impart them resistance against disinfectants and antibiotics. Scientists have been able to identify, isolate and replicate a mutated version of the ndvB gene. This gene when introduced into some of the replicating cells in a pseudomonas biofilm, rendered the other members of the biofilm vulnerable to common antibiotics. Such an approach can be adapted for application in dentistry to destroy resistant bacteria in a biofilm.

Use in Alveolar Remodeling

Alveolar remodeling is a natural phenomenon that occurs due to stress, injuries and inflammation of the periodontal tissue. The alveolar structures including the bone undergo active remodeling as a reaction to mechanical stimulation. The process of remodeling can be expedited by enhancing the expression of several factors that induce and maintain alveolar remodeling.

This can be achieved by the transfer of the LacZ gene into the periodontal tissue directly with the help of a plasmid. The integrated gene within the plasmid can be introduced into cells with the application of an electric impulse (electroporation).

Used in Antimicrobial Control Disease Progression

Host defense mechanisms can be boosted with the introduction of genes that contribute to host cell defense against pathogens. This boosting may be done with supplementation of genes encoding certain anti- microbial agents. These factors or genes can be introduced into the host cell through retroviral mediated in-vivo techniques into the host genome at areas susceptible to infections.

Some proprietary products are available that applies the defensin-2 gene for this kind of an effect. The above discussion on the application of gene therapy can be reviewed in terms of the basic designs of gene transfer into the cell. The approach adopted for gene delivery may be an in-vivo technique involving gene constructs trapped in physical or viral agents and delivered into the cell.

The ex-vivo method transfects cells in culture in-vitro and then introduces them into the target cells in the body. The protein-based methods apply the gene products to the required regions and the cell-based approach uses mesenchymal stem cells for activation of tissue repair.

Constraints and Limitations of Genetic Therapy

Though a lot has been written both in favor and against the application of gene therapy, the message is clearly home that a foolproof therapeutic package involving gene therapy still needs some more ground-work to become a practicable reality. The regulatory authorities have been rightfully alarmed by outcomes of certain trials and are skeptical about the safety as well as the feasibility of such therapies.

Planners have reiterated the need of extensive preclinical trials of novel therapies before they become standard modes of treatment. Other issues related to the confidentiality of genetic information, disclosure of susceptibility concerns and the risk statuses of individuals are a few of the ethical aspects that need to be addressed in context of gene therapy.

There are several systemic disorders associated with specific types of periodontal diseases. Treatments in such cases are basically framed on the logic of treating not only the defect within terms of the parameters of dentistry but treating the symptoms of the disorder as a whole.

Chronic and early-onset periodontitis need chemical and mechanical control of bacterial plaque. Severe congenital neutropenia or depletion in IgA levels my cause premature loss of teeth and need antibiotic prophylaxis along with chemical and mechanical control of bacterial plaque.

Conditions associated with hormonal changes and arising due to unresponsive bacteria call for extensive and rigorous bacterial control.

Diseases and traits that are genetically transmitted have been studied extensively and analyzed for their causative molecular defects and the modes of their inheritance. The frequency of occurrences of the coding elements of the genome has been studied along with that of noncoding sequences in the DNA.

Certain disease causing genes have always been found to occur along with certain noncoding sequences. They have been identified always to occur together and the details of this occurrence is analyzed in linkage studies. This phenomenon occurs perhaps due to the close proximity of these two segments in the genome that always segregate together in the gamete.

As stated earlier, molecular research has revealed that specific regions of the non-coding regions are intimately associated with the inheritance of a particular gene. HLA associations of the disease producing genes have also been discussed earlier. Several thousands of similar genes have also been found existing across different organisms. The sequences of these stretches of DNA have not been defiled or disturbed by time and evolution in the organisms.

The genes and allied segments in the genome are said to be highly conserved in terms of structure and function. The origin of these genes and their subsequent distribution in the nature can be studied by analyzing their inheritance and linkage patterns.

The virulence of certain microorganism as well as susceptibility to diseases in an individual is determined by the genetic make-up of the microorganism as well as the individual. Craniofacial birth defects, orthognathic disorders, abnormal tooth size and shape, cancers, temporomandibular joint diseases and several others are linked to outcomes of gene-gene, gene-environment interactions.

Though gene therapy seems to be the panacea for all genetic disorders, it has its own share of limitation and pitfalls. The technique of gene delivery is tedious and difficult. Even if the gene causing a disorder is identified and mutations are well-defined, an attempt to introduce the corrected version of the gene in a cell may not be successful.

The limitations range from difficulty to pinpoint the exact gene responsible for a disease (except for a single gene disorder), developing an ideal vector for a gene, identification of the site of delivery, compatibility of the environment in the host tissues and eventually the normal and desired expression of the inoculated gene in the system.

The success of gene transfer cannot be predicted successfully specially in cancers as there are multiple affected sites in the system, which makes the decision of selection of the target region difficult. Some of these problems can be circumvented with the understanding of the mechanisms of viral replication and gene regulatory pathways.

Issues related to the durability and integration of the transferred gene is of immense significance as the desired period for sustenance of therapeutic benefits from a gene transfer varies with the type of the disease being addressed. Genetic integration of the transferred material into the host genome provides a long-term replication as well as expression of the gene.

Such functional durability comes at the cost of certain risks of undesirable effects. Unwarranted and unexpected integration of genes at different locations may trigger and disastrous consequences. Multiple introduction of gene therapy is possible with physical agencies but frequent repetition of gene transfer using viral vectors is not recommended.

Precise introduction of genes is the prime requisite for delivering nonspecific apoptotic genes that kill cancer cells. These genes don’t need to integrate into the genome but become active anywhere inside the cell. Applications of these ‘suicide genes’ produce more immediate effects irrespective of the site of application.

The success of gene therapy also depends upon the degree of immune responses elicited by the host especially against viral vectors. Viral vectors elicit immune responses in the host against themselves if the host cell recognizes the vector as “foreign”. In fact development of such immune responses is desired in the host immune system if the therapy is directed against cancer cells in treating carcinomas.

Undesired immune responses reduce the efficacy of the therapy. Repeated applications of viral vector mediated gene delivery may cause increased immune mediated destruction of the viral vectors or may result in serious side effects. Usage of viral vectors may be a potential cause of toxicity, immune and inflammatory responses with the very first instance of its application.

Other than perhaps single gene disorders, more commonly occurring diseases like hypertension and diabetes are dependent on more than a single factor and hence the applicability of gene therapy in such situations is debatable. The other more contentious issues with gene therapy are related to ethical considerations like questions regarding the criteria that decide what is ‘normal’ and what defines ‘abnormality’.

‘Whether a disability can be viewed as a disease’ and ‘whether a somatic gene therapy is more ethical than germ line therapy’ are some of the probing questions that remain to be answered. The issues of the feasibility of developing such expensive treatment modalities and the affordability of these regimes by less affluent population are also unanswered. Majority of diseases in dentistry are difficult to treat with single gene transfers.

New interventions that combine gene therapy with other approaches such as stem cell therapy are fast emerging. Gene therapy has the potential to treat diseases such as cystic fibrosis, cancers, heart diseases and human immunodeficiency virus infection. However, no clinical trial of gene therapy has resulted in the development of a commercially available treatment till date.

Unsettled issues in gene therapy also include the effectiveness of delivery, longevity of the therapy and safety of the procedures. While patient groups are largely satisfied with the current disease- based approach to gene therapy research, scientists have called for more studies on vector safety, delivery techniques, identification the molecular causes of diseases and finding the reasons for uncertainty of outcomes of current applications.

Gene Therapy In Dentistry Summary

• Control of genetic diseases have been tried with several strategies applied both as prenatal as well as postnatal treatment modalities.
• Common strategies to treat genetic diseases include supplementing a gene product, treating with drugs, transplantation or removal of diseased tissue and stem cell therapy.
• Therapy at the level of genes is called gene therapy. Gene therapy may be applied to the germ line cells or directed towards somatic cell lines.
• Gene therapy involves the steps of identification of the defective gene, cloning of normal healthy gene, identification of target cell (tissue or organ) and insertion of a normal functional gene into the host DNA.
• Physical and chemical methods as well as viral vectors are used for gene transfer.
• Genes can be transferred directly into affected tissues (in-vivo process) or may be introduced into cells taken out of the body (biopsy) and then put back into the host (ex-vivo process).
• Ribozymes are certain types of RNA molecules that can act like an enzyme to cleave and destroy harmful mRNA transcripts.
• Gene therapy can be used in bone repair, in treating diseased salivary glands, for pain management and in conditions of periodontal diseases. Applications are also being tried to treat cancers of the head and neck region and for active alveolar remodeling.

Genetics Of Cancer

The term “cancer” is derived from the Greek word for crab. Hippocrates likened the spreading of cancerous tumor to the shape of a crab’s claws. Cancer is a disease characterized by uncontrollable and unwanted growth of body cells due to the loss of their normal regulatory controls.

Majority of cancers manifest in the form of solid tumors. A cancerous tumor is often a collection of many abnormal cells, most of which divide wildly. Cancerous tumors infiltrate neighboring tissues by forcing their way between normal cells and may spread to distant places in the body through blood or lymph vessels.

Characteristics Of Cancer Cells

Cancer cells are different from the normal cells. Cancer is a disorder involving dynamic changes in the structure as well as the function of the cellular genome in the cancer cells. Following changes are observed in the cancer cells:

• Unrestricted cellular proliferation-Cells affected with cancerous changes lose their usual control over growth and division. The unrestrained growth and division of the cancer cells hamper the normal functioning of the body as a whole by disrupting metabolic activity of the organism and also by causing local effects by the growing mass.
• Transformation-Cancerous cells are transformed cells. These abnormal cells are transformed and become independent of factors usually needed for normal cell growth and proliferation.
• Ability to invade-One of the potent properties of cancer cells is their ability to invade from their site of origin into the surrounding healthy tissue.
• Metastasis-Cancer cells characteristically scatter away from their origin and disseminate to distant parts of the body where they seed and proliferate.
• Suppression of apoptosis-The program for normal cell death (apoptosis), that usually operates in a healthy cell is altered and suppressed in cancer cells.
• Angiogenesis-Cancer cells have the ability to induce new vessel formation or neo- vascularization in the tumor mass to facilitate the availability of oxygen and nutrients.

Factors causing or aiding cancer can be grouped as environmental and genetic. Certain environmental components account for the occurrence of approximately 80% of all human cancers and are hence preventable.

Read and Learn More Genetics in Dentistry Notes

Factors Responsible for the Causation of Cancer

Cancer may develop either due to environmental as well as genetic factors.

Environmental Factors of Cancer

Chemicals: Many chemicals like polycyclic aromatic hydrocarbons (3, 4 benzpyrene), aromatic amines (B-naphthylamine), vinyl chloride and arsenical compounds are known carcinogens and may cause cancers of lung, skin, bladder and liver. Substances like tobacco and alcohol are associated with cancers (squamous cell carcinoma) of the oral cavity.

Radiations: Similarly ultraviolet light (exposure to sunlight) is proven to be carcinogenic for skin (malignant melanoma and basal cell carcinoma) in fair skin people. High dose of ionizing radiation is well known carcinogen especially in people working with radioactive materials. Ionizing radiation is responsible for leukemia and cancers of skin, thyroid, bone and breast. Melanomas are common forms of cancer that involve oral mucous membrane in addition to their usual site of occurrence, the skin.

Viral infection: Many viruses are considered strong carcinogenic agents. About 15% of all human cancers are due to viruses. Several human tumors have known viral etiology, e.g. infection with the human papilloma virus (HPV) causes carcinoma of cervix. HPV infection is incriminated in development of squamous cell carcinoma that constitutes about 95% of all oral cancers. Epstein-Barr virus is responsible for formation of Burkitt’s lymphoma and nasopharyngeal carcinoma. This virus is associated with oral lesions called hairy leukoplakia, especially in patients suffering from AIDS. Certain leukoplakias of the oral cavity may be precancerous conditions. Hepatitis C and B virus produce liver cancer and RNA retrovirus leads to T-cell leukemia and lymphoma.

Bacteria and other microorganisms: Cancer may also result from bacterial infection (H. pylori can produce lymphoma and gastric carcinoma), toxins of fungi (aflatoxins can cause cancer of liver) and parasites like schistosoma can cause bladder cancer.

Production of Cancer by Carcinogens

The Production of Cancer by a Carcinogen Involves a Multistep Process

In the first step the presence of a carcinogen causes a lesion in the cell’s genome (in the DNA of the target cell) that leads to the transformation in the cell. In the second step this transformed cell divides repeatedly (clonal proliferation). This uncontrolled cellular proliferation is the main event leading to formation of carcinoma.

In the third step the clonal proliferation of tumor cells acquire autonomous growth, i.e. they no longer require the stimulation by carcinogen or other intrinsic factors and rapidly proliferate themselves. In still later stages tumor cells acquire the ability to invade the surrounding tissue, metastasize to distance places in the body and induce vascularization of the tumor.

What is Cell Proliferation and How is it Controlled?

Unrestricted cell proliferation is the main characteristic of cancer. Carcinomatous changes in a cell are brought about by disruption in the normal mechanisms that control cellular proliferation and differentiation. Thus in order to understand the dynamics of cancer we need to understand what is cell proliferation and how it is regulated. Normal proliferation, differentiation and growth in cells are sequentially controlled by the following events:

• Binding of growth factors to specific receptors on the cell membrane.
• Activation of growth factor receptors that further activates signal transducing proteins on the inner surface of the plasma membrane.
• An appropriate signal is then transmitted to the nucleus through certain messenger proteins across the cytoplasm.
• DNA transcription is initiated by the activation of transcription factors that bind at specific regions on the genome to activate transcription.
• Cell enters into mitosis after passing through the checkpoints and eventually undergoes nuclear and cytoplasmic division.

The events mentioned above operate under strict genetic control. Abnormal proliferation of ceils may result from mutations that alter the functions of genes governing cellular proliferation. Uncontrolled cellular proliferation can be studied vis-à-vis the mechanisms of normal cellular life cycle.

Signal Transduction In Cell Proliferation

Several of growth factors (GFs) and growth factor receptors (GFRs) exhibit significant role in normal cellular growth and differentiation. Various types of these factors initiate specific course of action. It has been observed that factors like the epidermal growth factor (EGF) stimulates epidermal cells, fibroblast growth factor (FGF) stimulates fibroblasts, platelet derived growth factors (PDGF) stimulates proliferation of connective tissue, etc.

These factors bind to the receptors on the cell membrane in order to exert their action. The sequential activation of successive events through cascading pathways resulting in cellular activity, growth, differentiation or proliferation is termed as signal transduction. Thus extracellular growth factors trigger cellular events through complex pathways. Each of the steps in the pathway is controlled through specific genes and their activity.

Binding of a growth factor to its specific receptor leads to the activation of the receptor.

• A series of cytoplasmic proteins get activated by the receptor in a cascade of reaction. These proteins are called signal transducing proteins. Many of such proteins are present on the inner surface of plasma membranes.
• Two important signaling proteins are produced by the ras and abl genes.
• During the resting state of the cell the ras families of proteins bind to GDP (guanosine diphosphate) constitutively.
• On stimulation by growth factor receptors, inactive ras becomes active by releasing the attached GDP and binding to a new GTP (guanosine triphosphate) molecule.
• The activated ras further turns on cytoplasmic kinases that pass signals to the nucleus for cellular proliferation.
  [The life of activated ras proteins is very short. The enzyme guanosine triphosphatase (GTPase) hydrolyzes GTP to GDP and inactivates ras proteins thereby downregulating cytoplasmic kinases. As a result the cell no longer responds to a signal till further activation. The abl gene induces different signal transducer proteins. This gene is located on chromosome 9.
• Cytoplasmic kinases enter the nucleus and activate a large number of genes immediately and very early (myc, myb, jun, fos and rel gene). The activity in these genes further regulate transcription of specific DNA segments.
• The myc protein is the one to get frequently bound to specific DNA sites after a cell receives a signal for proliferation.
• The activity of myc proteins induces transcriptional activation of several growth related genes including cyclin D (see later in cell cycle). The quantity of myc protein reduces back to basal levels once the cell enters the cell cycle.

Signal Transduction (Genes And Cancer)

Defective signaling in growth regulating pathways can lead to abnormal growth. Overexpression of growth factors can result in nonneoplastic disorders like psoriasis. Abnormalities at the level of growth factor receptors can lead to conditions like insulin-resistant diabetes (insulin receptor) and dwarfism (fibroblast growth factor receptor). Development of carcinomas, however, involves multiple steps and show other features such as invasion and metastasis.

These multiple steps include unregulated expression of growth factors, receptors or components of other signaling pathways. As discussed in earlier chapters, abnormal expression of components regulating signaling pathways is caused by mutations in the responsible genes. These mutant genes are called oncogenes.

Growth Factors and Cancer

Genes coding for growth factors may acquire oncogenic properties after mutation. For example, the gene coding for PDGF after mutation over expresses the growth factor which give rise to cancers like osteosarcoma and astrocytona.

Growth Factor Receptors and Cancer

Genes coding for growth factor receptors have been found to be mutated in several carcinomatic conditions. Such mutations are believed to induce continuous signals for cell growth and proliferation, even in the absence of growth factors.

Signal-transducing Proteins and Cancer

The ras genes that produce signal transducing protein are susceptible to mutations. Such mutations are responsible for almost 30% of all human tumors. As a consequence of mutation the enzyme GTPase is unable to hydrolyze the active GTP back to inactive GDP. Thus the ras protein remains constantly active and the cell continues to proliferate. Similarly a mutation in the GTPase protein itself leads to a defective enzymatic action that fails to restrain the activated ras protein. This eventually results in cancer.

Transcription Factor and Cancer

Several early and immediate gene products link the activities of growth factors to other factors that results in transcription of the DNA. The myc gene binds to DNA and activates many transcription factor elaborating genes involved in growth. Mutations in transcription factors with overexpression contribute to sustained proliferation.

Cell Cycle Control

The Cell Cycle

The cell cycle includes all events of cell growth, cell activity, replication of the DNA content and cell division that gives forth the daughter cells. This process is divided into four sequential phases.

• G1 phase (Gap phase or presynthetic phase) – This phase starts immediately after completion of cell division. The chromosomes gradually become thinned and extended. Cells are responsive to growth signals. They may or may not enter the next cell division depending whether signals are positive or negative with respect to cell division. Cells like neurons that are highly differentiated and lose their capability to divide further are shifted to GO phase. Cells in the GO phase usually subserve their functions and perish after their life is over. These cells may reenter into mainstream G1 phase for replication under special circumstances.
• S phase (Synthetic phase) – It is called S phase as DNA replication or synthesis occurs in this phase.
• G2 phase (premitotic phase) – G2 phase is short where chromosome begins to get condensed in preparation for the cell division phase that comes next. All the above three phases described above constitute the interphase of the cell cycle. Cells usually spend the bulk of their functional lives in the interphase.
• M phase (Mitotic phase) – The M phase results in complete nuclear and cytoplasmic division of a cell into the daughter cells.

Cell Cycle Checkpoints

As cells transit from one phase to the next in the cell cycle, all the events during such transitions are scrutinized and regulated at a number of specific and regular points within the cell cycle. These locations are known as checkpoints. These check- points verify the structural integrity of the genome, ensure that the DNA is free of any breaks and monitor the cellular environment as a prerequisite for a given phase in the cell cycle. The check points of the cell cycle are shown below.

• Restriction point-This restriction point (R) occurs late in the G1 phase between the middle and the termination of the G1 phase. It is the time when a cell verifies whether it has appropriately been instructed by the growth signals to proceed to the S phase for DNA replication. Growth signals sufficient to trigger cells to go into S phase would induce replication of DNA or else the cells would shift to the GO phase.
• G1/S DNA damage checkpoint – The G1/S phase transition forms a major checkpoint for detection of any damage in the DNA molecule entering the synthetic phase.
• S phase DNA damage checkpoint – This checkpoint is strategically located at the later part of the S phase. Defective synthesis of DNA is detected at this stage and appropriately dealt with.
• G2/M checkpoint – Acts as a DNA damage check point.
• Centrosome duplication checkpoint- A defect in duplication of the centrosome or chromatid segregation arrests cells at the G2/M transition.
• Mitotic checkpoint – The M phase checkpoints observes the formation of normal mitotic spindles. The detection of any chromosome that is not attached at a spindle blocks the onset of anaphase.

How is Cell Division (Cycle) Controlled?

The steps in cell division are usually controlled by proteins called cdk-cyclin complexes. The cdks (cyclin dependent kinases) belong to a family of kinases. The kinases act as catalytic subunits and are named cyclin dependent kinases (cdks) as their activity is dependent on certain cyclins. Cyclins are types of regulatory subunits. The catalytic and regulatory subunits always occur as associated pairs.

Thus a specific cdk is fully activated only when its cyclin partner is expressed in association. As exemplified at specific cell cycle stages, the G1 phase cdk4 and cdk6 act in association with cyclin subunits D1, D2 and D3, while the cdc2/cyclin B complex (cdc = cell division cycle) is expressed in G2/M phase of cell cycle.

Multiple proteins actively regulate different stages of cell division. The cdks control the phosphorylation of regulatory proteins at different stages in the cell cycle progression, e.g. the retinoblastoma (RB) tumor suppressor gene product (pRb) is a key regulatory protein of the G1 phase that is phosphorylated by a cdk/cyclin complex.

• In the first part of the G1 phase (very early interphase) pRb is bound to E2F. The transcription factor E2F needs to be in an unbound form for the cells to transit form the G1 to the S phases.
• The pRb with E2F forms a complex that inhibits expression of other transcription factors needed for the initiation of S phase.
• Cells remain in the G1 phase or G0 phase in now unable to phosphorylate the pRb and the cells presence of the complex.
• The synthesis of D cyclin is activated subsequent to the action of growth factors. This event stimulates cells to reenter the cycle from GO or G1 phases.
• Cdks and cyclins couple to form active complexes regulating further steps in the cell cycle. The cdk 4/cyclin D and cdk6/cyclin D become active in the early phase of G1 and cdk2/cyclin E complex function in late phase of G1.

• As the cdk/cyclin complexes phosphorylate the protein pRb in the pRb and E2F complex, the trapped E2F is released.
• The free E2F subsequently activates transcription of genes that are essential for initiating the S phase.
• The inactivation of certain other genes caused by PRb-E2F complex is also disinhibited.
• Thus with the inactivation of pRb the cell now enters in S phase.

Alternatively the cdk-cyclin kinases also can be inactivated to retain a cell in the G0 or G1 phases. Such inactivation of cdk/cyclin complex can be achieved with the binding of certain inhibitory proteins to the cdk/cyclin complexes. The inactivated complexes are now unable to phosphorylate the pRb and the cells fail to transit into the S phase. There are two families of the cdk/cyclin inhibitor proteins.

• INK4 family (Inhibitors of cdk4 family) – INK4 family of proteins specifically bind and inactivate cdk4 and cdk6. The p16, p15, p18 and p19 are the four members of the family.
• Cip or Kip family (cdk interacting protein or kinase inhibitory protein). The p21, p27 and p57 are the three members of the family. p21 binds to all complexes of cdk2, cdk4 and cdk6 and forms the universal cdk inhibitor that can block all stages of G1 and S phase.

Cell Cyclic Control Genes And Cancer

The cyclin D-dependent kinases integrate the extracellular signals towards progression of the cell cycle. Alterations in pRb and cyclin D-dependent kinases may lead to inappropriate and unbalanced phosphorylation of pRb. This may result in uncontrolled signaling and proliferation of cells.

Deletion or mutation of the suppressor gene CDKN2 has been implicated in multiple cancer states. The RB (retinoblastoma) gene that acts as a tumor suppressor gene has been found to be associated with cancer. Retinoblastoma is a tumor of the retina seen in children. The retinoblastoma (RB) gene is located on the q arm of chromosome 13.

Retinoblastoma arises when both the copies of the RB genes are deleted or inactivated. Usually the child inherits one defective allele and acquires a fresh mutation in the normal allele in childhood. Thus these tumors are generally sporadic in occurrence resulting from new mutation; homozygous mutations can also be inherited giving rise to the condition.

A Condition like retinoblastoma arises due to the loss of protein pRb (product of gene RB) that leads to unrestrained cellular proliferation. Absence of pRb has been linked to osteosarcoma and lung cancer.

RB gene yields a nuclear phosphoprotein (pRb) that influences crucial activities in the cell cycle. The protein pRb is usually kept bound to the E2F group forming an inactive pRb/E2F complex in cells that don’t proceed towards cell division. Once the cdk/cyclin complex phosphorylates the pRb faction of the pRb/ E2F complex, it sets the E2F component free to bind and activate the next set of transcription factors.

Protein pRb can also bind certain viral tumor antigens like SV40T and E1A. In this situation pRb doesn’t bind E2F and remains as a pRb-tumor antigen complex. The free E2F helps the cell to pass from G1 to S phase as the viral antigen induces unrestrained growth by blocking normal activity of pRb.

Cell proliferation can be arrested if-

• pRb is not phosphorylated (remains coupled to E2F).
• D-cyclin is absent (disabled cdk complex).
• p16, p21 and p27 inactivates cdk-cyclin complex.

On the contrary the loss of the above functions may lead to unrestrained growth or tumor formation.

G1/S Checkpoint

Damage caused by double strand breaks (DSBS) in the DNA activates specific events at this check-point. Ionizing radiation or genotoxic chemicals usually cause such damage in the DNA strands. Escape of undetected damaged DNA on to daughter cells in somatic and germ cells may have devastating consequences.

Operation of the G1/S cell cycle checkpoint is governed by the tumor suppressor gene TP53. This gene gives rise to the protein p53 that acts as receiver of stress signals including DNA damage. Any damage to DNA leads to the activation of p53 which then acts as a transcription factor inducing cell destruction.

The levels of p53 are generally low in normal cells. To function as transcription factor p53 protein must be activated by phosphorylation and acetylation. The factor Mdm2 prevents phosphorylation and acetylation of p53 and removes p53 from the nucleus. This leads to degradation of p53 by proteosomes in the cytoplasm.

The level of p53 thus is kept low by continuous export of p53 by Mdm2 from nucleus to cytoplasm followed by degradation. On the other hand DNA damage itself results in phosphorylation and acetylation of p53. Once p53 is phosphorylated, Mdm2 cannot bind to the modified (activated) p53. The activated p53 remains in the nucleus.

Transcription of a number of genes is brought about by the p53 protein to trigger cell cycle arrest Cyclin B/Cdc 2 Cyclin A/Cdk 2 Cyclin E/Cdk 2 Cyclin D/Cdk 4,6 and apoptosis p53 induced gene p21C1P1 binds to cdk2/cyclin E resulting in the arrest of cell cycle at G1/S checkpoint.

Activities of p53 have earned it the name “Guardian of Genome”. Mutations in TP53 gene are responsible for about 50% cancers in humans. p53 is located on chromosome 17p13.1. Virtually all types of cancers are associated with defects in the p53 gene. Such defects or mutations generally exist in a homozygous pattern.

Mutant p53 alleles are inheritable. Individuals having such mutations are susceptible to develop malignant tumors. Carriers of heterozygous defects are said to have the Li-Fraumeni syndrome and may develop a varied type of tumors (carcinomas, lymphomas, brain tumors, sarcomas, etc).

p53 protein has a vital function of arresting the cell cycle on detection of breaks and damages in the DNA content of a cell. The protein gets accumulated in the nuclei and inhibits the cell from crossing over to the S phase.

As mentioned above, p53 induces the action of p21 to complete the arrest. p53 also helps in the repair of broken DNA molecules by activating certain other transcription factors and DNA repair enzymes. In the event of a complete repair, the cell is allowed to advance to the next step in the cycle.

In case of an incomplete or failed repair, p53 stops the cell division and induces apoptotic mechanisms in the cell.

S Phase

The S phase checkpoints are also invoked by structural changes in the DNA. Cell cycle is stopped with the action of the dephosphorylated pRb. Arrest of progression of cell cycle may also be brought about by p21 that blocks cdk.

G2/M Checkpoint

This checkpoint is located at the junction between the G2 and the M phase and it is triggered by DNA breaks in the genome. Cell cycle is inhibited at this stage by inactivation of the Cdc2/cyclin B complex. The inhibition of the complex stops the transit of a cell from G2 to mitosis. The action of the checkpoint is to maintain the Cdc2/cyclin B1 complex in an inactive state with the help of p53 protein.

The induction of p21 and its binding to Cdc2/cyclin B in the nucleus causes cell cycle arrest at G2/M checkpoint.

Viruses And Cancer Genes

The study of viral carcinogenesis has shed light on the genetic mechanisms involved in cancer formation. Retroviruses (RNA viruses) are the ones that cause most of cancers in animals and a very few in humans. Some of the DNA viruses also cause a few cancers in man. The understanding of replication in the retrovirus helps us analyze events occurring in cancer.

Retroviruses

The retroviral genome consists of a diploid, double stranded RNA molecule. These viruses are incapable of replication till they infect a cell and use the cellular machinery of the host to replicate its genome and assemble other constituents of its structure. The presence of the key reverse transcriptase enzyme forms a double stranded DNA copy from the viral RNA.

The transcribed DNA strands easily incorporate into the host DNA and are called “provirus”. A provirus constitutes three genes that are adequate for complete viral replication.

• gag – the gene codes for structural protein of the virus.
• pol – this segment codes for the enzyme reverse transcriptase. Genetics of Cancer 165
• env – the sequence codes for protein of the outer envelope.

The promoter and enhancer elements are integrated at each end of the genome. These elements are The promoter and enhancer elements are integrated constitutively associated with the provirus and are termed long terminal repeats (LTRS).

The normal replication of the host DNA also results in the replication of the viral genome by default. Transcription of the integrated viral genome, on the other hand, gives rise to different cellular components of the virus. All viral components assemble in the cell and come out as viral progeny in multiplied numbers after lysing or destroying the host cell.

Once a provirus is integrated in the host genome, it stays in the cell for its life; even passing to the daughter cells of the infected host cell. Proviruses have also been found integrated into DNA of gametes (eggs and sperms) where they reach after infecting the germ cells in an individual.

In addition to the gag, pol and env genes found in common retroviruses a potent fourth gene exists in certain viruses (as identified during the study of Rous sarcoma virus). This gene is capable of carcinomatous transformation in a host cell. It is called the src gene with its action of coding for a protein kinase and inducing cancerous transformation of host cell well-documented. A viral gene that can transform the infected host cell is termed oncogene.

Oncogenes carried on viruses are called viral oncogenes or V-onc whereas oncogenes located in host cell genome are called cellular oncogenes or C-onc genes.

Some DNA sequences in the host cell are homologous or identical to the viral oncogenes and thus are called proto-oncogenes. As discussed earlier proto-oncogenes regulate normal cell growth and do not cause cancer in normal circumstances. The cellular proto-oncogenes are potentially carcinogenic and can be transformed to act as oncogenes by point mutation, amplification and chromosomal translocation.

How Viral Oncogenes are Formed

It is interesting to note that retroviral oncogenes originates from cellular genes in the host. Any error in the replication of retroviral genome, after their integration in host genome, gives rise to retroviral oncogene. This viral oncogene is structurally similar to its cellular counterpart (the viral oncogene sis is almost similar to the gene for platelet-dependent growth factor [PDGF]) but different in its function.

Conversion of Proto-oncogenes to Cellular Oncogenes (c-onc)

Proto-oncogenes can be converted into cellular oncogenes in the following ways:

• By increase in the amount of proto-oncogene product: This can be achieved in two ways.
  • Integration of the viral oncogene to the host DNA close to a proto-oncogene may induce uncontrolled expression of proto-oncogene through the action of the LTRS of the v-onc.The LTR of the Epstein-Barr viruses have been observed to overexpress myc gene in infected human cells leading to Burkitt’s lymphoma.
  • Multiple copies of the proto-oncogenes can be formed in a cell through gene ampli- fication. Activity of several copies of the gene yields a large amount of the transcriptional product that leads to transformation of proto-oncogenes to cellular oncogenes.
    The N-myc gene is amplified manifolds in neurofibromatosis. C-myc, N-myc and L-myc amplifications are associated with lung carcinomas and c-neu or erb-B2 genes with types of breast carcinomas.
• Mutation in coding sequence:
  Oncogenes are also formed by mutations in the proto-oncogenes. As discussed, these proto- oncogenes are potential oncogenes and are activated through mutations to produce cancer.

• • Mutations in the ras gene account for approximately one-third of all human cancers.
• Chromosomal translocation:
  A good percentage of human cancers are caused due to translocation of chromosomes with rearrangement of the genome. Chronic myeloid leukemia and Burkitt’s lymphoma are two common examples of the group. The Philadelphia chromosome seen in the white blood cells of the CML patients is formed by rearrangement of chromosomal segments as a result of translocation A reciprocal translocation between the long-arm of chromosome number 22 and the 9th chromosomes transfers cellular abl oncogene from chromosome 9 that fuses with bcr (break-point cluster region) gene of chromosome 22. This fused gene (chimeric gene) manufactures protein that contains about 900 amino acids of ber region and 1100 amino acids of c-abl region. The resultant condition gives rise to abnormal proliferation of the cells.

Similar translocation between the long arm of chromosome 8 containing the c-myc gene and the chromosome 14 at the locus carrying the gene for immunoglobulin heavy chains is implicated in the formation of Burkitt’s lymphoma. This rearrangement brings the translocated c-myc gene under the regulatory influence of the immunoglobulin gene resulting in about 20 fold increase in the levels of c-myc transcription.

Apoptosis

Apoptosis or cell death is a programmed event to maintain a balance between the generation of new cells and the death of senescent or defective cells.

Apoptosis occurs due to several events that causes damage to the growth regulating genes, loss of check- point genes and also when the telomerase enzyme is unable to protect the integrity of the terminal part of the chromosomes after each cell division. (With each cell division, the tails of each chromosome gets shortened due to progressive loss of nucleotides to reach a certain limit where the cells automatically undergo self-destruction. This threshold is referred as the Hayflick’s limit. This phenomenon occurs due to gradual dwindling in the levels as well as activity of the telomerase enzyme that is needed to repair damaged ends of the chromosome).

Cells usually die when they grow old and are unable to furnish proteins like the telomerase enzyme that are needed to cross the checkpoints in the cell cycle. Old cells also suffer breaks in the DNA and are directed towards self-destruction. Cells also die if they are subject to sustained injuries such as heat, oxidative stress, UV irradiation damage or get killed when they become vulnerable and infected with a virus or other intracellular pathogen that destroys the cell.

Thus apoptosis is a form of programmed cell death initiated by extracellular or intracellular signals in which enzymes are activated that break down the cytoplasmic and nuclear skeleton, degrade the chromosomes, disintegrate the DNA and shrink the cells. Initiation of the process of apoptosis begins either with extracellular or intracellular signals.

Execution of cell death is effected with the release of caspases (cysteine containing aspartase specific protease). Caspases are the ultimate destroyers of cell. These are a family of proenzymes that are activated in a cascade. The targets of these proteases are the DNA, several cytoskeletal proteins, DNA repair enzymes, etc. The caspase family includes at least 13 proteins and is divided into 3 groups.

Morphological changes occur in the dying cells. Surrounding cells like macrophages remove the dead cells.

Genetics Of Cancer Summary

• Cancer cells are characterized by:
  • Uncontrolled proliferation.
  • Transformation to abnormal cells.
  • Capacity to invade surrounding tissues.
  • Property to metastasize to distant places.
  • uppression of apoptosis.
  • Induction of angiogenesis.
• Cancer is due to the loss of the normal mechanisms which control cellular proliferation and differentiation.
• Cellular proliferation is under the control of genes.
• Genes responsible for causing cancer are known as oncogenes.
• Tumor suppressor genes (TSG) apply brakes to unrestrained cell growth as they induce tumor suppressor activity. Thus cancer may develop due to loss of function (mutation) of TSGS.
• About 100 oncogenes and about 30 TSGs are now known.
• Signal transduction is a process by which extracellular growth factor regulate cell growth and differentiation by a complex pathway. [Growth factor activation of receptor cytoplasmic proteins (ras and abl) are activated – ras binds to GTP cytoplasmic kinases are activated Kinases enter the nucleus – activate myc which regulate transcription of DNA – cell cycle begins].
• Genes that code for growth factors and growth factor receptors, after mutation may acquire oncogenic properties.
• The mutation of ras gene (which codes for signal transducing protein) and myc gene coding for transcription factor may cause cancer.
• The cell division cycle is divided into four
• The transition from one phase to the next is sequential phases, i.e. G1, S, G2 and M phase. regulated by checkpoints.
• Cell cycle is controlled by cdk/cyclin complexes.
• Various proteins act as key regulatory proteins during cell cycle. For example the product of retinoblastoma (RB) tumor suppressor gene (pRb) is a regulator protein of G1 phase that is phosphorylated by cdk/cyclin complex.
• Cell cycle checkpoints are under genetic control and surviallence. In case DNA (gene) is damaged, cell cycle progression is checked till the damaged DNA (gene) is repaired.
• Any alteration (mutation) in these genes will lead to the formation of tumor due to uncontrolled cell proliferation.
• The maintenance of the G1/S cell cycle checkpoint is dependent on the tumor suppressor gene (TP53). The mutation of TP53 gene is responsible for about 50% cancers in human.
• The gene p21 when induced by p53 binds to cdk2/ cyclin E which leads to cell cycle arrest at G1/S checkpoint. Thus mutation of p53 and p21 may lead to nonfunctioning of G1/S, S and G2/M checkpoints leading to formation of tumor.
• Retroviruses (RNA viruses) and DNA viruses are responsible for causing cancers in man.
• The oncogenes present in viruses are called viral oncogenes (V-onc) those in host cells are called cellular oncogenes (C-onc). In host cells there are DNA sequences homologous to the viral oncogenes and are called proto-oncogenes. Proto- oncogenes are responsible for promotion of normal cell growth.
• Apoptosis is triggered by DNA damage (programmed cell death). A cascade of proteolysis is initiated with intracellular or extracellular activation of apoptotic pathway. The proteases involved are called caspases. Cell death is brought about by the action of caspases.

Genetics Of Malocclusion

Occlusion means alignment of the upper and lower teeth together. Normally all upper teeth fit slightly over the lower teeth. The upper teeth keep the cheeks and lips from being bitten and the lower teeth protect the tongue. Malocclusion of teeth denotes improper or misalignment of teeth. The maxillary and mandibular teeth are incorrectly positioned with relation to each other. Malocclusion also occurs because of altered relation between the upper and lower jaws.

Classification of Malocclusion

• Class 1 malocclusion is the most common variety of malocclusion. The bite is normal as per the permanent Ist molar relationship but the teeth are crowded or not positioned correctly. The upper teeth slightly overlap the lower teeth.
• Class 2 malocclusion is also called as retrognathism. It occurs when the upper jaw/ upper teeth are forwardly placed (lower teeth/ lower jaw are placed distally)
• Class 3 malocclusion is also called prognathism. It occurs when the lower jaw protrudes or just forward causing the lower jaw and teeth to overlap the upper jaw and teeth from beneath the upper jaw.

Malocclusion Causes

Malocclusion is caused due to:

Malocclusion Acquired Factors

• Alteration in the shape or size of jaws; if teeth in a small jaw jostle for space and grow crowded in small area pushing each other.
• Alteration in the shape or size of teeth.
• Tooth loss.
• Thumb or finger sucking, use of pacifier and mouth breathing (due to enlargement of tonsils) and tongue thrusting.

Read and Learn More Genetics in Dentistry Notes

Malocclusion Genetic Factors

• Inherited conditions include:
• Inheritance of too many or too few teeth.
• Inheritance of too much or too little space between the size and shape of upper and lower jaws. These teeth.
• Inheritance of irregular mouth and jaw size and shape.
• Abnormal formations of the jaws and face, e.g. cleft palate.

The etiology of malocclusion is a complex subject and not fully understood. The above brief description of etiology indicates that the bony factors (size and shape of maxillary and mandibular arches) and dental factors (size and shape of teeth, failure of eruption, supernumerary teeth and early loss of teeth) can be determined by both environmental and genetic factors.

For example, the failure of eruption of upper lateral incisor may be due to acquired as well as genetic causes. The presence of supernumerary teeth may lead to the failure of eruption of incisor. Supernumerary tooth again may feature due to an inheritance (as supernumerary tooth may also be present in a parent of the patient).

On the other hand failure of eruption of central incisor may be also due to early loss of many deciduous teeth (due to caries) that leads to forward drift of first permanent molar teeth resulting in the crowding of teeth. Thus it sometimes becomes difficult to assess the clear-cut role of acquired and genetic factors in the causation of malocclusion as there is a complicated interplay between various factors.

Malocclusion may result due to inheritance of disproportionate size of the teeth and jaw, resulting in crowding or spacing of teeth with a small jaw leading to crowding and large jaw giving abnormal spacing between teeth. The other important factor leading to malocclusion is the disproportion between skeletal variables are primarily genetic in nature.

It should be noted that genetic influence on the shape and size of jaws is not due to a single gene defect but is mostly determined by the additive effects of many genes (i.e. polygenic in nature). The environmental factors play an influential role on shaping of the genetic factors. Thus, the etiology of malocclusion is mostly multifactorial in nature (due to interaction between genetic and environmental factors).

Though it is simple to analyze the genetics of a single gene (Mendelian trait), analysis of multifactorial traits (cleft palate, cleft lip, caries, periodontitis and malocclusion) are difficult to analyze. For the analysis of the multifactorial inheritance one has to evaluate the role of genetic as well as environmental factors and the interaction between the two. The time-tested methods to analyze the role of genetic and environmental factors for a particular multifactorial disease in humans are familial and twin studies.

Family And Twin Studies

In familial studies one has to observe the similarities as well as differences between the mother and the child, father and the child and between sibling pairs. Correlation coefficients of the trait are obtained between parents and offspring or between sibling pairs and half sibs. For most measurements of facial skeletal dimensions correlation coefficients for parent-child pairs are about 0.5 which is the upper limit of correlation between first-degree relatives.

The correlations for parent-child in relation the dental char- acteristics range from 0.5 to 0.15. This is also reflected in the Harris and Johnson (1991) study which revealed high heritability of craniofacial (skeletal) characteris- tics and low heritability of dental characteristics. This indicated that malocclusion resulted mainly from facial skeleton deformities that could be inherited while pure dental variations were due to environmen- tal factors. Many other family studies have indicated the role of heredity in determination of the craniofa- cial and dental morphology (Korkhaus, 1930; Rubbrecht, 1930; Trauner, 1968 and Peck et al, 1998).

As stated earlier twin studies are useful in identifying the genetic and environmental factors determining multifactorial traits. Differences in features between monozygotic twin pairs implicate environmental factors while similarities points towards genetic influences as the primary causal factors of the disease. The similarities in disease features between dizygotic twin pairs are thought to result from environmental influences and genetic factors.

A comparison of the differences observed within twin pairs in the two categories should provide a measure of the degree to which monozygotic twins are more alike to each other than dizygotic twins between themselves in a pair. Studies with twins reared apart are more useful as compared to twins reared together in a common environment as these studies overcome the problems of twins displaying similarities because of being reared in a common environment.

Following is a brief review of family and twin studies in relation to the heritability of craniofacial and dental characteristics. Most of the familial and twin studies indicated that heredity plays a significant role in the etiology of malocclusion.

Lundstrom (1948) reported that characteristics like width and length of dental arch, height of palate, spacing and crowding of teeth, tooth size and degree of overbite are genetically determined. A study on triplets by Kraus et al (1959) investigated the cephalometric parameters and concluded that the morphology of craniofacial bones are under strict genetic control.

Nevertheless, environment plays a major role in determining how these bony elements combine with each other to produce normal occlusion or malocclusion. This observation explains why sometimes differences are observed between a pair of monozygotic twins in spite of having the same genetic constitution.

Markovic (1992) conducted a cephalo- metric twin study and concluded that 100% of monozygotic twin pairs demonstrated concordance for malocclusion while 90% of dizygotic twin pairs were discordant. This is strong evidence in support of genetic etiology of malocclusion.

Schulze and Weise (1965) reported the polygenic nature of mandibular prognathism (class 3 malocclusion). They found that the concordance in monozgotic twin pairs was very high as compared to dizygotic twins. Litton et al (1970) also investigated the poly- genic nature of class 3 malocclusion (mandibular prognathism). The Harris (1975) study indicated that craniofacial skeletal patterns of children with class 2 malocclusion were heritable. In a large familial group Nakasima et al (1982) studied the inheritance of class II and class 3 malocclusion.

High correlation coefficient values were seen between parents and their off- spring in the class 2 and class 3 groups. Thus, there appears to be a strong familial tendency in the development of class 2 and class 3 malocclusions. They concluded that the hereditary pattern must be taken into consideration in the diagnosis and treatment of patients with these classes of malocclusion. Environmental factors have also been suggested as contributory to the development of mandibular prognathism.

Among these are enlarged tonsils (Angle, 1907), nasal blockage (Davidov et al, 1961), congenital anatomic defects (Monteleone and Davigneaud, 1963), hormonal disturbances (Pascoe et al, 1960), endocrine imbalances (Downs, 1928), posture (Gold, 1949) and trauma/disease including premature loss of the first permanent molars (Gold, 1949).

Development of both the maxillary and mandibular arch shapes in an Australian twin study was found to be under genetic influence. However, authors (Richards et al, 1990) also have reported the existence of some independent variables that determine the shape of mandibular and maxillary archs. Hughes et al (2001) quantified the extent of variations in different occlusal features in Australian children of European descent with complete primary dentitions but no permanent teeth present in the mouth.

Occlusal traits including interdental spacing, incisoral overbite and overjet, arch breadth and arch depth were studied. Estimates for overbite and overjet were 0.53 and 0.28 respectively and estimates for arch dimensions ranged from 0.69 to 0.89. These results indicated a moderate to relatively high genetic contribution to the observed variations.

The aim of a study by Eguchi et al (2004) was to quantify the relative contributions of genetic and environmental factors to variations in dental arch breadth, length and palatal height in a sample of Australian twins. The study brought forth information that the heritability load for dental arch breadth ranged from 0.49 to 0.92, those for the arch length from 0.86 to 0.94 and those for palatal height were 0.80 and 0.81 respectively. These results indicate a high genetic contribution to the variation in dental arch dimensions in mainly teenage twins.

A twin study by Corruccini et al (1980) indicated the importance of environmental factors in the etiology of malocclusion. The researchers showed that heritability of overjet is almost zero. The study on Australian twins conducted by Townsend et al (1988) for occlusal variations has shown the importance of environmental influences in shaping overjet.

The cross bite was found to be determined by environmental factors. They also indicated that genetics may perhaps play an important role for overjet, a lesser role in overbite and least involved in determining molar relationship.

The masseter muscle electrical activity and its morphology were analyzed in twin studies and it was found that both its activity and morphology were genetically influenced. These twin studies revealed for the first time that soft tissue function and morphology could also be inherited (Lauweryns et al, 1992 and 1995).

A study by Dempsey et al (1996) on tooth size in Australian twins indicated a relatively strong genetic and environmental influence on the development of the canines and first premolars. The findings of the canine and first premolar mesiodistal dimensions support the evolutionary theory that indicates the presence of dominance variation in morphological features that have been subjected to strong selective pressure in the past.

Twin studies have shown that tooth crown dimensions are strongly determined by heredity (Osborne et al, 1958). The molecular genetics of tooth morphogenesis with the homeostatic MSX1 and MSX2 genes have been linked to stability in dental patterning. It has been reported that inheritance of tooth size fits the polygenic multifactorial threshold model.

Markovic (1982) found a high rate of concordance for hypodontia in monozygous twin pairs while he observed discordance patterns in dizygous twin pairs. This and other previous studies concluded that a single autosomal dominant gene with incomplete penetrance could explain such a mode of transmission.

Niswander and Sugaku (1963) analyzed data from family studies and have suggested that the development of supernumerary teeth, most frequently seen in the premaxillary region, appears to be genetically determined. The etiology of ectopic canines is controversial with divergent opinions regarding its genetic or environmental mechanism of occurrence.

Mossey et al, 1994 indicated existence of an association in inheritance between ectopic maxillary canines and class 2 malocclusion demonstrating the involvement of genetic factors in the inheritance of the trait. The study of Camilleri et al (2008) addressed the hypothesis that genetic factors play a role in the etiology of ectopic maxillary canines. Sixty-three probands were identified and information on the dental status of 395 of their relatives was determined.

Only two of seven pairs of monozygotic twins were concordant for ectopic canines. This is consistent with environmental or epigenetic variables affecting the phenotype. The low concordance rate is consistent with the low penetrance determined by segregation analysis studies further supporting the existence of environmental etiological factors.

Environmental influences during the growth and development of face, jaws and teeth are mainly exerted through pressures and forces acting on these structures. Mossey (1999) states, “it is difficult to determine the precise contribution from hereditary and environmental factors in a particular case.

For example, the simultaneous appearance of proclined maxillary incisors and digit sucking may lead to the assumption that the digit was the sole causative factor, but the effect of the digit may very well be either potentiated or mitigated by other morphological or behavioral features in that particular individual.

A similar argument may apply in cases of mouth breathing where the influence of the habit and associated posture is very much dependent on the genetically determined craniofacial morphology on which it is superimposed, and the reason for the habit developing may well be dependent on the morphology in the first place. These scenarios are classical examples of the interaction of genotype and environment, and ultimately success of treatment will depend on the ability to ascertain the relative contribution of each.”

Linkage Studies

Linkage studies identifying chromosomes or gene(s) responsible for craniofacial and dental variables of malocclusion in humans are very limited.

• The role of X and Y-chromosomes on craniofacial morphology has been investigated by Gorlin et al (1965). X and Y-chromosomes exert growth- promoting effects on human tooth crown size. The X-chromosome appears to regulate mainly enamel thickness while Y-chromosome affects both enamel and the dentine. Amelogenin is a matrix protein secreted by ameloblasts and it is thought to direct the growth of hydroxyapatite crystals. The gene for amelogenin is located on X and Y-chromosomes (Lau et al, 1989). The amelogenin gene is located on the distal portion of short arm of X-chromosome and to the pericentromeric region of Y-chromosome. The gene on the X-chromosome is the predominantly functional one. Its mutation leads to amelogenesis imperfecta. This dental deformity (amelogenesis imperfecta) is associated with malocclusion (vide infra).
• Mutation in the novel enamelin gene (ENAM) is responsible for autosomal recessive amelogenesis imperfecta and localized enamel defects. It was discovered by Hart et al (2003). In this study 20 consanguineous families with amelogenesis imperfecta were identified with probands suggesting an autosomal recessive transmission. Linkage studies indicated the presence of amelogenesis imperfecta gene on chromosome 4q region. The mutation of this gene resulted in generalized hypoplastic amelogenesis imperfecta phenotype and a class 2 open bite malocclusion. A strong association was thus established between amelogenesis imperfecta and malocclusion due to homozygous mutation in ENAM gene.
• Ravassipour et al (2005) conducted an investigation to evaluate the association of the AI enamel defect with craniofacial features characteristic of an open bite malocclusion. Open bite malocclu- sion was found to have occurred in individuals with AI caused by mutations in the AMELX and ENAM genes even though these genes are con- sidered to be predominantly or exclusively expressed in the teeth. The purpose of this investigation was to evaluate the association of the AI enamel defect with craniofacial features characteristic of an open bite malocclusion. Affected AI individuals with cephalometric values meeting criteria of skeletal open bite malocclusion were observed in all major AI types. The pathophysi- ological relationship between AI associated enamel defects and open bite malocclusion re- mains unknown.
• The study by Frazier-Bowers et al (2007) investigated whether the class 3 trait was present in several of the pedigrees affected with AI. Their results suggested that the class III trait factor co-segregated with AI in the experiment population.
• The Klinefelter males (47, XXY) show pronounced mandibular prognathism and reduction of cranial base angle (Brown et al, 1993). Similarly, 45,X females show imbalance of growth in the craniofacial skeleton (Peltomaki et al, 1989). They show retrognathic face with short mandible and flattened cranial base angle. There is a tendency to have a large maxillary overjet and crossbite. The X-chromosome seems to be responsible for altering the growth of cranial base by acting on cartilaginous joint at the base of the skull. This has a direct effect on the shape of the mandible.
• A linkage study for the growth of maxilla in mouse was conducted by Oh et al (2007). They found that the gene(s) regulating the shape of maxillary complex was situated on the mouse chromosome 12 at 44cM.
• A recent study by Fraziers-Bowers et al (2007) identified the chromosomal locus responsible for the class 3 trait. DNA samples were processed and subjected to a genome-wide scan and linkage analysis. The linkage analysis of these families revealed that a region on chromosome 1 was suggestive of linkage with the class 3 trait.
• The growth hormone receptor (GHR) gene was found to be associated with mandibular height in a Chinese population. Zhou et al (2005) evaluated the relationship between craniofacial morphology and single-nucleotide polymorphisms (SNPs) in GHR in a healthy Chinese population. Their results indicate that the GHR gene polymorphism 1526L is associated with mandibular height in the Chinese population.

As far as genetics of malocclusion is concerned, much progress is still awaited. We do not know the exact mechanisms of genetic or environmental inter- action that combine to produce malocclusion. Similarly, interactions between genetic and environmental factors are least understood. A clear understanding of mechanisms involving environmental factors would help in designing therapies along with the manipulation of environmental factors for orthodontic treatment. Multifactorial traits determined by the additive effects of many genes need to be investigated better to find out the exact number and specific locations of genes involved in their etiology.

More precise research tools and methods should be applied to understand aspects of genetics associated with malocclusion. It is hoped that sequencing of human genome and the use of single nucleotide polymorphisms (SNPs) will help to attain the desired levels of understanding.

Molecular Control Of Development

It is amazing to realize the degree of precision and detailing involved in the process of the development of a full-grown individual starting from the first cell of life, the zygote. The intricate mechanisms that control each of the steps in the process are nothing short of miracles of molecular engineering given the thousands of stages at which the process can veer towards undesired destination.

Thus though it may seem normal it is actually a matter of chance, so marvelously sustained as the usual’, that majority of the population walks around in normal physical and mental formats. The following sections of the chapter discuss the molecular mechanisms that guide and control the various processes of growth and differentiation in the developing embryo.

As detailing of each of the steps in molecular control of development is beyond the scope of this book, the description of the events would nevertheless enable the students of dentistry to understand the fundamental concepts of developmental genetics.

Embryonic development in humans or for that matter any kind of regulation imposed on the functioning of a cell, tissue or organ is enforced by expression of definite protein molecules. Eventually these are the genes that exert their influence on cellular functions by synthesis of a specific proteins needed for a particular function. The synthesis of proteins differs from cell-to-cell and within the same cell at different points of time. This provides the basic mechanism for control of any cellular process.

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The process of Growth in an organism is achieved by cell division through mitosis that multiplies the number of existing cells and also by increasing the amount intercellular matrix. The process of differentiation is the creation of new types of cells or tissues, which were not previously present. The differentiated cell possesses new morphological and functional characteristics, which distinguish it from other cells.

As described below, we now know that these characteristics result from the formation of new enzymes and proteins. Earlier workers tried to study the mechanism of differentiation by experiments on embryos of amphibia and chicks. Their work has produced many interesting results some of which are as follows:

It has been observed that certain regions of the embryo have the ability to influence the differentiation of neighboring regions. Interesting experiments have shown that these areas can induce formation of the same and specific tissues if they are implanted at areas outside their normal site of occurrences.

The primary organizer (identified at the dorsal lip of the blastopore) is the first organizer that is recognizable in the embryo. The failure of development of the primary organizer results in absolute failure of embryonic development. On the other hand if the dorsal lip of the blastopore is grafted on to a different site of another embryo, it induces the development of an entire embryo at the implanted site. Thus the signals that determine the initial organization in an embryo come from the primary organizer.

The effects of these organizers are brought about by enzymes or signaling proteins that are basically the product of gene transcription and translation. These signals may be in the form of (a) inductors which stimulate the tissue to differentiate in a particular manner; or (b) inhibitors which have a restraining There are stretches of DNA sequences called enhancers influence on differentiation.

Therefore the study of the controlling mechanisms can be termed Genetic control of development or described as Molecular control of development as it is now well-documented that the final control of the mechanisms of control rests with the genes involved in such control.

Though all the cells in the body contain the same complement of genes and other nuclear molecules, specialization of the structure and functions of a cell is determined by activation of only a certain number of genes in a particular type of cell. The process of protein synthesis involves two of the fundamental processes in cell biology; transcription and translation.

The basic process of transfer of genetic information begins with transcription of the mRNA molecule from DNA that occurs inside the nucleus followed by extrusion of mRNA outside the nucleus.

The sequential arrangement of codons on the mRNA is used for synthesis of proteins by translation occurring in the cytoplasm and involves protein synthesizing machinery in the cytoplasm, e.g. ribosomes, tRNA, etc.

In any given cell, at any given time (interphase), only a few of its genes are active and others are resting. Cells are said to be differentiated structurally and functionally because of expression of a small number of developmental regulatory genes (master genes) in them during specific time of embryonic development. The expression of such master genes initiate cascade of events in different cells imparting the cell and subsequently the tissues of the embryo their unique structural and functional identity.

Every differentiated cell contains two types of genes; the housekeeping genes and the specialty genes. The majority of genes (80-90%) in a cell are housekeeping genes which are required for basic cellular metabolic functions. These common genes are also widely expressed in other cell types of the body. The specialty genes are expressed to define the unique features of different cell types.

In higher organisms regulation of gene expression is quite complex and is brought about by the action of specialized molecules such as hormones or growth factors on the target cells. Regulation (either facilitation or suppression) of gene expression is effected through binding of a transcription factor to specific DNA segments in the promoter region of a gene (vide infra).

There are stretches of DNA sequences called enhancers that may be located within the noncoding sequences of the gene, or located upstream or downstream of the gene. These regions on the DNA can bind transcription factors and increase the rate of transcription. There are also similar regions which inhibit transcription and are called silencers. Transcription factors may bind to these specific regions on the DNA activating or inhibiting (turn on or turn off) gene expression.

Molecular Processes In Development

It is well known now that several genes and gene families play important role in the development of the embryo. Most of these genes produce transcription factors which control RNA transcription from the DNA template in the target cells. The transcription factors thus play an important role in gene expression as it can switch specific genes on and off by activating or repressing it.

It is believed that several transcription factors control gene expression, which in turn, regulates the fundamental embryological processes like induction, segmentation, migration, differentiation and apoptosis (programmed cell death) in embryonic cells till permanent cell lines are established in tissues. The above fundamental embryological processes are mediated by growth and differentiation factors, growth factor receptors and various cytoplasmic proteins.

Our existing knowledge regarding the molecular basis for embryonic development is mainly based on the Drosophila (fruit fly). However, evidences are now being gathered indicating that the basic body plan of mammalian embryo is under the control of many such similar genes as those in the fly that have been identified for controlling morphogenesis in Drosophila.

At the molecular level signaling is effected by protein molecules that act from outside the cells and can act locally or from a distance as intercellular signaling molecules. Many signaling molecules are called growth factors. Signaling molecules need to bind to receptor molecules that usually exist as trans- membrane proteins in the plasma membrane of the cells. Attachment of the signaling molecules to the receptor molecules cascade a series of events through which a molecular signal is relayed from the cell membrane to nucleus (signal transduction pathway) using related molecules.

Signal transduction is used by the cell to activate several mechanisms including generation of transcription factors which initiates gene expression in the nucleus. factors binds to the DNA at promoter or enhancer region of the specific gene and initiate the process of transcription. The transcription factors are important molecules that guide embryological development.

It is expected that mutations and disturbed expression of genes related to growth factors, receptors, or the transcription factors would be associated with various kinds of growth anomalies and cancers. The details of this phenomenon are discussed in appropriate sections of the book.

Growth and Differentiation Signaling Molecules

Proteins capable of stimulating cellular proliferation and cellular differentiation occur naturally and are termed growth factors. The epidermal growth factor (EGF), fibroblast growth factor (FGF) and the platelet derived growth factor (PDGF), stimulate the proliferation of epidermal cells, fibroblasts and the connective tissues, etc.

Growth factors typically act between cells in embryos through attachment to specific cell membrane receptors as intercellular signaling molecules. Several methods are adopted for execution of the effects of the signaling molecules. Growth factors can modify the expression or the effects of one another.

The signals are called hormones, which travel through blood to reach a distant place in the body. This system constitutes the Endocrine system.

Paracrine system act by signaling targets cells situated in the near vicinity of the signal executing cell.

The Juxtacrine mode of action requires that the effecter as well as the effected cells remain in cell-to-cell physical contact. The “gap junction” and “notch signaling are well known examples of juxtacrine system model of signaling. The notch signaling is described later in this chapter.

A few common growth and differentiation factor groups and their role in development are described below.

Growth Factors and their Functions

• Epidermal Growth Factor (EGF) Determines growth and proliferation of cells of ectodermal and mesodermal origin.
• Transforming Growth Factors (TGFs) TGF-B1 to TGF-B5 Forms the extracellular matrix, induces epithelial branching, myoblast proliferation.
  Bone Morphogenetic Factors (BMP 1 to 9) Helps bone formation, cell division, cell migration and apoptosis.
  Müllerian Inhibiting Factor (MIF) Regression of paramesonephric duct.
  Nodal         Formation of primitive streak, right- left axial fixation formation of mesoderm.
  Lefty          Determination of body asymmetry
  Activin       Proliferation of granulosa cells
  Inhibin      Inhibition of gonadotrophin

• Hedgehog proteins
  Sonic Hedgehog, Desert, and Indian. Shh control neural tube formation, somite differentiation, gut formation, limb development, and growth of genital tubercle.
• WNT Protein
  Midbrain development, somite and urogenital ultimately activates a zinc finger transcription factor, Gli. differentiation, limb patterning.
• Fibroblast Growth Factors (FGFs)
  Mesoderm differentiation, angiogenesis, axon growth, limb development, development of various parts of brain, liver induction, mesenchymal proliferation in jaw, induction of prostate gland, outgrowth of genital tubercle.,
• Insulin-like Growth Factors (IGFs)
  IGF-1 act as factor for bone growth, IGF-2 is a fetal chromosome number growth factor.
• Nerve Growth Factor (NGFs)
  Stimulate the growth of sensory and sympathetic neurons.

Abnormalities in the growth factor signaling pathway may lead to abnormal growth or cancer. The over expression of growth factors can lead to non- cancerous disorder like psoriasis. Mutation and over expression of PDGF gene may also cause cancers like osteosarcoma and astrocytoma. Mutation in growth factor receptors can lead to insulin-resistant diabetes (insulin receptor) and dwarfism (fibroblast growth factor receptor). Mutation and overexpression of these receptors are responsible for variety of cancers.

Growth Factor Receptors

Receptors are specialized protein molecules that recognize and bind specific signal molecules (ligands) such as growth factors and hormones. The transmembrane receptors are proteins situated across the plasma membrane of the cell. Receptors bind to the specific signaling molecules on the outer side of the membrane and activate certain molecules (G proteins, etc.) on the inner side of the membrane.

This is followed by a series of activation, mainly by phosphorylation, in some cytoplasmic proteins known as protein kinases, e.g. Tyrosine kinase, Protein kinase C, etc.

Other kinds of surface receptors also exist beside the transmembrane receptors. The notch receptor plays an important role in embryonic development. In this kind of signaling (juxtacrine signaling), a protein on one cell surface interacts with a receptor on an adjacent cell surface. Notch is a cell surface receptor, which has a long extracellular part and a smaller intracellular part.

Contact with the specific protein (delta or jagged) present on the surface of a nearby dominant cell activates the notch receptor. Attachment to one of these proteins causes the notch receptor to be broken in its intracytoplasmic domain. This broken portion acts as a transcription factor that regulates gene expression in that cell.

Thus one cell (called dominant) can influence transcription of an adjacent cell. Such example is visible in a developing neuron (dominant) that inhibits its surrounding cells to develop into glial cells. This phenomenon is termed as lateral inhibition.

Other growth factor receptors are responsive to molecules secreted by cells of the extracellular matrix (ECM) like glycoproteins, collagen, proteoglycans, etc. Receptors for fibronectin and laminin are called integrins.

For cell-to-cell communication, the gap junction channels are made up of connexin proteins.

Signal Transduction

The process by which a cell converts one kind of signal into another is called signal transduction. Binding of extracellular signaling molecules to receptors triggering a sequence of biochemical reactions inside the cell marks a signal transduction. These reactions are carried out by different enzymes as a chain of reactions and hence referred to as a “signal cascade”.

Extracellular growth factors regulate cell growth and differentiation by a complex pathway through signal transduction. Each sequential step in the pathway is genetically determined. The steps are sequenced as:

• Growth factor binding.
• Activation of the receptor.
• Activation of cytoplasmic proteins called signal transducing proteins; many of such proteins are situated on the inner surface of plasma membrane.
• Activation of one or more of the several cytoplasmic protein (kinase) systems.
• Formation of transcription factor.
• Effect of transcription factor: activation or inhibition the expression of a growth or a differentiation related gene.

We have already the idea that the processes in embryogenesis involve structures like ‘organizers’ that ‘induce’ the formation of specific types of cells from a common precursor. This process of induction is in fact carried out by organizers with the help of synthesis of several factors.

The synthesis of these important factors is under the control of certain well known genes that are involved with development of the embryo. One such gene is the PAX-6 gene which encodes for a transcription factor regulating several important events in embryogenesis including development of the eye.

Transcription Factors

A large number of transcription factors are common and found in all types of cells and across several organisms. However, few transcription factors are found only in certain types of cells or are active only during specific stages of development. The transcription factors regulate gene expression by acting on promoter or enhancer regions of specific genes.

These transcription factors are transcription regulatory protein molecules that bind to specific sites on the DNA. These proteins have ‘typical’ structural configurations at their binding sites with DNA. These sites are called ‘motifs’. Some of these ‘typical’ configurations are what we know as basic helix-loop- helix protein, zinc finger protein, etc.

Abnormalities in transcription pathways may lead to abnormal growth or cancer. The mutation of signal transducing proteins (e.g. ras gene) is responsible for almost 30% of human tumors. Mutations of genes which code for certain transcription factors are responsible for colon cancer, neuroblastoma, Burkitt’s lymphoma and lung cancer.

Specific abnormalities related to transcription factors are referred to in the section of the text dealing with molecular control of some important events in dental development.

Some of the important Transcription factors and their functions are discussed below:

• Basic helix-loop-helix protein is involved in myogenesis, neurogenesis, hematogenesis and the development of pancreas. This kind of transcription factors contain a short length of amino acids in which two alpha-helices are separated by an amino acid loop.
• Zinc Finger Proteins regulate expression of genes. The DNA binding domain in this protein is the zinc finger motif. The transcription protein is constituted by zinc ions binding to regularly placed cysteine and histidine units of the polypeptide chain. This results in puckering of the chain into finger-like structures (Fig. 8.2). These fingers configure to specific sites of the desired DNA helix. The kidney, gonads, hindbrain and white blood cells are some of the diverse examples of structures influenced by this transcription factor.

Anomalies in genes like GLI 3, WT1 and ZIC2 located in chromosomes 7p13, 11p13 and 13q32 respectively cause head, hand and foot abnormalities, Wilms’ tumor, ambiguous external genitalia and Holoprosencephaly. Mutations in the gene ZIC3 located at Xq26 may result in abnormal position of heart, liver and spleen.

• HOX genes regulate segmentation, patterning of the hind brain and formation of the axis of the embryo.

The HOX genes in humans encode a special class of transcription factors that regulate the sequential development of different body segments. Originally discovered in Drosophila this class of genes are called homeotic genes because mutations in these genes are capable of transforming one part of the body into another (e.g. growth of legs in place of antennae).

The regional morphogenic characterization of individual segments of Drosophila embryo is brought about by the expression of a group of homeotic genes. These genes determine which embryonic segment should bear antennae, wings or legs. These 8 homeotic genes are situated on chromosome number 3 and are arranged in two groups or clusters (Antennapedia and Bithorax). These genes are collectively called the homeotic complex or HOM-C.

Each of the 8 genes contains a highly conserved coding sequence of 180 base pair region of DNA (usually near their 3′ end) called the homeobox. The homeobox codes for a 60 amino acid protein called homeodomain. These homeodomains, thus, remain constitutively integrated within the bigger polypeptides coded by the homeotic genes.

These polypeptides synthesized by homeotic genes are transcription regulating factors. Homeo- domains within these polypeptides recognize and bind to specific DNA sequences of target genes.

The eight genes present in the homeotic complex express themselves in a selective sequence. That means the genes, which are cranial in position in the cluster or so as to say, located towards the 3′ end of the entire DNA material of the fly if put together in a 3′ to 5′ sequence, are expressed in the cranial segments or cranial regions of the developing embryo.

Successive downstream genes are expressed in the caudal structures of embryo giving each of the regions its structural identity in the craniocaudal axis. As stated before, the homeotic genes express polypeptide transcription factors. The products of a preceding or cranial gene regulate transcription in a succeeding or caudal homeotic gene.

Experimental mutation, suppression, or expression of the homeotic genes at different regions causes abnormal regional patterns in the embryo.

Such homeotic genes (clustered genes, each having a homeobox) similar to Drosophila are found in mammals and in humans. The human genes (called HOX genes) have same clustered organization, follow same order of gene arrangement within the cluster, their expression and functions are also in sequences as observed in Drosophila.

Quite interestingly, when the sequences within the homedomain and some short regions of the homeobox were compared, striking similarities were noticed across species. These sequences and their protein products (called ‘motifs’) have been strictly conserved through evolution. The amino acid sequences of homeodomains of Drosophila are up to 90% similar when compared with that of humans.

During hundreds of millions of years of evolution these genes have duplicated twice in man and hence human chromosomes have four copies of the clusters of homeobox genes. The genes (HOXA, HOXB, HOXC, and HOXD) arranged on four different chromosomes (Chromosome number 7, 17, 12 and 2). Genes in each group are numbered from 1 to 13 corresponding to the fly genes and each group can be placed in a vertical alignment.

Genes with same number but present on different chromosomes in the vertical alignment form a paralogous group. In humans, the HOXA and its paralogs are expressed in the cranial segments, the HOXB and its paralogs are expressed in the next caudal segment, the HOXC and its paralogs in the next segment and the pattern follows. The products of paralogs interact for a final result of expression within a segment.

There are 39 genes in all and each gene contains a homeobox region, which encodes for homeodomain protein. Similar to fruit fly, homeobox genes of humans are also expressed sequentially in craniocaudal direction during axis formation. The sequential expression of HOX genes correlates with the development of structures in craniocaudal sequence.

The HOX genes are responsible for cranial to caudal patterning of the derivatives of ectoderm, mesoderm and endoderm germ layers. HOX genes regulate the differentiation of somites, vertebrae and hindbrain segmentation. The expression of individual HOX gene may also occur in places like hair, blood cells and developing sperm cells.

This indicates that though the main function of HOX genes is to set up structures along the main axis of the embryo, but the individual gene may also guide the formation of specific structure, which may not lie along the body axis.

• PAX Genes (paired box genes) shape the development of sense organs (eye and ear) and the nervous system. These genes regulate cellular differentiation at the time of epithelial- mesenchymal transition.

The paired box gene is DNA sequence that encodes a 128 amino acid protein. This protein transcription regulating factor binds to the DNA at sites (domains) for activation of transcription. In humans the Pax gene family consists of 9 genes (Pax-1 to Pax-9). Details of the importance of these genes in dentistry is discussed later in the book.

Some of the developmental abnormalities associated with Pax genes are discussed below.

• The Pax-2 gene located on the 10th chromosome (10p25) when mutated results in renal malforma- tion and malformation of retine and optic nerve (renal-coloboma syndrome). Mutations in the Pax-3 (chromosome 2935), Pax- 6 (chromosome11p13) cause loss of hearing, areas of depigmentation in hair and skin and abnormal pigmentation of iris along with the absence of iris and sarcoma.
• SOX Genes (LEF-1, SRY type HMG) are expressed in many structures during develop- ment. Sox genes consist of over 20 members in the family. The Sox genes contain a 79 amino acid domain that is known as HMG (high mobility A BRIEF ACCOUNT OF THE MOLECULAR group) box. These genes show homology with Y-linked SRY gene. SRY gene plays a major role in male sex determination. The name of this group (SOX) was derived from SRY HMG box. The HMG domain activates transcription by bending DNA (hence, also called DNA bending protein) in such a way that other regulatory factors can also bind with promoter region of genes. The skeletal tissue and type II collagen development is linked to the Sox-9 gene. Mutation of Sox-9 on chromosome 17 result in bowing of long bones. A mutation of the Sox-10 on chromosome 22 is incriminated in Hirschsprung disease.
• POU Genes (Pit-1, Oct) Play a vital role in cleavage of the early embryonic cells. A Pou transcription factor is constituted by a homeodomain region and a second site on the factor that binds to the target DNA segment. The Pou gene family is named by the fist alphabets of few of the first genes identified i.e., Pit-1, Oct-1 and Unc-86.
  The development of anterior pituitary gland is related to the expression of the Pit-1 gene. The Oct-2 gene is expressed in the B-cell activating immunoglo- bulin synthesizing genes. The Unc-86 gene is involved in the development of nematode neuronal cells.
• Lim proteins regulate muscle differentiation. These genes constitute a large family and are associated with the development of all parts of body. Absence of Lim-1 protein results in headless embryo.
• T-BOX (TBX) Genes initiate the induction of mesoderm germ layer and specification of hind v/s forelimbs. Notochord differentiation is related to T-Box expression.
  Also called Brachyury T-box genes, encode tran- scription factors that play important roles in develop- ment of mammary glands, upper limb and heart. Mutation of TBX-3 on chromosome 12 causes hypo- plasia of mammary gland and abnormalities in upper limbs. Mutation in TBX-5 may cause arterial septal defects and absence of forearm.
• Dlx Genes (Dlx-1 to Dlx-7) are involved in morphogenesis of jaw and inner ear. The Dlx gene family consists of 6 members and is closely associated with HOX genes.

A Brief Account Of The Molecular Control Of Early Embryonic Development

In the preceding part of this chapter we have seen that the development of human body is regulated by sequential gene expression. In this process specific genes are expressed in sequence, one after the other, at different regions of the body resulting in development of dissimilar structures at different regions of the same body.

Finally the entire process of development results in structural and functional differentiation of highly specialized tissues and organs endowed with definite roles. These cascades of gene expression and the resultant sequence of embryological events are well-studied in the fly Drosophila that is very briefly discussed now.

Establishment of the Axes of Embryo

The process of axes differentiation and early embryonic development in Drosophila is one of the earliest events and under strict genetic control but in human this part of development occurs under lesser rigid genetic scrutiny.

In Drosophila the development of the anteroposterior, dorsoventral and right/left axes are under the control of a group of maternal genes, which are called maternal effect genes. These genes are expressed outside the egg (within the mother fly) even before fertilization. The products of expression of these genes are transcription factors called morphogens that subsequently act on future zygotic targets.

These products are carried into the egg where they diffuse unequally in the oocyte cytoplasm to establish gradients across the future anteroposterior axis of the egg. Due to the presence of such gradients proteins are differentially distributed in the common cytoplasm of the egg. These gradients determine the synthesis of specific proteins in the different segments of the embryo.

In the Drosophila body axes are established even before fertilization. In mammals body axes do not become fixed until the end of cleavage or early gastrulation. The formation of anteroposterior axis in human embryo is initiated by the cells of future anterior margin of the embryonic disk.

This area of disk expresses the genes (OTX2, LIM1, and HESX1) which are necessary for formation of the head even before gastrulation. The B-Catenin, BMP-4 and activin genes lead to the formation of primitive streak. These genes are first expressed in the cranial region of embryo. Once the primitive streak is formed the embryonic axes (craniocaudal, dorsoventral and right/left) are soon established.

Segmentation

The Drosophila embryo next divides into identical segments (Fig. 8.5). This is achieved by segmentation genes, which are subclass of genes called zygotic genes. The diffused ‘morphogens’ control the expression of segmentation genes. The segmentation is completed in three steps in the Drosophila embryo.

The segmentation gap genes control the first step of segmentation that divides the embryo into broad regions. Gap genes are controlled by maternal bicoid proteins.

The pair rule genes regulate the subdivision of the embryo in 7 segments along craniocaudal axis in the second step of segmentation. The pair rule genes are regulated by the products of genes regulating the previous step, the Gap genes.

Segmentation enters the third stage where the segment polarity genes like the Gooseberry, hedgehog, patched, wingless genes divide the embryo further into 14 segments. These segment polarity genes are controlled by the genes of the previous step, the pair rule genes. Similar segmentation genes used in developing humans have been identified.

Determination of Regional Characteristics

The process of segmentation is followed by development of regional characteristics in the newly formed segments of embryo. As discussed earlier, activation of the homeotic genes brings about specific characterization of individual segments of the developing embryo. These genes determine the growth of antennae, wings or legs in the appropriate segments of the fly.

The 8 Homeobox genes contains a highly conserved coding sequence of 180 base pair region of DNA (usually near their 3′ end) called the homeobox.

Similar to fruit fly, homeobox genes of humans are also expressed sequentially in craniocaudal direction during axis formation. The sequential expression of HOX genes correlates with the development of structures in craniocaudal sequence.

A number of other gene families that regulate development also contain similar homeobox domains synthesizing homeodomains but with different sequences in their genes, e.g. Paired, Pax (Pax-4 and Pax-6), POU, LIM, etc.

There is a direct relationship between vitamin A (retinol) and expression of HOX gene. Either too much or too little of retinoic acid (metabolite of Vitamin A) causes misexpression of HOXB-1. This may lead to abnormal development of legs, hindbrain and pharyngeal neural crest cells. The retinoic acid may cause extra pair of limb in frogs at the site of tail. This is an example of homeotic shift similar to formation of extra pair of wings in fruit fly.

Summary

• The process of Growth in an organism is achieved by cell division.
• The process of Differentiation is the creation of new types of cells or tissues.
• Thus the signals that determine the initial organization in an embryo come from the primary organizer.
• Molecular processes in development are governed by fundamental embryological processes like induction, segmentation, migration, differentiation and apoptosis (programmed cell death) in the embryonic cells.
• Cellular signals are relayed from the cell membrane to nucleus in sequential steps (signal transduction pathway) for the initiation as well as control of cellular processes.
• Several molecules are involved in the process of signal transduction like growth and differen- tiation signaling molecules, e.g. Epidermal Growth Factor (EGFS), Transforming Growth Factors (TGFs), Hedgehog proteins, the WNT proteins, etc.
• These Growth factors bind to their receptors to execute specific signal transduction activities.
• Transcription factors are the molecules that interact directly or indirectly with the genomic DNA to carry out final effects of cell signaling. Proteins like the Basic helix-loop-helix protein, Zinc Finger Proteins, HOX gene and Sox Gene proteins, etc. are important transcription factors.
• The process of early embryonic development includes the steps of establishment of the axes of embryo followed by segmentation of the embryo and determination of its regional characteristics.

Genetics Of Periodontitis

Periodontitis is a dental disorder involving inflammation and infection of the ligaments and bones that support the teeth and results from progressive and uncontrolled gingivitis.

Classification of periodontitis is based on the rate of disease progression. Periodontitis can be divided into two major types, e.g. aggressive (localized aggressive and generalized aggressive) and chronic.

Until recently, periodontitis was thought to be strictly determined by environmental factors like poor oral hygiene, smoking, low socioeconomic condition, bacterial infections of oral cavity, etc. alone. It is a common observation that given the same status of poor oral hygiene and sharing the same environmental factors, some people show severe periodontal disease while some of the same people suffer from mild or no disease.

This indicates the existence of individual differences in susceptibility to the disease and perhaps towards a genetic basis for the susceptibility. Scientists are trying to find out the genes responsible for the disease. It is now well known that periodontal disease is multifactorial (complex) and its susceptibility is influenced both by genetic and environmental factors.

Genes responsible for multifactorial disease are also known as susceptibility genes. However, these genes alone do not produce the disease unless they are exposed to the necessary environmental factors.

Periodontitis is also associated with many types of syndromes and genetic disorders. Periodontitis presents itself as one of the clinical manifestation of the syndrome. This establishes the genetic basis of periodontitis as these syndromes result due to mutation in a single gene/genes.

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Verification of the Genetic Basis of Periodontitis

Following genetic analytical methods have been used to evaluate the genetic basis of periodontitis.

Familial Aggregation Of Periodontal Diseases

Several members of the same family suffering from periodontitis is indicative of a genetic etiology. Many scientific studies have indicated familial aggregation for both aggressive (Boughman et al., 1988; Long et al, 1987; Marazita et al, 1994 and Hart and Korman, 1997) and chronic periodontitis (Hassell and Harris, 1995). Reports of the familial nature of chronic forms of periodontitis are less frequent.

This aggregation within families strongly suggests of a genetic predisposition to the disease. However, the familial aggregation of the disease may also reflect on the common environmental factors prevalent in the family, i.e. levels of oral hygiene, shared transmission of bacteria, same socioeconomic condition, sanitation, etc.

Therefore familial aggregation is only suggestive of a genetic basis but does not prove a definite genetic etiology. To substantiate evidence in favor of a genetic basis of familial aggregation, application of more specific tools such as segregation and twin studies are imperative.

Segregation Analysis

Segregation analysis is conducted in affected families to determine the mode of inheritance of a disease whether it follows an autosomal-dominant, autosomal- recessive, X-linked dominant, X-linked recessive or multifactorial mode of inheritance.

Many studies have been conducted to determine the mode of inheritance of diseases. These studies have indicated many patterns of inheritance in periodontitis. The most definitive segregation analyses in North American families were performed by Marazita and coworkers (1994) who studied more than 100 families segregating aggressive forms of periodontitis and found support for an autosomal-dominant transmission.

They concluded that autosomal-dominant inheritance with approximately 70% penetrance occurred for both Blacks and nonBlacks. It is believed that various forms of aggressive periodontitis are due to single gene defects that are inherited as autosomal- dominant disorders with incomplete penetrance. Some studies have indicated an autosomal recessive mode of inheritance (Saxen, 1980; Saxen and Nevanlinna, 1984). Few workers have also reported X-linked inheritance (Melnick et al, 1976) in the same context.

Why were these studies unable to point towards a single type of mode of inheritance? It might be because of the multifactorial or polygenic nature of the disease or probably due to the fact that the origin of periodontitis is heterogeneous in nature. There is a common precedent in genetics, with relation to heritable pathologic conditions, to show different modes of inheritance in different families. These findings reflect that different genetic loci are capable of causing the same disease in both dominant and recessive manners.

Even though segregation studies have shown different modes of inheritance, they all support the involvement of genes in the causation of the disease.

Twin Studies

Many human traits are multifactorial (polygenic). The modes of inheritance in which can be manipulated with environmental modifications. Twin studies allow evaluation of the importance of genetic as well as environmental factors in causation of a disease. As early as in 1940, Noack recognized similarities of features in periodontal conditions seen in identical twins. Many twin studies in the past were carried out on monozygotic adult twin groups. These twins were grouped as the reared-together and reared-apart sections. Following conclusions were drawn that:

• The periodontal conditions in identical twins were similar.
• A significant genetic influence on various parameters of periodontal diseases was found.
• Findings in the reared-together and reared-apart groups were similar which strongly suggested a genetic basis of the disease. This finding also indicated that environmental factors had no significant influence on development of the disease.

A study by Corey et al (1993) on monozygotic and dizygotic twins indicated a higher concordance rate for monozygotic twins (23% for monozygotic twins as compared to 8% for dizygotic twins). Concordance implied that both the twins of a pair were suffering from periodontitis. This strongly suggested the involvement of a genetic component in the etiology of periodontitis as we know that monozygotic twins possessed identical genes.

Michalowicz et al (1991) investigated alveolar bone height and probing depth in twins from the Minnesota study and showed significant variations in them, according to the differences in their genotype. The twin groups had similar smoking histories and oral hygiene practices. It was concluded that genetics plays a role in moulding the susceptibility to periodontal disease.

In another study Michalowicz (1994) found that monozygotic twins reared either together or apart have been found to bear a more similar type of periodontal disease experience than found between dizygotic twins. These findings indicate that genetic factors may influence the manifestations of periodontal disease.

In a more recent study (Michalowicz et al, 2000) of another large human twin cohort, hereditary factors accounted for approximately 50% of “adult period- ontitis”. The heritable component for periodontitis was not associated or influenced by behaviors such as smoking, utilization of dental care, and oral hygiene habits. This indicated that development of periodontitis was influenced by genes that mediated biological mechanisms.

Twin studies were also used to know about the effect of host genes on the composition of micro- bacteria in the oral cavity. The long-term colonization of bacteria in the oral cavity may be due to the genetic constitution of the host. Studies have revealed that adolescent twins were more similar in their oral microbiota than pairs of unrelated individuals (Moore et al, 1993).

However, the study of Michalowicz et al (1999) in adult twins indicated that neither the host genes nor the environment played a significant influence on the presence of bacteria in subgingival plaque. These two studies suggested that although the host genes influence initial bacterial colonization but this influences do not persist till adulthood.

Linkage Studies For Periodontitis

Linkage studies are conducted to localize the disease causing gene on a specific chromosome. Till date only very few linkage studies analyzing aggressive periodontitis are reported.

• Saxen and Koskimies carried out the first linkage study in 1984 on Finnish families to test the claim for association of aggressive periodontitis with HLA antigens. This study concluded that the gene responsible for aggressive periodontitis was not linked with HLA antigen in these families.
• Boughman et al reported the second linkage study in 1986 for a Maryland population. According to this study the gene responsible for aggressive periodontitis was situated on the long arm of chromosome number 4 (4q 11-13). The inheritance pattern of this gene was determined as autosomal- dominant. The gene was found to cosegregate with the gene responsible for dentinogenesis imperfecta. Both the genes were linked, i.e. present close to each other on the long arm of chromosome number 4.
• However, another study in 1993 (Hart et al.) on a different population of USA failed to locate the gene for aggressive periodontitis on chromosome number 4. This finding indicated that aggressive periodontitis was heterogeneous in nature, i.e. different genes may be responsible for different forms of aggressive periodontitits.
• Prepubertal periodontitis (PPP) is a rare and rapidly progressive disease of young children that results in destruction of the periodontal support of the primary dentition. The condition may occur as part of a recognized syndrome or may occur as an isolated finding. Both autosomal dominant and recessive forms of Mendelian transmission have been reported for PPP. Hart et al. (2000) have localized a gene of major effect for PPP on chromosome 11q14. This PPP candidate interval overlaps the region of chromosome 11q14 that contains the cathepsin C gene responsible for Papillon-Lefèvre and Haim-Munk syndromes.
• Recently in 2004, a linkage study by Li and coworkers mapped the gene responsible for localized aggressive periodontitis on the long arm of chromosome number 1 (1925).

Association Studies For Periodontitis

The susceptibility to a disease depends upon the genetic background of a person. Since periodontitis is an inflammatory disease, it is believed to be affected by genes that are involved in the regulation of inflammatory cell functions. Associations between periodontitis and genes responsible for control of inflammat response are of great value in understanding the genetics of periodontitis.

The immune system controls inflammatory responses against a disease. However, the immune response to a disease varies from person to person and the pattern of immune response in an individual is determined by the genetic make-up unique to the person. The clinical presentation of a disease, therefore, varies in different persons reflecting the diversity of immune response shown by each individual. The same concept is also applicable to developing specific and targeted medical treatment.

Thus if we are able to identify the variations in the genes involved in the control of inflammatory process and ultimately the disease, we can successfully identify the degree of susceptibility, severity and prognosis of the disease and perhaps design medical interventions custom made according to a particular etiology. In recent past many studies have identified the association between periodontitis and variation in the genes (single nucleotide polymorphism) responsible for regulation of immune and inflammatory response.

Association of HLA (Human Leukocyte Antigens) with Periodontitis

The human leukocyte antigen (HLA) complex plays an important role in immune response. Many autoimmune diseases are found to be associated with various HLA antigens. Genes for class I and II antigens are located on chromosome number 6. At present more than 150 HLA antigens are known. The HLA molecules are involved in antigen recognition of periodontal pathogens, interaction between T and B- lymphocytes and in production of IgG. Following positive association between HLA and periodontitis was found:

• A positive association was reported between aggressive periodontitis and HLA-A9 and B15 antigens (Sofaer, 1990). Persons having these two antigens are at 3.5 times higher risk of developing the disease as compared to those who are negative for these antigens. Thus HLA -A9 and B15 seem to represent susceptibility factors for aggressive periodontitis.
• Class 2 DR4 antigen is in association with type I diabetes mellitus (Rotter et al, (1992). Since periodontitis is a diabetes related complication, DR4 antigen and periodontitis are said to be in association with each other. The DR4 antigens were found to be more prevalent in patients with aggressive periodontitis than in controls (Katz, Goultschin, Benoliel et al., 1987). However, a few studies have been unable to find any association between the two.
• HLA-A2 and HLA-B5 antigens appear to be less prevalent in patients of aggressive periodontitis as compared to controls (Kaslick, West, Chasens et al, 1975). This indicates that these HLA antigens are protective in nature.

Though above studies have indicated a positive association, many other studies have also pointed towards a negative association between HLA and periodontitis. The cause of this discrepancy appears to be the involvement of environmental factors and racial differences acting as variables.

Association between Periodontitis and Interleukin-1 (IL-1) Gene Polymorphism

Interleukin-1 plays an important role in the initiation and progression of periodontal disease. Il-1 is found in two different forms; IL-1a and IL-1b. Genes for IL-1 are present on the long arm of chromosome number 2 (2q13). IL-1 is mainly produced by activated monocytes. There is an increased level of IL-1 in periodontal tissue which stimulates bone reabsorption, inhibits collagen synthesis, up regulates matrix metalloproteinase activity and synthesis of prostaglandin.

• An initial study (Korhman et al, 1997) reported a positive association between polymorphism in the genes encoding for IL-1 and IL-1B (composite genotype) and increased severity of periodontitis. This association was seen in nonsmokers. Genotype positive nonsmokers were 6.8 times more likely to have severe periodontal disease.
• Another study (Mc Guire and Nunn, 1999) reported that the IL-1 genotype increased the risk of tooth loss by 2.7 times and heavy smoking increased it by 2.9 times. The combined effect of heavy smoking and IL-1 genotype positive increased the risk of tooth loss for 7.7 times.
• A study carried out in 2001 by Axelsson indicated that genetic polymorphism of IL-1 and smoking seems to have synergistic risk factors. These factors when combined leads not only to tooth loss but also for alveolar bone loss. Nonsmokers are at low risk of tooth loss.

(Thus there are contrasting findings regarding interactions between genetic polymorphism and smoking to affect the risk of periodontitis. Though the IL-1 genotype and smoking habit seem to interact with each other, the nature and direction of this interaction is poorly understood. More studies with large sample size are needed to evaluate the exact correlation between smoking and IL-1 interactions).

• A variant of the IL-1B coding region with a single nucleotide base pair substitution is associated with a four-fold increase in IL-1B production.
• Generalized aggressive periodontitis is in linkage disequilibrium with an allele at IL-1B. This suggests that the gene for periodontitis is located close to the gene for IL-1B (Diehl et al, 1999).
• The IL-1B allele is found to be more prevalent in patients suffering from advance chronic period-ontitis.
• Though the association between periodontitis and polymorphism of IL-1 gene cluster was observed in about 30% of European population, it was observed so only in 2% of Chinese population (Armitage, 2000).
• Many studies have indicated the lack of association between interlukin-1 polymorphism and aggressive periodontitis.

Where do we stand now? Studies in both chronic and aggressive periodontitis have yielded mixed results. It is evident that to get a clear picture of the association between IL-1 polymorphism and various types of periodontitis we need more comprehensive studies.

Tumor Necrosis Factor (TNF-α) and Periodontitis

The TNF-a is the proinflammatory cytokinase which is involved in pathogenesis of periodontitis. The TNF-α gene is located on chromosome number 6. Two different polymorphisms have been reported in the promoter region of the gene (Fassmann et al, 2003). This results in increased production of TNF-α. The level of TNF-a molecules is high at the sites of active tissue destruction and low at healthy sites. The TNF-α is the proinflammatory cytokinase

Individuals expressing the TNF-a poly-morphisms manifest greater susceptibility to certain infections. Although till date TNF-a poly-morphism has not directly been linked to susceptibility to periodontitis, there is a strong possibility that such a link exists (Craandijk et al, 2004).

Association between Neutrophil IgG Receptor (FcyR) Polymorphism and Periodontitis

FcyRs are the group of receptors that are expressed on the cell surface of leukocytes binding to IgG antibodies. The interactions between FcyRs and IgG trigger a variety of immune responses like phagocytosis, endocytosis, antibody dependant cellular cytotoxicity and enhancement of antigen presentation.

Polymorphisms in the genes coding FcyRs (especially in FcyRII) receptors (Osborne et al, 1994) are common. The FcyRII receptor is the only receptor that recognizes bacteria opsonized with IgG2. These polymorphisms result in the expression of receptors of high, low and intermediate affinities. The low affinity receptors lead to a decreased immune function and subsequently to increased susceptibility to periodontal pathogen and to severe periodontitis even in presence of high level of antibodies.

Kobayashi et al (2000) found functional poly- morphisms of IgG Fc receptors (FcyR) in Japanese patients with AgP. They found that FcyRIIIb (NA2 allele) was linked with the aggressive form of periodontitis and possibly also linked to an FcyRIIIa allele. Kobayashi et al (2001) reported that 2 FcyRIII alleles may be associated with the degree of severity in chronic periodontitis in a Japanese population.

Meisel et al (2001) analyzed the association between FeyRIIIa (high-affinity receptor) and FcyRIIIb (low- affinity receptor) and chronic periodontitis (CP). Thus a large number of studies have recognized causal association between polymorphisms of FcyR genes and both aggressive and chronic periodontitic conditions.

The alleles of FcyR gene are linked with genes causing periodontitis. The genetic poly-morphism of FeyRII receptor is a promising marker for the susceptibility of periodontitis.

Association between IgG2 Production and Periodontitis

Immunoglobulin G2 (IgG2) is produced in response to periodontal infections. The production of IgG2 is under the control of specific genes. The levels of production of IgG2 vary from person to person. The reduction in the production of IgG2 during the course of periodontal infection may lead to an increased susceptibility to disease. On the other hand an increase in the production of IgG2 provides sufficient protection against the disease. Marazita et al, (1994, 1996) studied families with aggressive periodontitis and found a major locus accounting for approximately 62% of the variance of IgG2 production.

Patients with periodontitis and normal subjects vary greatly in their capacity to produce IgG2. Patients with high titers of IgG2 antibodies have significantly less attachment loss than do patients with low titers. It is observed that patients with localized aggressive periodontitis have high titers of IgG2 compared to patients with generalized periodontitis. This indicates that IgG2 provides sufficient protection against the spread of disease and tries to limit the same.

Associations between Polymorphism in Matrix Metalloproteinase, Cathepsin C and Vit D Receptor with Periodontitis

The matrix metalloproteinase (MMP) are enzymes involved in connective tissue destruction. Single nucleotide polymorphisms are observed in MMP genes and are designated MMP-1, MMP-2, MMP-3 polymorphisms. Very few studies have evaluated the correlation between MMP polymorphisms and periodontitis (Holla et al 2005; Itagaki et al, 2004).

A recent study has indicated a decreased level of Cathepsin C activity in chronic periodontitis. Cathepsin C is a lysosomal enzyme that plays an essential role in immune and inflammatory processes and hence may play an important role in periodontitis (Hewitt et al, 2004). A mutation of the cathepsin C gene leads to Papillon-Lefore syndrome (see later).

Polymorphism in Vit D receptor has been associated with aggressive periodontitis. A very few studies, though, have verified their association.

Syndromic Form Of Periodontitis

It has been observed that there is a clear association between some type of genetically determined syndromes/disorders and periodontitis. These syndromes increase the susceptibility of the individual to periodontitis by interfering with the structural integrity of periodontal tissue or periodontitis may be a concomitant feature of the syndrome. These syndromes are due to mutations in a single gene (monogenic or Mendelian syndromes). Transmission of the disease from one generation to the next follows

Mendelian patterns of inheritance as autosomal- dominant (AD), autosomal-recessive (AR) or X-linked dominant or recessive (XLD or XLR) traits.

As stated above, periodontitis presents itself as a part of a wider spectrum of clinical manifestations of a number of syndromes that occur as a result of mutations in genes. This itself points towards the clearest and most direct clinical evidence that periodontitis has strong genetic etiology. Following is a brief description of some of the genetic disorders where periodontitis is observed as a clinical manifestation.

Neutrophil Functional Dispiders

Leukocyte Adhesion Deficiency Syndromes (LAD Syndromes)

Polymorphonuclear leukocytes play an important role in the restriction of the bacterial infection. There are several adhesion receptors on the surface of polymorphonuclear leukocytes. Adhesion receptors are necessary for the proper functioning of leukocytes (phagocytosis and chemotaxis). If the circulating leukocytes have defective or reduced adhesion surface receptors they will not adhere to vascular endothelium.

As a result leukocytes would not be accumulated at the site of inflammation where they are needed to combat the infection. This leads to an increased susceptibility to an infectious disease as exemplified by the leukocyte adhesion deficiency syndrome (LADS). Recurrent bacterial infections, impaired pus formation and impaired wound healing clinically characterize the syndrome. A number of these infections are associated with increased susceptibility to periodontitis. LAD occurs in two forms, LAD syndrome type I and LAD syndrome type 2.

Inheritance: Both types of LAD syndromes are inherited as AR.

Mutation in LAD type 1: The mutation occurs in the B2 integrin chain (ITGB2) gene -21q22.3 (Arnaout et al, 1990). At present more than 20 mutations of the integrin B2 gene are known. The gene mutation causes defects in the integrin receptors of leukocytes. This mutation leads to defects in cell adhesion and chemo- taxis ultimately resulting in increased susceptibility to severe infections including prepubertal aggressive periodontitis.

Mutation in LAD type 2: This is a rare variety of the LAD syndrome and is usually associated with psychomotor retardation (mental retardation, short height and recurrent infection). The syndrome is caused due to a mutation in the guanosine 5′- diphosphate-fucose transporter 1 (SLC 35C1) gene (Lowe et al, 1990; Ishikawa et al, 2005). The infectious episodes and severity of disease is much milder as compared to LAD type I. This syndrome results in chronic periodontitis.

Chédiak-Higashi Syndrome

The syndrome is clinically characterized by a decreased pigmentation of eyes and hair, photophobia, nystagmus and susceptibility to infection.

Inheritance: This syndrome is inherited as an autosomal recessive (AR) trait.

Mutation: The mutation occurs in lysosomal trafficking regulator (LYST) gene-1q42.1-q42.2 (Nagle et al, 1996). This leads to an abnormal transport of vesicles to and from neutrophil lysosomes or to a defect in the ability of cells to produce lysosomes (Charette et al, 2007). Chédiak-Higashi syndrome is associated with severe periodontitis unresponsive to conventional periodontal treatment. However, periodontitis is only seen in its severe form with the syndrome.

Deficiency in Neutrophil Number (Neutropenias)

Neutropenia is defined as an abnormally low number of circulating neutrophils. The condition is associated with increased susceptibility to infections such as aggressive periodontitis. Table 14.1- includes various types of neutropenias and associated gene mutations in brief.

Genetic Defects of Structural Components

Papillion-Lefèvre Syndrome (PLS)

This syndrome is characterized by severe early onset periodontitis, which affects both primary and permanent dentitions and hyperkeratosis of palmar and plantar surfaces.

Inheritance: Autosomal recessive inheritance (AR).

Mutation: It is due to mutations in the cathepsin C gene which is located on chromosome 11 (11q14-q21). At present more than 50 mutations have been identified in cathepsin C gene. Cathepsin C is a cysteine protease that plays a role in degrading proteins and activating proenzymes in immune and inflammatory cells. Inactivation of cathepsin C results in failure to cleave and activate the neutrophil serine proteases cathepsin G, neutrophil elastase and proteinase 3 (Pham et al, 2004).

It was observed that in some of the PLS patients aggressive periodontitis were associated with a kind of virulent microorganism. Elimination of this microorganism prevented the periodontal destruction. This suggested that the periodontitis was not a direct effect of mutation of the gene but the gene mutation increased the susceptibility of a person to infection by the specific microorganism instead.

Ehlers-Danlos Syndrome (EDS)

This syndrome is associated with connective tissue disorders. EDS is characterized by defective formation of collagen fibers. The abnormal collagen in the patient leads to fragility and hyperextensibility of the skin, hypermobility of joints, easy bruising and is also associated with early onset of periodontitis. Though at least 17 different types of EDS have been reported but early onset periodontitis has been associated only with two of its subtypes: EDS type 4 and EDS type 8.

Inheritance: Both the types 4 and type 8 EDS are inherited as autosomal-dominant (AD) conditions.

Mutation: The mutation leading to the type 4 EDS was found to be linked to the gene responsible for the synthesis of type 3 (COL 3A1) collagen (Superti-Furga et al, 1988). This leads to the defect in synthesis of collagen 3 resulting in decreased levels of collagen 3.

Mutations causing type 8 EDS, though, have not yet been identified. This disease has been, nevertheless, linked to chromosome number 12 (12p13). The association of early onset periodontitis with type 8 EDS is more common as compared to that of type 3.

Hypophosphatasia

These patients have decreased levels of alkaline phosphatase in the serum. The condition is associated with abnormal bone mineralization, skeletal abnormalities and cementum hypoplasia. These patients present a severe loss of alveolar bone and premature loss of primary teeth. Pulp chambers may also get enlarged. Lack of connective tissue attachment to bone is responsible for early exfoliation of the primary teeth. Hypophosphatasia may be considered as an etiology of aggressive periodontitis. The condition leads to premature loss of primary teeth and occasionally permanent teeth.

(Mode of) inheritance: Autosomal dominant or recessive.

Mutation: Mutation occurs in the ALPL (alkaline phosphatase, liver/kidney/bone) gene. The gene is localized on the short arm of chromosome 1 (1p36.1- p34). Mutations result in abnormal production of alkaline phosphatase leading to poor mineralization of tissues.

Kindler Syndrome

It is caused due to a mutation in the Kinderlin gene (KIND1) (Jobard et al, 2003). The gene is expressed in many tissues including epidermal keratinocytes. In skin it plays a role in cell adhesion process. Patients of Kindler syndrome show multiple dermatological findings (hyperkeratosis, pigmentation, eczema, skin fragility, etc.), the syndrome is associated with severe aggressive periodontitis both in the primary and secondary dentition.

(Mode of) inheritance: Autosomal recessive (AR) in nature.

Mutation: Mutation occurs in the Kindlin gene (KIND1) situated on the short arm of chromosome number 20 (20p13).

From the above description one can conclude that isolated periodontitits is clearly behaves as a multifactorial disease. Researchers have shown associations between complex form of periodontitis and abnormalities in genes (single nucleotide polymorphism) responsible for regulation of immune and inflammatory responses. Further research is needed to examine the role of both environmental and genetic factors in the causation of periodontitis.

We have also seen that monogenic (single gene) inheritance of periodontitis is possible but these incidences are associated with certain syndromes. We have successfully located many mutant genes (refer table) and understood to some extent the mechanisms of these mutations that disrupt normal homeostasis in the periodontum.

Though a large number of studies are being conducted to identify the genetic basis of periodontitis, we are still far away from determining the risk factors, prevention and treatment of aggressive and chronic periodontitis, genetically.

 

Genetics Of Dental Caries

Dental caries is the medical term for tooth decay or cavities. Tooth decay is one of the most common of all disorders of teeth.

Bacteria, that are normally present in the mouth, convert all foods especially sugar and starch into acids. Bacteria, acid, food debris and saliva combine in the mouth to form a sticky substance called plaque that adheres of the teeth. Tooth decay begins if this plaque is not removed throughly and routinely. As stated earlier some of the plaque forming bacteria converts sugar and carbohydrates (starches) in the foods we eat into acids.

These acids dissolve minerals on the surface of the tooth. This erodes the enamel or creates pits on the enamel that are two small to see at first but they get larger over time. Cavities are usually painless until they grow very large and affect nerves or cause a tooth fracture. A tooth abscess can develop. Untreated tooth decay also destroys the internal structure of the tooth (pulp) and ultimately causes loss of the tooth.

The etiology of dental caries has been studied for many years. Multiple factors contribute to a persons risk for caries.

• Environmental factors: Diet, oral hygiene, fluoride exposure and the cariogenic bacteria.
• Host factors: Salivary flow, salivary buffering capacity, position of teeth relative to each other, composition of tooth enamel and host immune response.

In spite of all that is known about this disease there are individuals who still appear to be more susceptible to caries and those who are extremely resistant to dental caries regardless of the environmental risk factors to which they are exposed.’

Similar to periodontics, malocclusion and cleft lip or cleft palate, dental caries is also to be a multifactorial (complex) disease. Though dental caries are seemingly caused out of interaction between environmental and genetic factors, yet the disease is to a great extent influenced by environmental factors. Most scientists agree that the genetic component of dental caries has a minor one in comparison to the overall effect of the environment.

Read and Learn More Genetics in Dentistry Notes

Twin Studies

How We Come to Know that Genetics Plays a Role in the Etiology of Caries

Twins studies were carried out to investigate the role of genetics in the etiology of caries. In case of multi- factorial diseases where genetic and environmental factors play important role in the causation of the disease, twin studies can be used as useful tools to evaluate the roles of the genetic component of the disease. Presence or absence of the trait or disease in a large number of the two types of twins (mono and dizygotic varieties) is calculated in percentage.

Diseases in which the percentage of twin pairs, where both the twins of the pair are affected, is greater in the monozygotic group as compared to the dizygotic group, the diseases can be confirmed to have a definite genetic etiology. The genetic basis of a disease is tested in monozygotic twins who are reared together in the same environment or separated after birth and reared in two different environments. All the similarities in these twins would expectedly be due to com- mon genes and all the dissimilarity will be due to environmental factors.

The early twin studies carried out at the beginning of the 20th century provided some indications that inheritance played a role in caries but the evidence were not conclusive. They only pointed that inheritance was only a contributor to the process (Bachrach et al, 1927; Horowitz et al, 1958; Mansbridge et al, 1958 and Goodman et al, 1959). Twin studies conducted later in the century compared the incidence of caries in monozygotic twins and dizygotic twin groups.

Many studies detected a statistically significant genetic component in the susceptibility to caries and demonstrated that caries experience in monozygotic twins had a greater concordance (Bordoni et al, 1973, Hassell et al, 1995; Townsend et al, 1998). These studies concluded that not only environmental factors clearly have a greater influence but that genetic factors also contribute to the causation of dental caries (Niswander et al, 1975).

A major advance in the understanding of the role of inheritance and the incidence of dental caries was achieved by analyzing twins reared apart. These patients had an average age greater than forty and did not share similar environments from shortly after birth until the time of analysis. The analysis of twins raised apart provides the strongest evidence of a genetic contribution to the incidence of dental caries (Borass et al, 1988 and Conry et al, 1993).

The analyses of dental caries incidence in monozygotic and dizygotic twins also indicate that a large number of different genes contribute to the observed outcomes. Although the twin studies provided some strong evidence of genetic contribution to the risk of dental caries, none provided any evidence of linkage to specific genes (Shuler, 2001).

Risk Factors In Dental Dental Caries

The most important components contributing to the risk of dental caries are as under:

• Microorganisms present in the oral cavity and the host immune response
• Cariogenic diet
• Role of saliva in protection against caries • Morphology of tooth and composition of enamel matrix
• Gene(s) for dental caries.

Many studies have indicated that even if all the environmental factors are identical and monitored to be under controlled and standard conditions, variability in the susceptibility to dental caries still exists and differs within the tested individuals.

This indicates that certain environmental factors are more cariogenic for some individuals as compared to others. This may be explained by the existence of genetic variability among individuals. The genetic influences modify the expression of disease in the individual.

Microorganism Of The Oral Cavity And Host Immune Response

Dental caries will not occur if the oral cavity is free of bacteria. These bacteria are organized into dental plaque. Of the many types of bacteria in the mouth the most caries active appear to be Streptococcus mutans, Lactobacillus spp., Veillonella spp. and Actinomyces spp. These bacteria can be transferred from the mother to child and are present at varying levels in all human mouths. A variety of carbohydrates provide substrates for these organisms to grow on.

Most research on the bacteriology of dental caries has focused on the ubiquitous S. mutans and its ability to ferment sucrose (Loesche, 1986). This organism preferentially ferments sucrose to produce significant amounts of acid and extracellular polysaccharide (plaque). However, most researchers now agree that other organisms present in the mouth are capable of plaque formation and acid production from a variety of fermentable carbohydrate substrates besides sucrose which is present in the normal mixed diet.

The individual’s genotype may influence the likelihood of intraoral colonization of cariogenic bacteria. Scientists have shown that persons may be caries-resistant or caries-susceptible for a particular strain of bacteria. Even if cariogenic bacteria are present in the oral cavity the caries-resistant person usually doesn’t develop caries. This confirms the presence of important genetic elements influencing susceptibility to dental caries.

Streptococcus mutans was first isolated from human carious lesions in 1924 by Clark. This indicated that dental caries had a bacterial etiology and were transmissible infections. S. mutans isolates have been divided into eight serotypes. Human isolates represent serotype c, e and f. The S. mutans serotype c being the most prevalent streptococci isolated from human dental plaque.

Salivary immunoglobulin A (sIgA) is the major antibody present in the saliva. It is the host’s first line of immune defense against Streptococcus mutans. Salivary IgA is synthesized and secreted by plasma cells located in salivary glands. The host’s immune system protects individual from caries by producing various types of antibodies. In humans majority of these antibodies are of the IgA type but types IgG and IgM are also present in the saliva. Salivary IgA acts in the following ways to restrict infection:

• It neutralizes bacterial exotoxins.
• It neutralizes enzymes contributing to the disease processes such as glucosyltransferases. (Caries only form when this enzyme acts in presence of sucrose. Glucosyltransferases breakdown sucrose into its components like simple sugars known as fructose and glucose. Mutans streptococci use these sugars to form plaque. The enzyme glucosyltranferase could be used in making effective vaccines for the reduction in dental caries. Certain components of these enzymes when injected into an individual produce antibodies against the enzymes and inactivate bacterial activity to reduce plaque formation. In the past this enzyme was used as an antigen and was injected in the salivary glands of rats. The immunized rats produced the antibody IgA in their saliva and had fewer dental caries. Thus in future this enzyme may be used as vaccine in humans against caries).
• Inhibits the attachment of bacteria on epithelial or tooth surface.

Because of the above function of the salivary IgA most of the caries vaccines induce salivary IgA antibody response to S. mutans antigens (Han, 2007).

Human studies in the past have measured the levels of antibodies present in blood serum and saliva of caries patients. Some of these studies found a negative association between IgA and caries activities while other reports have shown a positive association. Few reports found no correlation between the two. Thus the association between the levels of IgA and the development of dental caries has been studied with conflicting results.

However, the number of papers is growing that report increases in immunoglobulin IgA contents in the saliva in cases with high caries experience (Weyna et al, 1979 and Dens et al, 1995). One group of research workers studied pair-matched patients between a group of IgA deficient patients and a section of immunocompetent normal subjects by age and plaque index. During a two years period they observed less caries experience in immunodeficient patients than found in normal controls (Robertson et al, 1980). Another study observed that IgA-deficient children showed caries scores lower than those of healthy children (Fernandes et al, 1995).

Association between HLA Antigens and Susceptibility to Dental Caries

Current evidences support the relationship between immune complex genes (HLA) and caries and the association of different levels of cariogenic bacteria and enamel defects. Many studies are now available which show the association between increased risk for caries and immune complex (HLA) genes.

One study reported the strong association between HLA DRw6 loci and DMFS index (Lehner et al, 1981). The same immune complex locus showed a low dose response to Streptococcus mutans antigens. However, few studies could not detect a relationship between HLA DR type and dental caries (De Vries et al, 1985 and Acton et al, 1999).

Celiac disease is an autoimmune disorder of the small intestine that occurs in genetically predisposed people of all ages. Symptoms include chronic diarrhea, weight loss and fatigue. The vast majority of celiac patients have one of the two types of HLA DQ. This gene is a part of the MHC class II antigen-presenting receptor.

The gene is located on the short arm of the sixth chromosome and as a result of this linkage the locus has been labeled CELIAC1. Celiac disease patients exhibit an increased incidence of dental caries. This might be due to the fact that these patients have defective enamel that predisposes the tooth to dental caries (Aine et al, 1990; Aine, 1996 and Aguirre et al, 1997).

However, the cause of this defective enamel in celiac disease patients has not been well-understood. These patients show significant positive correlation between their HLA type and presence of the enamel defect. The HLA-DQ2 and DQ 8 is strongly associated with enamel defects and dental caries in celiac patients.

The association between HLA complex and caries has indicated that a few genes in the HLA complex are responsible for dental caries resulting from altered enamel development and also due to low dose response to cariogenic bacteria (Lehner et al, 1981), i.e. less aggressive immune response to bacterial invasion.

We still do not know whether specific genes dedicated to the development of enamel or responsible immune response to cariogenic bacteria are located close enough (linked) to certain HLA complex. It is expected that the existence of any such association would be determined in the near future.

It is well known that individuals with immune deficiency diseases are susceptible to dental caries and have a greater frequency of harboring S. mutans than do normal persons, e.g. as seen in HIV infection.

Cariogenic Diet

The dietary components that contribute most to the process of caries formation are fermentable carbohydrates. These need to be retained in the mouth long enough to be metabolized by oral bacteria (principally Streptococcus mutans) to produce acid. The acid attacks the tooth enamel and gradually dissolves it (demineralization). A repair process known as remineralization offsets this demineralization process. The balance between remineralization and demineralization deter- mines the occurrence of caries.

The presence of fat in experimental diets has been shown to affect cariogenicity of sugars. The effects have been ascribed to enhance clearance of sugars from the mouth. It is also conceivable that several fatty acids express a potent antibacterial effect. The presence of calcium and phosphorus has been shown to influence the cariogenicity of foods; the effect, however, is restricted to the food containing the minerals. Evidence suggests that pyridoxine (vitamin B6) may exert a cariostatic (stopping caries) effect by enhancing decarboxylation activity in dental plaque.

Studies have indicated that the persons suffering from hereditary fructose intolerance are free from caries (Newburn et al, 1980 and Saxen et al, 1989). They are free from caries because there is absence of sugar in their diet and not because a hereditary fructose intolerance provides some kind of resistance to the production of caries.

Very few studies are available that has investigated the heritability of caries in relation to sucrose. A twin study in 2003 was aimed to determine heritability estimates for dental caries traits and sucrose sweet-ness preference. Results indicated that variations in dental caries traits and sucrose sweetness preferences have a significant genetic contribution mediated independently (Bertz et al, 2003).

Many studies have revealed that higher and more frequent sugar intake may increase the risk of caries formation in children. The high sugar intake reflects a preference for sweet substances. Inherited behavior and taste thresholds may play an important role in the frequency of carbohydrate intake. Genetic sensitivity to taste may be associated with a preference for or rejection of some food by children.

Many studies indicate that children belonging to the group “non- tasters” (with high threshold for taste) were sweet likers and prefer strong tasting food (Verma et al, 2006) while children belonging to group “tasters” are sweet dislikers and preferred weak tastes. The incidence of dental caries was significantly higher in nontasters as compared to tasters.

Role Of Saliva In Protection Against Caries

Saliva is body’s natural protective mechanism against decay. It contains salivary proteins that adsorb strongly onto the teeth, protecting enamel against acid dissolution. This adsorbed protective layer is referred to as the pellicle. Salivary proteins also act as antibacterial agents. Saliva is the primary resource of calcium, phosphate and fluoride; materials used to remineralize the enamel.

Saliva also acts quickly to clear away food debris from the mouth and to buffer the organic acids that are produced by the bacteria. Saliva is therefore a very vital and complex material in the prevention of dental caries. Salivary dysfunction can lead to rapid deterioration of dental enamel. Salivary dysfunction may occur as a result of certain medications or as side effects of medical treatments such as radiotherapy.

There is a strong correlation between the composition of saliva and the production of caries. The formation of dental plaque is the result of interactions between environmental and genetic factors. The caries- susceptible plaque is formed due to presence of certain chemicals in saliva. A group of saliva proteins known as proline-rich proteins (PRPs) are responsible for early plaque and pellicle formation (Mayhall, 1970 and Bennick et al, 1983).

At present eight different kinds of PRPs are known that are thought to be produced by a cluster of genes located on the short arm of chromosome number 12 (Goodman et al, 1985 and Mamula et al, 1985). People show variations in the type of PRPS produced in their saliva due to variations in their genotype at these regions. Some people with certain protein genotypes (especially Pa+ and Pr22) are more susceptible to dental caries (Yu et al, 1986), On other hand individuals with genotypes Pa- and Pr11 are resistant to the dental caries. Similarly low levels of salivary calcium and phosphate have been shown to be associated with the increased risk of caries.

Different individuals respond in a different way to specific biochemical differences in oral environment depending on their genetic constitutions. For example there is significant difference between monozygotic and dizygotic twins in terms of salivary flow, pH, and salivary amylase activity when compared between the two groups.

On the contrary, both the monozygotic twins of a pair will respond similarly (because of same genetic constitution) to these factors whereas such similarity may be lacking between the two individuals of a dizygotic pair (because of difference in their genetic constitutions).

Literature is nearly equally divided both in favor of and against the anticaries role of salivary immuno- globulins, especially sIgA. Many studies have indicated inverse relations between sIgA and caries (Camling et al, 1987 and Rose et al, 1994). Few studies have also reported increased levels of sIgA (Prakash et al, 1994). However, some studies have indicated no correlation at all (Kristila et al, 1994).

Xerostomia or decreased secretion of saliva (due to pathological dysfunction of salivary glands) has been demonstrated to be responsible for increased rate of caries. Studies indicate that a low salivary flow rate (less than 1 ml/min after salivary stimulation) is associated with an increased risk of caries. Sjögren’s syndrome is an autoimmune disorder in which abnormally activated immune cells attack and destroy exocrine glands that produce tears and saliva.

The primary and secondary variants of Sjögren’s syndrome are found to be associated with increased caries risk (Ravald et al, 1998). This is due to the fact that Sjögren’s patients have a decreased flow rate of saliva. Similarly, the condition of scleroderma related xerostomia is also associated with caries tooth (Wood and Lee, 1988).

Several medical conditions including therapeutic radiations administered to the head and neck regions (Nasman et al, 1994) and pharmacological agents with xerostomic side effects (Ryberg et al, 1990) lower salivary flow rate dramatically to pathological levels and elevate the patient’s risk of caries. The evidence therefore indicates that normal salivary flow rate is strongly protective against caries and clinicians should identify individuals with reduced salivary output to modify their treatment.

Dental caries has a higher prevalence rate in females as compared to males. A recent study indicated that biochemical composition of saliva and salivary flow rates are modified in women due to hormonal fluctuations during events such as puberty, menstruation and pregnancy. This makes the oral environment significantly more cariogenic for women when compared to men.

Saliva is a major carrier of topical fluoride. The concentration of fluoride in the ductal saliva, as it is secreted from salivary glands, is low. This concentration of fluoride is not likely to resist cariogenic activity. Drinking fluoridated water, brushing with fluoride toothpaste or using other fluoride dental products, on the other hand, can raise the concentration of fluoride in the saliva present in the mouth to about 100- to 1,000-folds.

Saliva and the extraneous sources thus serve as important sources of fluoride that gets concentration in the plaque and aid tooth remineralization. Fluoride concentrated in plaque and the saliva inhibits demineralization of the sound enamel and enhances the remineralization or recovery of demineralized enamel (Featherstone, 1999). Fluoride also inhibits dental caries by impairing cellular mechanisms of cariogenic bacteria.

High caries prevalence has been reported for individuals suffering from the deletion at 22q11; the High caries prevalence has been reported for 22q11 Deletion Syndrome (22q11 DS). It was observed that patients with 22q11 DS had impaired salivary secretion rates, higher numbers of cariogenic bacteria, increased salivary protein concentrations and reduced output of electrolytes in the saliva compared to the controls. This indicated that the salivary function is affected in 22q11 DS explaining increased caries risk seen in these subjects (Klineberg et al, 2007).

Morphology Of Tooth And Composition Of Enamel Matrix

The morphology of teeth related to their shapes, sizes, pit and fissure morphology, enamel structure and composition, arch forms, dental spacing and order of the teeth are some of the important factors that regulate the “washing” effects of saliva and thereby may profoundly influence the production of caries.

These factors are in fact largely determined by hereditary factors. Both kinds of twin studies (“twins reared together” and “reared apart”) have indicated that the morphologies of teeth, arch forms, dental spacing, malocclusion, etc. have strong genetic contributions.

Teeth are composed of a thin layer (1-2 mm) of dental enamel, which forms the hard protective coating over the tooth. This layer mainly consists of calcium, phosphate and other ions in a structure known as “hydroxyapatite”. Dental enamel is porous and is susceptible to acid dissolution during the process of demineralization. Many genes are known which are active in the formation of enamel (AMELX, ENAM, KLK-4 and MMP20 which encode various proteins like ameloblastin, amelogenin, enamelin, tuftelin- 1, and tuftelin interacting protein 11).

Certain variations in some of these enamel matrix genes may be associated with enhanced caries susceptibility. Some of these genes are linked to specific syndromes where the process of tooth development itself is altered. This is due to an alteration in the proteins responsible for biomineralization of the enamel matrix. Patients with alterations in the morphology of teeth and formation of the enamel are susceptible to caries. Syndromes, which are associated with caries and well-defined altered craniofacial phenotypes, are usually determined by mutation in a single gene.

These syndromes are Turner’s syndrome (Takala et al, 1985), the fragile X syndrome (Shellhart et al, 1986), ectodermal dysplasia, cleft lip and/or cleft palate (Dahllot et al, 1989), diastrophic dysplasia, etc. We do not know much about specific genes responsible for these disorders yet.

Nonsyndromic forms of Amelogenesis Imperfecta (AI) present with abnormal formation of the enamel. The enamel is composed mostly of minerals that are modified and regulated by the activities of the proteins in it. AI is caused due to malfunction of proteins in the enamel: Ameloblastin, enamelin, tuftelin and amelogenin (Refer Chapter 10).

People afflicted with amelogenesis imperfecta have teeth with an abnormal color. The teeth have a higher risk for dental cavities. The AMELX, ENAM, KLK-4 and MMP20 genes provide instructions for making proteins that are essential for normal development of tooth. Mutations in the AMELX, ENAM, MMP20, and KLK-4 genes have been found causative for amelogenesis imperfecta (nonsyndromic form).

Mutations in any of these genes alter the final structure of these proteins or completely prevent synthesis of any protein at all (Refer Chapter 10). As a result tooth enamel is abnormally thin or soft and has high a risk of developing dental caries.

The Search Of Candidate Gene(s) For Dental Caries

Very recently (postgenomic) quite a few studies have tried to locate the candidate gene for caries but with little success. A candidate gene is a gene known to be located in a region of interest in the genome. Product/s of the candidate gene has/have biochemical or other properties the presence or absence of which can be directly related to a disease. Findings form the following significant studies are as under:

• A study on mice indicated that major gene(s) responsible for the regulation of susceptibility to dental caries or resistance are located on chromosomes number 1, 2, 7 and 8 (Nariyama et al, 2004).
• Genome wide genotype data and DMFT scores in a large number of families were evaluated (Vieira et al, 2008). Low caries susceptibility loci were found on chromosomes number 5 (5q13.3), 14 (14q11.2) and X (Xq27.1). The high caries susceptibility genes were identified on chromosome number 13 (13q31.1) and 14 (14q24.3). The presence of genes for caries on the X chromosome may account for the sex differences observed in the incidence of caries. This study was the first of genome wide scans introduced for dental caries.
• In a recently concluded study single nucleotide polymorphism (SNP) assays were performed for 6 candidate genes. The candidate genes selected for this study were the amelogenin (AMELX), ameloblastin (AMBN), tuftelin (TUFT1), enamelin (ENAM), tuftelin-interacting protein (TFIP11) and kallikrein 4 (KLK4) genes. There were no significant associations concluded between single candidate genes and caries susceptibility. A significant interaction between tuftelin and S. mutans was however observed (Slayton et al, 2005).
• The Osteopontin (OPN) gene plays an important role in mineralization. In a recently conducted study OPN was chosen as candidate gene with respect to caries susceptibility as OPN gene was found to be associated with incidences of enamel hypoplasia in primary dentition. Results indicated an association between the OPN gene and caries in the primary dentition (Willing et al, 2006).
• A new study applied scanning of single- nucleotide polymorphism (SNP) markers with relation to selected candidate genes (ameloblastin, amelogenin, enamelin, tuftelin-1 and tuftelin interacting protein 11) that influence enamel formation. One copy of a rare amelogenin allele was found to be associated with caries experience. This result suggested that variations in amelogenin may contribute to caries susceptibility (Deeley et al, 2008).

In conclusion one may say that the search for genetic variables in the etiology of dental caries has just begun.

 

Genetics Of Cleft Lip And Cleft Palate

Molecular Regulation Of The Development Of The Palate

The palate is formed by the fusion of one median palatine process (primary palate) and two lateral palatine processes. The median palatine process is formed by the fusion of right and left medial nasal processes. The fused medial nasal processes form median part of upper lip, the part of upper jaw (which carries four incisors) and the primary palate.

The two lateral palatine processes are formed by shelf-like outgrowths from the maxillary processes in the 6th week of development. The growth of shelf-like process depends upon the interaction between ectoderm and mesenchyme. The following important genes play important role in the development of the palate. The sonic hedgehog (SHH), bone morphogenetic proteins (BMP), fibroblast growth factors (FGF) and members of the transforming growth factor B (TGF B) gene superfamily determine the formation of the palate (Carlson, 2004).

• The mesenchyme of the palatal shelf expresses MSX-1 that stimulates BMP-4 signaling in the mesenchyme.
• This leads to expression of SHH signaling in the apical ectoderm.
• SHH further induces BMP-2 signaling in the underlying mesenchyme.
• Both BMP-2 and BMP-4 stimulate mesenchyme proliferation leading to the growth of the shelf like palate.
• Epidermal growth factor (EGF) stimulates glycosaminoglycan production within the palatal

As the right and the left palatal shelves start fusing with each other in the midline, they are covered by epithelium. Some of these fused midline epithelial cells soon disappear by the process of apoptosis, while some other cells transform themselves from epithelial to mesenchymal cells.

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This transformation of cells is mediated due to the release of transforming growth factor-83 (TGF-B3). It is well known that TGF-B3 is expressed in the epithelium just before fusion. Mutation of TGF-B3 gene leads to formation of isolated cleft palate.

(The TGF-B3 knock out mouse exhibits an isolated cleft palate due to failure of palatal shelf fusion. Although the palatal shelves otherwise develop normally, they show a marked reduction in the filopodia present on the midline epithelial cell surface and show a down-regulation of chondroitin sulphate proteoglycan on the apical surface of the epithelia. Both of these are required for efficient midline epithelial adhesion).

Thus, it is clear that a tight control of a cascade of genes is required to complete normal palatogenesis. Anteriorly each palatal shelf joins with the primary palate. The fusion between two palatal shelves occurs anteroposteriorly and the process is completed by the 11th or 12th week.

Etiology Of Cleft Lip And Cleft Palate

Cleft lip with or without cleft palate (CL/P) and cleft palate (CP) are common birth defects that represent a major public health burden both in the social and medical contexts. The prevalence of CL/P varies from 0.3 to 2.5 per 1000 livebirths Worldwide. Cleft lip and palate (CLP) and cleft lip (CL) comprise 45 and 25% of all children respectively born with an oral cleft.

The population-based studies have shown that 50 to 70% of CL/P births had no other major malformation while upto 30% are associated with other anomalies. 10 to 15% of all CL/P cases report a positive family history. The median cleft lip and lateral facial cleft are rare conditions.

The etiology of CL/P is complex and thought to involve genetic influences with variable interactions from environmental factors. The etiological factors of cleft lip and cleft palate can be grouped as under:

Nongenetic-This includes various environ- mental (teratogenic) risk factors which may cause CL/P.

Genetic-Genetic causes include:

• Syndromic: Here the cleft is associated with other malformations. Usually, it is due to a single gene (monogenic or Mendelian) disorder. The clefting may also occur due to other chromosomal abnormalities due to multiple gene involvement.
• Nonsyndromic: Here the cleft is mostly an isolated feature and occurs in the vast majority of individuals having a cleft-lip or palate (up to 70% cases). In this form of cleft neither a recognized pattern of malformation nor a known cause for the disorder can be identified.

Overall, environmental factors are considered much less important agents than genetic factors in the etiology of oral clefts.

Nongenetic (Environmental) Risk Factors

Scientists have investigated many nongenetic environmental factors (teratogens) that increase the possibility of clefting. The lack of total concordance found in monozygotic twins (Spritz, 2001) indicated environmental etiology. Following is a list of these teratogens:

• Drugs
  • Vasoactive drugs – Pseudoephedrine, aspirin, ibuprofen, amphetamine, cocaine.
  • Anticonvulsant drugs – Phenobarbitol, trimethadione, valproate, and dilantin.
  • Anticancer drug – Aminopterin.
  • Antinausea or vomiting drug – Diphenhydrinate.
  • Others – Corticosteroids, isotretinoin (Accutane), sulfasalazine, naproxen.
• Maternal smoking-Leads to embryonic hypoxia (Wyazynski et al, 1997).
• Alcohol intake-May also lead to fetal alcohol syndrome (Jones et al, 1973; Goslin et al, 2001).
• Maternal fever
• Exposure to chemicals-Xylene, toluene, acetone, benzene and pesticides.
• Nutrition-Folic acid and vitamin A deficiency during pregnancy (Hartridge et al, 1999). 7. Altitude Hypoxia (Castilla et al, 1999).
• Traumatic stress by releasing hydrocortisone.

Syndromic Form Of CL/P And CP

It is monogenic (Mendelian) form of clefting. This form of cleft is associated with syndrome (linked to varieties of other congenital anomalies). Since these syndromes are determined by the mutation in single gene they are of a high-risk type. Over 300 syndromes are known to be associated with cleft lip or palate as an associated feature. The syndromic CL/P can be subdivided into following categories (Table 12.1):

• CL/P associated with syndromes caused by mutation in single gene.
• CL/P associated with syndromes caused by chromosomal abnormalities.
• CL/P associated with syndromes caused due to known teratogens.
• CL/P associated with syndromes whose cause is not known.

About 300 syndromes are known to have clefting of the lip or palate as an associated feature. The following table describes in brief a few well-known syndromic forms of CL/P.

Besides above-mentioned syndromes, X-linked Optiz syndrome (due to mutation in MIDI gene on chromosome Xp22) and mutation in the MSX1 gene and mutation in TBX22 genes are also known to be responsible for syndromic form of clefting.

Genetic Mechanism for Syndromic Form of Clefting

We have started, though recently, to understand genetic mechanisms implicated in some of the syndromic forms of CL/P.

DiGeorge syndrome (arising due to a deletion in chromosome 22q11) results from the failure of migration of neural crest cells in third and fourth branchial arches. These individuals also have associated CP (Seambler, 2000). We now know that the mutation in TBX22 gene encompasses almost all the common features of DiGeorge syndrome (Lindsay et al, 2001).

• The geneTCOF1 in Treacher-Collins syndrome encodes the protein called treacle. The exact function of treacle is unknown at present. It is speculated that treacle is involved in shuttling proteins between nucleolus and cytoplasm of the cell (Marsh et al, 1998). At present we do not know how this function can cause various malformations observed in Treacher-Collins syndrome.
• The mutation of SHH (Sonic hedgehog) gene leads to holoprosencephaly (Belloni et al, 1996). Holoprosencephaly is associated with midline defects like incomplete or deformed forebrain division, cyclopia, etc. The SHH signaling peptides play the role in midline patterning of human embryo.
• The genetic mechanism of CP in Stickler syndrome can be explained on the basis of defective collagen biosynthesis. Stickler syndrome (type I) occurs due to mutation in COL2A1 gene which encodes for type II collagen (Snead et al., 1999). Stickler syndrome (type II) has also been noted to occur due to mutation in the COL11A1 gene encoding type XI procollagen.

Nonsyndromic Clefting

Nonsyndromic cleft lip and/or palate (NSCLP) is a malformation characterized by an incomplete septation between the nasal and oral cavities without any associated anomalies. The distinction between syndromic and nonsyndromic clefting is very easy. In case of syndromic clefting there is always the presence of other associated anomalies in addition to CL/P and CP.

In cases of NSCLP the clefting is not associated with any other anomaly. Nonsyndromic orofacial clefts (OFC) are one of the most common malform- ations among livebirths and are composed of two separate entities: cleft lip with or without cleft palate (CL/P) and isolated cleft palate (CP).

The nonsyndromic clefting is said to be polygenic in nature. It is produced out of interaction between a number of genes; each producing a small effect that add up together to create the clefting. In other words, a cleft occurs when the total genetic liability of an individual reaches a certain minimal level termed the threshold.

It should be noted that every individual carries some genes that predisposes cleft formation but if the liability due to these genes is less than the threshold or the critical levels, no cleft results.

NSCLP is also said to be a complex multifactorial trait with interactions between genetic and environmental factors playing an important role in its causation. As the etiology of NSCLP is complex, many reports in the literature are contradictory (Murray, 1995).

The genetics of nonsyndromic clefting has been investigated by following methods:

• Familial and Segregational Analysis: Many studies have shown an increased risk of clefting in siblings. The risk was found to be 30% higher in siblings as compared to normal population prevalence. This clearly shows that NSCLP is genetically determined. The mode of inheritance of nonsyndromic cleft lip with or without cleft palate (NSCLP) is still a matter of dispute. Many studies have indicated towards a multifactorial mode of inheritance while others are of opinion that the inheritance has a mixture of monogenic and multifactorial patterns.
• Twin Studies: NSCLP has shown higher concordance rates in monozygotic twins. The concordance rate in monozygotic twins is approximately 25 to 45% as opposed to 3-6% in dizygotic twins. On the other hand, lack of complete concordance was also found similar to any other multifactorial trait. This suggested involvement not only of genetic but also of environmental factors in the causation of non- syndromic clefting.
• Linkage Analysis: Linkage analysis is used to map a disease (mutant) gene to specific region on a chromosome. Today we know that mutations in many candidate genes or loci and their chromosomal locations are associated with non- syndromic clefting. (A candidate gene is a gene known to be located in a region of interest in the genome. The product(s) of a candidate gene has/ have biochemical or other properties suggesting that it may be the gene being related to a condition).
• Association studies: Both kinds of the approaches, i.e. candidate gene approach and genome wide association studies are reported for nonsyndromic clefting. Association studies have identified many genes for the clefting.

The following candidate genes have been identified as etiological factors of nonsyndromic CL/P.

It has been observed that some of the genes that are responsible for causing syndromic forms of clefting are also responsible for causing nonsyndromic form of clefting. These overlapping genes are MSX-1, IRF 6, PVRL1, CATCH22, TGFß3 and TBX22. It is now becoming apparent that the same genes contribute to the population of nonsyndromic clefts perhaps through variable penetrance or through the action of different modifiers.

In conclusion, one may say that genetic analysis of CL/P is quite confusing as mutation screening of specific candidate genes, association studies and even genome-wide scans have largely failed to reveal the exact molecular basis of human clefting. There is also overlapping of genes determining syndromic and non- syndromic forms of CL/P.

Greater efforts are necessary in order to have a complete picture of the main factors involved in lip and palate malformation. These elements will permit us to better understand and better treat patients affected by clefting.

 

Genetics Of Craniofacial Disorders And Syndromes

Molecular Regulation Of The Development Of Face

During the beginning of the 4th week of embryonic development the face is represented by stomatodeum situated beneath the developing brain. The stomato- deum is separated from primitive gut by bucco- pharyngeal membrane. This membrane breaks down at the end of 4th week. Surrounding structures of stomatodeum ultimately form the face.

These structures are the frontonasal process, the medial and lateral nasal processes, the maxillary and mandibular processes. All these facial processes are covered by the ectoderm beneath which lies the mesoderm (mesenchyme). The mesenchyme of upper face comes from neural crest cells from the forebrain and midbrain areas while that of the mandible come from neural crest cells of midbrain and hindbrain region.

The genetic control of the early development of the face is guided as per sequential events given below.

• The frontonasal process is formed by the synthesis of retinoid acid in the ectodermal cells covering the forebrain. Retinoic acid is responsible for the maintenance of the fibroblast growth factor -8 (FGF-8) signals and sonic hedgehog (Shh) signals (in the forebrain and in frontonasal ectoderm) (Carlson, 2004).
• Shh and FGF-8 molecules stimulate neural crest cells to proliferate in the frontonasal process.
• After the 5th week of development the proli- feration of frontonasal process slows down and maxillary, mandibular, medial and lateral nasal processes start growing rapidly. Growth of all these processes results from interactions between the overlying ectoderm and underlying mesoderm. Here again the active signaling molecules in the ectoderm are FGF-8 and Shh. These signals stimulate growth of the mesenchyme.

• The growth of the maxillary process is due to establishment of a signaling center in the mandibular arch. FGF- 8 is the molecular signal for maxillary process formation.
• Next the homeobox containing Msx-1 gene is expressed in the mesenchyme of all the facial processes.
• The transcription factor Otx-2 is expressed in the first arch (maxillary and mandibular processes). This gene characterizes the precursors of the first arch. (It should be noted that the Hox genes are not expressed in the first arch. However, they are expressed in all the other pharyngeal arches).
• Further development of the mandibular process is strictly under genetic control. The medial region of the mandibular process responds to FGF-2 and FGF-4 local epithelial signals and stimulates growth of the underlying mesenchyme. These signals are mediated through Msx-1 factors. Growth of lateral region of mandibular process is due to FGF-8 signals. These signals are mediated by bone morphogenetic proteins- Bmp-4 and Bip-7 which are produced in the lateral regions of the mandibular process.
• The development of the mandibular arch in the proximal to distal direction depends upon the expression on Dlx group of transcription factors.
• Dlx-1 and Dlx-2 are expressed most proximally in the mandibular process; Dlx-5 and Dlx-6 are expressed more proximally and Dlx-3 and Dlx-7 are expressed most distally.
• Dlx-1 and Dlx-2 are also expressed in the maxillary process.

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During the 7th week the maxillary processes increase in size and move medially. The maxillary process now fuses with the medial nasal process on each side. Hence, the upper lip is formed by the fusion of two maxillary and two medial nasal processes. Stomatodeum is now bound above by the upper lip.

The two mandibular processes grow medially and fuse in the midline to form the lower lip and lower jaw. The fused mandibular processes now form the lower margin of stomatodeum.

To begin with, the lateral nasal process and maxillary process are separated by a deep furrow, the nasolacrimal groove. This groove extends upto the developing eye. The ectoderm of the floor of nasolacrimal groove forms a solid epithelial cord. The cord gets detached from the surface epithelium and gets canalized to form lacrimal sac and nasolacrimal duct at a later stage.

After formation of the basic facial structures by the end of 7th embryonic week, these structures are invaded by mesenchymal cells of the 1st and 2nd pharyngeal arches. The first arch mesenchymal cells form muscles of mastication and are innervated by the trigeminal (5) nerve. The mesenchymal cells of second arch form the muscles of facial expression and are thus innervated by the facial (7) nerve.

Ectodermal Dysplasia

Ectodermal dysplasias (EDs) are heritable conditions characterized by abnormal development of two or more ectodermal structures such as the hair, teeth, nails and sweat glands. Besides these defects related to embryonic ectoderm, an affected person may also show defects of cranial and facial structures, digits and some other parts of the body.

The disease is caused due to defects in the cutaneous and oral embryonic ectoderm and as such it may simultaneously affect many structures that are derived from the ectoderm. Therefore, each person with ectodermal dysplasia may have a different combination of ectodermal defects.

One may have hair and nails affected while another may involve sweat glands and teeth. Each combination is considered a distinct type of ectodermal dysplasia. More than 192 distinct disorders have been defined till date.

Incidence: 1 case per 10,000 births to 1 in 17,000 births.

Pathophysiology

Sweat gland defects-People with ectodermal dysplasia may not sweat or may have decreased sweating due to the lack of sweat gland development. Children with the disease may have defective body mechanisms that control fevers because the skin cannot sweat and control temperature properly. Affected adults are unable to tolerate a warm environment.

Defects of other glands derived from ectoderm– Hypoplasia of the salivary and lacrimal glands may occur. In some patients mucous glands may be absent in the upper respiratory tract and in the bronchi, esophagus, and duodenum. These defects may result in oral dryness, absence of tears and difficulty in swallowing.

Hair defects: Affected individuals tend to have sparse scalp and body hair (hypotrichosis). The hair is often light-colored, brittle and slow-growing.

Abnormal nails-Abnormal nail plate formation may result in brittle, thin, ridged or grossly deformed nails.

Abnormal teeth-Abnormal morphogenesis or absence of multiple teeth may occur.

Additional features-This includes a prominent forehead, thick lips and a flattened bridge of the nose. Sometimes affected person may also show thin, wrinkled and dark-colored skin around the eyes; chronic skin problems such as eczema and a bad- smelling discharge from the nose (ozena).

Following are few very common EDs:

Hypohidrotic ED

This is also known as EDA (anhidrotic ED) or Christ- Siemens-Touraine syndrome. It is the most common form of ED. The condition is mostly (in 95% of cases) inherited as an X-linked recessive trait. As a consequence only males are affected and females are carriers. However, due to lyonization and mosaic expression of the abnormal X-chromosome, females may show mild features of EDA.

Less commonly (in 5% cases) the condition is inherited as an autosomal dominant and autosomal recessive disoder.

Hypohidrotic ED Clinical Features

The affected person shows typical facial features represented by a prominent forehead, thick lips, broad nose, sunken cheeks, low set ears and wrinkled and dark eyelids. Teeth are either less in number or completely absent. If present, teeth are conical or pegged-shaped. Patients may have very less, dry and light hair. The skin is dry due to lack of sweat gland formation and these patients may acquire eczematous conditions of the skin. Tears are often absent.

Genetics of Hypohidrotic ED

The EDA, EDAR, and EDARADD genes provide instructions for manufacturing proteins that work together during embryonic development. These proteins form the parts of a signaling pathway that is critical for interaction between the two cell layers, the ectoderm and the mesoderm.

In the early embryo these cell layers form the basis for the genesis of many of the body’s organs and tissues. Ectoderm- mesoderm interactions are essential for the formation of several structures that arise from the ectoderm including the skin, hair, nails, teeth, and sweat glands.

The EDA gene provides instructions for making a protein called ectodysplasin A (Bayes, et al 1998 and Monreal, et al 1998). This protein is part of a signaling pathway that plays an important role in the development of ectodermal appendages (hair, teeth and sweat glands) before birth. The ectodysplasin-A has an important role to play in ectodermal- mesodermal interactions during embryonic development.

Defects in the molecular structure of this protein inhibit the action of enzymes necessary for normal development of ectoderm (Chen, et al 2001). More than 60 mutations have been identified in the EDA gene. The gene is located on the long (q) arm of the X-chromosome between positions 12 and 13.1 (Xq12-q13.1). The hypohydrotic ED is inherited as an X-linked recessive trait.

The EDAR gene provides instructions for making a receptor protein called the ectodysplasin A receptor. This protein is a part of the signaling pathway that plays an important role in development before birth. The ectodysplasin A receptor interacts with a protein called ectodysplasin A (produced from the EDA gene).

Ectodysplasin A attaches to this receptor on the cell surface like a key in its lock. When these two proteins are connected they trigger a series of chemical signals that affect cell activities such as division, growth, and maturation. Gene EDR is located on long arm of chromosome number 2 between 11 and 13 positions. Mutations of this gene cause hypohidrotic ectodermal dysplasia which is inherited as autosomal recessive trait (Shimomura, et al 2004).

The EDARADD gene provides instructions for making a protein called the EDAR-associated death domain protein (Headon, et al 2001). This protein is part of a signaling pathway active in developmental events before birth. The EDARADD protein interacts with another protein, called the ectodysplasin A receptor which is produced from the EDAR gene. This interaction occurs at a region called the death domain.

This domain configuration is present in both the proteins. The EDARADD protein acts as an adapter, which means it assists the ectodysplasin A receptor in triggering chemical signals within cells. These signals affect cell activities such as division, growth and maturation. Before birth this signaling pathway controls the formation of ectodermal structures such as hair follicles, sweat glands, and teeth.

The EDARADD gene is located on the long arm of chromosome number 1 at 43th position (1q43). Mutation of this gene causes hypohidrotic ED, which is inherited as autosomal recessive inheritance.

Mutations in the EDA, EDAR, or EDARADD gene prevent normal interactions between the ectoderm and the mesoderm and thus impair the normal develop ment of hair, sweat glands and teeth. The improper formation of these ectodermal structures leads to the characteristic features of hypohidrotic ectodermal dysplasia.

Some mutations in the EDA gene represent alterations or substitutions in single DNA building blocks (base pair parameters) whereas other mutations insert or delete genetic material in the gene. These changes lead to the production of a nonfunctional version of the ectodysplasin A protein.

This abnormal protein cannot trigger chemical signals needed for normal interactions between the ectoderm and the mesoderm. Without these signals hair follicles, teeth, sweat glands and other ectodermal structures do not form properly leading to the characteristic features of hypohidrotic ectodermal dysplasia.

Hidrotic ED

This condition is also known as the Clouston syndrome. It is inherited as an autosomal dominant disease. The gap junction proteins help in the communication and interaction between cells. The hidrotic ED mutation is present in the GJB6 gene located in the long arm of chromosome number 13 in its pericentriolar region (13q). This gene encodes the gap junction protein connexin 30 (Cx30) (Jerome, et al 2000 and Guilherme, et al 2004).

Hidrotic ED Clinical features

Scalp hair is very sparse, fine and brittle. Alopecia is common. Eyebrows are thinned or absent. Nail dystrophy is commonly seen. Persistent paronychial infections are frequent. Fingers and toes are abnormal, i.e. they are either more in number or fused with each other. Bulbous fingertips may be present. Patients may have normal face, normal teeth and normal sweating.

AEC (Hay-Wells) Syndrome

Its full name is ankyloblepharon-ectodermal dysplasia- clefting. It is inherited as an autosomal dominant trait of variable expressivity. It is due to a mutation in the p63 gene, which is located on the long arm of chromosome number 3 at position 27. The syndrome is caused by heterogenous missense mutation in p63 (McGrath, et al 2001).

AEC (Hay-Wells) Syndrome Clinical Features

Patients exhibit characteristic facial features like ankyloblepharon (congenital adhesion of the upper and lower eyelids by fibrous bands), sunken maxilla, broad nasal bridge and cleft palate. Absence or sparse hair in the scalp, absence or malformation of nails and pegged teeth are presented in the disease. Mild hypohydrosis is also common.

EEC Syndrome

EEC is an acronym of ectrodactyly-ectodermal dysplasia-cleft lip/cleft palate syndrome. It is inherited as an autosomal dominant trait of low penetrance and variable expressivity. The disease is due to a mutation in the gene p63 located on long arm of the chromosome number 3 at position 27 (Bokhoven, et al 2001).

EEC Syndrome Clinical Features

Affected persons may show severely malformed hands and feet (lobster claw deformity). Some may show hypoplastic metacarpals and metatarsals. Cleft lip and palate are very common.

Other features include dry and coarse hair; abnormal nails, mild hypohydrosis, enamel hypoplasia and microdontia.

Associated defects include blepharophimosis, deafness and abnormalities of the genitourinary tract.

EEC Syndrome Treatment

• Wigs can be used or special hair care taken for hair loss.
• Patient should always remain in a cool environment to prevent dryness of skin. Skin care products should be used to prevent dryness of the skin and for prevention from exposure to heat.
• Dental evaluation should be conducted every 6 to 12 months. Dental treatment like simple restoration of dentures or dental implants may become necessary in many patients.

Holoprosencephaly

The face and the forebrain (prosencephalon) of an embryo normally begin to develop in the 5th and 6th weeks of pregnancy. The development of face is related to the development of the forebrain. If the forebrain fails to segment into normal right and left hemispheres, it may result in deformities of the face. The abnormal development of brain and face is known as holoprosencephaly (HPE).

Following three types of the HPES are described in the literature:

Alobar-This type of HPE is features complete failure of separation of right and left cerebral hemispheres. There is single lobe and a single ventricle. This is a very severe form of HPE and the affected usually die during intrauterine life or in their early infancy. This variety is associated with severe craniofacial abnormalities.

Semilobar-This type of HPE is characterized by incomplete separation of cerebral hemispheres. This is associated with milder facial abnormalities.

Lobar-It is marked by substantial but still incomplete separation of the hemispheres. Separation is seen posteriorly but part of frontal lobe may remain fused together. Sometimes patients may have a nearly normal brain.

The diagnosis of the various types of HPE can be easily made with the help of a CAT scan or an MRI.

Holoprosencephaly Signs and Symptoms

The most severe forms of holoprosencephaly produce seizures and mental retardation. Midline structures of brain like the corpus callosum and septum pellucidum are not developed. The two thalamic lobes may be fused into one. Olfactory tracts and bulbs may remain absent in some cases. The circle of Willis is not well developed usually.

Typical facial defects involve the eyes, nose and upper lip. In some cases the nose may be entirely missing. However, there is no strict association between the types of HPE and the degree of severity of the facial defects in each of the types. Yet some associations are commonly seen:

• Facial defects associated with alobar type of HEP-The most severe of the facial defects is cyclopia; an abnormality characterized by the development of a single eye located in the area normally occupied by the root of the nose and a missing nose or a nose in the form of a proboscis (a tubular appendage) located above the eye. Premaxillary agenesis with median cleft lip, ocular hypotelorism, flat nose and sometimes bilateral cleft lip may be present with cyclopia.
• Facial defects associated with semilobar type of HPE-Bilateral cleft lip with the median process representing the philtrum-premaxilla anlage. There may be midline cleft lip and/or palate. Nose may show a flat nasal tip, absence of nasal septum and/or a flat nasal bridge. Ocular hypotelorism is very common. Microcephaly is also seen in many affected persons. Sometimes a person affected with semilobar type of HPE may have relatively normal facial appearance.
• Facial defects associated with lobar type of HPE-The least severe presentation in the spectrum of facial anomalies is the median cleft lip which is also called premaxillary agenesis. Bilateral cleft lip with a median process, ocular hypotelorism and a flat nose may be the only facial abnormalities. Person may also present with relatively normal facial appearance.

Microforms of HPE that can be observed in relatives of probands with HPE include the following:

• Microcephaly/Single central maxillary incisor/ Ocular hypotelorism/Anosmia/hyposmia (resulting from absence of olfactory tracts and bulbs)/Iris coloboma/Absent superior labial frenulum/Midface hypoplasia.

Causes of HPE

Etiology of HPE is heterogeneous. It is due to the fact that both environmental and genetic factors have been identified as causative agents.

HPE Environmental Causes

• Maternal diabetic mellitus during pregnancy • Alcohol
• Retinoic acid
• Hypocholesterol.

HPE Genetic Causes

• Chromosomal Abnormalities
  • Trisomy 13 (Patau’s syndrome) and Trisomy 18 (Edward’s syndrome)
  • Deletion and duplication of various chromosomes.
• Single Gene Abnormalities
  The mutation in a single gene causes two types of HPE, viz. Syndromic HPE (associated with various syndromes) and nonsyndromic HPE.

Nonsyndromic HPE

The nonsyndromic gene mutations responsible for HPE are usually inherited as autosomal dominant traits. The following table indicates genetics of autosomal dominant nonsyndromic HPES

SHH-The human sonic hedgehog gene (SHH) encodes a secreted protein (sonic hedgehog protein). This protein is involved in establishing cell fates at several points during development. It is expressed in the Hensen’s node and floor plate of the neural tube. Thus it is one of the primary inducers of the ventral neural tube.

Various types of mutations of the SHH gene are known (frame shift, heterozygous deletion, miss-sense mutations, etc). Mutation of sonic hedgehog gene leads to faulty production of sonic hedgehog protein that ultimately results in abnormal development of the forebrain and face (Roessler, et al 1996).

ZIC2-This gene encodes zinc finger protein 2. It plays a major role in mediating the response to sonic hedgehog protein signaling. Mutation of ZIC2 causes holoprosencephaly (Brown, et al 1998).

SIX3-This gene encodes the homeobox protein SIX3. It participates in midline forebrain structuring and in formation of eyes. It is present in the anterior region of the neural plate and midline ventral forebrain. Its mutation interferes with the development of brain and eye (Wallis, et al 1999).

TGIF-It modulates the TGIF beta pathways. Thus TGIF links the NODAL signaling pathway leading to the bifurcation of the human forebrain and the establishment of ventral midline structures (Gripp, et al 2000).

PTCH 1 – This gene encodes for the patched 1 receptor protein. This protein is the receptor for SHH.

Treatment of HPE

The treatment of persons suffering from HPE is basically the management of its symptoms.

• Hormonal replacement in case of pituitary dysfunction.
• Antiepileptic drugs for seizures.
• Surgical repair of abnormalities associated with face such as cleft lip/palate.

Mandibulofacial Dysostosis (Treacher Collins-Franceschetti Syndrome)

The syndrome is characterized by disorders of craniofacial developmental events. The disease is hereditary in nature.

Mandibulofacial Dysostosis Incidence: From 1 in 40,000 to 1 in 70,000 live births.

Mandibulofacial Dysostosis Clinical Features

Some of the important clinical features are as under:

• Down slanting palpebral fissure, coloboma of lower eyelids.
• Hypoplasia of malar bones and occasional absence of palatine bones.
• Hypoplasia or sometimes agenesis of mandible. Microstomia.
• Cleft palate and malocclusion of teeth.
• Malformation of the external ear. Sometimes middle and internal ears (auditory ossicles, cochlear and vestibular apparatus) are also affected. This leads to deafness.
• Occasional heart defects.
• Patients are of normal intelligence and have normal reproductive life.

Mandibulofacial Dysostosis Genetics

Inheritance: The inheritance shows an autosomal dominant (AD) pattern with complete penetrance and variable expressivities.

The syndrome is due to the mutation in the gene called the Treacher Collins-Franceschetti syndrome 1 (TCOF1) gene. It is located on the long arm of chromosome number 5 (5q31.3-q33.3). Approximately about 150 mutations have been identified in the TCOF 1 gene.

The TCOF1 gene codes for a protein called treacle which is required for normal craniofacial development. Mutation of gene leads to the absence or reduced production of treacle in the cells (Edwards, et al 1997). Researchers believe that a loss of this protein triggers certain apoptotic signals in cells important for the development of facial bones that self-destruct themselves (undergo apoptosis).

This abnormal cell death leads to Treacher Collins syndrome. As per other views towards the syndrome, it occurs due to the failure of migration of neural crest cells in the 1st and 2nd branchial arches. This leads to dysplasia of musculoskeletal derivatives of these arches.

Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is a disorder exhibiting defective endochondral and intramembranous bone formation.

Cleidocranial Dysplasia Incidence: 1 per million individuals worldwide.

Cleidocranial Dysplasia Clinical Features

Following abnormalities of bone formation are Inheritance: The CCD is an autosomal dominant observed.

Skull

Closure of the fontanels is delayed with the presence of many sutural bones are in the cranial sutures. Though head is brachycephalic in shape, bossing of frontal, parietal and occipital regions gives the skull a large globular shape.

Shoulder Girdle

Clavicles are either completely or partially absent. The patient can approximate the two shoulders in front of the chest (Fig. 11.8). Sometimes anomalies in other bones of the body may also be seen, e.g. in the pelvis, vertebral column, etc. with shortening of metatarsals, metacarpals and long bones.

Face

Maxilla, lacrimal and zygomatic bones are under- developed. Cleft palate is occasionally present. Paranasal air sinuses are underdeveloped. Sometimes the patients may have enlarged mandible as compared to the normal.

CCD patients may show a delayed eruption of permanent teeth. They may have supernumerary or impacted teeth. Patients are of normal intelligence but slightly short in height as compared to other normal members in the family.

Genetics

Inheritance: The CCD is an autosomal dominant (AD) disorder.

Narahara et al (1995) observed CCD in association with a translocation defect involving chromosomes 6 and 18. It is now believed that CCD is due to a mutation of the gene called core binding factor alpha-1 (CBFA1). This gene belongs to the RUNT transcription factor family and hence the other name of this gene is also runt related transcription factor 2 (RUNX2).

The gene CBFA1 is located on the short arm of chromosome number 6 (6p21) (Mundlos, et al 1995 and Mundlos, et al 1997). CBFA1 gene controls differentiation of precursor cells into osteoblasts and is thus essential for membranous as well as endochondral bone formation.

Researchers believe that the CBFA1 protein acts as a “master switch” regulating a number of other genes involved in the development of cells that build bones (osteoblasts). Thus CBFA1 gene plays an important role in osteoblastic differentiation.

Some mutations result in change of just a single building block (amino acid) of the CBFA1 protein (nucleotide alteration). Other mutations introduce a premature stop signal that result in an abnormally short protein. Occasionally the entire gene may be missing due to an anomaly.

This leads to the shortage of the functional protein (haploinsufficiency of CBFA1) that interferes with normal bone and cartilage development resulting in the signs and symptoms of cleidocranial dysplasia. Thus it may be concluded that CCD is caused due to mutations in the CBFA1 gene and that a heterozygous loss of function (autosomal dominance) is sufficient to cause the disease.

Apert Syndrome (ACROCEPHALOSYNDACTYLY)

It is a congenital genetic defect, which affects the first branchial arch (the precursor of maxilla and mandible). This syndrome is characterized by:

• Craniosynostosis (premature fused cranial suture).
• Craniofacial anomalies like midface hypoplasia
• Syndactyly (fused fingers and toes).

Apert Syndrome Incidence: 1 per 2, 00,000 live births.

Apert Syndrome Clinical Features

• Craniosynostosis involves the coronal suture resulting in a prominent forehead, flat occiput and brachycephaly.

• Down slanting palpebral fissure.
• Low set ears with occasional loss of hearing. • Hypertelorism and exophthalmos.
• Depressed nasal bridge and a short wide nose.
• Maxilla is not well-developed. High arched palate.
• V-shaped maxillary dental arch, malocclusion, supernumerary teeth (Fig.11.10), cleft palate or bifid uvula.
• The digits of hands and feet are either webbed or fused (syndactyly). The severity of the fusion varies with a minimum 3 of digits on each hand the premature fusion of the coronal, sagittal and and foot fused.
• Cardiovascular and GIT anomalies are common.
• Patients may be of normal intelligence but usually retarded.

Apert Syndrome Genetics

The inheritance of the syndrome is autosomal dominant (AD) in nature or the disorder arises fresh due to new mutations in an individual.

Apert syndrome results from mutations in the fibroblast growth factor receptor 2 (FGFR2) genes. This gene is located on the long arm of chromosome number 10 (10q 26). 98% of mutations of FGFR2 genes are substitution mutations such as Ser 252 Trp (between Serine and Tryptophan), Pro 253 Arg (Proline and Arginine) (Wilkie, et al 1995).

Such fibroblast growth factor receptor 2 mutations cause an increase in the number of precursor cells of osteogenic pathways. This again leads to increased subperiosteal bone formation and premature ossification of the calvaria ending in early closure of the cranial sutures.

Syndactyly of Apert syndrome may be due to keratinocyte growth factor receptor (KGFR) mediated effect. The optic disk pallor is more severe in Pro 253 Arg mutation while cleft palate and visual impairment is seen more in the Ser 252 Trp substitution.

Sometimes Apert syndrome may also be caused by deletion/translocation of the short arm of chromosome 2 to long arm of chromosome 11 or 12.

Some other syndromes like Pfeiffer syndrome and Crouzon syndrome (craniofacial dysostosis) also show features of craniosynostosis. These are allelic disorders with overlapping features between the related disorders (Wilkie, et al 1995).

Crouzon Syndrome (Craniofacial Dysostosis)

Crouzon syndrome or craniofacial dysostosis is similar to the Apert syndrome except that it is not associated with syndactyly. Crouzon syndrome is a genetic disorder also known as branchial arch syndrome. Specifically this syndrome affects the first branchial (or pharyngeal) arch; structures that are precursors of the maxilla and mandible.

The main feature of this syndrome is its craniosynostosis or occasionally the lambdoid sutures. The pathological fusion begins in the first year of life and is completed by the second or third year. The order and rate of fusion in the sutures determine the degree of deformity and disability.

Crouzon Syndrome Incidence: 1 in 25000 persons.

Crouzon Syndrome Clinical Features

• Signs of the disease originate from the early closure of cranial sutures.
• Coronal and sagittal sutures are obliterated.
• Short and broad head (brachycephaly).
• Bulging eyes (exophthalmos) due to shallow eye sockets after early fusion of surrounding bones of the orbit.
• Hypertelorism (greater than normal distance between the eyes), divergent squint.
• Progressive optic nerve atrophy results from raised intracranial tension which leads to subsequent visual impairment.
• Under-development of maxilla (insufficient growth of the midface results in protrution of the chin despite normal growth of the mandible). Due to maxillary hypoplasia Crouzon patients generally have a considerable and permanent underbite and subsequently cannot chew using their incisors (they do not use their incisors to take a bite from a sandwitch). For this reason the Crouzon patients eat in an unusual way.

• They may show a narrow/high-arched palate, posterior bilateral crossbite, hypodontia (missing teeth), malocclusion and an increased spacing between the teeth.
• Most Crouzon patients also have noticeably shorter humerus and femur bones in proportion to the rest of their bodies when compared to members of the general population.
• Deafness.
• Mental retardation is a frequent feature.

Crouzon Syndrome Genetics

Crouzon Syndrome Inheritance – Autosomal dominant (AD). Sometimes the disorder arises due to new mutations.

The syndrome is due to a mutation in the fibroblast growth factor receptor genes (FGFR-2) which is mapped to the chromosome locus 10q25-10q26. This gene provides instructions for furnishing a protein called the fibroblast growth factor receptor 2.

Among its multiple functions this protein signals transfor- mation of immature cells into bone cells during embryonic development. Mutations in the FGFR2 gene probably over-stimulate signaling by the FGFR2 protein and result in the bones of the skull fusing prematurely (Rutland, et al 1995).

Crouzon syndrome with acanthosis nigricans is always due to an Ala 391Glu mutation within the transmembrane region of the FGFR3 gene. Acanthosis nigricans is a brown to black, poorly defined, velvety hyperpigmentation of the skin.

Crouzon syndrome exhibits locus heterogeneity with causal mutations in FGFR2 and FGFR3 in different affected individuals.

Crouzon Syndrome Treatment

Surgery is typically used to stop the closure of sutures and prevent damage to the growing brain by compression exerted by the non-expansible skull. Blindness and mental retardation are typical outcomes in neglected cases. Once treated for the cranial vault symptoms, Crouzon patients generally live a normal lifespan.

Pfeiffer Syndrome

Pfeiffer syndrome is a genetic disorder characterized by premature fusion of certain skull bones (cranio- synostosis). This early fusion prevents the skull from growing normally and affects the shape of the head and face. Pfeiffer syndrome also affects bones in the hands and feet.

Pfeiffer Syndrome Incidence: 1 in 100,000 individuals.

Pfeiffer Syndrome Clinical Features

• Pfeiffer syndrome result from the premature fusion of the skull bones.
• High forehead.
• Wide set eyes.
• Underdeveloped upper jaw (maxillae).
• Dental problems due to crowded teeth and often a high palate.
• A beaked nose.
• Poor vision.
• Hearing loss in about 50% of children.
• The thumbs and great toes are wide (broad) and bend away from the other digits.
• Unusually short fingers and toes (brachydactyly) are also common and there may be some webbing or fusion between the digits (syndactyly).

Pfeiffer syndrome is divided into three subtypes as type 1, type 2 and type 3. Type 1 or “classic” Pfeiffer syndrome includes individuals with mild manifes- tations including brachycephaly, midface hypoplasia and finger and toe abnormalities. Most individuals with type 1 have normal intelligence and a normal lifespan.

Types 2 and 3 are more severe forms of Pfeiffer syndrome often involving problems with the nervous system. Type 2 consists of a cloverleaf-shaped skull, extreme proptosis, finger and toe abnormalities, elbow ankylosis or synostosis, developmental delay and neurological complications.

Type 3 is similar to type 2 but without a cloverleaf skull. Clinical overlap between the three types may occur.

Pfeiffer Syndrome Genetics

Pfeiffer Syndrome Inheritance: It is inherited as an Autosomal Dominant (AD) entity.

Mutations in the FGFR1 and FGFR2 genes cause Pfeiffer syndrome.

FGFR1 gene is located on the short arm of chromosome number 8 (8p11.2-p11.1) and FGFR2 is located on the long arm of chromosome number 10 (10q26) (Lajeunie, et al 1995).

The FGFR1 and FGFR2 genes play an important role in signaling the cell to respond to its environment perhaps by division or cell maturation. A mutation in either of the genes causes a prolonged signaling promoting an early maturation of bone cells in a developing embryo and premature fusion of bones in the skull, hands and feet.

Type 1 Pfeiffer syndrome is caused by mutations in either the FGFR1 or FGFR2 genes. Types 2 and 3 are affected by mutations in the FGFR2 gene. Mutations in the FGFR1 usually present a milder phenotype of the disease.

Pfeiffer Syndrome Treatment

Treatment includes multiple-staged surgery of craniosynostosis. Midfacial surgery is performed to reduce the exophthalmos and the midfacial hypoplasia.

Differential Diagnosis

The main differential diagnoses include syndromes that are characterized by craniosynostosis. Mutations in the same FGFR (FGFR1, FGFR2 or FGFR3) can result in different variants of craniosynostosis syndromes thereby implicating a common pathological mecha- nism with a common FGFR gain of function mecha- nism resulting in Pfeiffer, Apert, Muenke, and Beare- Stevenson syndromes.

• Pfeiffer and Apert syndromes are noteworthy for some similarities between them but the two disorders essentially are genetically distinct entities.
• Crouzon syndrome has similar phenotype as the Pfeiffer syndrome but lack the hand and foot anomalies.
• Phenotypic overlap occurs with Muenke syndrome which is caused by a specific FGFR3 mutation.

Cherubism

Cherubism is a rare genetic disorder characterized by abnormal bone tissue in the lower part of the face.

Cherubism Incidence: The condition is very rare.

Cherubism Clinical Features

The disease begins in early childhood. Swelling of the lower face starts around the third or fourth year of life and progresses until the late teens. The lower jaw (mandible) and upper jaw (maxilla) become enlarged as the bone is replaced by painless cyst like growths. These growths give the cheeks a swollen and rounded appearance. X-ray reveals multilocular cystic changes in the mandible and maxilla.

The abnormal growths are gradually replaced with normal bone in early adulthood. As a result many affected adults have a normal facial appearance. The deciduous dentition may be shed spontaneously and prematurely. The permanent teeth are also defective in their number that may be more or less than the normal. Most people with cherubism have almost no signs and symptoms affecting other parts of the body.

Cherubism Genetics

Cherubism Inheritance: The mode of inheritance in cherubism is autosomal dominant (AD). Usually the affect person has only a single affected parent or some time the disease is due to new (de novo) mutation.

Cherubism is due to mutation in the SH3BP2 (SH3-domain binding protein 2) gene located on the short arm of chromosome number 4 at the 16.3 position (4p16.3). About 11 different mutations are known in the SH3BP2 gene which give rise to cherubism (Ueki, et al 2001).

Mutations in the SH3BP2 gene lead to the production of an overactive protein. The overactive protein likely causes inflammation in the jaw bones and triggers the production of osteoclasts, which are bone eating cells that breakdown bone tissue during bone remodeling.

An excess of these osteoclasts contributes to the destruction of bone in the upper and lower jaws. A combination of bone loss and inflammation likely underlies the cyst-like growths so characteristic of cherubism.

Cherubism Treatment

Treatment of cherubism is not well-established. It is usually a self limiting disease. Surgical treatment should be designed on individual basis depending on the functional and esthetic need of the patient.

Van Der Woude Syndrome

Van Der Woude Syndrome syndrome is sometimes also known as lip-pit IRF6 protein is active in cells that give rise to tissues syndrome.

Van Der Woude Syndrome Clinical Features

• People with this disorder are born with a cleft lip, a cleft palate or both.
• Affected individuals usually have depressions (pits) near the center of the lower lip which may appear moist due to the presence of salivary and mucous glands in the pits.
• Some individuals may show mucous cysts of lower lip.
• It is a common form of syndromic cleft lip and palate and accounts for 2% of all cleft lip and palate cases.
• Affected individuals may have hypodontia and usually possess normal intelligence. Incidence: The incidence of this syndrome is 1 in 35,000 to 1,00,000 individuals worldwide.

Van Der Woude Syndrome Genetics

Inheritance: Van Der Woude syndrome (VDWS) is inherited as an autosomal dominant (AD) trait or sometimes of sporadic inheritance. The syndrome has 80% penetrance and variable expression.

It is caused by mutations of the IRF6 (interferon regulatory factor 6) gene located on the long arm of chromosome 1 (1q32-q41). About 60 different mutations of IRF6 gene are known currently. The IRF6 protein is active in cells that give rise to tissues in the head and face. It is also involved in the development of other parts of the body including the skin and genitals.

Mutations in the IRF6 gene that cause van der Woude syndrome prevent one copy of the gene in each cell from producing the functional protein. A shortage of the IRF6 protein affects the development and maturation of tissues in the skull and face. These abnormalities underlie the signs and symptoms of van der Woude syndrome that include mouth) and pits or mounds in the lower lip.

The cleft lip, cleft palate (an opening in the roof of the action of certain modifier genes on IRF6 function. marked phenotypic variation may be caused by the Intriguing linkage studies have suggested that a 17p11.2-p11.1 may influence the degree of phenotypic second modifying gene mapped to chromosome expression of a gene defect at this locus.

Most reported cases of VWS have been linked to chromosome 1q32-q41 (VWS1) but a second VWS locus (VWS2) has been mapped to 1p34 (Koillinen, et al 2001). Direct sequence analyses of genes in this region gene encoding interferon regulatory factor-6 (IRF6).

(1932-q41) have identified specific mutations in the Mutation analysis demonstrated that popliteal pterygium syndrome can be caused by mutations in syndrome. Many scientists have also reported that the same gene and is therefore allelic to van der Woude VDWS can also be caused due to a microdeletion involving 1q32-q41 in families with this syndrome.

Van Der Woude Syndrome Treatment

• Examination and genetic counseling by a pediatric geneticist (dysmorphologist) should be suggested. Surgical repair of cleft lip and cleft palate or other anomalies may be required.
• Surgical excision of lip pits is often performed for cosmetic reasons even in less severely affected individuals.

Gorlin-Goltz Syndrome

Jarisch and White first reported the Gorlin syndrome in 1894 that was later described in detail by Gorlin in 1960.

Gorlin-Goltz Syndrome Clinical Features

The clinical features that are considered for diagnosing Gorlin syndrome are classified into two groups of criteria; major criteria and minor criteria.

Gorlin-Goltz Syndrome Major Criteria

• Two or more basal cell carcinomas in persons cell growth and division. As a result cells proliferate younger than 20 years.
• Odontogenic keratocysts of the jaw.
• Bifid, fused ribs.
• Calcification of the falx cerebri.
• Palmar or plantar kits.

Gorlin-Goltz Syndrome Minor Criteria

• Syndactyly of digits.
• Macrocephaly.
• Hypertelorism, frontal bossing, cleft lip or palate. is affected with Gorlin syndrome.
• Scoliosis, vertebral anomalies.

Gorlin-Goltz Syndrome Incidence: The prevalence of Gorlin syndrome is estimated to be 1 in 57,000 people.

Gorlin-Goltz Syndrome Genetics

Mutations in the PTCH1 gene cause Gorlin syndrome. This gene encodes for a protein called Patched-1 which functions as a receptor protein. The Patched-1 receptor proteins have specific sites into which certain other proteins called ligands (e.g. Sonic Hedgehog) fit like keys into locks. Ligands and their receptors together trigger signals that affect cell development and function.

Patched-1 prevents cell growth and division PTCH1 gene acts as a tumor suppressor gene which (proliferation) until Sonic Hedgehog is attached. The means it keeps cells from proliferating too rapidly or in an uncontrolled way. Mutations in this gene prevent the production of Patched-1 or lead to the production of an abnormal version of the receptor.

An altered or missing Patched-1 receptor cannot effectively suppress cell growth and division. As a result cells proliferate uncontrollably to form tumors that are characteristic of Gorlin syndrome.

The genetic locus for Gorlin syndrome is located on chromosome bands and subbands 9q22.3-q31. More than 50 germline mutations in PTCH are described. About 40% of cases of Gorlin syndrome represent new mutations in affected individuals.

Inheritance: Gorlin syndrome is inherited in an autosomal dominant trait. Usually one of the parents is affected with Gorlin syndrome.

Waardenburg Syndrome (WS)

It is an inherited disorder often characterized by varying degrees of hearing loss and changes in skin and hair pigmentation.

Waardenburg Syndrome (WS) Clinical Features

• Eyes are of two different colors. One eye is usually brown and the other blue. Sometimes one eye has two different colors.
• Difference in eye color is associated with hearing impairment.
• People with WS may also have distinctive hair coloring such as a patch of white hair or premature gray hair as early as age 12.
• Some individuals may show white patches on the skin.
• Cleft lip and cleft palate may also be associated with WS.
• Some individuals may show intestinal or spinal disorders.

Waardenburg Syndrome (WS) Incidence: About 1 in 10,000 to 1 in 20,000 individuals. mutations in the PAX3 gene. Mutations in the MITF

Types of WS or Types of Waardenburg Syndrome (WS)

Though there are about four different types of Waardenburg syndrome but most common types of WS identified by scientists are Type I and Type II. The type of WS is determined by the distinctive features present in a person.

Type 1 WS-It is represented by persons having wide- set eyes (hypertelorism) due to a prominent, broad nasal root (dystopia canthorum). Hearing impairments occur in about 20% of individuals with this type of Waardenburg syndrome.

Type 2 WS-Persons who do not have a wide set eyes but who have many other WS characteristics are described as having Type II WS. About 50% of persons with WS Type II have a hearing impairment or are deaf. Type II WS can be further classified into 4 different sub types.

Waardenburg Syndrome (WS) Genetics

Inheritance: This condition is usually inherited in an autosomal dominant (AD) pattern. A small percentage of cases result from new mutations in the gene. Some cases of type II Waardenburg syndrome appear to have an autosomal recessive pattern of inheritance. Most often the parents of a child with an autosomal recessive disorder are not affected but are carriers of the disease.

The genes that cause Waardenburg syndrome are involved in the formation and development of several types of cell including pigment-producing cells called melanocytes. Melanocytes make a pigment called melanin that contributes to the coloration of skin, hair, and eye and also plays an essential role in the normal function of the inner ear.

Mutations in any of these genes disrupt the normal development of melanocytes leading to abnormal pigmentation of the skin, hair, and eyes and problems with hearing. In addition to melanocyte development these genes are important for the development of nerve cells in the large intestine.

Mutations in any of these genes result in hearing loss, changes in pigmentation, and intestinal problems Some individuals may show intestinal or spinal related to Hirschsprung disease. disorders.

Type 1 Waardenburg syndrome is caused by and SNAI2 genes are responsible for type 2 Waardenburg syndrome.

Paired box 3 (PAX3) gene is active in cells called neural crest cells. These cells migrate from the Paired box 3 (PAX3) gene is active in cells called developing spinal cord to specific regions in the embryo. The protein made by the PAX3 gene directs the activity of other genes (such as MITF) that signal neural crest cells to form specialized tissues or cell types such as limb muscles, bones in the face and skull (craniofacial bones), some nerve tissue and pigment-producing cells called melanocytes.

Melanocytes produce the pigment melanin, which contributes to hair, eye, and skin color. Melanocytes are also found in certain regions of the brain and inner ear.

Microphthalmia-associated transcription factor (MITF) gene helps to control the development and function of pigment-producing cells called melano- cytes. The official name of this gene is “micro- phthalmia-associated transcription factor” and “MITF’ is the gene’s official symbol. The MITF gene is also known by a few other names listed below. The Snail 2 (SNAI2) gene probably plays a role in the formation and survival of melanocytes.

The mapping analysis by Farrer, et al (1994) indicated that WS type I is linked to PAX gene situated on chromosome number 2; WS IIA is linked to mutation in gene MITF located on short arm of chromosome 3 (3p): WS IIB is due to mutation in gene located on short arm of chromosome 1 (1p) and WS IIC is due to mutation in gene situated on short arm of chromosome number 8 (8p).

Waardenburg Syndrome (WS) Treatment

No effective treatment is available for persons with Waardenburg syndrome.

Osteogenesis Imperfecta

The osteogenesis imperfecta (OI) is a serious inherited disorder. The bones formed are defective, i.e. they are brittle and easily fractured. This condition results due to an abnormality in the formation of type 1 collagen. It is formed either in less quantity or poor quality. 90% of body collagen is type 1. This type of collagen fibers is found in bones, capsule of organs, cornea, dentin, sclera, tendon, dermis, fascia, the dura and ligaments.

Incidence: the disease is observed in 7 per 100,000 persons. Type 2 and 4 are more common than the rest.

The disorder is classified in various types, i.e. OI type 1 to OI type V8. The genetic cause of OI type 5 and 6 are not known. The types 1 to 4 are inherited as autosomal dominant and type 7 and 8 as autosomal recessive traits. Clinical features of only first four types of OI are described in brief:

OI, type 1-It is the mildest form and may remain undetected till the occurrence of the first fracture in a child. The collagen formed in this type of OI is of normal quality but produced in insufficient quantity resulting in the bones getting fractured easily and the spine showing slight curvature defects. Joints are loose or sub-laxed.

The sclera looks blue (sometimes purple or gray) instead of white as the sclera is very thin and the choroidal veins present beneath the sclera gives a blue look to the sclera. Sometimes Type 1 OI is associated with DGI characterized by opalescent teeth.

OI, type 2-This is also known as congenital OI. It is the most severe form of OI and it is lethal at birth or shortly thereafter. As most cases die within first few Mutation of COL1A1 Gene years, this type of OI may not be encountered in dental practice. The collagen produced in neither of good quality nor of sufficient quantity. This leads to severe bone deformity and small stature in persons who survive the disease.

OI, type 3 – This type shows progressive deformities with increasing age, i.e. neonates present with mild symptoms that bloom into severe symptoms with age. The collagen produced is of sufficient quantity but of poor quality. Bone deformities are severe and bone gets fractured easily. Joints are loose. Sclera shows discoloration.

OI, type 4-Collagen produced is of sufficient quantity but poor quality. Affected person is of short stature with deformed and fractured bones and spinal curve disorders. There may be early loss of hearing.

Genetics of Ol or Osteogenesis Imperfecta

The formation of collagen begins as procollagen molecules. Each rope-like procollagen molecule is made up of three chains, the two proalpha 1 chains and one proalpha 2 chain.

The proalpha 1 chain is produced by gene COL1A1 (Collagen type 1 alpha 1) and proalpha 2 chain is produced by gene COL1A2 (collagen type 1 alpha 2) (Byers, et al 1991 and Byers, 1993). Gene COL1A1 is located on the long arm of chromosome number17 between 21.3 and 22.1. Gene COL1A2 is located at 7q22.1.

Two other genes are also involved in the synthesis of collagen type I, i.e. CRTAP (Cartilage associated Two other genes are also involved in the synthesis protein) and LEPRE 1 (Leucine proline-enriched proteoglycan 1). The CRTAP gene codes instruction for making a protein called cartilage associated protein. This protein is critical for normal folding and assembly of collagen. It plays an important role in bone development.

This gene is located on short arm of chromosome number 3 at position 22.3. The LEPRE1 gene is also known as P3H1. It provides the code for making an enzyme called prolyl-3 hydroxylase 1. This enzyme modifies an amino acid called proline in collagen molecules. Proline is necessary for proper folding and assembly of collagen. This gene is located on the short arm of chromosome number 1 at position 4.1.

Mutation of COL1A1 Gene

The mild mutation of this gene leads to OI type 1. There is a reduction in the production of proalpha 1 chain thus reducing the amount of collagen 1 produced. A severe mutation of this gene leads to production of type 2 to type 4 varieties of OI. Deletion of segments of DNA from COL1A1 gene results in nonfunctional proalpha1 chain.

Some mutations may replace the amino acid glycine with some other amino acid. Sometimes mutation may also interfere with the assembly of collagen molecules. Thus the mutation on gene COL1A1 leads to the formation of abnormal types 1 collagen.

Mutation of COL1A2 Gene

This gene codes for proalpha 2 chain of collagen molecule. The mutation of COL1A2 gene produces abnormal collage type 1. The mutation of this gene leads to severe types of OI (type 2, type 3 and type 4).

Mutation of CRTAP Gene

Mutation of this gene leads to type 7 variant of OI. Cartilage associate protein is not formed due to the underlying mutation. This results in the production of abnormal cartilage.

Mutation of LEPRE 1 Gene

The mutation leads to abnormal production of enzyme prolyl-3 hydroxylase 1 which ultimately leads to incorrect folding and assembly of collagen molecules. The mutation of this gene is associated with OI type 8.

Osteogenesis Imperfecta Modes of inheritance-Type 1 to type 4 OI are inherited as autosomal dominant entities while type 7 and 8 are inherited as autosomal recessive ones.

Osteogenesis Imperfecta Treatment

No genetic cure is available at present. Putting metal rods in these bones can prevent fractures of long bones.