Genetics and malocclusion

GENETICS AND MALOCCLUSION

PART-1 : BASIC GENETICS & ITS SIGNIFICANCE IN MALOCCLUSION AND CRANIOFACIAL ANOMALIES

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INTRODUCTION HUMAN CHROMOSOMES DNA: THE HERIDITARY MATERIAL GENETIC CODE STRUCTURE OF GENES DEVELOPMENTAL GENE FAMILIES HOMEOBOX GENES AND ITS IMPORTANCE TWIST GENES GROWTH FACTORS AND ITS SIGNIFICANCE MUTATION AND ITS TYPES MUTATIONAL TRACKING AND MOLECULAR APPROACHES PATTERNS OF INHERITANCE POLYGENIC AND MULTIFACTORIAL INHERITANCE SEGREGATION AND LINKAGE ANALYSIS TWINNING AND SIGNIFICANCE OF TWIN STUDIES SIGNIFICANCE OF GENETIC STUDIES FOR MALOCCLUSION

AND CRANIOFACIAL ANOMALIES


INTRODUCTION

The human genome contains approximately 3 billion nucleotides, making up about 100,000 alleles, which in turn are contained on 46 chromosomes. Transcription of these chromosomes releases the information necessary to synthesize some 6000 proteins. These proteins make up the trillion cells giving rise to the nearly 4000 anatomical structures that constitute a single human being. Mutation, the accidental alteration of the genome, may result in heritable conditions or syndromes affecting any aspect of growth and development.


Several questions need to be answered before a complete understanding can be gained about how genetic factors influence a feature or disorder. These include:

How important are genetic effects on human differences?

What kinds of action and interaction occur between gene products in the pathways between genotype and phenotype?

Are the genetic effects on a trait consistent across sexes?

Are there some genes that have particularly outstanding effects when compared with others?

Whereabouts on the human gene map are these genes located? www.indiandentalacademy.com

HUMAN CHROMOSOMESSTRUCTURE AND CLASSIFICATIONIn humans the normal cell nucleus contains 46 chromosomes, made up of 22 pairs of autosomes and a single pair of sex chromosomes XX in the female and XY in the male. The Y chromosome is much smaller than the X.Each chromosome is composed of DNA double helix and the packaging of DNA into chromosomes involves several orders of DNA coiling and folding. In addition to the primary coiling of the DNA double helix, there is secondary coiling around spherical histone beads forming what are called nucleosomes. There is a tertiary coiling of the nucleosomes to form the chromatin fibres which form long loops on a scaffold of non-histone acidic proteins, which are further wound in a tight coil to make up the chromosome as visualized under the light microscope, the whole structure making up the so-called solenoid model of chromosome structure. www.indiandentalacademy.com

MORPHOLOGY Each chromosome consists of two identical strands known as chromatids, or sister chromatids. These sister chromatids are joined at a primary constriction known as the centromere. Centromeres consist of several hundred kilobases of repetitive DNA and are responsible for the movement of chromosomes at cell division. Each centromere divides the chromosome into short and long arms designated p (=petite) and q (=grande) respectively. Morphologically chromosomes are divided into,

Metacentric centromere located centrallyAcrocentric centromere located at terminal endSubmetacentric centromere in intermediate position


CHROMOSOME NOMENCLATUREEach chromosome arm is divided into regions and each region is subdivided into bands numbering always from the centromere outwards. A given point on a chromosome is designated by the chromosome number, the arm (p or q), the region and the band, e.g. 15q12. Sometimes the word region is omitted so that 15q12 would be referred to simply as band 12 on the long arm of chromosome15. X- Chromosome

short & long armwww.indiandentalacademy.com

CHROMOSOME BANDINGMost commonly circulating lymphocytes from peripheral blood are used for studying human chromosomes. Several different staining methods can be utilized in identifying individual chromosomes characterized by light and dark bands under microscope.

METHODS OF CHROMOSOME ANALYSIS


Gene map of the human genome

KARYOTYPE ANALYSISDetailed analysis of the banding pattern of the individual chromosomes is carried out. The banding pattern of each chromosome is specific and can be shown in the form of a stylized ideal karyotype known as an idiogram. A formally presented karyotype or karyogram will show each chromosome pair in descending order of size.


NUCLEOTIDESNucleic acid is composed of a long polymer of individual molecules called nucleotides. Each nucleotide is composed of a nitrogenous base, a sugar molecule and a phosphate molecule. The nitrogenous bases fall into two types, purines and pyrimidines. The purines include adenine and guanine; the pyrimidines include cytosine, thymine and uracil. There are two different types of nucleic acid, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). DNA and RNA both contain the purine bases adenine and guanine and the pyrimidine cytosine but thymine occurs only in DNA while uracil is only found in RNA.


DNA : THE HERIDITARY MATERIALDNA molecules have a very distinct and characteristic three-dimensional structure known as the double helix. In 1953 the structure of DNA was discovered by Watson and Crick working in Cambridge using x-ray diffraction pictures taken by Franklin and Wilkins.

X-ray diffraction pictures of the double helix show repeated patterns of bands that reflect the regularity of the structure of the DNA.


The double helix executes a turn every 10 base pairs. The pitch of the helix is 34A so the spacing between bases is 3.4A. The diameter of the helix is 20A. The double helix is said to be 3 antiparallel. One of the strands runs in the 5’3’ direction and the other 3’5’ direction. The double helix is not absolutely regular and when viewed from the outside a major groove and a minor groove can be seen. These are important for interaction with proteins, for replication of the DNA and for expression of the genetic information.


COMPLEMENTARY BASE PAIRINGThe bases of the two polynucleotide chains interact with each other. Thymine always interacts with adenine and guanine with cytosine (i.e. A-T and G-C). The way in which the bases form pairs between the two DNA strands is known as complementary base pairing. Complementary base pairing is essential for the expression of genetic information and is central to the way DNA sequences are transcribed into mRNA and translated into protein.

TYPES OF DNA SEQUENCESAnalysis of human DNA have shown that approximately 60-70% of the human genome consists of single or low copy number DNA sequences. The remainder of the genome, some 30-40% consists of either moderately or highly repetitive DNA sequences.


NUCLEAR DNA(A) Nuclear genes

(i) Unique single copy genes(ii) Multigene families – e.g. the HOX homeobox gene family.

Classical gene familiesGene super families

(B) Extragenic DNA(i) Tandem repeat

SatelliteMinisatellite

TelomericHypervariable

Microsatellite(ii) Interspersed

Short interspersed nuclear elementsLong interspersed nuclear elements

MITOCHONDRIAL DNATwo rRNA genes22 tRNA genes13 genes coding for proteins involved in oxidative phosphorylation. www.indiandentalacademy.com

TECHNIQUES OF DNA ANALYSISMany methods of DNA analysis involve the use of nucleic acid probes and the process of nucleic acid hybridization.

NUCLEIC ACID PROBESNucleic Acid probes are usually single stranded DNA

sequences which have been radioactively or non-radioactively labeled and can be used to detect DNA or RNA fragments with sequence homology. DNA probes can come from variety of sources including random genomic sequences, specific genes, cDNA sequences or oligonucleotide DNA sequences produced synthetically based on the knowledge of the protein amino acid sequence.


A DNA probe can be labeled by a variety of processes, including isotopic labeling with 32 P and non-isotopic methods using modified nucleotides containing fluorophores, e.g.fluorescein of rhodamine. Hybridization of a radioactively labelled DNA probe with complementary DNA sequences on a nitrocellulose filter can be detected by autoradiography while DNA fragments which are fluorescently labelled can be detected by exposure to the appropriate wavelength of light, e.g. fluorescent in situ hybridization.


NUCLEIC ACID HYBRIDIZATIONNucleic acid hybridization involves mixing DNA from two sources which have been denatured by heat or alkali to make them single stranded and then, under the appropriate conditions, allowing complementary base pairing of homologous sequences. If one of the DNA sources has been labelled in some way, i.e. is a DNA probe, this allows identification of specific DNA sequences in the other source. The two main methods of nucleic acid hybridization most commonly used are Southern and Northern blotting.


Southern blottingSouthern blotting, named after Edwin Southern who developed the technique, involves digesting DNA by a restriction enzyme which is then subjected to electrophoresis on an agarose gel. This separates the DNA or restriction fragments by size, the smaller fragments migrating faster than the larger ones.


The DNA fragments in the gel are then denatured with alkali, making them single stranded. A ‘permanent’ copy of these single stranded fragments is made by transferring them on to a nitrocellulose filter which binds the single-stranded DNA, the so called Southern blot. A particular target DNA fragment of interest from the collection on the filter can be visualized by adding a single stranded 32 P radioactively labelled DNA probe which will hybridize with homologous DNA fragments in the Southern blot which can then be detected by autoradiography.


Northern blottingNorthern blotting differs from southern blotting by the use of mRNA as the target nucleic acid in the same procedure; mRNA is very unstable because of intrinsic cellular ribonucleases. Use of ribonuclease inhibitors allows isolation of mRNA which, if run on an electrophoretic gel, can be transferred to a filter. Hybridizing the blot with a radiolabelled DNA probe allows determination of the size and quantity of the mRNA transcript, a so called Northern blot. In a gene disorder in which a mutation has not been identified in the coding sequences, an alteration in the size of the mRNA transcript suggests the possibility of a mutation in a non-coding region of the gene such as the splice junction of the intron-exon border. Northern blotting can also be used to demonstrate the differential pattern of expression of a gene in different tissues or at different times of development.


GENETIC CODEThe DNA sequence of a gene is divided into a series of units of three bases. Each set of three bases is called a codon and specifies a particular amino acid. The four bases in DNA and RNA can combine as a total of 43 = 64 codons which specify the 20 amino acids found in proteins. Because the number of codons is greater, all of the amino acids, with the exceptions of methionine and tryptophan, are encoded by more than one codon. This feature is referred to as the degeneracy or the redundancy of the genetic code


Codons which specify the same amino acid are called synonyms and tend to be similar. Variations between synonyms tend to occur at the third position of the codon, which is known as wobble position. The degeneracy of the genetic code minimizes the effects of mutations so that alterations to the base sequence are less likely to change the amino acid encoded and possible deleterious effects on protein function are avoided. Of the 64 possible codons, 61 encode amino acids. The remaining three, UAG, UGA, and UAA, do not encode amino acids but instead act as signals for protein synthesis to stop and as such are known as termination codons or stop codons. The codon for methionine, AUG, is the signal for protein synthesis to start and is known as the initiation codon. Thus all polypeptides start with methionine although this is sometimes removed later.


GENOTYPE AND PHENOTYPEConsideration of the heritability of a particular feature or trait requires a consideration of the relationship between genotype and phenotype. Genotype is defined as the genetic constitution of an individual, and may refer to specified gene loci or to all loci in general. An individual’s phenotype is the final product of a combination of genetic and environmental influences. Phenotype may refer to a specified character or to all the observable characteristics of the individual.


STRUCTURE OF GENESA gene is a unit of information and corresponds

to a discrete segment of DNA with a base sequence that encodes the amino acid sequence of a polypeptide. Genes vary greatly in size from less than 100 base pairs to several million base pairs. In humans there are an estimated 50-100000 genes arranged on 23 chromosomes. The genes are very dispersed and are separated from each other by sequences that do not contain genetic information.; this is called intergenic DNA. The intergenic DNA is very long, such that in humans gene sequences account for less than about 30% of the total DNA.


Only one of the two strands of the DNA double helix carries the biological information and this is called the template strand or sense or coding strand, which is used to produce an RNA molecule of complementary sequence which directs the synthesis of a polypeptide. The other strand is called the nontemplate strand or antisense or noncoding strand.www.indiandentalacademy.com

Gene promotersExpression of genes is regulated by a segment of DNA sequence present upstream of the coding sequence known as the promoter. Conserved DNA sequences in the promoter are recognized and bound by the RNA polymerase and other associated proteins called transcription factors that bring about the synthesis of RNA transcript of the gene.


Introns and ExonsIn genes coding information is usually split into a series of segments of DNA sequence called exons. These are separated by sequences that do not contain useful information called introns. The length of exons and introns varies but the introns are usually much longer and account for the majority of the sequence of the gene. Before the biological information in a gene can be used to synthesize a protein, the introns must be removed from RNA molecules by a process called splicing which leaves the exons and the coding information continuous.


DEVELOPMENTAL GENE FAMILIES1) Segmentation genes2) Paired-box genes (PAX)3) Zinc finger genes4) Signal transduction (‘Signalling’) genes5) Homeobox genes (HOX)

SEGMENTATION GENESInsect bodies consist of series of repeated body segments which differentiate into particular structures according to their position.


Three main groups of segmentation determining genes have been classified on the basis of their mutant phenotypes.(A) Gap mutants – delete groups of adjacent segments(B) Pair-rule mutants – delete alternate segments(C) Segment polarity mutants – cause portions of each segment to be deleted and duplicated on the wrong side.

1) Hedgehog (Vertebrates) Sonic Hedgehog Desert Hedgehog Indian Hedgehog

2) Wingless


Hedgehog morphogens are involved in the control of left-right asymmetry, the determination of polarity in the central nervous system, somites and limbs, and in both organogenesis and the formation of the skeleton.

In humans, Sonic hedgehog (SHH) plays a major role in development of the ventral neural tube with loss-of-function mutations resulting in a serious and often lethal malformation known as holoprosencephaly where the facial features shows eyes close together and there is a midline cleft lip due to failure of normal prolabia development.


PAIRED-BOX GENES (PAX)The mammalian Pax gene family consists of nine

members that can be organized into groups based upon sequence similarity, structural features, and genomic organization. The four groups include A) Pax1 and Pax9 B) Pax2, Pax5, and Pax8 C) Pax3 and Pax7 and D) Pax4 and Pax6 ZINC FINGER GENESThe term zinc finger refers to a finger-like loop projection which is formed by a series of four amino acids which form a complex with a zinc ion. Genes, which contain a zinc finger motif, act as transcription factors through binding of the zinc finger to DNA.


SIGNAL TRANSDUCTION GENESSignal transduction is the process whereby

extracellular growth factors regulate cell division and differentiation by a complex pathway of genetically determined intermediate steps. Mutations in many of the genes involved in signal transduction can cause developmental abnormalities. Fibroblast growth factor receptors (FGFRs) belong to the category of signal transduction genes.


HOMEOBOX GENES (HOX) AND ITS IMPORTANCESince their discovery in 1983, the homeobox genes were originally described as a conserved helix-turn-helix DNA motif of about 180 base pair sequence, which is believed to be characteristic of genes involved in spatial pattern control and development. The protein domain encoded by the homeobox, the homeodomain, is thus about 60 amino acids long. Proteins from homeobox containing, or what are known as HOX genes, are therefore important transcription factors which specify cell fate and establish a regional anterior/posterior axis.


The first genes found to encode homeodomain proteins were Drosophila developmental control genes, in particular homeotic genes, from which the name "homeo"box was derived. However, many homeobox genes are not homeotic genes; the homeobox is a sequence motif, while "homeotic" is a functional description for genes that cause homeotic transformations. Four homeobox gene clusters (HOXA, HOXB, HOXC, and HOXD) that comprise a total of 39 genes have been identified in humans. Each cluster contains a series of closely linked genes. In each HOX cluster there is a direct linear correlation between the position of the gene and its temporal and spatial expression. These observations indicate that these genes play a crucial role in early morphogenesis. Lower number HOX genes are expressed earlier in development and more anteriorly and proximally than are the higher number genes. www.indiandentalacademy.com

HOX Cluster

Number of genes

Chromosome location

HOXA(=HOX1)

11 (1-7, 9-11, 13) 7p

HOXB(=HOX2)

10 (1-9, 13) 17q

HOXC(=HOX3)

9 (4-6, 8-13) 12q

HOXD(=HOX4)

9 (1, 3, 4, 8-13) 2q

Homeobox gene clusters in humans

The 39 human HOX genes are organized in four clusters on four chromosomes. They are derived from a single ancestral cluster from which the single HOM-C complex in Drosophila is also derived.


Antennapedia = labial, proboscipedia, Deformed, Sex combs reduced, AntennapediaBithorax = Ultrabithorax, abdominalA, AbdominalBwww.indiandentalacademy.com

Cluster duplication during evolution has led to the concept of paralogous groups of HOX genes. Thus, groups of up to four genes derived from a common ancestral gene in the primitive cluster can be identified based upon sequence homology. The paralogues can exhibit similar experssion domains along the antero-posterior axis of the embryo leading to the concept of functional redundency between genes.


HOMEODOMAINThe homeodomain is a DNA-binding domain, and many homeobox genes have now been shown to bind to DNA and regulate the transcription of other genes. Thus homeodomain proteins are basically transcription factors, most of which play a role in development.The homeodomain is a common DNA-binding structural motif found in many eukaryotic regulatory proteins. Homeodomain proteins are involved in the transcriptional control of many developmentally important genes, and 143 human loci have been linked to various genetic and genomic disorders.


X-ray crystallographic and NMR spectroscopic studies on several members of this family have revealed that the homeodomain motif is comprised of three α-helices that are folded into a compact globular structure. Helices-I and II lie parallel to each other and across from the third helix. This third helix is also referred to as the “recognition helix”, as it confers the DNA binding specificity if individual homeodomain proteins. The homeodomain has been evolutionarily conserved at the structural level. This is most evident upon examination of divergent members of the homeodomain family.


TWIST GENESChromosomal rearrangements and linkage analysis have mapped the locus for TWIST, the human homologue of the Drosophila twist gene, located in chromosome 7p21- p22. The TWIST gene contains a basic helix-loop-helix (bHLH) motif that suggests that the TWIST gene product acts as a transcription factor. The HLH region of this motif is important for homo- or heterodimerization, whereas the basic domain is essential for binding of the dimer complex to a target DNA binding sequence(s). TWIST genes were identified in Drosophila and Drosophila TWIST gene affects the expression of a fibroblast growth factor receptor (FGFR) homologue DFR1. In humans mutations in TWIST amino acid sequence and FGFRs results in craniosynostosis.


GROWTH FACTORSGrowth factors constitute an important class of signaling molecules. The effects of growth factors are always mediated through binding of the factor to specific cell surface receptors. During embryonic development many growth factors have been shown to act as signals between tissue layers and they also act as signals during organogenesis. One mechanism whereby growth factors regulate development is through stimulation of homeobox genes.


Growth factor families1) Transforming growth factor- beta (TGF-β)

a) TGF-β 1-5b) Bone morphogenetic protein (BMP) 2-8c) Growth and Differentiation factor (GDF)

1-72) Epidermal growth factor (EGF)

a) EGFb) TGF-αc) Amphiregulind) HB-EGF


3) Fibroblast growth factor (FGF)FGF 1-8

4) Insulin like growth factor (IGF)IGF 1-2

5) Platelet derived growth factor (PDGF)PGDF A, B

6) Neurotrophinsa) Nerve growth factor (NGF)b) Brain-derived neutrotrophic factor (BDNF)c) Neurotrophin (NT) 3-4

Of all these factors, FGFs and BMPs are important for craniofacial development.


FIBROBLAST GROWTH FACTORS (FGFs) AND ITS RECEPTORS (FGFRs)

FGFs comprise a family of 22 genes encoding structurally related proteins (Ornitz and Itoh 2001). FGF1 (acidic-FGF, αFGF) and FGF2 (basic-FGF, βFGF) are the main factors. Studies of human disorders & gene knock-out studies in mice show the prominent role for FGFs is in the development of the skeletal system in mammals. Six subfamilies of FGFs, grouped by sequence similarities, tend to share biochemical and functional properties and are expressed in specific spatial and developmental patterns.


Four distinct FGF receptor tyrosine kinase molecules (FGFR1, FGFR2, FGFR3 and FGFR4) bind and are activated by most members of the FGF family. Alternative mRNA splicing produces FGF receptors with unique ligand binding properties. Alternative splicing is mostly tissue-specific, producing epithelial variants (b splice forms) and mesenchymal variants (c splice forms).

GENE Chromosome location

FGFR1 8p11

FGFR2 10q25

FGFR3 4p16www.indiandentalacademy.com

FGF activity and specificity are further regulated by heparan sulfate oligosaccharides, in the form of heparan sulfate proteoglycans. Heparin/ Heparan sulfate, FGF, and an FGF receptor (FGFR) associate to form a trimolecular complex. Heparan chains, themselves, have unique tissue-specific modifications that are required for, and may actually regulate, functional ligand–receptor interactions.


The expression of FGFs and FGFRs is temporally and spatially regulated during craniofacial development. FGFs and FGFRs play an important role in intramembranous as well as endochondral bone formation. During intramembranous bone formation, Fgf2, Fgf4, and Fgf9 are expressed in sutural mesenchyme in early craniofacial skeletogenesis, suggesting that they may be involved in regulating calvarial osteogenesis. Fgf18 and Fgf20 are also expressed in developing calvarial bones.

Mutations in FGFs and FGFRs tend to cause craniosynostosis



MUTATIONA mutation is defined as a heritable alteration or change in the genetic material. A mutation arising in a somatic cell cannot be transmitted to offspring, whereas if it occurs in gonadal tissue or a gamete it can be transmitted to future generations. TYPES OF MUTATIONSMutations occur in two forms: 1) Point mutations - involve a change in the base present at any position in a gene 2) Gross mutations - involve alterations of longer stretches of DNA sequence. The location of the mutation within a gene is important. Only mutations that occur within the coding region are likely to affect the protein. Mutations in noncoding or intergenic regions do not usually have an effect.


POINT MUTATIONSMissense mutationsThese point mutations involve the alteration of a single base which changes a codon such that the encoded amino acid is altered. Such mutations usually occur in one of the first two bases of a codon. The redundancy of the genetic code means that mutation of the third base is likely to cause a change in the amino acid. The effect of a missense mutation on the organism varies. Most proteins will tolerate some change in their amino acid sequence. However, alterations of amino acids in parts of the protein that are important for structure or function are more likely to have a deleterious effect and to produce a mutant phenotype.


Nonsense mutationsThese are point mutations that change a codon for an amino acid into a termination codon. The mutation causes translation of the messenger RNA to end prematurely resulting in a shortened protein which lacks part of its carboxyl-terminal region. Nonsense mutations usually have a serious effect on the activity of the encoded protein and often produce a mutant phenotype.


Frameshift mutationsThese result from the insertion of extra bases or the deletion of existing bases from the DNA sequence of a gene. If the number of bases inserted or deleted is not a multiple of three the reading frame will be altered and the ribosome will read a different set of codons downstream of the mutation substantially altering the amino acid sequence of the encoded protein. Frameshift mutations usually have a serious effect on the encoded protein and are associated with mutant phenotypes.


GROSS MUTATIONSDeletionsThese involve the loss of a portion of the DNA sequence. The amount lost varies greatly. Deletions can be as small as a single base or much larger in some cases corresponding to the entire gene sequence.InsertionsIn this case the mutation occurs as a result of insertion of extra bases, usually from another part of a chromosome.


RearrangementsThese mutations involve segments of DNA sequence within or outside a gene exchanging position with each other. A simple example is inversion mutations in which a portion of the DNA sequence is excised then re-inserted at the same position but in the opposite orientation.

Gross mutations, because they involve major alterations to gene sequences, invariably have serious effect on the encoded protein and are frequently associated with a mutant phenotype.


FUNCTIONAL EFFECTS OF MUTATIONS ON THE PROTEINMutations exert their phenotypic effect in one of two ways, either through loss- or gain- of function.Loss-of-function mutationsLoss-of-function mutation can result in either reduced activity or complete loss of the gene product. The former can be the result of either reduced activity or of decreased stability of the gene product and is known as a hypomorph, the latter being known as a null allele or amorph. Loss-of function mutations in the heterozygous state would, at worst, be associated with half normal levels of the protein product.


HaploinsufficiencyLoss-of function mutations in the heterozygous state in which half normal levels of the gene product result in phenotypic effects are termed haploinsufficiency mutations. There are number of autosomal dominant disorders where the mutational basis of the functional abnormality is the result of haploinsufficiency, in which, homozygous mutations result in more severe phenotypic effects.


Gain-of-function mutationsGain-of-function mutations, as the name suggests, result in either increased levels of gene expression or the development of a new function(s) of the gene product. Mutations which alter the timing or tissue specificity of the expression of a gene can also be considered to be gain-of-function mutations. Gain-of-function mutations are dominantly inherited and the rare instances of gain-of-function mutations occurring in the homozygous state are associated with a much more severe phenotype, which is often a prenatally lethal disorder.


MUTATION TRACKING AND MOLECULAR APPROACHESWith marked advances in molecular genetic technology in recent years, gene mapping techniques are now providing powerful approaches for locating genes associated with various diseases and disorders. Functional cloning uses the protein sequence and thereby the putative corresponding DNA sequence to clone the relevant gene, or by extracting the messenger RNA (mRNA) from the tissue to produce a complementary DNA (cDNA). This cDNA corresponds to the DNA sequence of the coding regions (exons) of a gene. Positional cloning, also known as reverse genetics, is used to identify the location of the mutant gene on a particular chromosome by virtue of its co segregation with polymorphic DNA markers. The first generation of these markers was termed restriction fragment length polymorphisms (RFLPs). www.indiandentalacademy.com

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs)Variation in the nucleotide sequence of the human genome is common, occurring approximately once every 200bp. RFLPs arise as a result of minor alterations in the DNA sequence on pairs of chromosomes. The DNA, usually obtained from peripheral blood leucocytes, is digested with a restriction enzyme, which recognizes particular DNA sequences and cuts at a certain point in the sequence. The resulting DNA fragments are then separated in an agarose gel where the distance they migrate depends upon their size, shorter fragments migrating further than larger fragments over a given period of time. The DNA is then transferred from the gel to a nylon membrane (Southern blotting) where it can be probed by markers.


The markers are DNA fragments which have been mapped to parts of chromosomes. Because of the variation in cutting sites, in an ideal situation the probe will bind to two different sized fragments of DNA. The probe is labelled using a radioisotope and appears as one or more bands on an autoradiograph.The different bands are referred to as alleles, and by following the segregation of these alleles with the disease, the position of the gene is established. The limitation of RFLPs is that individuals are frequently homozygous at a given marker, that is, they have two alleles of the same size.


To establish linkage (the position of the diseased gene in relation to the RFLPs) affected individuals need to be heterozygous, that is, have two alleles of different sizes. Linkage analysis depends upon having a sufficient number of meioses, either in one or more large pedigrees or multiple smaller pedigrees. It is difficult to establish linkage without a number of three-generation (or more) pedigrees. Linkage also relies upon the fact that, at meiosis, recombination events occur on the chromosomes. Thus, some individuals will inherit exact copies of their parents’ chromosomes while others will inherit chromosomes which represent rearrangements of the original chromosomes. These recombination events are the key to mapping of a gene.


HYPERVARIABLE TANDEM REPEAT DNA LENGTH POLYMORPHISMSThe different classes of tandemly repeated DNA sequences in the human genome have been useful clinically in mutation tracking. VARIABLE NUMBER TANDEM REPEATS (VNTRs)More recently, a new generation of polymorphic markers has been employed. These variable number of tandem repeat (VNTR) markers rely upon variations in the number of repeat sequences in non-coding regions of chromosomes. The VNTRs may be either dinucleotide repeats (repeats of two DNA bases, usually cytosine and adenine) or tri-, tetra-, or penta-nucleotide repeats.


VNTRs have an advantage over RFLPs in that the number of repeats is (in theory) infinitely variable and these markers are more likely to be heterozygous. VNTRs obviate the need for Southern blotting. They are identified using the polymerase chain reaction (PCR) which uses primer sequences flanking the variable segment to amplify the DNA using a thermal cycler. The resulting amplified DNA fragments are then separated by electrophoresis in a polyacrylamide gel and revealed by autoradiography. Detection systems other than radioactive systems are now available and some of these processes can be automated.


Cosegregation of a disease with one or more DNA markers can be confirmed by statistical analysis. The measures of cosegregation are the LOD score (logarithm of the odds for linkage as opposed to no linkage) with a value of three being regarded as significant, this indicating a one-thousand-fold likelihood of linkage. The other measure is the recombination fraction, which is an indication of the distance from the marker to the gene. With a high LOD score and a low recombination fraction the researcher can be fairly certain that the gene responsible for the disease has been localized.


The next stage is to clone the gene and numerous techniques are available to accomplish this. If the disease has been localized to a small area of a chromosome, the current strategy would be to use the markers either side of the disease (flanking markers) to probe a yeast artificial chromosome (YAC) library and this, in turn, can be used to screen other libraries containing smaller fragments of DNA such as cosmid libraries.


Typical YACs are considerably larger than cosmids so this approach enables the relevant section of DNA to be analysed on a smaller scale. Other techniques that can be used include identification of the coding parts at the beginning of genes (CpG islands) and exon trapping. Once the gene has been isolated it can then be sequenced and the coding regions (exons) and non-coding regions (introns) identified. Following this, mutations in affected individuals can be identified using techniques such as single strand chain polymorphisms (SSCP) or direct sequencing.


PATTERNS OF INHERITANCEAn important reason for studying the pattern of inheritance of

disorders within families is to enable advice to be given to members of a family regarding the likelihood of their developing it or passing it on to their children.

A trait or disorder which is determined by a gene on an autosome is said to show autosomal inheritance, whereas a trait or disorder determined by a gene on one of the sex chromosomes is said to show sex-linked inheritance.

1) Autosomal inheritance Autosomal dominant Autosomal recessive

2) Sex-linked inheritance X-linked dominant X-linked recessive Y-linked inheritance


AUTOSOMAL DOMINANT INHERITANCEAn autosomal dominant trait is one which manifests in the heterozygous state, i.e. in a person possessing both an abnormal or mutant allele and the normal allele. It is often possible to trace a dominantly inherited trait or disorder through many generations of a family. This pattern of inheritance is sometimes referred to as ‘vertical’ transmission. Features include,

1) Males and females affected in equal proportions2) Affected individuals in multiple generations3) Transmission by individuals of both sexes, i.e.

male to male, female to female, male to female and female to male


Variable expressivityThe clinical features in autosomal dominant disorders can show striking variation from person to person. This difference in involvement between individuals is referred to as variable expressivity.Reduced penetranceIn some individuals heterozygous for certain autosomal disorders, the presence of the mutation can be undetected clinically, representing so-called reduced penetrance or what is known as the disorder ‘skipping a generation’. Reduced penetrance is thought to be the result of the modifying effects of other genes, as well as being due to interaction of the gene with environmental factors. An individual who is heterozygous for a dominant mutation but has no features of the disorder is said to represent non-penetrance.


AUTOSOMAL RECESSIVE INHERITANCERecessive traits and disorders are only manifest when the mutant allele is present in a double dose, i.e. homozygosity. Individuals heterozygous for a recessive mutant allele show no features of the disorder and are perfectly healthy, i.e. they are carriers. It is not possible to trace an autosomal recessive trait or disorder through the family, i.e. all the affected individuals in a family are usually in a single sibship, that is, they are brothers and sisters. This is sometimes referred to as ‘horizontal’ transmission.


Mutational heterogeneityIndividuals have been identified who have two different mutations

at the same locus and are known as compound heterozygotes, constituting what is known as allelic or mutational heterogeneity. Most individuals affected with an autosomal recessive disorder are probably, infact, compound heterozygotes rather than true homozygotes, unless their parents are related when they are likely to be homozygous for the same mutation by descent, having inherited the same mutation from a common ancestor.

Three features, which suggest the possibility of autosomal recessive inheritance, were,1) The disorder affects males and females in equal

proportions.2) It usually affects individuals in one generation in a single

sibship, i.e. brothers and sisters, and does not occur in previous and subsequent generations.

3) Parents can be related, i.e. consanguineous.www.indiandentalacademy.com

SEX-LINKED INHERITANCESex-linked inheritance refers to the pattern of inheritance shown by genes which are located on either side of sex chromosomes. Genes carried on the X chromosome are referred to as being X-linked, while genes carried on the Y chromosome are referred to as exhibiting Y-linked or holandric inheritance.


X-linked dominant inheritanceThese are disorders, which are manifest in the heterozygous

female as well as in the male who has the mutant allele on his single X-chromosome. X-linked dominant inheritance superficially resembles that of an autosomal dominant trait because both daughters and sons of an affected female have 50% chance of being affected.

Features include,1) Males and females are affected but often females are

affected in excess.2) Severity is less in females compared to males.3) Affected males can transmit the trait to all his

daughters but to none of his sons.


X-linked recessive inheritance:An X-linked recessive trait is one determined by a gene

carried on the x chromosome and usually only manifests in males. A male with a mutant allele on his single X-chromosome is said to be hemizygous for that allele.

Features of X-linked recessive inheritance were,1) Males usually only affected.2) Transmitted through unaffected heterozygous carrier

females affect males, as well as by affected males to their obligate carrier daughters with a consequent risk to male grand children through these daughters.

3) Affected males cannot transmit the disorder to their sons.

Y-linked inheritanceY-linked or holandric inheritance implies that only males are affected. An affected male transmits Y-linked traits to all his sons but to none of his daughters. www.indiandentalacademy.com

POLYGENIC AND MULTIFACTORIAL INHERITANCE

POLYGENIC INHERITANCEPolygenic or quantitative inheritance involves the inheritance and expression of a phenotype being determined by many genes at different loci, with each gene exerting a small additive effect. Additive implies that the effects of the genes are cumulative, i.e. no one gene is dominant or recessive to another. MULTIFACTORIAL INHERITANCEAs it is likely that many factors, both genetic and environmental, are involved in causing these disorders they are generally referred to as showing multifactorial inheritance. In multifactorial inheritance environmental factors interact with many genes to generate a normally distributed susceptibility.


HERITABILITYHeritability can be defined as the proportion of the total phenotypic variance of a condition which is caused by additive genetic variance. Heritability is expressed either as a proportion of 1 or as a percentage. Heritability is estimated from the degree of resemblance between relatives expressed in the form of a correlation coefficient which is calculated using statistics of the normal distribution. Alternatively, heritability can be calculated using data on the concordance rates in monozygotic and dizygotic twins.


SIB-PAIR ANALYSISIf affected siblings inherit a particular allele more or less often than would be expected by chance, this indicates that that allele or its locus is involved in some way in causing the disorder.The strength of this relationship can be quantified by determining the ratio of the expected to observed proportions of affected sib-pairs which share zero alleles at the relevant locus.

SEGREGATION AND LINKAGE ANALYSISSegregation analysis refers to the study of the way in which a disorder is transmitted in families so as to establish the underlying mode of inheritance. It is a statistical method for determining the mode of inheritance of a particular phenotype from family data, particularly with the aim of elucidating single gene effects or so-called major genes.


With increasing computer power, models have been developed to detect the contribution of individual genetic loci that have large effects against a background of polygenic and environmental effects. Once evidence of major genes has been detected, linkage analysis provides a means of determining where individual genes are located within the genome. Until recently, however, application of these methods to clarify the genetic basis of dental disorders has been limited by the difficulties of obtaining data from large family pedigrees and also in identifying appropriate polymorphic marker loci.


TWINNINGTwins can be identical or non-identical monozygotic

(MZ) or dizygotic (DZ) – depending on whether they originate from a single conception or from two separate conceptions.MONOZYGOTIC TWINSMonozygotic twins originate from a single egg which has been fertilized by a single sperm. A very early division, occurring in the zygote before separation of the cells which make the chorion, results in dichorionic twins. Division during the blastocyst stage from days 3 to 7 results in monochorionic diamniotic twins. Division after the first week leads to monoamniotic twins.


DIZYGOTIC TWINSDizygotic twins result from the fertilization of two ova by two sperm and are no more closely related genetically than brothers and sisters. Hence they are sometimes referred to as fraternal twins. Dizygotic twins are dichorionic and diamniotic although they can have a single fused placenta if implantation occurs at closely adjacent sites.


SIGNIFICANCE OF TWIN STUDIESThe classical twin approach for separating the effects of nature and nurture involves comparing identical (monozygous) twins and non-identical (dizygous) twins. Differences between monozygous (MZ) twin pairs reflect environmental factors, whereas differences between dizygous (DZ) twin pairs are due to both genetic and environmental factors. Therefore, greater similarities between MZ twin pairs compared with DZ twin pairs can be interpreted as reflecting genetic influences on the feature(s) being studied.


Apart from comparisons of monozygous and dizygous twins, there are other twin models that provide insights into the contributions of genetic and environmental factors to observed variability. The monozygous co-twin model involves comparisons of monozygous twins where each member of a pair has been exposed to different environmental effects. For example, identical twins might be treated with different appliances to correct similar malocclusions and the outcomes compared.Monozygous twins are assumed to have identical genotypes, so their offspring are genetically related as half-sibs but are socially first cousins. A nested analysis of variance similar to that used in analysing data from half and full-sibling litters in animal studies can be applied to provide estimates of genetic and environmental effects. www.indiandentalacademy.com

SIGNIFICANCE OF GENETIC STUDIES FOR MALOCCLUSION AND CRANIOFACIAL ANOMALIESDental occlusion reflects the interplay between a number of factors including tooth size, arch size and shape, the number and arrangement of teeth, size and relationships of the jaws, and also the influences of the soft tissues including lips, cheeks and tongue.The term ‘malocclusion’ is generally used to refer to variations from normal occlusal development, and although in some instances it is possible to specify the cause of a particular malocclusion, for example, genetic syndromes, embryological defects, or trauma, most malocclusions represent variations from normal development for which there is no apparent cause. There have been several excellent reviews of genetic studies of craniofacial development and morphology.


Smith and Bailit (1977), in their comprehensive review of the problems and methods in studies of the genetics of dental occlusion, listed five main research objectives:1) Elucidating modes of inheritance2) Detecting the effects of admixture and inbreeding3) Performing linkage analyses4) Estimating heritabilities5) Comparing population differences


Modes of inheritanceOcclusal variation appears to conform to a multifactorial mode of inheritance, although strong familial similarities may be due to single major genes. For example, the famous ‘Hapsburg jaw’ seen in consecutive generations of an Austrian royal family may have been caused by a small number of segregating major genes. It is also possible that epistatic factors, that is, the interaction between genes at different loci, may play a more important role than most researchers have thought. Admixture and breeding effectsMany research workers suggested that racial admixture increases the occurrence of malocclusion. The notion that admixture might lead to an increased frequency of malocclusion in humans appears to have originated from the work of Stockard and Johnson (1941) in which gross deformities of the jaws of dogs were produced by cross-breeding different inbred strains.


The X and Y-chromosomesPattern profiles of dental crown size show the dosage effect of the sex chromosomes, with both the X and Y-chromosomes appearing to exert growth-promoting effects on human tooth crown size. The X chromosome appears to mainly regulate enamel thickness. On the other hand, the Y chromosome seems to affect both enamel and dentine. The X and Y-chromosomes also seem to influence craniofacial growth and development.It is suggested that the X chromosome may alter morphology of the cranial base by affecting growth at the synchondroses, that is, cartilaginous joints, and it also appears to have a direct effect on mandibular shape.


HeritabilityEarly traditional twin studies (Lundstrom 1954) and intrafamilial comparisons (Stein 1956) indicated that occlusal traits were under reasonably strong genetic control. However, more recent reports in twins (Corrucini 1980) and in first-degree relatives (Harris 1991) have emphasized the importance of environmental factors. Population differencesVariations in dental occlusion between different human populations have been described and interpreted in genetic terms. Midfacial growth, alveolar bone development and tooth migration associated with vigorous masticatory function tends to provide space for unimpeded emergence and alignment of permanent teeth in Aboriginals. Recent studies have shown that occlusal variation increased significantly in the Yuendumu people within one generation after adoption of a more westernized diet.


Manifestation of a malocclusion is the culmination of a hierarchy of subclinical molecular, biochemical, physiologic, and metabolic markers of risk. Any one of these can be modified by the environment, which makes the clinical expression remote from gene action. This is the essence of why dentofacial structure is not suitable for analyses with Mendelian models.Heritability is a one-dimensional descriptive statistic that does not address the mode of inheritance of malocclusions. The long-term goal should be to identify factors that affect the frequency and/or severity of the phenotype. Calculation of heritability estimates is a preliminary step that should be followed by tests for causative agents. Within clinical orthodontics, the preliminary goal has been to define the relative contributions of genetics and the environment. www.indiandentalacademy.com

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Genetics and malocclusion

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