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Genetics in orthodontics-sakthi


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Genetics in orthodontics-sakthi

  2. 2. INTRODUCTION The development of human craniofacial complex is an extraordinary combination of evolutionary genetics, embryogenesis and precise disposition of 4 germ layers, morphogenesis, organogenesis and maturation. Of the 25,000 genes in human genome around 17,000 genes are implicated in craniofacial development. Hence a thorough knowledge of genetics is important to know the intricacies of craniofacial growth and development. 2
  3. 3. “The genetic messages encoded within our DNA molecules will provide the ultimate answers to the chemical underpinnings of human existence and in the 21st century all biology will be gene based and all biologists will be geneticists”
  4. 4. Basic Terminologies BASIC TERMINOLOGIES 4
  5. 5. Science of genetics: Concerned with inheritance of traits, whether normal or abnormal and with the interaction of genes and the environment. Molecular Genetics The branch of genetics that deals with hereditary transmission and variation on the molecular level. 5
  6. 6. • Chromosome A threadlike linear strand of DNA and associated proteins in the nucleus of eukaryotic cells that carries the genes and functions in the transmission of hereditary information. 6
  7. 7. According to position of centromere: • Metacentric • Submetacentric • Acrocentric • Telocentric 7
  8. 8. CHROMOSOME IDEOGRAMS: • A consistent numbering system is essential for mapping chromosomes. In Paris 1971 a mapping system known as the International System for Cytogenetic Nomenclature (ISCN) was established.
  9. 9. • THE RULES OF THE ISCN NUMBERING SYSTEM 1. Numbering of a chromosome begins at its centromere. 2. Chromosomes are assigned a long arm and a short arm, based on the position of their centromere. The shorter arm of the chromosome is known as the p, or petite arm, from the French word for "small." The longer arm is known as the q. Chromosomal regions that are present on the short arm will begin with the designation p, while regions on the long arm will begin with q. 3. By convention, the p arm of the chromosome is always shown at the top in a karyotype. Arm Region Band Subband p 2 1 1 q 2 1 1 1 2 1 2 3 2 3 2 1 2 1 5 4 3 2 1 4 Chromosome 17 3 1 2 3 1 2, 3 4 1 2 3 17q11.2
  10. 10. 4. Each arm of the chromosome is divided into regions. The numbers assigned to each region gets larger as the distance from the centromere to the telomere increases 5. Depending on the resolution of the staining procedure, it may also be possible to detect additional bands within each region, which are designated by adding a digit to the number of the region, increasing in value as the distance from the centromere increases.
  11. 11. Ideogram of the medium-sized sub-metacentric chromosome 12 (400) • The position of the centromere separates the p and q arms (hatched area). • This ideogram describes the pattern of Giemsa staining at a fairly low resolution (about 400 total number of bands in a karyotype). • At this resolution, the long q arm of chromosome 12 is subdivided into two main regions: 12q1 and 12q2. Region 12q1 is further subdivided into five sub-regions: 12q11 through 12q15, each of which corresponds to a band detected by Giemsa staining. • We refer to these subdivisions as "12q one-one" through "12q one-five" (not as "12q eleven" through "12q fifteen"). The more distal 12q2 region is subdivided into sub-regions 12q21 through 12q24. Sub-region 12q24 is further subdivided into regions 12q24.1 through 12q24.3.
  12. 12. Ideogram of the medium-sized sub-metacentric chromosome 12 at higher resolution • When chromosomes that are in prometaphase are stained a higher resolution is obtained, because prometaphase chromosomes are slightly less condensed than metaphase chromosomes. • At this higher level of resolution approximately 850 bands are distinguishable in a karyotype.
  13. 13. • Chromatid Either of two parallel filaments joined at the centromere which make up a chromosome, and which divide in cell division, each going to a different pole of the dividing cell and each becoming a chromosome of one of the two daughter cells 13
  14. 14. • An autosome is a nonsex chromosome. It is an ordinarily paired type of chromosome that is the same in both sexes of a species. For eg, in humans, there are 22 pairs of autosomes. • Non-autosomal chromosomes are usually referred to as sex chromosomes, allosomes or heterosome 14
  15. 15. Homologous chromosomes Pair of chromosomes, one inherited from each parent, that have corresponding gene sequences and that pair during meiosis 15
  16. 16. The smallest human chromosome is chromosome 21 and the largest one is chromosome 1. This is one reason why Down‟s syndrome (trisomy 21) is the most common trisomy 16
  17. 17. By comparison to the X chromosome, the much smaller Y chromosome has only about 26 genes and gene families. Out of 26 only 9 gene families are involved in sperm production . Only one of the Y chromosome genes, the SRY gene, is responsible for male anatomical traits. When any of the 9 genes involved in sperm production are missing or defective the result is usually very low sperm counts and subsequent infertility. 17
  18. 18. 18
  19. 19. • DNA The molecule that encodes genetic information in the nucleus of cells. It determines the structure, function and behaviour of the cell 19
  20. 20. • RNA A polymeric constituent of all living cells and many viruses, consisting of a long, usually single-stranded chain of alternating phosphate and ribose units with the bases adenine, guanine, cytosine, and uracil bonded to the ribose. The structure and base sequence of RNA are determinants of protein synthesis and the transmission of genetic information. 20
  21. 21. • Gene A segment of a DNA molecule that contains all the information required for synthesis of a protein. It is the biological unit of heredity transmitted from parent to progeny. 21
  22. 22. Alleles: Different forms of genes at the same locus or position on the chromosome Homozygous: – If both copies of genes are identical Heterozygous: – If the two copies of genes differ 22
  23. 23. Locus The position that a given gene occupies on a chromosome 23
  24. 24. The largest gene identified so far is the dystrophin gene (responsible for Duchenne‟s muscular dystrophy) . It has 80 coding regions and encodes only a 3,700 amino acid-long protein. Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (231). 24
  25. 25. • Mode of inheritance: – Dominant: • If the trait of the disease manifests itself when the affected person carries only one copy of the gene responsible – along with one normal allele – Recessive: • If two copies of defective genes are required for expression of trait 25
  26. 26. • Genotype: – Genetic constitution of an individual, and refers to specified gene loci or to all loci in general • Phenotype: – Specified character or to all observable characteristics of the individual 26
  27. 27. • Mitosis The process by which a cell divides and produces two daughter cells identical to parent cell. • Meiosis The process of cell division in sexually reproducing organisms that reduces the number of chromosomes in reproductive cells from diploid to haploid, leading to the production of gametes in animals and spores in plants. 27
  28. 28. 1. Any genetically determined characteristic. 2. A distinctive behavior pattern.
  29. 29. Autosomal Dominant Disorders In this type of disorder, the mutant gene is located on one chromosome of the autosomal chromosome pair and the comparable gene or the homologous chromosome (partner chromosome) is normal e.g. Treacher Collin syndrome.
  30. 30. Autosomal Recessive Disorders Autosomal recessive disorders are inherited from clinically .normal parents who have the same mutant gene on one chromosome of the homologus chromosome pair (i.e. each not only a single dose of the mutant gene).
  32. 32. X-linked Recessive Disorder  Genes located in the X-chromosomes represent only in a single dose in the XY males.  These are X-linked recessive disorder, is fully expressed in a male who has only a single X-linked recessive gene.  A female with the single X-linked recessive gene is phenotypically normal carrier because of the presence of a normal partner gene at the same locus on the second Xchromosome.
  33. 33. Therefore with some exception only males are affected with X-linked disorders. The mutant gene is either transmitted from a carrier mother to her affected son, or the affected male represent a fresh mutation. Eg. Haemophilia.
  34. 34. X-linked Dominant Disorders In this group the X-linked gene is a dominant gene, expressing itself in a single dose. Therefore both females and males in each generation will be affected.
  35. 35. 37
  36. 36. • Gregor Johann Mendel (18221884) was an Augustinian priest and scientist, and is often called the father of genetics for his study of the inheritance of traits in pea plants. Mendel showed that the inheritance of traits follows particular laws, which were later named after him. 38
  37. 37. – Studied segregation of traits in the garden pea (Pisum sativum) beginning in 1854. – Presented his paper on “Experiments with Plant Hybridization” in 1866 39
  38. 38. • Why Mendel chose pea plant? 1. The garden pea was easy to cultivate and had relatively short life cycle 2. The plant had distinguishing characteristics such as flower color and pea texture 3. Because of its anatomy, pollination of plant was easy to control and cross fertilization could be accomplished artificially 40
  39. 39. Mendel‟s Experiments: Focused on 7 well-defined garden pea traits by crossing different phenotypes one at a time Counted offspring of each phenotype and analyzed the results mathematically 41
  40. 40. 42
  41. 41. Mendel‟s first law: The Principle of Segregation: The two members of a heredity factor pair segregate from each other in the formation of gametes. Now: Two members = alleles Hereditary factor = gene 43
  42. 42. Dihybrid cross F2 generation ratio: 44
  43. 43. Mendel‟s second law: The Principle of Independent Assortment: During gamete formation, members of one heredity factor pair segregate into gametes independently from other heredity factor pairs. Now: Two members = alleles Hereditary factor = gene 45
  44. 44. BEYOND MENDEL Partial dominance The phenotype of the heterozygote falls between those of the two homozygotes. The phenotype shows the ratio of 1:2:1 Eg: Four o‟ clock plant(Mirabilis jalapa) 46
  45. 45. AREAS OF GENETICS CLASSICAL GENETICS Mendel’s principles Mitosis & Meiosis Sex determination Sex Linkage Chromosomal mapping Cytogenetics MOLECULAR GENETICS Structure of DNA Chemistry of DNA Transcription Translation DNA cloning & genomics DNA mutation & repair EVOLUTIONARY GENETICS Quantitative genetics Hardy Weinberg equilibrium Assumptions of equilibrium Evolution Speciation 47
  46. 46. STEPPING INTO ERA OF MOLECULAR GENETICS • Evidence for DNA as genetic material In 1928 E.Griffith in his experiments with S.pneumoniae found that there is transforming factors which converted nonlethal strains to lethal ones 48
  47. 47. • In 1952 Chase Hershey published & a research that supported the notion that DNA is the genetic material using bacteriophages 49
  48. 48. RNA as genetic material Many of the viruses carry RNA as genetic material This RNA can be: Double stranded-reoviruses Single stranded-measles ,rubella ,polio Retroviruses-HIV1 & HIV2 50
  49. 49. PRION • A prion is a nonliving, self-replicating infectious agent made of protein. It can reproduce with the aid of its host's biological machinery, like a virus. "Prion" is short for "proteinaceous infectious particle.“ • Prions found in animals exclusively infect the brain, are fatal and untreatable. • Prions are responsible for the outbreak of Mad Cow Disease in Britain . • The prion protein was not isolated until 1982, when Stanley B. Prusiner discovered it and coined the term. 51
  50. 50. STRUCTURE OF DNA 52
  51. 51. • In 1953 Watson & Crick published a paper in Nature which suggested the structure of DNA. This paper which first put forth the correct structure of DNA is a milestone in modern era of molecular genetics 53
  52. 52. • DNA is made of chains of nucleotides • It is a double helix with sugar phosphate backbones on the outside and the bases on inside • The diameter of helix is around 20A 54
  53. 53. • The two strands of DNA are antiparallel to each other • One strand has a 5‟phosphate and other has 3‟hydroxyl group 55
  55. 55. • A nucleotide is formed when base attaches to the 1‟ end of the carbon of sugar and a phosphate attaches to the 5‟ of same sugar • Nucleotides take their name from the base present in it 57
  56. 56. • Nucleotides are linked together by phosphodiester bond formed between the 5‟ C of one nucleotide & 3‟ OH group of adjacent molecule • Sugar+ base=nucleoside • Hence nucleotide is nucleoside phosphate 58
  57. 57. • Adenine binds to thymine with 2 hydrogen bonds • Cytosine binds to guanine with 3 hydrogen bonds 59
  58. 58. Chargaff‟s Rule There is 1:1 correspondence between the purines and pyrimidines i.e. amount of adenine equals thymine and cytosine equals guanine RNA does not follow this rule that is amount of adenine & uracil are not equal neither the amount of cytosine and guanine 60
  59. 59. • Why not 2 purines or pyrimidines together ? Not enough space (20 Å) for two purines to fit within the helix and too much space for two pyrimidines to get close enough to each other to form hydrogen bonds between them. • But why not A with C and G with T? Only with A & T and with C & G are there opportunities to establish hydrogen bonds between them. The ability to form hydrogen bonds makes the base pairs more stable structurally. 61
  60. 60. • Types of DNA B TYPE • Most common • Right handed helix • Bases are perpendicular to main axis • 10 bases per turn • Helix diameter 20A 62
  61. 61. A TYPE • If the water in DNA increases to 75% it changes to A form • Bases tilt wrt long axis • 11 bases per turn • Helix diameter 23A 63
  62. 62. Z TYPE • Left handed helix • Backbone formed zigzag hence called Z • Found in conditions when cytosine is methylated or there is high salt concentration • Helix diameter 18A • 12 bases per turn 64
  63. 63. DNA REPLICATION 65
  64. 64. • The process of DNA replication provides an answer of how genetic information is transmitted from one generation to other • The synthesis of both complimentary DNA strands occurs from 5‟-3‟ end • 5 main enzymes involved in this process -helicase -primase -DNA polymerase 1 -DNA polymerase 3 -ligase 66
  65. 65. • Continuous replication takes place on the 3‟-5‟ end and this strand is called leading strand • Discontinuous form of replication takes place on the 5‟-3‟ end and this strand is referred as lagging strand • The discontinuous replication takes place in small fragments called Okazaki fragments (after R.Okazaki who first saw them) 67
  66. 66. Why RNA is used to prime DNA synthesis? Probably, making use of RNA primers lowers the error rate of DNA replication because priming is basically an error prone process since nucleotides are initially added without a stable primer configuration. Therefore a RNA primer is first put and removed. Resynthesis by polymerase I is in a much more stable primer configuration and thus makes very few errors 68
  67. 67. DNA Replication • Origins of replication 1. Replication Forks: hundreds of Y-shaped regions of replicating DNA molecules where new strands are growing. 3’ 5’ Parental DNA Molecule Replication Fork 3’ 5’
  68. 68. • Origins of replication 2. Replication Bubbles: a. Hundreds of replicating bubbles (Eukaryotes). b. Single replication fork (bacteria). Bubbles Bubbles
  69. 69. • Strand Separation: 1. Helicase: enzyme which catalyze the unwinding and separation (breaking HBonds) of the parental double helix. 2. Single-Strand Binding Proteins: proteins which attach and help keep the separated strands apart.
  70. 70. • Strand Separation: 3. Topoisomerase: enzyme which relieves stress on the DNA molecule by allowing rotation around a single strand. Enzyme DNA Enzyme free
  71. 71. • Priming: 1. RNA primers: before new DNA strands can form, there must be small pre-existing primers (RNA) present to start the addition of new nucleotides (DNA Polymerase). 2. Primase: enzyme that polymerizes (synthesizes) the RNA Primer.
  72. 72. • Synthesis of the new DNA Strands: 1. DNA Polymerase: with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction. 5’ 3’ Nucleotide DNA Polymerase RNA Primer 5’
  73. 73. 2. Leading Strand: synthesized as a single polymer in the 5’ to 3’ direction. 5’ 3’ 5’ Nucleotides DNA Polymerase RNA Primer
  74. 74. 3. Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously against overall direction of replication. Leading Strand 5’ 3’ DNA Polymerase 3’ 5’ RNA Primer 5’ 3’ 3’ 5’ Lagging Strand
  75. 75. 4. Okazaki Fragments: series of short segments on the lagging strand. DNA Polymerase RNA Primer 5’ 3’ Okazaki Fragment 3’ 5’ Lagging Strand
  76. 76. 5. DNA ligase: a linking enzyme that catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands. Example: joining two Okazaki fragments together. DNA ligase 5’ 3’ Okazaki Fragment 1 Lagging Strand Okazaki Fragment 2 3’ 5’
  77. 77. The process of making an identical copy of a section of duplex (double-stranded) DNA, using existing DNA as a template for the synthesis of new DNA strands. 79
  78. 78. • Conservative: both the strands are conserved and act as template for new strands 80
  79. 79. • Semiconservative: One strand of DNA act as template for the formation of daughter of daughter DNA 81
  80. 80. • Dispersive Some parts of parental DNA are conserved and other are new daughter DNA 82
  83. 83. TRANSCRIPTION It is the process whereby the DNA sequence in a gene is copied into mRna. It is the first step in gene expression 85
  84. 84. Types of RNA • mRNA:It carries the information from the DNA to the ribosomes in cytoplasm • tRNA: It brings the amino acids to the ribosomes where protein synthesis takes place • rRNA:It is a structural and functional part of ribosomes 86
  85. 85. • The process of transcription is controlled by RNA polymerase. It adds up the appropriate complementary ribonucleoside to the 3‟ end of the RNA chain • The transcribed mRNA strand is called sense strand and the DNA template strand is called antisense strand 87
  86. 86. • The DNA region that RNA polymerase associates immediately before beginning transcription is known as promoter.It contains information for transcription initiation and are the sites where the gene expression is controlled • The polymerase moves down the DNA until the RNA polymerase reaches a stop signal or terminator sequence. 88
  87. 87. Introns These are segments of DNA within genes that are transcribed into RNA but are not translated into protein sequences. They are removed from the RNA before its transport into cytoplasm Discovered by Sharp & Roberts in 1977 The segments between introns are called exons 89
  88. 88. • Posttranscriptional modifications  mRNA splicing The non coding introns are excised & the non continuous exons are spliced together  5‟ capping Methylated guanine nucleotide is added to the 5‟ end of the molecule to facilitate mRNA transport to the cytoplasm & its attachment to ribosome.  Polyadenylation The cleavage of the 3‟ end of the mRna molecule from the DNA involves the addition of approximately 200 adenylate residues,the so called polytail, after cleavage of the RNA .It facilitates transport of mrna to cytoplasm 90
  89. 89. TRANSLATION • The formation of proteins from RNA is called translation • All proteins are synthesized from only 20 amino acids • Amino acids consist of: 91
  90. 90. • Transfer RNA is shaped like a clover leaf with three loops. It contains an amino acid attachment site on one end and a special section in the middle loop called the anticodon site. The anticodon recognizes a specific area on a mRNA called a codon 92
  91. 91. Attachment of amino acid to Trna The function of Trna is to ensure that each AA incorporated into a protein corresponds to a particular codon in Mrna The amino acid attach to Trna by enzyme known as aminoacyl-Trnasynthetases and such RNA is said to be „charged‟ 93
  92. 92. • Translation can be divided into 3 stages  Initiation  Elongation  Termination 94
  93. 93. • INITIATION Initially there is formation of initiation complex : 30S subunit of RNA Mrna Charged methionine(AUG) Trna Initiation factors(IF-2 & IF-3) The 30S subunit has 3 sites present in it where the Trna attaches: A site (aminoacyl site)-First step P site(peptidyl site) E site(exit site) 95` ```````````
  94. 94. • ELONGATION Now the first tRna moves to the P site and 2nd tRna gets attached to the Asite Next imp step is peptide bond formation between the amino acids attached to 2 tRnas Peptidyl transferase in the 50S subunit acts as an enzyme for the formation of bond between carboxyl end of one & amino end of other amino acid The next step is translocation in which an enzyme called translocase which physically moves the mRna & its associated tRna. So the first attached RNA moves to E site & new one to A site 96
  95. 95. • TERMINATION Termination of protein synthesis occur when one of the 3 nonsense codons appear at A site. These are:  UAG(amber)  UAA(ochre)  UGA(opal) When a nonsense codon enters A site a release factor(RF-1 or RF-2) recognizes it and cause hydrolysis of bond between peptide chain & tRna at Psite 97
  96. 96. After the release factor act, the ribosome has completed the task of translating mrna to polypeptide. Finally release of all factors and dissociation of 2 subunits takes place 98
  97. 97. GENETIC CODE • The genetic code is the set of rules by which information encoded in genetic material is translated into proteins • The unit of information is CODON = genetic 'word„ a triplet sequence of nucleotides • 3 nucleotides = 1 codon = 1 amino acid 99
  98. 98. Characteristics of genetic code  Reading in Frames Codon is defined by the initial nucleotide from which translation starts. For example, the string GGGAAACCC, if read from the first position, contains the codons GGG, AAA and CCC; and if read from the second position, it contains the codons GGA and AAC. Every sequence can thus be read in reading frames, each of which will produce a different amino acid 100
  99. 99.  Degenerate code The genetic code is degenerate that is a given amino acid may have more than one codon.The genetic code has redundancy but no ambiguity. For example, although codons GAA and GAG both specify glutamic acid (redundancy), neither of them specifies any other amino acid (no ambiguity). 101
  100. 100.  Universal The genetic code is almost universal. Mitochondrial genes When mitochondrial mRNA from animals or microorganisms is placed in a test tube with the cytosolic protein-synthesizing machinery (amino acids, enzymes, tRNAs, ribosomes) it fails to be translated into a protein. The reason: mitochondria use UGA to encode tryptophan (Trp) rather than as a chain terminator. When translated by cytosolic machinery, synthesis stops where Trp should have been inserted. 102
  101. 101. RECOMBINANT DNA 103
  102. 102. It is a process of combining DNA of 2 different species Also called as gene cloning & genetic engineering Recombinant DNA is a tool in understanding the structure, function, and regulation of genes and their products 104
  103. 103. Purpose of rDNA  Identification of mutations & diagnosis of affected and carrier states for hereditary diseases  Production of large quantities of a gene product (protein or RNA) for easier study of those molecules  Isolation of large quantities of pure protein Insulin, factor VIII, factor IX, growth hormone erythropoietin  Increased production efficiency for commercially made enzymes and drug  Correction of genetic defects in complex organisms, including humans. 105
  104. 104. CLONING 106
  105. 105. Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. The copied material, which has the same genetic makeup as the original, is referred to as a clone. 107
  106. 106. There are 3 different types of cloning: • Gene cloning, which creates copies of genes or segments of DNA • Reproductive cloning, which creates copies of whole animals • Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. 108
  107. 107. • Ian Wilmut with his colleagues working on a project on July 5 1996 cloned the first mammal a sheep named Dolly • Dolly took 277 tries to create, and other labs were unable to reproduce the results. 109
  108. 108. • Celebrity Sheep Has Died at Age 6 Dolly, the first mammal to be cloned from adult DNA, was put down by lethal injection Feb. 14, 2003. Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis. • Although most Finn Dorset sheep live to be 11 to 12 years of age, postmortem examination of Dolly seemed to indicate that, other than her cancer and arthritis, she appeared to be quite normal. 110
  109. 109. DISADVATAGES • Losing the diversity of genes • The great diseases and leading to extinction • Ethical issue • Process with loopholes 111
  110. 110. All the countries except for South Africa have banned reproductive cloning In these countries only therapeutic cloning is allowed 112
  111. 111. DEVELOPMENTAL GENE FAMILIES 1) 2) 3) 4) 5) Segmentation genes Paired-box genes (PAX) Zinc finger genes Signal transduction („Signalling‟) genes Homeobox genes (HOX) SEGMENTATION GENES Insect bodies consist of series of repeated body segments which differentiate into particular structures according to their position.
  112. 112. 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
  113. 113. 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 lossof-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.
  114. 114. 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 GENES The 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.
  115. 115. SIGNAL TRANSDUCTION GENES Signal 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.
  116. 116. HOMEOBOX GENES (HOX) AND ITS IMPORTANCE Since 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.
  117. 117. HUMAN GENOME PROJECT 119
  118. 118. • Begun formally in 1990, the U.S. Human Genome Project was a 13-year effort coordinated by the U.S. Department of Energy and the National Institutes of Health. 120
  119. 119. • • • • • • Project goals were to identify all the approximately 20,000-25,000 genes in human DNA determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases improve tools for data analysis transfer related technologies to the private sector address the ethical, legal, and social issues (ELSI) that may arise from the project. 121
  120. 120. . In the spring of 2000 J. Craig Venter CEO of Celera Genomics & Francis Collins Director of National Institute Of Health‟s Human Genome Research jointly announced the working draft of human genome . The project originally was planned to last 15 years, but rapid technological advances accelerated the completion date to 2003 There are 3 billion base pairs in human genome Around 25000-30000 genes 122
  121. 121. Benefits: • Molecular medicine • Energy sources and environmental applications • Risk assessment • Anthropology, evolution, and human migration • DNA forensics (identification) • Agriculture & livestock breeding 123
  122. 122. MUTATIONS Mutations are change in the base pair sequences of a particular organism Causes of Mutation • Radiation • Chemical • Age 124
  124. 124. Silent mutation Most amino acids are encoded by several different codons. For example, if the third base in the TCT codon for serine is changed to any one of the other three bases, serine will still be encoded. Such mutations are said to be silent because they cause no change in their product and cannot be detected without sequencing the gene . 126
  125. 125. Missense mutations With a missense mutation, the new nucleotide alters the codon so as to produce an altered amino acid in the protein product. Eg: sickle-cell disease The replacement of A by T at the 17th nucleotide of the gene for the beta chain of hemoglobin changes the codon GAG (for glutamic acid) to GTG (which encodes valine). 127
  126. 126. Nonsense mutation With a nonsense mutation, the new nucleotide changes a codon that specified an amino acid to one of the STOP codons (UAA, UAG, or UGA). Therefore, translation of the messenger RNA transcribed from this mutant gene will stop prematurely. The earlier in the gene that this occurs, the more truncated the protein product and the more likely that it will be unable to function. 128
  127. 127. • Frameshift/Indel Indels involving one or two base pairs (or multiples) can have devastating consequences to the gene because translation of the gene is "frameshifted". by shifting the reading frame by one nucleotide, the same sequence of nucleotides encodes a different sequence of amino acids. The mRNA is translated in new groups of three nucleotides and the protein specified by these new codons will be worthless. 129
  128. 128. TOOLS FOR MOLECULAR BIOLOGY • 1) Restriction enzymes : Genomic DNA can be cut into a number of fragments by enzymes called restriction enzymes which are obtained from bacteria. Eg. : Enzyme EcoRI. • 2) Gel electrophoresis : As DNA is negatively charged molecule, the genomic DNA that has been digested with a restriction enzyme can be separated according to size and charge by electrophoresing DNA through gel matrix. • Pulsed field gel electrophoresis.
  129. 129. • 3) Southern blotting and DNA probes : • Southern blotting allows the visualization of individual DNA fragments. • DNA probes are useful to indicate where the fragment of interest lies. • 4) Northern blotting and western blotting : • Northern blotting is used to visualize RNA fragments on to membrane. • Western blotting is used to visualize proteins.
  130. 130. Southern blotting and DNA probes.
  131. 131. • 5) Polymerase chain reaction : Minute amounts of DNA can be amplified over a million times within a few hours using this invitro technique
  132. 132. 6) DNA cloning Recombinant DNA technique, showing incorporation of foreign DNA into plasmid. Ampicillin resistant genes can be used to distinguish transformed E. coli cells
  133. 133. • 7) DNA libraries These are pools of isolated and cloned DNA sequences that form a permanent resource for further experiments. 2 types of libraries : – Genomic libraries -contains almost every sequence in the genome. – cDNA libraries - contain sequences derived from all mRNAs expressed in that tissue. • 8) DNA sequencing : Used to identify the exact nucleotide sequence of a piece of DNA.
  134. 134. Polymerase Chain Reaction is an in vitro technique for the amplification of a specific sequence of DNA Which is used for further testing.
  135. 135. Kary Mullis (1987) Cetus Corporation (A Biotech Company of United States) Nobel Prize 1993
  136. 136. Components of the reaction mixture Template DNA. Primers (forward and reverse) dNTPs Taq DNA Polymerase Buffer solution Divalent cations Sterile deionized water
  137. 137. TEMPLATE DNA It contains the DNA region to be amplified Range - 1-2 µl ( for a total reaction mixture of 10 µl)
  138. 138. Primers Short Single stranded oligonucleotides They are complementary to the 5' or 3' ends of the DNA region Range - 1 µl ( for a total reaction mixture of 10 µl) TTAACGGCCTTAA . . . TTTAAACCGGTT AATTGCCGGAATT . . . . . . . . . .> and <. . . . . . . . . . AAATTTGGCCAA TTAACGGCCTTAA . . . TTTAAACCGGTT
  139. 139. PCR Primer Design Guidelines Primer Length: Optimal length of PCR primers is 18-22 bp TTAACGGCCTTAA….. TTTAAACCGGTT AATTGCCGGAATT........>
  140. 140. Primer Melting Temperature: (Tm) Temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Range - 52-58 C Formula Tm = 4 (G+C) + 2 (A+T) (GCAT no. of respective nucleotides in the primer)
  141. 141. GC Content 40-60%. GC Clamp Presence of G or C bases within the last five bases from the 3' end of primers Promotes specific binding at the 3' end due to the stronger bonding of G and C bases
  142. 142. Steps in PCR Initialization Denaturation Annealing Extension / Elongation Final elongation Final hold
  143. 143.  INITIALIZATION STEP Heating the reaction to a temperature of 94-96°C for 1-9 minutes.  DENATURATION:   94-98°C for 20-30 seconds. Denaturation of DNA template by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
  144. 144.  ANNEALING: 50-65°C for 20-40 seconds Stable DNA-DNA hydrogen bonds are formed  The polymerase binds to the primer-template hybrid and begins DNA synthesis.  
  145. 145. EXTENSION/ELONGATION STEP   75-80°C At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template by adding dNTPs in 5' to 3' direction.
  146. 146. Final elongation  70-74°C for 5-15 minutes  To ensure that any remaining single-stranded DNA is fully extended. Final hold  4-15°C for an indefinite time  short-term storage of the reaction
  147. 147. ALLELE SPECIFIC PCR • Selective PCR amplification of the alleles to detect single nucleotide polymorphism (SNP) • Selective amplification is usually achieved by designing a primer such that the primer will match or mismatch one of the alleles at the 3‟ end of the primer.
  148. 148. ASSYMETRIC PCR • It is used for DNA sequencing • The two primers are used in the 100:1 ratio so that after 20-25 cycles of amplification one primer is exhausted thus single stranded DNA is produced in the next 5-10 cycles
  149. 149. REAL TIME PCR • Quantitative real time PCR (Q-RT PCR) • It is used to amplify and simultaneously quantify a target DNA molecule
  150. 150. REAL TIME PCR
  151. 151. HELICASE DEPENDENT AMPLIFICATION Constant temperature is used rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
  152. 152. INTERSEQUENCE SPECIFIC PCR ISSP A PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.
  153. 153. INVERSE PCR A method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences of various genomic inserts.
  154. 154. ANCHORED PCR • When sequence of only one end of the desired segment of gene is known,the primer complimentary to the 3' strand of this end is used to produce several copies of only one strand of the gene.
  155. 155. RT-PCR (REVERSE TRANSCRIPTION PCR) It is used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript. RACE-PCR Used to obtain 3' and 5' end sequence of cDNA transcripts
  156. 156. Comparison PCR - Polymerase Chain Reaction and Gene Cloning Parameter PCR Gene cloning 1. Final result Selective amplification of specific sequence Selective amplification of specific sequence 2. Manipulation In vitro In vitro and in vivo 3. Selectivity of the specific segment from complex DNA First step Last step 4. Quantity of starting material Nanogram (ng) Microgram (m) 5. Biological reagents required DNA polymerase (Taq polymerase) Restriction enzymes, Ligase, vector. bacteria 6. Automation Yes No 7. Labour intensive No Yes 8. Error probability Less More 9. Applications More Less 10. Cost Less More 11. User’s skill Not required Required 12. Time for a typical experiment Four hours Two to four days
  157. 157. APPLICATION OF PCR        Cloning a Gene encoding a known protein Amplification of old DNA Amplifying cloned DNA from Vectors Rapid Amplification of cDNA ends Detecting Bacterial or Viral Infection ● AIDS infection ●Tuberculosis (Mycobacterium tuberculosis)
  158. 158.  Genetics Diagnosis  Diagnosing inherited disorders  Cystic fibrosis  Muscular dystrophy  Haemophilia A and B  Sickle cell anaemia  Diagnosing cancer  Blood group typing.
  159. 159. Problems with PCR • Polymerase errors Polymerase lacks exonuclease activity • Size limitations PCR works readily with DNA of lengths two to three thousand basepairs • Non specific priming
  161. 161. • It is a technique for correcting defective genes that are responsible for disease development • There are four approaches: 1. A normal gene inserted to compensate for a nonfunctional gene. 2.An abnormal gene traded for a normal gene 3.An abnormal gene repaired through selective reverse mutation 4.Change the regulation of gene pairs 167
  162. 162. 168
  163. 163. • In the lab, a virus is stripped of its disease-causing genes, while the genes that enable it to infect cells are retained. • A therapeutic gene is inserted into this virus. This allows the virus to "infect" cells with the therapeutic gene. This viral vector is injected into the specific diseased tissues. • The virus attaches to the diseased cells and gets sucked into the cell by process called endocytosis. • The virus breaks apart inside the cell and the genetic material from the virus enters the nucleus of the cell. If the procedure is successful, the cell begins to produce the proteins encoded by the newly delivered therapeutic gene. 169
  164. 164. Gene of the Moment: p53 Cancer is a major focus for gene therapy research at the moment. The role of p53 is to keep a check on the cell cycle. If anything damages the DNA in a cell, p53 stops the cell cycle and tries to repair it, or if the damage is beyond repair, it induces apoptosis. If the gene for p53 has been mutated and no longer works, damaged DNA and cells can go unchecked. In the case of cancer, tumours can develop. Many cancer gene therapies focus on repairing or adding the p53 gene into the cancer cells in order to induce apoptosis 170
  165. 165. Advantages of Gene Therapy • Enabling people to have children where natural conception is impossible (a more effective treatment of infertility). • The potential for discovering cures for incurable diseases - leading to less pain and suffering. • Sex selection to prevent genetic diseases. • Increased availability of organs for transplant. • Increased procreative autonomy (choice over some of the genetic characteristics that one's future child will possess). 171
  166. 166. Disadvantages • Short Lived – Hard to integrate therapeutic DNA into genome of rapidly dividing cells which prevent gene therapy from long time action – Would have to have multiple rounds of therapy • Viral Vectors – patient could have toxic, immune, inflammatory response – also may cause disease once inside • Multigene Disorders – Heart disease, high blood pressure, Alzheimer‟s, arthritis and diabetes are hard to treat because you need to introduce more than one gene 172
  167. 167. EVOLUTION OF GENETICS IN ORTHODONTICS • Charles de Lourde, of England, who wrote in 1840 “Irregularity is due . . . to heredity, where the child inherits the jaws of one parent and the teeth of another.”
  168. 168. AJO 1957 … A STUDY OF THE FAMILY-LINE TRANSMISSION OF DENTAL OCCLUSION MILTON B. ASBELL, CAMDEN, N. J. • Heredity may be an important factor in many forms of malocclusion, a project was undertaken to determine the genetic backgrounds in a series of cases of malocclusion in white boys, aged 9 to 14 years. • RESULTS: Three types of transmission may be noted, which are as follows : (1) Repetitive trait: This type of transmission is characterized by the recurrence of a single morphologic trait within the family line over several generations. 2) Discontinuous or assortative trait: This type of transmission is characterized by the recurrence of a single morphologic trait, within the family line over several generations. This indicates a hereditary endowment arising in either the maternal side or the paternal side, plus the shift or possible gene recombination within the family line of the opposite sex. 3) Mixed trait: This type of transmission is characterized by different morphologic traits within either family line of several generations;
  169. 169. AJO FEB 1958 … A REVIEW OF THE GENETIC INFLUENCE ON MALOCCLUSION - HAROLD J. NOYES, • I am intrigued with the science of genetics and impressed with its tremendous growth in the past few years, I feel that as yet it is essentially academic with respect to the clinical practice of orthodontics and of only occasional value as a tool in the diagnosis and treatment of malocclusion. • Only in prognosis does it have a measure of value, and here one must be cautious when predicting anything that could not be forecast from comprehensive orthodontic records.
  170. 170. AJO AUG 1966 … Genetic and environmental factors in dento facial morphology FRANS ,VAN DER LINDEN • The interaction between genetic and environmental factors starts at conception and continues until the end of life. • During fetal life the contact of the genetic composition with the environment is rather limited. On the other hand, all components outside the genes (the protoplasm of the ovum, for example) are considered environmental. • RESULTS: • A permanent interaction between genetic and environmental factors, both of a continually altering nature, determines the dento facial morphology in every moment of life. • Genetic factors seem to have the greatest influence, and environmental factors appear to be of minor importance. • The stability of the result of orthodontic treatment depends mainly on a new state of balance in the interaction between genetic and environmental factors.
  171. 171. AJO MAY 1972 … Effect of molecular genetics and genetic engineering on the practice of orthodontics J. A. Salzmann • Genetic engineering has opened a new approach to the diagnosis, prevention, and control of diseases and malformations. This holds forth great promise for the future of orthodontics. • Techniques of genetic engineering include amniocentesis, chromosome karyotyping, recognition of chromosome aberrations and their relation to specific dentofacial anomalies and malocclusion, the aborting of harmful genes, and the introduction of desirable genes into the early forming embryo. • These techniques eventually will make possible the prevention of many antenatal, congenital, and postnatal genetically induced dentofacial anomalies, including dental malocclusion.
  172. 172. CONCLUSION • Malocclusion and jaw relation of genetic origin can be successfully treated orthodontically, except in extreme cases that involve the over-all morphology of the bones of the face and require surgical intervention, We modify the direction of dentofacial growth when we correct dental malocclusion and, therefore, can change or forestall abnormalities of genetic origin. • When the orthodontist corrects a malocclusion he is, in effect, changing the genetic expression of his patient.
  173. 173. AJO AUG1982… Hereditary factors in the craniofacial morphology of Angle’s Class II and Class III malocclusions • Attempted to assess the role of heredity in the development of Angle’s Class II and Class Ill malocclusions by comparing craniofacial morphologic differences between parents with Class II offspring and those with Class Ill offspring and by analyzing the parent-offspring correlatfons within each Class II and Class III malocclusion group. • RESULTS: • There appears to be a strong familial tendency in the development of Class II and Class Ill malocclusions. • We conclude that the hereditary pattern must be taken into consideration in the diagnosis and treatment of patients with these classes of malocclusion
  174. 174. AJODO MAR 1997 … A heritable component for external apical root resorption in patients treated orthodontically • External apical root resorption (EARR) is a common and occasionally critical problem in orthodontic patients. • Mechanical forces compress the periodontium, leading to localized resorption of cementum that exposes dentin to destruction by clastic activity. Factors controlling occurrence and extent of EARR are poorly understood, but there may be a familial (genetic) factor in susceptibility. • RESULTS: • Heritability estimates were fairly high, averaging 70% for three roots, although low for the mandibular incisor, probably because of little variation. No evidence was found for a sex or age difference in susceptibility. • Quantification of a transmissible component suggests it would be useful to search for the biochemical factors controlling the familial differences in susceptibility.
  175. 175. AJODO JUNE 2000… The genetics of human tooth agenesis: New discoveries for understanding dental anomalies-Heleni Vastardis • The important role of genetics has been increasingly recognized in recent years with respect to the understanding of dental anomalies, such as tooth agenesis. The lack of any real insight into the cause of this condition has led us to use a human molecular genetics approach to identify the genes perturbing normal dental development. • RESULTS: • With the use of “the family study” method, evidence is produced showing that other genetic defects also contribute to the wide range of phenotypic variability of tooth agenesis. Identification of genetic mutations in families with tooth agenesis or other dental anomalies will enable preclinical diagnosis and permit improved orthodontic treatment.
  176. 176. AJODO APR 2011… Incidence and effects of genetic factors on canine impaction in an isolated Jewish population • Introduction: The etiology of palatal canine impaction is multifactorial and includes a genetic contribution. The aim of this study was to find the incidence and effects of genetic factors on palatally impacted canines in a genetically isolated community of ultraorthodox Hassidic Jews of Ashkenazi decent • Conclusions: • Our results imply that genetics plays a signi ficant role in maxillary canine palatal impaction. • A genetically isolated Hassidic Jewish community can be a useful group to study the effects of genetic factors on various dental anom-alies, including palatally displaced canines.
  177. 177. GENE THERAPY IN ORTHODONTICS Condylar cartilage Different studies done on rats by Rabie et al have demonstrated that use of functional appliances causes transient upregulation of a number of genes like PTHrP, Ihh, Runx2, collagen typeX, VEGF 183
  178. 178. Mandibular Appliance Modulates Condylar Growth through Integrins Marques et al JDR 2008 Objective: Test the hypothesis that chondrocytes respond to forces generated by a mandibular propulsor appliance by changes in gene expression, and that integrins are important mediators in this response Result: Immunohistochemical analyses demonstrated that the use of the appliance for different periods of time modulated the expression of fibronectin, integrin subunits, as well as cell proliferation in the cartilage confirming that force itself modulates the growth of the rat condylar cartilage, and that integrins participate in mechanotransduction. 184
  179. 179. Gene therapy to enhance condylar growth using rAAVVEGF(recombinant adeno associated virus- vascular endothelial growth factor) Dai and Rabie AO 2008 Objective: To test the hypothesis that the introduction of specific vascular growth inducing genes would favorably affect mandibular condyle growth in rats over a limited experimental period Result: Enhancement of mandibular condyle growth occurred in backward and upward direction in VEGF group rather than control group 185
  180. 180. Expression of Vascular Endothelial Growth Factor and the Effects on Bone Remodeling during Experimental Tooth Movement Kohno et al JDR 2003 Aim: To investigate the effect of rhVEGF injection on the rate of tooth movement and the comparison of the numbers of osteoclasts induced by the injection of rhVEGF and rhM-CSF. Result: • The amount of tooth movement in the rhVEGF injection group was larger than that in controls. The reason may be because a large number of osteoclasts induced by rhVEGF appeared and produced a large amount of bone resorption, leading to a increase in tooth movement 186
  181. 181. Local RANKL gene transfer to the periodontal tissue accelerates orthodontic tooth movement Kanzaki.H et al Gene Therapy 2006 Aim: To test that local RANKL gene transfer into the periodontal tissue would accelerate tooth movement Result It was demonstrated that transfer of the RANKL gene to the periodontal-tissue activated osteoclastogenesis and accelerated the amount of experimental TM. Local RANKL gene transfer might be a useful tool not only for shortening orthodontic treatment, but also for moving ankylosed teeth where teeth, fuse to the surrounding bone. 187
  182. 182. Orthodontics 2047- Genetically Driven Treatment Plans  Gene therapy for sutural growth disturbances: • Mutations in FGFR2 have been linked to several human craniosynostosis disorders, including Pfeiffer, Apert, and Crouzon syndromes. • In cases of craniosynostosis involving mutations in FGFR2, temporarily blocking FGFR2 signaling in the preosteoblasts within the sutural mesenchyme or providing a different anti proliferation signal to these cells would allow normal sutural growth without surgical intervention.
  183. 183. Gene therapy for mandibular growth. • studies of rats by Hagg and colleagues have demonstrated that the use of functional appliances causes transient upregulation of a number of genes ( PTHrP, Indian hedgehog, Runx2, collagen type X, and VEGF) in the mandibular condylar cartilage • Identification of the specific genes involved in patients‟ response to functional appliances will be able to help the orthodontist predict an appliance‟s chances of success in a given individual. • The genes responsible for mandibular growth and safe methods of transducing genes into tissues, gene therapy may become the standard of care for the treatment of mandibular-deficient malocclusions
  184. 184. Gene therapy for orthodontic tooth movement. • Two elegant studies by Kanzaki and col-leagues have used gene therapy with OPG and RANKL to accelerate and inhibit orthodontic tooth movement in a rat model. • The authors concluded: “Local RANKL gene trans-fer might be a useful tool not only for shortening orthodontic treatment, but also for moving ankylosed teeth where teeth fuse to the surrounding bone”
  185. 185. Genetic counseling Genetic counseling is the process by which patients or relatives, at risk of an inherited disorder, are advised of the consequences and nature of the disorder, the probability of developing or transmitting it, and the options open to them in management and family planning in order to prevent, avoid or ameliorate it. 191
  186. 186. Aims :  Obtaining a full and careful history.  Establishing an accurate diagnosis.  Drawing a family tree is essential.  Estimating the risk of a future pregnancy being affected of carrying a disorder.  Information giving  Continued support and follow up.  Genetic screening – includes prenatal diagnosis, carrier detection. 192
  187. 187. • Patients with a great variety of diseases and syndromes are now referred for evaluation and counseling. • Genetic evaluation and counseling is a team affair & is a practical method of calculating risk figures, intended for information regarding the unborn,. • The decision taken by the parents after the counselling session must leave them satisfied instead of placing them in a state of dilemma 193
  188. 188. CONCLUSION The past 40 years have seen rapid biomedical advances leading to treatment modalities that could not have predicted decades ago. Clinically relevant discoveries in orthodontics during that period have occurred mainly in material science and appliance design. Although progress in those fields will continue to affect orthodontic profession advances in genetic testing, gene therapy, pharmacogenomics, mechanogenomics and stem cell therapy are likely to produce most dramatic changes in orthodontic treatment in next 40 years. 194
  189. 189. • Although genetic screens for various diseases currently exist, future progress in identifying the functions of genes in facial development & the mutations that affect these functions could change orthodontic practice • For eg. the analysis of genetic background of “responders” to growth modification would allow orthodontists to apply appropriate treatment methods judiciously thus reducing treatment time for average patient 195
  190. 190. REFERENCES Principles of genetics:Robert H Tamarin The Heritability of malocclusion I:BJO 1999 The heritability of malocclusion II:BJO 1999 Orthodontics in year 2047 genetically driven treatment plans:JCO 2007 • Currents concepts in biology of orthodontic tooth movement:AJO 2006 • Genetics of cleft lip and palate: syndromic genes contribute to the incidence of non-syndromic clefts:Human molecular genetics 2004 • • • • 196
  191. 191. • Gene Therapy to Enhance Condylar Growth Using rAAV-VEGF;AO 2008 • Construction of modern head current concepts in craniofacial development :JO 2000 • Expression of Vascular Endothelial Growth Factor and the Effects on Bone Remodeling during Experimental Tooth Movement:JDR 2003 • Local RANKL gene transfer to the periodontal tissue accelerates orthodontic tooth movement : Gene Therapy 2006 • McNamara, J.A. and Bryan, F.A.: Long-term mandibular adap-tations to protrusive function: An experimental study in Macaca mulatta, Am. J. Orthod. 92:98-108, 1987. 197
  192. 192. • Rabie, A.B.; Tang, G.H.; Xiong, H.; and Hägg, U.: PTHrP reg-ulates chondrocyte maturation in condylar cartilage, J. Dent. Res. 82:627-631, 2003. • Tang, G.H.; Rabie, A.B.; and Hägg, U.: Indian hedgehog: A mechanotransduction mediator in condylar cartilage, J. Dent. Res. 83:434-438, 2004. • Tang, G.H. and Rabie, A.B.: Runx2 regulates endochondral ossification in condyle during mandibular advancement, J. Dent. Res. 84:166-171, 2005. • Kanzaki, H.; Chiba, M.; Arai, K.; Takahashi, I.; Haruyama, N. Nishimura, M.; and Mitani, H.: Local RANKL gene transfer to the periodontal tissue accelerates orthodontic tooth movement, Gene Ther. 13:678-685, 2006. • Kanzaki, H.; Chiba, M.; Takahashi, I.; Haruyama, N.;Nishimura, M.; and Mitani, H.: Local OPG gene transfer to periodontal tissue inhibits orthodontic tooth movement, J. Dent. Res. 83:920-925, 2004.