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M1 – Genetics Origin and Nature of Genetic Variation Dr. Pandya 10/3/2008 1 2 3 4 OBJECTIVES 1. Origin of genetic variation at protein and DNA level. 2. Concept of mutation/polymorphism. 3. Molecular basis for different types of mutation. 4. Methods used to detect genetic variation/mutations. II. CLASSIFICATION I. MUTATION: Ultimate source of genetic variation. DEFINITION: Any permanent change in the genetic material. FREQUENCY: Mutation rate (μ) is the frequency of such change and is usually expressed as the number of mutations/locus/gamete/generation. 1. Mutations associated with base pair changes in single genes (e.g., insertions, deletion, substitution) 2. Others: a. Chromosomal rearrangements which may include several genes (deletion, duplication, translocation, etc.) b. Chromosomal mis-segregation resulting in aneuploidy involving the entire chromosome e.g. trisomy 21. These are the most common human mutations. 5 6 III. FREQUENCY IV. TYPES OF MUTATIONS 1. Gene mutations: 10-6 – 10-8 /locus/generation. 1. Germline Mutations: • These can be transmitted • Occur during spermatogenesis/oogenesis • Implication for counseling of recurrence risk. 2. Chromosomal: 10-2 – 10-4/cell division ORIGIN 1. Errors occurring during DNA replication. 2. Somatic Mutations: 2. Changes induced by mutagens e.g. radiation, chemicals. • Antibody diversity • Relevance to cancer 7 8 MOLECULAR BASIS OF MUTATIONS SIGNIFICANCE The characterization of specific mutations permits screening for genetic disorders in families at risk as well as in the general population for certain diseases. 1. EVOLUTION – Mutation is the ultimate source of genetic variation that is required for evolutionary change. (Each zygote has approximately 100 new base pair combinations not present in the genome of either parent.) A. Nucleotide substitutions (point mutations) in a DNA sequence can have a variable effect depending on the location and nature of the mutation. 2. HEALTH RISK – Most recognized mutations are harmful (teratogenesis, carcinogenesis), but some may be advantageous. Mutations can be viewed as the price a species must pay for the privilege of evolving. B. Deletions and Insertions of one or more base pairs, or involving a substantial segment of a gene or an entire gene. 9 MISSENSE SUBSTITUTIONS 10 NONSENSE MUTATIONS • A single base pair substitution • Amino acid substitutions due to a single base pair change • Introduction of a premature “stop” codon • e.g. Sickle cell disease which is caused by single nucleotide change from AÜT in the beta globin gene. • A form of beta thalassemia is caused by a premature “stop” codon at position 39. GAG to GTG • This mutation causes a hereditary anemia common in patients of Mediterranean (especially Sardinian) origin. • Results in substitution of glutamic acid to valine at the sixth position of the polypeptide denoted as E6V codon 39 gln Ü stop CAG Ü UAG • Not all mis-sense mutations are deleterious. 11 12 RNA SPLICING MUTATIONS Splice Site mutation • A single bp change involving intron /exon splice sites or cryptic sites. • Beta-Thalassemia syndromes have mutations that alter the normal splice acceptor or donor sites • Activate cryptic splice sites that compete with the correct site. • A splicing mutation can also occur secondary to deletion or insertion of one or more than one base pair. 13 “Synonymous” Substitutions 14 Substitutions in the non coding sequence. Ordinarily, base pair substitution within intron or outside the 5' or 3' end of the gene would be expected to have no effect unless they alter splice site, a regulatory sequence or mRNA processing site. Some base pair substitutions may change the codon for an amino acid but do not alter the polypeptide sequence. AGG to AGA (arginine) Such a mutation can have phenotypic effects by activating cryptic splice sites that compete with the correct site. 15 16 Frameshift mutations • Number of bases involved is not a multiple of 3 •The resulting protein could either be shorter or longer than normal MUTATIONS INVOLVING MORE THAN A SINGLE BASE •e.g., beta thalassemia, Hb Tak, Tay Sachs disease. 17 18 Gene Duplications & Deletions Codon deletions and insertions Hb Lepore: Some patients with moderate to severe β-thalassemia have an unusual product with a δβ fusion gene which occurs by a process of non- homologous / unequal crossing over. • Number of bases involved is a multiple of 3 • e.g. Cystic Fibrosis. • A 3-base pair deletion at amino acid position 508, deletes the codon for phenylalanine • The ΔF508 mutation accounts for 70% of all mutant CF alleles. • This mutations results in synthesis of an abnormal gene CFTR gene product 19 The alpha thalassemias are a group of hereditary anemias which can be due to deletion of one or more tandemly duplicated -globin loci on chromosome16 20 UNSTABLE PREMUTATIONS • AKA as unstable dynamic mutations • A class of trinucleotide repeats • Variation within a restricted range is found in the general population as a normal polymorphism. • When the number of repeats exceeds a threshold the gene may become unstable & exhibit phenotypic effects CCG CCG CCG… x 4-50 repeats CAG CAG CAG… x 2-12 repeats 21 22 POLYMORPHISM EXAMPLES OF POLYMORPHISM DEFINITION: Genetic polymorphism is defined as the occurrence of multiple alleles at a locus in the population where no allele occurs with a frequency of greater than 0.99. 1. Phenotype: e.g. eye color 1. Average frequency of loci heterozygous for alleles which determine structurally different polypeptides is 12% - 18%. 2. Protein polymorphisms 3. DNA polymorphism 2. Average frequency of base pairs that are heterozygous in a diploid individual is 1 in 270. 23 24 BLOOD GROUP POLYMORPHISM PROTEIN POLYMORPHISM a. Blood groups e.g. ABO, Rh and MNS blood group systems b. Galactosemia locus • Red blood cell antigens are useful genetic markers in family population studies and in linkage analysis. • These are the first example of genetically determined protein variation. • Relevance in clinical medicine. c. Alpha, antitrypsin locus d. HLA system (Human Leukocyte Antigen) 25 26 GENETICS OF ABO AND RH SYSTEMS GENE PRODUCT ANTIGEN GENE PRODUCT ANTIGEN H and A A-transferase H-transferase PRECURSOR H B-transferase O No transferase GENOTYPE PHENOTYPE • ABO blood groups determined by a locus on chromosome 9. AA OA A AB AB • A, B, and O alleles / examples of multiallelism. BB B • The three alleles determine four phenotypes; A, B, AB and O. • Molecular differences in the glycosyltransferase gene responsible for the A, B and O alleles: H and B OB Unchanged H OO O Diagram of the pathways of biosynthesis Of the H, A, and B antigens. Alleles h and O have no detectable effect. - four nucleotide sequence difference between A and B alleles. Pathways of Biosynthesis of The H, A, and B Antigens - O allele has a single base pair deletion in the ABO gene coding region i.e. frame shift mutation. 27 • • Discovery of Rh system and its role in hemolytic disease of the newborn is 28 a major contribution of genetics to medicine. DNA POLYMORPHISMS a. Base pair substitution (1) RFLP (2) others (detected by denaturing gel electrophoresis) b. Variable number tandem repeats (VNTR) c. Minisatellites/microsatellites (di- tri- and tetranucleotide repeats) d. Single nucleotide polymorphism (SNP) 29 30 Comparison between the two categories of markers VNTR MICROSATELLITE Each repeat unit 20 to several hundred bp in length Each repeat units 2,3,4,5 bp in length No. of repeats range from 2 to 20 No. of repeats variable Detection by Southern analysis due to large size Detection by polymerase chain reaction due to small size Uneven distribution; clustered near chromosome ends Even distribution in the genome Less polymorphic More polymorphic 31 32 Applications of Polymorphisms Single Nucleotide Polymorphism • A variation of a single nucleotide in the genome • Frequency of at least 1% in the population • Occur every 1000 bp • Do not alter a restriction site unlike RFLP • Bi-allelic, but compensated by the numbers • Dense SNP maps of genome available 33 35 • • • • Mapping genes by linkage analysis Forensic applications i.e. paternity testing Organ transplantation for HLA matching Hapmap allows association studies for complex disorders • Pharmacogenetics 34