Genetic Variation in Individuals and Populations: Mutation and Polymorphism Chapter 9 Thompson and Thompson (only mutation) Dr. M. Fardaei 1

Outlines: Mutation Mechanisms responsible for mutation Types of Mutation in Human Genetic Disease Silent, Missense, Nonsense, RNA Splicing Mutations Mutation Nomenclature 2

Translation Protein Regulatory region DNA: Exon3Exon2 Exon1 Intron1 Intron2 Protein Modification Transcriptin Pre-mRNA: Exon3Exon2Exon1 Intron1 Intron2 Processing Exon3Exon2Exon1 mRNA: GU…..A...AG AUG UAA, UAG, UGA ORF 5’-UTR3’-UTR 3

Regulatory region DNA: Exon3Exon2Exon1 Intron1 Intron2 Transcriptin Pre-mRNA: Exon3Exon2Exon1 Intron1 Intron2 Splicing Donor sites (SD) Splicing Acceptor sites (SA) 4

Types of Mutation and Their Estimated Frequencies Class of Mutation Mechanism Frequency Examples Genome mutation Chromosome missegregation /cell division Aneuploidy Chromosome mutation Chromosome rearrangement 6*10 -4 /cell division Translocations Gene mutation Base-pair mutation base pair/cell division Point mutation 5

Genome mutations: - Arising from errors in chromosome segregation during meiosis or mitosis. - Very common in cancer cells - Involving only a part of a chromosome, such as: duplications, deletions, inversions, and translocations Chromosome mutations: Gene mutations: Mutations that alter individual genes. - Germ line mutation: heritable - Somatic mutation: only certain tissues, cannot be transmitted. 6

Gene mutations, including: Base-pair substitution, Insersion Deletion 5’…ACTG…3’5’…AGCTG…3’ 5’…ACTG…3’5’…AATG…3’ 5’…ACTG…3’5’…ATG…3’ 7

Genomes are dynamic entities that change over time as a result of : Mutation (small-scale sequence alterations) and Recombination (larger scale rearrangements) Terminology for describing point mutations: Transition: Substitution of one purine for the other (A G, G A) or one pyrimidine for the other (T C, C T). Transversion: The replacement of a purine for a pyrimidine (or vice versa). C A, A C, G C,A T, C G, G T, T A,T G, 8

Specifically when they are located immediately 5' to a guanine (5'-CG-3'). Hot Spot of Mutation The major form of DNA modification in the human: Methylation Cytosine NH2 5-methylcytosine Deamination Thymidine 9

Result: The CG doublet gives rise to C T or G A transitions...TG…...AC… Replication...CG…...GC…...TG…...GC…...CG…...GC…...CG…...GC… CH3 MethylationDeamination Hot Spot of Mutation The major form of DNA modification in the human: 10

Mechanisms responsible for mutation: - Errors introduced during DNA replication - Failure to repair DNA damage - Spontaneous chemical processes such as depurination, demethylation, or deamination - Reaction with chemical mutagens, exposure to UV or ionizing radiation. 11

Spontaneous chemical processes- Depurination per cell in a day. 12

13 Ethidium bromide: Ethidium bromide and other intercalating agents are flat molecules that can slip between base pairs in the double helix, and increasing the distance between adjacent base pairs. Usually associated with insertion mutations Reaction with chemical mutagens analogs Example: Ethidium bromide and Base analogs

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15 Base analogs 5-Bromo U (homo. of T) Keto Enol A G 5-bromouracil versus thymine during replication

 UV radiation of 260 nm induces dimerization of adjacent pyrimidine bases, especially if these are both thymines, resulting in a cyclobutyl dimer.cyclobutyl dimer UV radiation 16

Purine dimers are much less common. UV-induced dimerization usually results in a deletion mutation when the modified strand is copied. UV radiation 17

Types of Mutation in Human Genetic Disease Nucleotide Substitutions (Point Mutations) Deletion and Insertions 18

Types of Mutation in Human Genetic Disease Nucleotide Substitutions (Point Mutations), Silent mutations Missense mutations Nonsense mutations 19

Deletion and Insertions Addition or deletion of small number of bases If number of bases involved is not a multiple of 3, causes frameshift If number of bases involved is a multiple of 3, causes loss or gain of codon… Larger gene deletions and duplications (mediated by DNA sequence homology either within or between DNA strands) Insertions of L1 or Alu element (disrupt transcription or interrupts the coding sequence) Types of Mutation in Human Genetic Disease 20

Gene A Deletion Involving Recombination Between Repeated Sequences Deletion: Gene A Unequal crossing over Duplication: 21

22 Expansion of nucleotide repeat sequences CAG CAG CAG CAG CAG 5’ 3’ CA CA CA CA CA CA CA 5’ 3’ Microsatellite: (1-4) n 22

Deletion and Insertions Expansion of trinucleotide repeat sequences Types of Mutation in Human Genetic Disease 23

24 Expansion of trinucleotide repeat sequences 24

Types of Mutation in Human Genetic Disease Nucleotide Substitutions (Point Mutations) Deletion and Insertion Regulatory mutations affecting transcription factor binding, transcriptional control, or other aspects of gene expression. RNA processing mutations: Destroy consensus splice sites, Cap sites, Polyadenylation sites Abnormal splicing often leads to frameshift mutations and premature stop codons. 25

RNA Splicing Mutations Two general classes of splicing mutation have been described. Mutations that affect the required bases at either the splice donor or acceptor site interfere with normal RNA splicing at that site. Types of Mutation in Human Genetic Disease A second class of splicing mutations involves intron base substitutions, such mutations create alternative donor or acceptor sites that compete with the normal sites during RNA processing. 26

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Mutation Nomenclature 1. The position: genomic DNA or in a cDNA sequence by the prefix g. or c., respectively. 2. The A of ATG is designated +1. The next base upstream is -1 (there is no 0). 3. First the original base then the nucleotide number of that base and then > and then the new nucleotide at that position. Example, c.G1444>A 4. In an intron (IVS, intervening sequence) (GT…..AG) +1, +2, …..-2, -1, Example: g.IVS33+2T>A, g.IVS33-2A>T 29