Chapter 7 Gene Mutation and DNA Polymorphisms

1. Chapter 7 Gene Mutation and DNA Polymorphisms

2. A C ACG C A . . . T C GAG T C . . . A CACG C A . . .T C GGG T C . . . The Challenge after Human Genome Project Understanding the link between DNA sequence (Genotype) Environment Biology (Phenotype) 100% genetic 100% environmental

3. Genetic Variations Chromosome numbers Segmental duplications, translocations and deletions Sequence Repeats Transposable Elements Short deletions and insertions Tandem Repeats Nucleotide Insertions and Deletions (Indels) Single Nucleotide Polymorphisms (SNPs) Mutations Sizable Minor

4. Human genetic disease: a consequence of variationGenetic variation is responsible for the adaptive changes that underlie evolution Some changes improve the fitness of a species.Other changes are maladaptive.For the individual in a species, these maladaptive changes represent disease.Molecular perspective: mutation and variation

5. Why is there such a diversity of diseases? -- many regions of the genome may be affected -- there are many mechanisms of mutation -- genes and gene products interact with their molecular environments -- an individual interacts with the environment in ways that may promote disease

6. Outline of this chapter • Gene Mutation • DNA Polymorphism

7. The End

8. Gene Mutation • Definition • Major Types • Mutation Detection

9. Definition A gene mutation is a change in the nucleotide sequence that composes a gene. This is a change or variation from the most common or wildtype sequence. Somatic mutations Germline mutations

10. Somatic mutations • are mutations that occur in cells of the body excluding the germline. • Affects subsequent somatic cell descendants • Limited to impact on the individual and not transmitted to offspring

11. Germline mutations • are mutations that occur in the germline cells • Possibility of transmission to offspring

12. Somatic vs. germline mutations Mutations can occur in either somatic or germline tissue, but only germline mutations can be passed on to one’s offspring.

13. Causes of Mutation

14. Causes of Mutation

15. During an average human lifetime there are an estimated 1017 cell divisions. about 2 × 1014 divisions are required to generate the approximately 1014 cells in the adult. As each cell division requires the incorporation of 6 × 109 new nucleotides, error-free DNA replication in an average lifetime would require a DNA replication-repair process with an accuracy great enough so that the correct nucleotide was inserted on the growing DNA strands on each of about 6 × 1026 occasions.

16. Major Types • Point mutations • silent mutation • missense mutation • nonsense mutation • splicing mutation • Rearrangements • frameshift mutation • codon deletion • large deletion and insertion • deletions and duplications • trinucleotide expansion

17. Point mutation • substitution of one base with another

20. What are the consequences? • Silent mutation • Missense mutation • Nonsense mutation • Splicing mutation

23. Silent Mutation • no change in amino acid CGT - CGC

24. Missense mutation • A point mutation that exchanges one codon for another causing substitution of an amino acid • Missense mutations may affect protein function severely, mildly or not at all. CGT AGT

25. Hemoglobin • Linus Pauling, 1949 • Four globular proteins surrounding heme group with iron atom: two beta chains and two alpha chains • Function is to carry oxygen in red blood cells from lungs to body and carbon dioxide from cells to lungs

26. mutant allele wildtype allele wildtype phenotype mutant phenotype Single base change in hemoglobin gene causes sickle cell anemia

27. Nonsense mutation • A point mutation changing a codon for an amino acid into a stop codon (UAA, UAG or UGA). 5’ ATG GGA GCT CTA TTA ACC TAA 3’ met gly ala leu leu thr stop 5’ ATG GGA GCT CTA TGA ACC TAA 3’ met gly ala leu stop

28. Nonsense mutation • Premature stop codons create truncated proteins. • Truncated proteins are often nonfunctional. • Some truncations have dominant effects due to interference with normal functions.

29. Splicing Mutations • is a mutation that alters the exon-splicing pattern. • “GT/AG” rule • Disruption of existing splice sites • intron is not removed from mRNA • Creation of novel splice sites in exons

33. Insertion or deletion mutations • The genetic code is read in triplet nucleotides during translation. • Addition or subtraction of nucleotides not in multiples of three lead to a change in the reading frame used for translation. Amino acids after that point are different, a phenomenon called a frameshift. • Addition or subtraction of nucleotides in multiples of three leads to addition or subtraction of entire amino acids but not a change in the reading frame.

34. Frameshift Mutation 5’ ATG GGA GCT CTA TTA ACC TAA 3’ met gly ala leu leu thr stop 5’ ATG GGGAGCTCT ATTAAC CTA A 3’ met gly ser ser ile asn leu ….

35. Insertion or deletion of codons 5’ ATG GGA GCT CTA TTA ACC TAA 3’ met gly ala leu leu thr stop 5’ ATG GGA TTA TTA GCT CTA TTA ACC TAA 3’ met gly leu leu ala leu leu thr stop

36. Fusion gene Gg Ag yb d b/ d b Gg Ag yb d b reverse-Lepore Gg Ag yb d b Lepore Gg Ag yb d/b One gene is constructed from two segments from two different gene due to unequal crossing over between wrong pairing nonsister chromatids

42. Trinucleotide Expansion(dynamic mutation) Repeat expansions (increases in the copy number) of trinucleotide are responsible for the heritable diseases Disease-causing trinucleotide expansions have occurred: in the promoter region, in the 5’-untranslated region, in the 3’-untranslated region, in introns, in the coding parts of exons. adult onset, progressive neuro- or neuromuscular diseases.

43. Mechanism:Microsatellite instability is thought to occur by slipped strand mispairing during replication.

44. 12-MER REPEAT EXPANSION

45. Fragile X syndrome is a genetic disorder on the Xq27~28 that occurs in both males and females, but males are typically affected more severely because they only have one X chromosome. • Broad forehead; Long , rectangle face;Prominent jaw • Large, prominent ears. • Enlarged testicle after puberty (males). • Flat feet. • Flexible joints. • Mental retardation Fragile X mental retardation 1 ( FMR1) gene is responsible for this disease.

46. Expansion of CGG repeat in FMR-1 gene causes fragile X syndrome Normal: 6~50 Promutaion/premutation: 50~200 (more repeats than normal, but not enough to cause disease symptoms) Full mutation: >200

47. Triplet repeat disorders

48. Gene mutations (functional changes) Loss-of-function (usually recessive) Null– complete absence of a gene product or its function Leaky– partial absence of a gene or its function Gain-of-function (usually dominant) A mutation that confers a new function to the gene product Misregulated (usually recessive) A mutations in a promoter, enhancer, or regulatory elements of the mRNA (untranslated regions or splice site junctions)

49. Mutation Detection • Sequencing is “gold standard” • Methods to detect known mutations • Methods to detect unknown mutations

50. Detecting Known Mutations • Insertion or deletion large fragments – by Southern small fragments – by PCR • Point mutation Restriction site altered by mutation • RFLP or PCR/restriction enzyme digestion No restriction site altered by mutation • Allele specific oligonucleotide (ASO) probe