Chapter 11

1. Chapter 11 Gene Mutation

2. Mutation • A 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. • A mutant allele is an allele that differs from the common allele in the population (also called the wildtype allele). • A mutant phenotype refers to a phenotype that differs from the common or wildtype phenotype. • Mutations are not good or bad, just different from the majority in the population.

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

4. 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

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

6. Hemoglobin genotype causes sickle cell anemia phenotype • Sickle cell anemia was the first illness understood at the molecular level: • mutation encodes valine in place of glutamic acid. • Phenotype associated with homozygotes: • Altered surface of hemoglobin allows molecules to link in low oxygen conditions and creates sickle shape of red blood cells. • Sickling of red blood cells causes anemia, joint pain, and organ damage when RBC become lodged in small blood vessels.

7. Different sites in a gene can mutate and cause distinct phenotypes • Some beta hemoglobin mutations resulting in too few protein molecules cause thalessemia. • Excess of alpha hemoglobin compared to beta hemoglobin leads to iron release which kills RBC and destroys heart, liver and endocrine glands. • heterozygous mutation => milder thalassemia minor • homozygous mutation => more severe thalassemia major

8. Collagen • Comprises: • 60% of protein in bone and cartilage • a significant proportion of skin, ligament, tendon, tooth dentin and connective tissue. • Has a precise structure: • triple helix of two alpha1 and one alpha2 proteins • the longer precursor called procollagen is trimmed to form collagen

9. Different collagen mutations => Distinct disorders

10. Alzheimer disease • Mutations in presenilin1 cause early onset autosomal dominant Alzheimer disease • Presenilin protein is a receptor anchored in the Golgi membrane • Monitors beta amyloid usage • 30+ missense mutations in presenilin result in beta amyloid accumulation.

11. Genotype to disease phenotype • Cystic fibrosis diseaseCFTR protein • Duchenne muscular dystrophy dystrophin protein • Familial hypercholesterolemia LDL receptor protein • Hemophilia A Factor VIII protein • Huntington disease huntingtin protein Mutation: Many different mutations, common missing amino acid Mutation: Deletion of gene Mutation: Deficient LDL receptors lead to cholesterol buildup Mutation: Absent or deficient factor Mutation: Extra nucleotides in gene result in extra amino acids Phenotype: Lung infections, pancreatic insufficiency Phenotype: Loss of muscle function Phenotype: High blood cholesterol, early heart disease Phenotype: Slow or absent blood clotting Phenotype: Uncontrollable movements, personality changes

12. Spontaneous mutation • De novo or new mutations • Not caused by exposure to known mutagen • Errors in DNA replication • DNA bases have slight chemical instability • (exists in alternating forms called tautomers)

13. Spontaneous mutation rate • Rate differs for different genes • Size dependence • Sequence dependence • Hot spots • On average 1 in 100,000 chance of acquiring a mutation in a gene each round of replication. • Each individual has multiple new mutations. Most by chance are not in coding regions of genes.

14. Determining mutation rate • Estimates of spontaneous mutation rate can be derived from observation of dominant traits. • For autosomal genes, • mutation rate= number of cases • 2 ( # of individuals)

15. Mutation rates of genes causing disease

16. Mutations in pathogens • Bacteria and viruses undergo mutation • Mutation in bacteria can lead to antibiotic resistance. • Overuse and incomplete course of treatment increases chances of antibiotic resistance arising. • Viruses mutate rapidly. • Influenza vaccines are reassessed each season to accommodate viral changes. • Rapid mutation of HIV virus makes treatment difficult.

17. Mutational hot spots exist • Short repetitive sequences • pairing of repeats may interfere with replication or repair enzymes • Palindromes • often associated with insertions or deletions • Duplications of larger regions • mispairing during meiosis

18. Small or large insertion or deletions Palindromes can cause small insertion or deletions Duplications can cause large insertion or deletions

19. Induced mutations • Chemicals and radiation can cause mutations. • Chemicals causing mutations are called mutagens. • Chemicals causing cancer are called carcinogens. • Alkylating agents remove a base • Acridine dyes add or remove base • Xrays break chromosomes • delete few nucleotides • UV radiation creates thymidine dimers

20. Ames test • is an in vitro test of the mutagenicity of a substance using Salmonella bacteria with mutation in gene for histidine. • Bacteria are exposed to test substance. • Growth of bacteria on media without histidine is recorded. • Bacteria only grow if mutations have occurred. • Rate of mutation is determined. • Substance can be mixed with mammalian liver tissue prior to testing to mimic toxin processing in humans.

22. Normal THE ONE BIG FLY HAD ONE RED EYE Missense THQ ONE BIG FLY HAD ONE RED EYE Nonsense THE ONE BIG Frameshift THE ONE QBI GFL YHA DON ERE DEY Deletion THE ONE BIG HAD ONE RED EYE Insertion THE ONE BIG WET FLY HAD ONE RED EYE Duplication THE ONE BIG FLY FLY HAD ONE RED EYE generation 1 THE ONE BIG FLY HAD ONE RED EYE generation 2 THE ONE BIG FLY FLY HAD ONE RED EYE generation 3 THE ONE BIG FLY FLY FLY HAD ONE RED EYE Type of mutation Example Expanding

23. Point mutation • A point mutation is a change of a single nucleotide to one of the other three possible nucleotides • Transition • purine replaces purine • A -> G or G -> A • pyrimidine replaces pyrimidine • C -> T or T -> C • Transversion • purine replaces pyrimidine or • pyrimidine replaces purine • A or G -> T or C • T or C -> A or G

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. • Hemoglobin mutation • glutamic acid -> valine causes sickle cell anemia

25. Nonsense mutation • A point mutation changing a codon for an amino acid into a stop codon (UAA, UAG or UGA). • Premature stop codons create truncated proteins. • Truncated proteins are often nonfunctional. • Some truncations have dominant effects due to interference with normal functions. • Most common cause of factor XI deficiency is a nonsense mutation change glutamic acid to a stop. Short protein cannot function in clotting.

26. 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.

27. Insertion or deletion mutations • Deletion is the removal of sequences. • Two-thirds of Duchenne musular dystrophy cases are large deletions. • Insertion is the addition of sequences. • Gaucher disease is caused by a single base insertion creating a frameshift. • A tandem duplication is a particular form of insertion in which identical sequences are found side by side. • Charcot-Marie-Tooth disease is caused by a tandem duplication of 1.5 million bases

28. Expanding repeats • Insertion of triplet repeats leads to extra amino acids. • Some genes are particularly prone to expansion of repeats. • Number of repeats correlates with earlier onset and more severe phenotype. • Expansion of the triplet repeat and coincident increase in severity of phenotype occur with subsequent generations, a phenomena termed anticipation.

29. Myotonic dystrophy: a triplet repeat disease • 5 -37 copies of CTG repeat normal phenotype • 50-1000 repeats myotonic dystrophy • Genes with 40+ copies are unstable and can gain (or less commonly lose) repeat copies in successive generations.

30. Triplet repeat disorders

31. Noncoding repeats can cause phenotypes In myotonic dystrophy the expanded repeat is not in the exon. The expansion may affect the exportation of the mRNA from the nucleus.

32. Pseudogenes • A pseudogene is a DNA sequence reminiscent of a gene but which is not translated (may or may not be transcribed). • Pseudogenes may have evolved from original functional gene by duplication and acquired mutation. • Crossing over between a pseudogene and a bona fide gene can disrupt gene expression.

33. Different mutations may cause the same disorder Mutations in the LDL receptor disrupt function leading to increased blood cholesterol and early heart disease.

34. Mutations in different parts of a gene may have distinct impacts

35. Prion disorders • Prion disorders are caused by mutation in the prion gene which leads to an abnormally shaped prion protein. • The mutant form of the protein can convert normal prion proteins to mutant protein shapes. • Mutant protein occurs in two ways: • A mutation in the gene can be inherited. • The mutant protein can be transmitted like an infection from tissue with the mutant protein. • In cows a mutant prion protein causes mad cow disease. • Humans can obtain the protein by eating beef with mutant prions and develop Creutzfeldt-Jakob disease.

36. Prions change shape Neuron Nucleus Cytoplasm

37. Prions change shape Intracellular membrane Abnormal PrP Neuron Normal PrP Nucleus Cytoplasm

38. Prions change shape Intracellular membrane Neuron Converted molecule Normal PrP Nucleus Cytoplasm

39. Prions change shape Intracellular membrane Neuron Nucleus Cytoplasm

40. Prions change shape Intracellular membrane Neuron Nucleus Cytoplasm

41. Prions change shape Intracellular membrane Neuron Nucleus Cytoplasm

42. Prions change shape Intracellular membrane Neuron Nucleus Cytoplasm

43. Prions change shape Intracellular membrane Neuron Nucleus Cytoplasm

44. Prions change shape Intracellular membrane Neuron All abnormal PrP Nucleus Cytoplasm

45. Not all mutations impact protein function • Silent mutations are mutations that do not alter the • amino acid encoded. AAA and AAG both encode the amino acid lysine. A mutation from AAA to AAG in a gene alters the DNA sequence but protein sequence remains unchanged. These codons are called synonymous codons.

46. Not all mutations impact protein function • Missense mutations are those that alter the encoded amino acid to another amino acid. • The alteration creates a nonsynonymous codon. Some nonsynonymous mutations are conservative; chemically similar amino acid may not alter function The impact of a missense mutation is not predictable from protein sequence alone.

47. Not all mutations impact protein function • Conditional mutations are those that only produce a phenotype under particular conditions or environments. G6PD enzyme is used to respond to oxidants, chemicals that strip electrons from other molecules. High levels of oxidants occur when eating fava beans or taking antimalarial drugs. Conditions Individuals with mutations in G6PD Low oxidants no phenotype High oxidants red blood cells burst, anemia

48. DNA Repair • Errors in DNA replication or damage to DNA create mutations. • Most errors and damage are repaired by the cell. • The manner in which DNA repair occurs depends upon the type of damage or error. • Different organisms vary in their ability to repair DNA. • In humans, mutations in DNA replication occur in 1 in 100 million bases.

49. Mismatch repair • Mismatch repair occurs when enzymes detect nucleotides that do not base pair in newly replicated DNA. • The incorrect base is excised and replaced. • The detection of mismatches is termed proofreading.

50. Excision repair • Damaged DNA is removed by excision of the bases and replacement by a DNA polymerase. • Nucleotide excision repair • Replaces up to 30 bases • used in repair of UVB and some carcinogens • Base excision repair • Replaces 1-5 bases • Repairs oxidative damage