1. MUTATION AND POLYMORFISM Genetics and genomics for ED students 20.02.2015.

2. Genetic variability • is increased by – mutation – sexual reproduction meiosis (generation of gametes) - homologous recombination (crossing over) - independent assortment of homologous chromosomes fertilisation - significance

3. DNA Mutationcausing any change) DNA variants, alleles (any coding or non-coding sequence) Genetic (DNA) polymorphism

4. Significance of mutation(for all species) Without mutation, evolution would not be possible. This is because mutations provide the "raw material" upon which the mechanisms of natural selection can act.

5. Regarding the variants…. • normal or wild variant (allele) is the most frequent in a • population • polymorphism (or polymorphic) is the variant (allele) if its • frequency is › 1 % in the population • (formerly:having no effect on phenotype) • mutation (or mutant) is the variant (allele) if its • frequency is ‹ 1 %in the population • (formerly: disease causing, it has a • negative connotation)

6. Long way from mutation to polymorphism Appearance of new variant by mutation Survival of rare allele Increase in allele frequency after population expand New allele is fixed in population as novel polymorphism

7. Classification of mutation types • by the cause • by the site • by the function • by the fitness • by the size

8. By the cause mutations may be • Spontaneous • Induced

10. Spontaneous chemical reactions in bases • Tautomerization • Depurination • Deamination • Errors in DNA related processes • Replication • Recombination • Repair Spontaneous mutation

11. E.g. Tautomers of adenine Frequent rare Amino group Imino group T - A (Purine-Purine) results

12. Depurination (hydrolysis)

13. Deamination Mutation hot spot Repaired Not repaired DNA methylation (regulation of DNA functions, see epigenetics)

14. Induced mutation Some environmental agent = mutagen • physical - radiation • Heat • UV • Ionizing • chemicals • Natural toxins • Synthetic substances • Laboratory substances • Pollutants • Chemoterapeutics

15. Natural substances Psoralen Aflatoxin Aspergillus sp.

16. Laboratory chemicals: acridine orange, ethidium bromide, propidium jodide Fluorescent dyes BrdU - thymine analogue A senescent endothelial cell stained with the fluorescent dye acridine orange to visualise the lysosomes. Acrylamide Polyacrylamide

17. Pollutants E.g. benzpyrene Metabolized to epoxides in liver polyaromatic hydrocarbons (PAH) DNA adduct Mutation

18. Biological warfare agent Mustard gase Iranian victim (end of 20th century) I. World war victim (beginning of 20th century)

19. Correction of DNA errors DNA polymerase with proofreading ability Repair mechanisms nuclear but not mitochondrial DNA

20. DNA repair mechanisms Direct repair the change is reversed no template is needed mainly in prokaryotes • Excision repair template is needed in eukaryotes

21. Repair of single strand damage(complementer strand is used as template)

22. Xeroderma pigmentosum is caused by the defective nucleotide excision repair enzymes

23. Repair of double strand breaks (DSB) may result loss of nucleotides = deleterious Sister chromatid (after S phase) or like in meiosis, homologous chromosome is used as template = safety

24. Multicellular cell cycle M-phase cytokinesis mitosis M G1 G2 Go G2 Restriction point - Growth factors - anchorange Checkpoints: Restriction point G2 M (spindle) S Interphase

25. Function and activity of checkpoint machinery G1 G2 M DNA damage free kinetochors not complete DNA replication sensor protein kinases transducer effector s t o p of c e l l c y c l e repair

26. Ataxia telangiectasia (ATM=sensor) Its mutation causes rare, neurodegenerative, inherited disease (AR), that affects many parts of the body and causes severe disability, characterized by radiosensitivity and different tumors.

27. Role of BRCA (transducer) proteins in DNA repair BRCA mutations are found in breast and ovarian tumors.

28. p53

29. Site of mutations - in the organism Somatic-in somatic cells localized- inherited within cells of an organism (mosaicism: tumors, antibody diversity, etc.) higher in dividing cells Generative- in primordial germ line inherited from one generation to the next one

30. And nondisjunction of sex chromosomes

31. Site of mutations - in a gene may be 1 2 UTR UTR 3 4 1/ Promoter mutations  decreased transcription 2/ Exon mutations  amino acid change or truncated protein (stop) see later 3/ Intron mutations  errors in splicing 4/ Polyadenylation site mutations  decreased mRNA stability 5 5 UTR  disturbed ribosome binding Mutations of other regulatory sequences (enhancers, silencers) also may influence transcription. B.R. Korf: Human Genetics and Genomics,2006

32. Splicing mutations B.R. Korf: Human Genetics and Genomics,2006

33. Splicing mutations B.R. Korf: Human Genetics and Genomics,2006

34. Different mutations of a gene may lead to different malfunctions of the protein(=CFTR) Most frequent

35. Function and mutations Back mutation or reversion is a point mutation that restores the original sequence and hence the original phenotype. Lethal mutations are mutations that lead to the death of the organisms which carry the mutations. Gain-of-function mutations- change the gene product such that it gains a new and abnormal function. These mutations usually have dominant phenotypes. Loss-of-function mutations- gene product having less or no function. Phenotypes associated with such mutations are most often recessive. Exception is when the reduced dosageof a normal gene product is not enough for a normal phenotye (this is calledhaploinsufficiency). Dominant negative mutations- the altered gene product acts antagonistically to the wild-type allele. These mutations are characterised by a dominant phenotype. In humans, dominant negative mutations have been implicated in cancer (e.g. mutations in genes p53,ATM).

36. Fitness and mutations • Most are neutral – during evolution later may be harmful • or beneficial • Some are beneficial – • - harmful one mutates back to wild • - getting beneficial function • – diversity of antibody • - CCR532 – HIV resistency • - sickle cell anemia – malaria • resistency • Some are harmful – causing diseases (all monogenic • inherited diseases)

37. Size of mutations Large Genomemutation= change of chromosome number Medium Chromosome mutations = change of chromosome structure Small gene mutations = ranging from a change of single nucleotide to a whole gene (not visible) Affecting the lenght of DNA Deletion (single base or shorter-longer sequences) Insertion (single base or shorter-longer sequences- repetitive more insertion than deletion No effect on the length of DNA nucleotide substitution Cytogenetics

38. Repetitive insertions • Tandem repeats • Satellite DNA • pericentromeric heterochromatin • Minisatellite (VNTR) • 10-60 bp • Telomere • Microsatellite (STR=short tandem repeats) • 2- some bp • good markers of kinship • Repeat number expansion diseases • Interspersed repeats: • SINEs (Short Interspersed Elements), • LINEs (Long …) e. g. L1

39. Microsatellite (STR = short tandem repeats) 1-4 bp Trinucleotide (triplet) repeats are very frequent only few of them cause disease

40. Trinucleotide repeats may be either in coding(C) or noncoding (NC) region NC C NC C (huntingtin) (Huntingtin) C coding NC noncoding

41. Polyglutamine Polyalanine disorders disorders • CAG repeats • Neurodegenerative disorders • Different proteins • Gain of function mutations • Variable length • Expansion • Replicational slippage

42. Replication slippage

43. Huntingtin MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQPPPP PPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLHRPK KELSATKKDRVNHCLTICENIVAQSVRNSPEFQKLLGIAHELFLLCSDD... • 350 kD protein • ubiquitously expressed • function unknown • correlation between repeat • size and age of onset and • the severity of disease (Huntington chorea) Huntington healthy

44. Polyglutamine Polyalanine disorders disorders • CAG repeats • Neurodegenerative disorders • Different proteins • Gain of function mutations • Variable length • Expansion • Replicational slippage • GCX repeats • Developmental abnormalities • Transcription factors • Loss of function mutations • Constant length • Stable • Uneven crossing over

45. Uneven crossing over

46. Uneven sister chromatid exchange

47. Polyalanine disorder Disorder Gene • Holoprosencephaly ZIC2

48. Deletion or insertion of a single nucleotide (InDel) It is a frameshift mutation if number of nucleotide is not a multiple of three, and in-frame if number of nucleotide is a multiple of three

49. DNA mRNA protein Mutant protein

50. Medium InDel mutations Deletion Pl. Hypodontia (Deletion of Pax 9) Insertion (retro)transposons Eg. L1 hemophilia A L1 is a LINE: Long Interspersed Elements