Mutasions

1. Mutasions By: Mahdi Bijanzadeh MD, PhD. bijanzadeh-m@ajums.ac.ir

2. Mutations At the end of this session, you should answer this questions: 1. What is mutation? 2. How many types of mutation are there? 3. How many types of chromosome disorder are there? 4. What is non-synonymous mutation? 5. What is repair mechanism of DNA polymerase?

3. Mutation • Definition: Heritable alteration or change in nucleotide sequence or genetic material. • Polymorphism: 1%, sometime without phenotype affects • 99.9% similarity in Human nuclear DNA sequencing: • This 0.1% is responsible for spectrum of phenotype differences in Human being: evolution, non-pathogens (more common) to pathogen: • Anatomy, physiology, food intolerance, drug reaction, drug side effects, infection susceptibility, cancer susceptibility, even personal disorders, sport tendency & art taste.

4. Mutation Types: • 1. Genome: in chromosome number (e.g. aneuploidy). • 2. Chromosome or Macro: in chromosome structure. • 3. Gene: in single gene. They can also classified as: • 1. Somatic: no transfer to next generation, in a lot of cancers. Accidently in some cells → somatic mosaicism. • 2. Gonadal: can transfer to next generation, unless it affects fertility or survival into adulthood. Estimated that each individual carries up to 6 lethal or semilethal recessive mutant alleles that in homozygous state would have very serious effects.

5. Mutation Etiology: • 1. Exposure to mutagenic agents (mutagens). • 2. Spontaneously: errors in DNA replication and repair.

6. Genome mutations • The most common in Human. • 1/25-50 cell meiosis divisions, excepted in killer abortions. • Can change no. & presentation of hundreds to thousands cells. • Usually incompatible with survival & normal reproduction: doesn’t transmit to next generation.

7. Chromosome mutations • Only in a part of chromosome. • in 1/1700 cell divisions • Spontaneously or due to chromosome abnormal meiotic disjunction. • Due to duplication, partial triplication, deletion, inversion & translocation. • Can change no. & presentation of hundreds cells. • Similar to genome mutations usually incompatible with survival & normal reproduction: doesn’t transmit to next generation.

8. Gene mutations • In nuclear or mitochondrial DNA. • A nucleotide to millions nucleotides. • For more than thousand of Human genetic diseases. • Dominant or recessive autosomal or X-linked disorders.

9. Types of chromosome abnormalities 1. Numerical abnormalities (genome mutations): Heteroploid: Euploid or Polyploidy (n): Triploid, Tetraploid Aneuploid: most common, 5% pregnancies Trisomy: 21( Down Syndrome) 18, 13: less no. of genes Monosomy: almost lethal, except: X 2. Structural abnormalities (chromosome mutations). 3. Different cell lines (genome mutations).

10. Genome mutations • Euploid or Polyploid:contain multiples of haploid number of chromosomes such as 69, triploidy, or 92, tetraploidy. • In humans, often cause spontaneous miscarriages, but survival beyond midpregnancy is rare. Only a few triploid live births have been described and all died soon after birth.

11. Genome mutations • Aneuploid • Trisomy: Presence of an extra chromosome. - Most cases of Down syndrome due to an additional 21; hence, Down syndrome often known as trisomy 21. - Other compatible with survival to term: Patau syndrome (trisomy 13) & Edwards syndrome (trisomy 18). - Most other autosomal trisomies result in early pregnancy loss, with trisomy 16:a particularly common finding in first-trimester spontaneous miscarriages.

12. Genome mutations * The presence of an additional sex chromosome (X or Y) has only mild phenotypic effects: • Klinefelter syndrome: 47,XXY: 1/1000 live birth, infertile male, long limbs, motion disorders, 10-20 IQ, more X more findings. • 47, XXX: 0.1% of all women, 10-20 IQ, sometimes quite oppositional behavior. • 47, XYY: 0.1% of all men, 10-20 IQ, emotional immaturity and impulsive behavior, long status.

14. Genome mutations • Monosomy: The absence of a single chromosome. • Monosomy for an autosome is almost always incompatible with survival to term, for an X or a Y: in a 45,X karyotype: Turner syndrome:infertile female, short status. • Similar to trisomy can result from meiosis nondisjunction. - Disomy: one gamete receives 2 copies of homologous - Nullisomy: other corresponding daughter gamete will have no copy of the same chromosome. - Anaphase lag: caused by loss of a chromosome as it moves to pole of the cell during anaphase.

15. Genome mutations • Different cell lines or Mixoploidy: Mosaicism: 2 or more from same origin (zygote) generally from mitotic nondisjunction 1-2% of Down syndrome. Chimerism: 2 or more from different origins (zygotes) in human: bispermic blood gonadal: Freemartins

16. Chromosome mutations Structural abnormalities: chromosome rearrangements result from chromosome breakage with subsequent reunion in a different configuration. They can be: 1. Balanced: chromosome complement is complete, with no loss or gain of genetic material, are generally harmless with exception of rare cases that one of breakpoints damages important functional gene. Carriers are often at risk of producing children with unbalanced complement. 2. Unbalanced: chromosomal complement contains an incorrect amount of chromosome material and clinical effects are usually serious.

17. Chromosome mutations • Translocation: transfer of genetic material from one chromosome to another, its types: - Reciprocal: break occurs in each of 2 chromosomes with segments exchanged to form 2 new derivative chromosomes. - Robertsonian: a particular type of reciprocal that breakpoints located at, or close to, centromeres of 2 acrocentric chromosomes.

18. Chromosome mutations • Robertsonian translocation or centric fusion: from breakage of 2 acrocentric chromosomes (numbers 13, 14, 15, 21, & 22) at or close to centromeres, with subsequent fusion of their long arms. Short arms are lost no clinical importance, as they contain genes only for rRNA, there are multiple copies on various other acrocentric chromosomes. Because no loss or gain of important material: functionally balanced rearrangement. • The total number is reduced to 45. • The overall incidence ~ 1 in 1000, with by far the most common is 13q 14q.

19. Chromosome mutations • Genetic counseling: • Robertsonian Down syndrome: embryo inheriting 2 normal 21 (from each parent) plus a translocation chromosome involving a 21, parents of this child have high risk for further affected children (same trisomy 21). • The female carrier of a 13q21q or a 14q21q Robertsonian runs ~ 10% for having baby with Down syndrome, whereas for male carriers risk is 1-3%. • For unfortunate carrier of a 21q21q Robertsonian all gametes will be either nullisomic or disomic 21  all pregnancies will end spontaneously miscarriage or in birth of a child with Down syndrome. Rare risk> 50%

21. Chromosome mutations • Deletion: loss of part of a chromosome monosomy for that segment of chromosome. A very large deletion usually incompatible with survival to term, as a general rule: any deletion resulting in loss of > 2% of total haploid genome will be lethal. Now recognized as two levels: 1. Large chromosomal deletion: visualized under light microscope: Wolf-Hirschhorn & Cri du chat, which involve loss of material from 4p & 5p, respectively. Severe learning difficulties, often with FTT, better recognized by FISH and microarray-CGH.

22. Chromosome mutations 2. Submicroscopic microdeletions: Prader-Willi & Angelman syndromes, microdeletion 15q11-13, occurring on paternally and maternally inherited chromosome 15, respectively (genomic imprinting). • Prader-Willi: extremely floppy (hypotonic) & poor feeding in infancy, later hyperphagia & obesity, with mild-to-moderate learning difficulties. • Angelman by inappropriate laughter, convulsions, ataxia & severe learning difficulties. • Identified with high-resolution prometaphase cytogenetics augmented by FISH studies.

23. Chromosome mutations • Insertion: a segment of one chromosome inserted into another chromosome. If inserted material moved from elsewhere in another chromosome, karyotype is balanced. Otherwise causes an unbalanced chromosome complement. • Carriers of a balanced deletion-insertion rearrangement are at 50% risk of producing unbalanced gametes, because random chromosome segregation at meiosis, result in 50% of gametes inheriting either deletion or insertion, but not both.

24. Chromosome mutations • Inversion: a two-break rearrangement involving a single chromosome that a segment is reversed in position (i.e., inverted). 1. Pericentric: if inversion segment involves centromere. 2. Paracentric: if it involves only one arm of chromosome. • Balanced rearrangements & rarely cause problems in carriers, unless one of breakpoints disrupted an important gene. Pericentric inv.9: common variant or polymorphism: heteromorphism, no any functional importance; others can lead chromosome imbalance in offspring, with important clinical consequences.

25. Chromosome mutations • Ring chromosome: when a break occurs on each arm of a chromosome leaving two 'sticky' ends on central portion that reunite as a ring. Two distal chromosomal fragments are lost, so if involved chromosome is an autosome, effects are usually serious. • Often are unstable in mitosis, so it is common to find a ring chromosome in only a proportion of cells. Other cells in the individual are usually monosomic because of absence of ring chromosome.

26. Chromosome mutations • Isochromosome: shows loss of one arm with duplication of other. The most probable explanation for formation is that centromere has divided transversely rather than longitudinally. • The most commonly encountered consists of two long arms of the X chromosome. This accounts for up to 15% of all cases of Turner syndrome.

27. Gene mutations • In nuclear or mitochondrial DNA. • Contains: substitution: Transition (the most common) Transvertion deletion insertion • Dynamic mutations: expansion of trinucleotide repeat sequences, in coding or non-coding regions. Fragile X syndrome: CGG in 5’UTR CpG: Hot spot

28. Gene mutations

30. Rearrangements: 1. Balanced: inversion, translocation 2. Unbalanced: substitutiondeletion, duplication, insertion

31. Gene mutation diseases • Hemoglobinopathies: • β-thalassemia • α-thalassemia (deletion of an α-globoline gene)

32. Structural effects of mutations on the protein • 1. Synonymous or silent: single base, sp. third codon. selectively neutral. • 2. Non-synonymous: less common, more dangerous. Missens: single base-pair coding a different a.a. conservative or non- conservative (abn. Hb.) Nonsens: stop-codon, non-sense mediated decay. Frameshift: not a multiple of three disrupt reading frame and adverse effect on function. Most results a premature stop codon downstream to mutation or may lead to expression of a truncated protein, unless mRNA degraded by nonsense-mediated decay.

33. Mutations in non-coding DNA: no phenotype effects, exceptions: promoter and regulatory regions miRNA or siRNA binding sites within UTRs. • Splicing Mutations: in highly conserved splice donor (GT) & splice acceptor (AG) sites aberrant splicing: loss of coding sequence (exon skipping), retention of intronic sequence, may lead to frameshift mutations. Cryptic splice sites activationorExon splicing enhancer mutation.

34. Functional effects of mutations on the protein: • Loss-of-function:  hypomorph  amorph or null allele Autosomal or X-linked recessive inherited Haplo-insufficiency: familial hypercholesterolemia. • Gain-of-function: dominantly inherited  expression or development of new function  alter timing or tissue specificity of expression • Dominant-negative: loss of protein activity or function due to mutant gene in heterozygous state. common in dimers or multimers proteins: structural proteins such as collagens can lead to Osteogenesis imperfecta.

35. Mutations Types of mutations (based on etiology): • 1. Spontaneous mutations: naturally occurring mutations, are thought to arise through chance errors in chromosomal division or DNA replication. • 2. Induced mutations: environmental agents that cause mutations are known as mutagens. These include natural or artificial ionizing radiation and chemical or physical mutagens.

36. Mutagens 1. Ionizing radiation(natural or artificial) • Dosimetry: measurement of radiation dose expressed in relation to amount received by gonads. • The number of mutations produced by irradiation is proportional to dose/ No threshold below which irradiation has no effect/ Cumulative effects. 2. Chemicals or physical mutagens: mustard gas, formaldehyde, benzene, some basic dyes & food additives.. • Formation of DNA adducts, chromosome breaks, or aneuploidy.

37. Chromosome instability • Chromosome instability syndromes: A group of rare inherited conditions associated with chromosomal instability and breakage. They often lead to an increased tendency to develop certain types of malignancies. • Defect in caretaker genes →mutation surveillance and prevention → AR chromosomal instability syndrome → high risk of mutations → ↑risk of cancer particularly Leukemia, skin cancers.

38. Chromosome instability • Important points: clinical radiolographies heterozygots: common, malignancies • Chromosome study is an important diagnosis method. • Some chromosome instability syndromes: • Fragile X syndrome • Xerodermapigmentosum • Ataxia telangectasia • Bloom syndrome • Fanconi anemia

39. Repair mechanisms 1. DNA polymerase: no pairing at 3’ → stops polymerase, shifts 3’end to exonuclease → adds a wrong base.

40. Repair mechanisms • 2. DNA Excision-repair systems: cutting of injured DNA, inserted whole space by DNA polymerase and ligase. • - Base-excision repair • - Mismatch-excision repair • - Nucleotide excision repair

41. - Base-excision repair • C→T is the most common point mutation. • Recognition of wrong T by DNA Glycosilase → • Exertion of T and hydrolysis its bind with glocuse → • Strand cutting by AP Endonuclease → • Repair DNA polymerase replaces place by C. • Should be carried out before duplication, otherwise un-repairable.

42. - Mismatch excision repair • Mismatch, deletion or insertion of one or little nucleotides ( randomly inserted by DNA polymerase). • After DNA replication. • Recognition of wrong strand: ? May be attached pr.s • Cutting of mismatched strand → repair

43. -Nucleotide excision repair • injured bases (chemical adducts) → bad DNA figuration. • Recognized by slide proteins along double helix.

44. Repair mechanisms • 3. Recombination • Croosing over: replacing of homologues DNAs on maternal & paternal chromatids → zygote. • For dangerous bi-strand cutting (due to Ionic radiation or some anticancer drugs) → bad gene sequence & function. • 1) Nonhomologous end-joining • 2) Homologous end-joining

45. What is mutation? How many types of mutation are there? How many types of chromosome disorder are there? What is non-synonymous mutation? What is repair mechanism of DNA polymerase?