Different classes of mutations – mutation detection

1. Different classes of mutations –mutation detection Vincenzo Nigro Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli Telethon Institute of Genetics and Medicine (TIGEM)

2. For mutations other than point mutations, sex biases in the mutation rate are very variable. However, small deletions are more frequent in females. • The total rate of new deleterious mutations for all genes is estimated to be about three per zygote. This value is uncertain, but it is likely that the number is greater than one.

3. the number of male germ-cell divisions

4. Relative frequency of de novo achondroplasia for different paternal ages

6. The effect of an allele • null or amorph = no product • hypomorph = reduced amount / activity • hypermorph = increased amount / activity • neomorph = novel product / activity • antimorph = antagonistic product / activity

7. amorph or hypomorph (1) • deletion • the entire gene • part of the gene • disruption of the gene structure • by insertion, inversion, translocation • promoter inactivation • mRNA destabilization • splicing mutation • inactivating donor/acceptor • activating criptic splice sites

8. Point mutations, which involve alteration in a single base pair, and small deletions or insertions generally directly affect the function of only one gene

9. amorph or hypomorph (2) • frame-shift in translation • by insertion of n+1 or n+2 bases into the coding sequence • by deletion of n+1 or n+2 bases into the coding sequence • nonsense mutation • missense mutation / aa deletion • essential / conserved amino acid • defect in post-transcriptional processing • defect in cellular localization

11. Loss of function mutations in the PAX3 gene (Waardenburg s.)

13. Classical splicing: conserved motifs at or near the intron ends.

16. hypermorph • trisomia • duplication • amplification (cancer) • Chromatin derepression (FSH) • trasposition under a strong promoter • leukemia • overactivity of an abnormal protein

17. neomorph • generation of chimeric proteins • duplication • amplification (cancer) • missense mutations • inclusion of coding cryptic exons • usage of alternative ORFs • overactivity of an abnormal protein

18. antimorph • missense mutations • inclusion of coding cryptic exons • usage of alternative ORFs

23. Gene conversion

24. Human Gene Mutation Database • The Human Gene Mutation Database (HGMD) • Locus-Specific Mutation Databases • Springer LINK: Human Genetics - Mutations Submission Form • Nomenclature for the description of sequence variations (mutation nomenclature)

25. nucleotides are designated by the bases (in upper case); A (adenine), C (cytosine), G (guanine) and T (thymidine) • nucleotide numbering; • nucleotide +1 is the A of the ATG-translation initiation codon, the nucleotide 5' to +1 is numbered -1; there is no base 0 • non-coding regions; • the nucleotide 5' of the ATG-translation initiation codon is -1 • the nucleotide 3' of the translation termination codon is *1 • intronic nucleotides; • beginning of the intron: the number of the last nucleotide of the preceeding exon, a plus sign and the position in the intron, e.g. 77+1G, 77+2T (when the exon number is known, IVS1+1G, IVS1+2T) • end of the intron: the number of the first nucleotide of the following exon, a minus sign and the position upstream in the intron, e.g. 78-2A, 78-1G (when the exon number is known, IVS1-2A, IVS1-2G)

26. Description of nucleotide changes • substitutionsare designated by a “>”-character • 76A>C denotes that at nucleotide 76 a A is changed to a C • 88+1G>T (alternatively IVS2+1G>T) denotes the G to T substitution at nucleotide +1of intron 2, relative to the cDNA positioned between nucleotides 88 and 89 • 89-2A>C (alternativelyIVS2-2A>C) denotes the A to C substitution at nucleotide -2 of intron 2, relative to the cDNA positioned between nucleotides 88 and 89

27. deletions are designated by "del" after the nucleotide(s) flanking the deletion site • 76_78del (alternatively 76_78delACT) denotes a ACT deletion from nucleotides 76 to 78 • 82_83del (alternatively 82_83delTG) denotes a TG deletion in the sequence ACTTTGTGCC (A is nucleotide 76) to ACTTTGCC • insertionsare designated by "ins" after the nucleotides flanking the insertion site, followed by the nucleotides insertedNOTE: as separator the "^"-character is sometimes used but this is not recommened (e.g. 83^84insTG) • 76_77insT denotes that a T was inserted between nucleotides 76 and 77 • variability of short sequence repeats, e.g. in ACTGTGTGCC (A is nt 1991), are designated as 1993(TG)3-6 with nucleotide 1993 containing the first TG-dinucleotide which is found repeated 3 to 6 times in the population.

28. insertion/deletions (indels) are descibed as a deletion followed by an insertion after the nucleotides afected • 112_117delinsTG (alternatively 112_117delAGGTCAinsTG or 112_117>TG) denotes the replacement of nucleotides 112 to 117 (AGGTCA) by TG • duplicationsare designated by "dup" after the nucleotides flanking the duplication site, • 77_79dupCTG denotes that the nucleotides 77 to 79 were duplicated • inversionsare designated by "inv" after the nucleotides flanking the inversion site • 203_506inv (or 203_506inv304) denotes that the 304 nucleotides from position 203 to 506 have been inverted

29. changes in different alleles (e.g. in recessive diseases) are described as "[change allele 1] + [change allele 2]" • [76A>C] + [76A>C] denotes a homozygous A to C change at nucleotide 76 • [76A>C] + [?] denotes a A to C change at nucleotide 76 in one allele and an unknown change in the other allele • two variations in one allele are described as "[first change + second change]" • [76A>C + 83G>C] denotes an A to C change at nucleotide 76 and a G to C change at nucleotide 83 in the same allele • NOTE: current recommendations use the ";"-character as a separator (i.e. [76A>C; 83G>C])

30. A pedigree of digenic inheritance showing how retinitis pigmentosa occurs only in individuals who have inherited one mutation in each of ROM1 and RDS. Heterozygotes for either mutant allele are asymptomatic

31. Triallelic inheritance In the consanguineous pedigree NFB14 both the affected (03) and the unaffected (04) individuals carry the same mutation (A242S) in the Bardet–Biedl syndrome gene, BBS6. Only the affected sibling is homozygous for a nonsense mutation (Y24X; X indicates a stop codon) in BBS2.

32. Triallelic inheritance Three mutations at two loci are necessary for pathogenesis in this pedigree, as the affected sibling (03) has three nonsense mutations (Q147X in BBS6, and Y24X and Q59X in BBS2) and the unaffected sibling (05) has two nonsense BBS2 mutations, but is wild-type for BBS6..

33. A similar model involving proteins B and D, which are members of the same multi-subunit complex but do not interact directly

34. Non-allelic complementation Mutations at one locus (mutated proteins are indicated by asterisks) are not sufficient to disrupt the formation of the complex between proteins A and B, although the strength of the interaction might be reduced (dashed line). A further mutation in protein B causes disruption of the complex (red cross), resulting in a detectable phenotype

35. DNA analysis • Today, in most laboratories the identification of unknownmutations in candidate genes, causing human diseases, is performed through manual scanning of PCR products in affected individuals, often with accurate preliminary selection • Tomorrow, after the identification of all human genes and the sequencing of the genome, DNA mutation scanning in the population will have a significant role in identifying sequence variations among individuals

36. Sequencing • With the ongoing reduction of costs (today about 5€/run), direct automated sequencing of PCR products has already been successfully applied for mutation detection. • Sequencing is often thought of as the 'gold standard' for mutation detection. • This perception is distorted due to the fact that this is the only method of mutation identification, but this does not mean it is the best for mutation detection

37. Sequencing problems • FALSE POSITIVE • when searching for heterozygous DNA differences there are a number of potential mutations, together with sequence artifacts, compressions and differences in peak intensities that must be re-checked by sequencing with additional primers and increased costs • FALSE NEGATIVE • loss of information farther away or closer to the primer • sequencing does not detect a minority of mutant molecules in a wild-type environment

38. Strategy for mutation detection • The gene is known or unknown? • Which is the size of the gene? • How many patients must be examined? • Expected mutations are dominant or recessive? • Mutations have already been identified in this gene? • There are other members of the same gene families (or pseudogenes) in the genome?

39. Dimension of the mutation detection study Number of patients Gene size X Number of controls

40. mutations are identified? NO YES General strategy for mutation detection frequent mutations are known? screening of recurrent mutations mutation scanning NO YES SEQUENCING

41. 5’ OH 5’ OH 5’ OH 5’ OH • each primer allele specific contains: • an obligate mismatch in the last but two 3’- OH base • a specific mismatch in the last 3’- OH base

42. MIX 2 MIX 1 Mut 1 Wt 2 Mut 3 Wt 4 Mut 5 Wt 6 Mut 7 Wt 8 Mut 9 Wt 10 Mut 11 Wt 12 Wt 1 Mut 2 Wt 3 Mut 4 Wt 5 Mut 6 Wt 7 Mut 8 Wt 9 Mut 10 Wt 11 Mut 12 Multiplex ARMS MIX 1 MIX 2 *

43. Current mutation detection techniques • SSCP (single strand conformation polymorphism) • HA (heteroduplex analysis) • CCM (chemical cleavage of mismatch) • CSGE (conformation sensitive gel electrophoresis) • DGGE (denaturing gradient gel electrophoresis) • DHPLC (denaturing HPLC) • PTT (protein truncation test) • direct sequencing

44. SSCP(single-strand conformation polymorphism) • Single-stranded DNA when placed in a non-denaturing solution folds into a specific structure determined by its sequence • Differences as little as 1 base can generate different conformations • This is visualized by a difference in electrophoretic mobilityof at least one strand • Structure of ss DNA changes under different physical and chemical conditions e.g. temperature, ionic strength, presence of denaturing agents, etc.

45. SSCPsingle strand conformation polymorphism • Sensitivity 150-bp fragment > 85%400-bp fragment > 60% (75% with two gels) • Detects both missense and nonsense mutations • Post PCR time: 36-72 hours (gel preparation, loading and run; autoradiography, analysis of results) • Use of radioactivity preferred • No special equipment required • DNA or mRNA as starting templates

46. SSCP • The simplest and fastest PCR product screening techniques, like SSCP (single strand conformation polymorphism) often gives unsatisfactory results for its low sensitivities(when testing G/C-rich and/or long PCR fragments, when using one condition of gel) • The recurrence of false negatives may invalidate the screening efforts, since mutations can be • truly absent • unnoticed in any of the fragments under study Thus, it could be necessary to re-screen all samples using a different technique

47. SSCP

48. Variations of SSCP DOVAM-S Detection of virtually all mutations • Selected 5 different SSCP conditions with different buffers and gel matrices ddF Dideoxy fingerprinting • Sequencing followed by non-denaturing electrophoresis

49. Mutation detection by heteroduplex analysis: the mutant DNA must first be hybridized with the wild-type DNAto form a mixture of two homoduplexes and two heteroduplexes

50. Heteroduplex analysis