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 the entire gene part of the gene part of the gene  disruption of the gene structure by insertion, inversion, translocation by insertion, inversion, translocation  promoter inactivation  mRNA destabilization  splicing mutation inactivating donor/acceptor inactivating donor/acceptor activating criptic splice sites 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 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 by deletion of n+1 or n+2 bases into the coding sequence  nonsense mutation  missense mutation / aa deletion essential / conserved amino acid essential / conserved amino acid defect in post-transcriptional processing defect in post-transcriptional processing defect in cellular localization 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 conserved motifs at or near the intron ends.

16. hypermorph  trisomia  duplication  amplification (cancer)  Chromatin derepression (FSH)  trasposition under a strong promoter leukemia 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) The Human Gene Mutation Database (HGMD) The Human Gene Mutation Database (HGMD)  Locus-Specific Mutation Databases Locus-Specific Mutation Databases Locus-Specific Mutation Databases  Springer LINK: Human Genetics - Mutations Submission Form Springer LINK: Human Genetics - Mutations Submission Form Springer LINK: Human Genetics - Mutations Submission Form  Nomenclature for the description of sequence variations (mutation nomenclature) Nomenclature for the description of sequence variations (mutation nomenclature) 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 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; non-coding regions; the nucleotide 5' of the ATG-translation initiation codon is -1the nucleotide 5' of the ATG-translation initiation codon is -1 the nucleotide 3' of the translation termination codon is *1the nucleotide 3' of the translation termination codon is *1 intronic nucleotides; 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)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)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  substitutions are designated by a “>”-character 76A>C denotes that at nucleotide 76 a A is changed to a C 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 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 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   insertions are designated by "ins" after the nucleotides flanking the insertion site, followed by the nucleotides inserted NOTE: 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 112_117delinsTG (alternatively 112_117delAGGTCAinsTG or 112_117>TG) denotes the replacement of nucleotides 112 to 117 (AGGTCA) by TG   duplications are designated by "dup" after the nucleotides flanking the duplication site, 77_79dupCTG denotes that the nucleotides 77 to 79 were duplicated  inversions are 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 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] + [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 [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 [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. 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.. 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 unknown mutations 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 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 loss of information farther away or closer to the primer sequencing does not detect a minority of mutant molecules in a wild-type environment 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. Gene size Number of patients X Number of controls Dimension of the mutation detection study

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

41. 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 5’ OH

42. MIX 2 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 MIX 1 MIX 2 * Multiplex ARMS

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 mobility of 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. SSCP single 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 truly absent unnoticed in any of the fragments under study 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 DNA to form a mixture of two homoduplexes and two heteroduplexes

50. Heteroduplex analysis

51. Variations of HA CSGE conformation sensitive gel electrophoresis  Mildly denaturing conditions induce conformational changes (bends) in ds DNA  This increase differential migration patterns for homo- and heteroduplexes UHG universal heteroduplex generator  with multiple mismatches to have an higher chance of detection

52. CSGE conformation sensitive gel electrophoresis  Sensitivity 300-bp fragment > 95% 500-bp fragment > 80%  Detects both missense and nonsense mutations  Post PCR time: 24-36 hours (gel preparation, loading and run; staining, analysis of results)  Use of radioactivity not necessary  No special equipment required  DNA or mRNA as starting templates

53. DGGE DGGE denaturing gradient gel electrophoresis  Sensitivity 300-bp fragment > 98% 500-bp fragment > 90%  Detects both missense and nonsense mutations  Post PCR time: 24-36 hours (gel preparation, loading and run; staining, analysis of results)  Use of radioactivity excluded  Special equipment required  Cumbersome preparation of the gel

55. DGGE  The sensitivity is adequate, but the set-up work load is much heavier  DGGE is thus well suited for the repetitive analysis of a given DNA region, following a careful optimization  The ideal application for DGGE is the diagnosis of a monogenic disorder in many patients by testing a small gene (i.e., beta globin)  For most research projects this technique is unsatisfactory, since too many PCR products must be optimized to test a few genes

56. Chemical cleavage of mismatch

57. PTT protein truncation test  Sensitivity 1000-bp fragment > 85%  Detects only nonsense mutations  Post PCR time: 48-72 hours (translation/trascription, gel preparation, loading and run, analysis of results)  Use of 35S radioactivity  No special equipment required  mRNA as starting template

60. Applications of PTT (% of truncating mutations)  Polycystic Kidney Disease PKD1 95%  Familial Adenomatous Polyposis APC 95%  Ataxia telangiectasia ATM 90%  Hereditary breast and ovarian cancer BRCA1-290%  Duchenne Muscular Dystrophy DMD 90%?  Fanconi anemia FAA80%  Hereditary non-polyposis colorectal cancer hMSH1-2 70%-80%  Neurofibromatosis type 2 NF2 65%  Hunter Syndrome IDS50%  Neurofibromatosis type 1 NF1 50%  Cystic Fibrosis CFTR15%

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

62. TMHA DHPLC temperature modulated heteroduplex analysis denaturing HPLC  Fully automatic  Sensitivity for a 300-bp fragment: > 99%  Detects both missense and nonsense mutations  Post PCR time: 3-40 minutes (annealing of samples, machine set up, analysis of results)  Use of radioactivity excluded  Requires a special expensive device

63. DHPLC strategy  Integrated analysis by PCR and DHPLC of all DNA samples from both isolated and familial cases of muscular dystrophy  It is convenient to carry out simultaneous analysis of many samples, including controls  All DNA are checked for mutations in a single DNA fragment, then we proceed to study another fragment  The costs of the analysis can be reduced to 1/10 of the cheapest sequencing procedure with comparable sensitivity

64. POOLED PLATES A+B PLATE A PLATE B DHPLC analysis

65. Case 1  The gene is known  It is composed of 5 small size exons  There are 10 patients, sons of consanguineous parents  Expected mutations are homozygous  Mutations have never been identified in this gene  There is no other member of the same gene families (or pseudogenes) in the genome

66. Case 2  The gene is known  The putative function of the gene product is to serve as a transcription factor  Expected mutations are dominant  Mutations have never been identified in this gene  There are other members of the same gene families (or pseudogenes) in the genome