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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) 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. the number of male germ-cell divisions Relative frequency of de novo achondroplasia for different paternal ages 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 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 Point mutations, which involve alteration in a single base pair, and small deletions or insertions generally directly affect the function of only one gene 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 Loss of function mutations in the PAX3 gene (Waardenburg s.) Classical splicing: conserved motifs at or near the intron ends. hypermorph trisomia duplication amplification (cancer) Chromatin derepression (FSH) trasposition under a strong promoter leukemia overactivity of an abnormal protein neomorph generation of chimeric proteins duplication amplification (cancer) missense mutations inclusion of coding cryptic exons usage of alternative ORFs overactivity of an abnormal protein antimorph missense mutations inclusion of coding cryptic exons usage of alternative ORFs Gene conversion 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) 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) Description of nucleotide changes substitutions are 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 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 TGdinucleotide which is found repeated 3 to 6 times in the population. 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 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 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]) 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 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. 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.. A similar model involving proteins B and D, which are members of the same multi-subunit complex but do not interact directly 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 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 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 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 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? Dimension of the mutation detection study Number of patients Gene size X Number of controls General strategy for mutation detection screening of recurrent mutations frequent mutations are known? YES mutations are identified? NO NO YES SEQUENCING mutation scanning 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 5’ OH 5’ OH Multiplex ARMS MIX 1 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 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 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 SSCP (single-strand conformation polymorphism) Single-stranded DNA when placed in a nondenaturing 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. 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 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 SSCP 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 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 Heteroduplex analysis 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 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 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 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 Chemical cleavage of mismatch 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 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 BRCA1-2 90% Duchenne Muscular Dystrophy DMD 90%? Fanconi anemia FAA 80% Hereditary non-polyposis colorectal cancer hMSH1-2 70%-80% Neurofibromatosis type 2 NF2 65% Hunter Syndrome IDS 50% Neurofibromatosis type 1 NF1 50% Cystic Fibrosis CFTR 15% Dimension of the mutation detection study Number of patients Gene size X Number of additional controls Number of controls Number of additional controls Number of additional controls 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 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 PLATE B PLATE A POOLED PLATES A+B DHPLC analysis 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 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