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Molecular diagnosis of heterogeneous genetic diseases: the example of muscular dystrophies

Molecular diagnosis of heterogeneous genetic diseases: the example of muscular dystrophies. Vincenzo Nigro Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli. Telethon Institute of Genetics and Medicine (TIGEM). What is a mutation?. A variation of the DNA sequence

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Molecular diagnosis of heterogeneous genetic diseases: the example of muscular dystrophies

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  1. Molecular diagnosis of heterogeneous genetic diseases: the example of muscular dystrophies Vincenzo Nigro Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli Telethon Institute of Genetics and Medicine (TIGEM)

  2. What is a mutation? A variation of the DNA sequence • that is only found in affected individuals • that is never found in non affected individuals • that accounts for the pathological process/status • that, when corrected in time, disease is rescued

  3. ..that is only found in affected and that is never found in non affected incomplete penetrance that is more often found in affected than in non affected...

  4. CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACCCCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTTGCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCGGCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAGGCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAAAAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTCTAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAAAGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCTCAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAATGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCTTCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGGGGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCAGGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTGTTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCATTTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAAGGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATCCCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTGCTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTTGTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAATAAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGGCAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACATCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTTAAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATGTGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAACACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAATTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCTGTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAGTGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAGAAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTATAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTTGTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAGGGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGGAGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCCTGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAACTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGATCTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTCCCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACCCCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTTGCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCGGCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAGGCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAAAAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTCTAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAAAGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCTCAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAATGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCTTCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGGGGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCAGGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTGTTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCATTTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAAGGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATCCCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTGCTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTTGTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAATAAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGGCAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACATCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTTAAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATGTGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAACACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAATTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCTGTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAGTGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAGAAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTATAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTTGTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAGGGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGGAGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCCTGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAACTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGATCTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACCCCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTTGCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCGGCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAGGCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAAAAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTCTAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAAAGAATGTTAACTATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCTCAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAATGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCTTCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGGGGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCAGGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTGTTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCATTTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAAGGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATCCCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTGCTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTTGTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAATAAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGGCAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACATCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTTAAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATGTGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAACACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAATTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCTGTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAGTGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAGAAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTATAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGCTCCAAAGCTCTTGCTTTGTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAGGGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGGAGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCCTGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAACTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGATCTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC 50.000 private variants = innocuous differences belonging to one family

  5. 1-allele diseases • monoallelic mutations may be responsible for dominant or X-linked disorders • new random mutations are the rule with an unpredictable pattern of distribution

  6. Gender effect in mutations • For mutations other than point mutations, sex biases in the mutation rate are very variable • Small deletions are more frequent in females • Germline base substitution mutations occur more frequently in males than in females, especially in older males • Point mutations at some loci occur almost exclusively in males, whereas others occur ten times more than in females

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

  8. Relative frequency of de novo neurofibromatosis for different paternal ages

  9. the number of male germ-cell divisions

  10. 2-allele diseases • novel mutations are rare, usually mutations have a long history (100-1000 generations) • mutations have an ethnical signature with a predictable pattern of distribution and frequency • biallelic mutations may be responsible for autosomal recessive disorders • polymorphisms and private variants are more easily discriminated vs true mutations

  11. 2-allele diseases • consanguineity is a risk factor for homozygosity • high carrier frequency is a risk factor for compound heterozygosity

  12. 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

  13. Dominant or recessive phenotype?

  14. Loss of function mutations in the PAX3 gene (Waardenburg syndrome)haploinsufficiency

  15. amorph / 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

  16. amorph / 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

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

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

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

  20. Mutation detection • mutation scanning • or resequencing methods for identifying previously unknown mutations • genotyping • methods for scoring previously known mutations or single nucleotide polymorphisms (SNPs)

  21. Key questions for mutation detection strategy • expected mutations are monoallelic or biallelic? • is the gene well recognized for that disease? • is the mutation pattern known? (deletion, dup, small mutations, etc.) • which is the complexity of the gene? • how many patients must be examined? • how many controls should be examined? • how many mutations and how many variations have already been identified in this gene? • are there more members of the same gene family (or pseudogenes) in the genome?

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

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

  24. DMD Duchenne Muscular Dystrophy - 1/3,500 boysOnset -- Early childhood - about 2 to 6 years • Laboratory -- CK (50x to 1.000x), LDH5, ALT, AST, aldolase increase Symptoms -- Generalized weakness and muscle wasting affecting proximal limb muscles first. Calves often enlarged. Heart involvementProgression -- Disease progresses slowly but will affect all voluntary muscles. Survival possible beyond late twenties • BMD Becker Muscular Dystrophy - 1/10,000 boysOnset -- Adolescence or adulthoodSymptoms -- Almost identical to Duchenne but often much less severe. Heart involvementProgression -- Slower and more variable than DMD with survival well into mid to late adulthood

  25. Carrier of a balanced reciprocal X-autosome translocation

  26. Dystrophin gene: page1/185

  27. Dystrophin gene: page 2/185

  28. Dystrophin gene: page3/185

  29. Dystrophin gene: page185/185

  30. Telethon-UILDM 250/300 DMD/BMD Qualitative test rejected Quantitative test more DNA 80plex-PCR Point mutations Deletions duplications mRNA study Family tests

  31. Log-PCR = 4 multiplex-PCR (2x20+2x18) with uniform spacing and gel position according to chromosomal position DMD patient : groups A, B BMD patient : groups C, D Deletion ex 17-43 Duplication ex 13-23 C D A B DMD BMD 1: del ex 43 2: del ex 11, 17, 19, 21 3: del ex 17, 19, 21 4: del ex 50, 52 5: del ex 7, 11, 17, 19 6: del ex 61 1: no del 2: del ex 8, 12, 18, 20, 22 3: del ex 12, 18, 20, 22 4: del ex 46, 51 5: del ex 6, 8, 12, 18 6: del ex 62 1 2 3 4 5 6

  32. large deletions in 377/506 DMD/BMD 74.5%

  33. large duplications in 51/506 patients 10.1%

  34. SALSA MLPA probes

  35. Hybridysation • The MLPA probemix is added to denatured genomic DNA • The two parts of each probe hybridise to adjacent target sequences

  36. Ligation 3. Probes are ligated by a thermostable ligase

  37. PCR amplification • A universal primer pair is used to amplify all ligated probes The PCR product of each probe has a unique length (130 480 bp)

  38. Separation and quantification by capillary electrophoresis Each peak is the amplification product of a specific probe. Samples are compared to a control sample. A difference in relative peak height or peak area indicates a copy number change of the probe target sequence

  39. Male Female Triple X 283 bp 346 bp Detection of Chr X copy number X

  40. MLPA discriminates sequences that differ in only a single nucleotide and can be used to detect known mutations. Mismatch Perfect match Mismatch at the probe ligation site  No ligation, no amplification product Ligation of the two probe oligonucleotides  Amplification product

  41. M M Methylated Target Unmethylated Target Denaturation and Multiplex probe hybridization Ligation and Digestion with methylation sensitive endonucleases M M MS-MLPA Only undigested (methylated) and ligated probes are exponentially amplified

  42. Limb-girdle weakness proximal weakness: most common • Lower extremities • difficulty climbing stairs • arising from a low chair or toilet • getting up from a squatted position • Upper extremities • trouble lifting objects over their head • brushing their hair • distal weakness • difficulty opening jars, inability to turn a key in the ignition, or tripping due to foot drop • cranial weakness • dysarthria, dysphagia or ptosis

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