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Ana Terron Kwiatkowski MRCPath I course, London 17/9/2010

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Ana Terron Kwiatkowski MRCPath I course, London 17/9/2010

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  1. A large amount of work in a molecular diagnostic laboratory results from the detection of trinucleotide repeat expansions associated with a number of neurological disorders. In practical terms, what tests may be required and what will they determine and how would this influence the issuing of a report? Ana Terron Kwiatkowski MRCPath I course, London 17/9/2010

  2. Methods of trinucleotide repeat detection 1- PCR with flanking primers - Sequence highly repetitive + GC rich - Shorter alleles may be preferentially amplified - Use of polymerases that promote amplification of long templates with high-fidelity and reduce smear, DMSO - Use controls - Products detected in agarose gels/ EtBr/ UV - Rapid method for detection of normal-premutation-small expansions (<120 repeats)

  3. Multiplex SCA gene amplification • - 5 common SCA1, SCA2, SCA3, SCA6 + SCA7 • Chimeric primers, Fl-labelled, Long Template PCR • Products detected by capillary electrophoresis (automated gene analyser) • - Specific gene alleles identified by product size and label: 1-2 CAG repeat difference can be detected • - Somatic mosaicism observed • - Up to 15 loci multiplexed successfully • Rapid method for screening • - Additional tests required for resolving larger alleles (SCA2, SCA7) in at-risk patients • (may appear as homozygous normal)

  4. 2- Triplet primed PCR • High sensitivity + specificity • Small amount of DNA required • - Reliable amplification of alleles 100 repeats • - Not accurate size of large expansions • Rapid testing/ screening Warner et al. 1996; J Med Genet 33:1022-26

  5. 3- Southern-blotting • - Accurate sizes of large expansions • - Methylation status can be evaluated • Method (FraX): • DNA digested with two enzymes (one methylation specific, EcoRI+EagI) • digests separated by electrophoresis and blotted onto membrane • blot hybridised with 32P-labelled sp probes (StB12.3)  bands detected by autorad • 2 bands (XX normal): • 2.8Kb active X + 5.2Kb inactive X (methylated) • - Labour intensive, require large amount of DNA • Use of radioactivity. Other detection methods could be used. • - FraX PND: Methylation usually not established in CVS! Heitz et al. 1992; J Med Genet 29:794-801

  6. 4- Linkage analysis Indirect analysis Use of microsatellite markers for the disease locus to detect the inheritance of the risk haplotype in offspring Fluorescent Multiplex PCR Requires samples from family, including affected one mutation should be detected by another direct method in at least at least affected relative Used in occasional unusual or difficult cases when Southern blot failed PND or exclusion test

  7. Reporting the results of triplet nucleotide repeat disorders Screening PCRs: Homozygous alleles need to be confirmed/ resolved by other method (could have an expanded allele not detected by PCR) Mosaicism: detection of 2 normal/premutation bands does not exclude presence of full expansion Anticipation/ reduced penetrance ↑Repeat size: ↓age onset and ↑severity No prediction of the presentation should be made from repeat size Interpretation of the results Consider reason for referral: symptomatic +/-FH, ?carrier/ predictive testing (FH) Report should include - Results: 1- Exclude diagnosis 2- Consistent with diagnosis Risk to offspring (AD/ AR/ X-linked) 3- Mutation/premutation carrier Risk of expansion in offspring - Recommend patient to be referred to Clinical Genetics - Offer testing to at-risk family members - Offer prenatal diagnosis (if the case) following appropriate genetic counselling recommended in Fragile X

  8. Reporting the results : Fragile X

  9. Reporting the results : Huntington disease Results from TP PCR test Normal  27 CAG repeats 27-35 repeats - potentially unstable, rare expansions into affected range  36 repeats confirm diagnosis 36-39 repeats frequently associated with later onset symptoms Predictive testing: 2 alleles normal - patient will not develop HD - risk of passing HD-CAG repeat to offspring excluded 1 allele within pathogenic size range + 1 normal allele: patient will develop HD patient’s offspring at 50% risk of inheriting HD pathogenic allele Exclusion test: Linkage analysis Individuals at 25% risk whose parent does not want to be tested/ informed (PND)

  10. Reporting the results : Friedreich ataxia

  11. References • Chong et al. 1994; Am J Hum Genet 51:522-26. • Rousseau et al. 1991; New England J Medicine 325:1673-81. • Warner et al .1993. Molecular and Cellular Probes 7:235-39. • Warner et al. 1996; J Med Genet 33:1022-26. • Tassone et al. 2008; J Mol Diagnostics 10:43-49. • Dorschner et al. 2002; J Mol Diagnostics 4:108-113. • CMGS. Practice guidelines for molecular diagnosis of Fragile X • Best practice guidelines for molecular analysis of Huntington Disease. DRAFT on CMGS.

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