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Myotonic Dystrophy

Myotonic Dystrophy. Case Report Sandra Ramos. Introduction Genetics of DM1. Dystrophia Myotonica Protein Kinase ( DMPK ) Located at Chr 19q13.2-13.3 Contains 15 exons spanning 13kb genomic DNA 98% DM is due to a CTG triplet expansion in the 3’ UTR (~500bp from the poly A tract)

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Myotonic Dystrophy

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  1. Myotonic Dystrophy Case Report Sandra Ramos South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  2. IntroductionGenetics of DM1 • Dystrophia Myotonica Protein Kinase (DMPK) • Located at Chr 19q13.2-13.3 • Contains 15 exons spanning 13kb genomic DNA • 98% DM is due to a CTG triplet expansion in the 3’ UTR (~500bp from the poly A tract) • Expansion also present in promoter region of SIX1 South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  3. Expansion classification • Normal range: 5 to 37 repeats • Intermediate repeats: 38 to 49 repeats (rare and may be unstable) • Expansion range: 50 to >1000 repeats • Anticipation: increasing severity and decreasing age of onset in successive generations • Symptoms are generally more severe in larger expansions, no clear phenotype-genotype correlations • Intergenerational & somatic instability of repeat size • Estimated increase ~50-80 rpts per year (Monckton et al. 1995) South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  4. Primary Analysis - PCR Detectable range • Normal : 5 to 37 CTGs - Intermediate : 38 to 49 CTGs - (very) small expansion : 50 up to ~140 CTGs • Secondary Analysis- Southern BlotDouble digest with Eco RI and Bgl I.- Eco RI normal: 9/10 Kb - Bgl I normal: 3.4 KbExpansion sizes (approximate sizes seen in Bgl I digest): - Small : 100 to 200 - Medium: 200 to 700 - Large > 700 South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  5. Case Report • Newborn with poor feeding and hypotonia referred for congenital DM testing, no family history • Large expansion detected ~ 900 repeats • Mother referred for carrier status, found to have very small expansion - 69 repeats • Asymptomatic 4 year old sibling of affected baby referred for carrier testing (sample in lithium heparin) Ethical? South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  6. Fluorescent PCR analysis Mother Affected Sibling Patient PCR showed ONE normal sized allele only South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  7. Southern Blot analysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 GR 10 Kb 9 Kb 865 CTG 550 CTG 200 CTG 3.4 Kb Bgl I Eco RI Expansion ? Patient (lanes 5 and 13) no visible expansion on Eco RI digest but possible expansion seen in Bgl I digest South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  8. Fluorescent PCR analysis FATHER N/N MOTHER N/EXP N/- EXP PATIENT PCR repeated with father but not informative, patient may be homozygous for 13 repeats or have normal 13 from father and expansion from mother South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  9. 1 2 3 4 5 6 7 10 Kb 9 Kb Southern Blot analysis Eco RI Repeat Southern Blot, Eco RI digest only. Patient 1 (lane 3), very faint band no expansion visible South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  10. - More blood was requested to repeat Southern Analysis but parents were reluctant to take more blood from the child. What can we do now? Genetic linkage analysis is a statistical method that is used to associate functionality of genes to their location on chromosomes.  The main idea is that markers which are found in vicinity on the chromosome have a tendency to be inherited together when passed on to offspring  Using a microsatellite marker close to the DMPK gene, we looked whether the patient had inherited the high risk allele South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  11. DM1 Gene X75b LINKAGE ANALYSIS Graphic view of DM1 gene and linked marker X75b. South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  12. Φ 1 2 3 4 5 6 Φ 1 2 1 2 3 3 4 4 Polyacrylamide gel analysis • Father (lane 1) has alleles 2 and 4 • Patient (lanes 2 and 3) has alleles 1 and 4 • Mother (lane 4) has alleles 1 and 3 • Affected sib (lane 5) has alleles 2 and 3 South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  13. Linkage Analysis • Mother has alleles 1 and 3 • Affected sibling has inherited allele 3 from mother • High risk allele is 3 • Patient inherited allele 1 from mother, therefore patient has not inherited the allele associated with myotonic dystrophy South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  14. PEDIGREE FATHER MOTHER DM(CTG)n 12/13 DM(CTG)n13/69 X75b(TG) 2/4 X75b(TG) 1/3 PATIENT AFFECTED SIBLING DM(CTG)n 13/13DM(CTG)n 12/EXP X75b(TG) 1/4X75b(TG) 2/3 South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  15. Conclusion • PCR analysis was not informative Southern blot analysis was not consistent, maybe due to blood being taken in wrong tubeMicrosatellite analysis using the marker X75b that is closely-linked to the myotonin kinase gene showed that the patient has not inherited the high-risk allele. South West Thames Regional Genetics Service - Molecular Genetics Laboratory

  16. Thank you for your attention! South West Thames Regional Genetics Service - Molecular Genetics Laboratory

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