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Aisha Ansari Edinburgh Molecular Genetics

Sequencing of the Hypertrophic Cardiomyopathy (HCM) genes using an automated high throughput strategy. Aisha Ansari Edinburgh Molecular Genetics. Hypertrophic Cardiomyopathy (HCM). Prevalence is 1 in 500 Autosomal dominant inheritance

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Aisha Ansari Edinburgh Molecular Genetics

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  1. Sequencing of the Hypertrophic Cardiomyopathy (HCM) genes using an automated high throughput strategy Aisha Ansari Edinburgh Molecular Genetics

  2. Hypertrophic Cardiomyopathy (HCM) • Prevalence is 1 in 500 • Autosomal dominant inheritance • Clinical features: LVH, heart failure, cardiac arrhythmias and SCD • Annual mortality rate of ~1% Image from: www.bestsyndication.com

  3. Structure of the Cardiac Sarcomere Image from: http://gilead.org.il/hcm/sarcomere.jpg

  4. Genetics of HCM • 16 different sarcomere and myofilament-related genes • >450 mutations described • Most mutations missense • Most family specific • Mutation hotspots rare • Up to 5% patients > 1 pathogenic variant

  5. Mutation Distribution

  6. MYBPC3 MYH7

  7. Project Aims • Aim: to provide a screening service for 6 of the commonly associated HCM genes • MYH7, MYBPC3, TNNT2, TNNI3, TPM1 & MYL2 (coding 112 exons)

  8. Mutation Distribution

  9. Project Aims • Aim: to provide a screening service for 6 of the commonly associated HCM genes • MYH7, MYBPC3, TNNT2, TNNI3, TPM1 & MYL2 (coding 112 exons) • Introduce high throughput sequencing • Miniaturise reaction volumes • Evaluate Biomek NX robot • Evaluate magnetic bead cleanup

  10. Primer design • Initial design with MutScreener program • 5 possible primer pairs • Redesign if SNP under primer • All primers checked • NGRL Manchester SNP check • Primer placement • BLAST & BLAT • RepeatMasker

  11. 28 29 30 31 MYBPC3 exons 28-31 • 12µl reaction • 40ng of DNA • Standardised PCR conditions • shows primer-dimer (PD) • Reducing primer conc. removed PD 28 29 30 31 MYBPC3 exons 28-31 • 50µl reaction • Commercial buffer • 1µl of DNA • Standardised PCR conditions

  12. 384 plate sequencing (5µl) • 2µlPCR product • 25 cycles • 1.5µl PCR product • 35 cycles

  13. 384 well sequencing (5µl) • 2µlPCR product • 25 cycles • QV20: 156 - 298 • CRL: 151 - 305 • 1.5µl PCR product • 35 cycles • QV20: 297 - 357 • CRL: 305 - 353

  14. Variants identified • 10 variants detected in 18 patients • MYH7 • 2 missense variants (1 of them reported as pathogenic) • 1 splice variant • 1 deletion • 1 silent variant • MYBPC3 • 2 missense variants • 1 nonsense (reported as pathogenic) • 1 splice variant • MYL2 • 1 missense variant

  15. Normal C Variant A/C Patient 45755 – MYL2 exon 3 • Missense variant • c.141C>A, p.Asn47Lys • Associated with rapidly progressing late onset mid-ventricular hypertrophy

  16. Costing breakdown (per patient) • Consumables • £111.95 • Use of 3730 (at MRC) • £122.63 • Staffing (1.5x clinical scientist & 1 MTO) • £631.40 • Repeat rate (10%) • £86.60 • Cost estimate per patient = £952.58

  17. Future Service Design • 7 patients/plate & zero • Full screen = 9.4x96 well plates/7patients • Combined into 384 plates for sequencing • 4.7x384 plates • >1500 sequencing reactions

  18. Future Work • Referral criteria • Screening all 6 genes • Reporting guidelines • Minimising repeats • Implement pre-PCR robotics • Unclassified variants • Data management • Backlog (TPM1 & MYL2) • GLEAM – other genes?

  19. Acknowledgements • Austin Diamond, Judith Pagan, Jon Warner, Paul Westwood & Nicola Williams • Dr Vicky Murday (Glasgow) for patient samples • Stewart McKay – MRC HGU Edinburgh • Everyone else in the molecular lab • Edinburgh clinical genetics

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