1 / 19

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

loren
Download Presentation

Aisha Ansari Edinburgh Molecular Genetics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related