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 GeneLocus % of cases MYH714q1244 MYBPC311p11.235 TNNT21q327 TNNI319q13.45 TPM115q22.12.5 MYL212q24.32 MYL33p211 ACTC115q141 TTN2q31<1 CSPR311p15.1<1 TCAP17q21<1 MYOZ24q26<1 VCL10q22.1<1

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 GeneLocus % of cases MYH714q1244 MYBPC311p11.235 TNNT21q327 TNNI319q13.45 TPM115q22.12.5 MYL212q24.32 MYL33p211 ACTC115q141 TTN2q31<1 CSPR311p15.1<1 TCAP17q21<1 MYOZ24q26<1 VCL10q22.1<1

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

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

13. 384 well sequencing (5 µl) µl  2µl PCR product  25 cycles  QV20: 156 - 298  CRL: 151 - 305 µl PCR product  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. Patient 45755 – MYL2 exon 3  Missense variant  c.141C>A, p.Asn47Lys  Associated with rapidly progressing late onset mid-ventricular hypertrophy A/CA/C Normal Variant C

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

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

18. Future Work Referral criteria Screening all 6 genes Reporting guidelines Reporting guidelines Minimising repeats 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