Sequencing of the Hypertrophic Sequencing of the Hypertrophic Cardiomyopathy (HCM) genes Cardiomyopathy (HCM) genes

using an automated high using an automated high throughput strategythroughput strategy

Aisha AnsariAisha Ansari

Edinburgh Molecular GeneticsEdinburgh Molecular Genetics

Hypertrophic Cardiomyopathy Hypertrophic Cardiomyopathy (HCM)(HCM)

Prevalence is 1 in 500Prevalence is 1 in 500

Autosomal dominant Autosomal dominant inheritance inheritance

Clinical features: LVH, Clinical features: LVH, heart failure, cardiac heart failure, cardiac arrhythmias and SCDarrhythmias and SCD

Annual mortality rate Annual mortality rate of ~1%of ~1%

Image from: www.bestsyndication.com

Structure of the Cardiac SarcomereStructure of the Cardiac Sarcomere

Image from: http://gilead.org.il/hcm/sarcomere.jpg

Genetics of HCMGenetics of HCM

16 different sarcomere and myofilament-16 different sarcomere and myofilament-related genes related genes

>450 mutations described >450 mutations described Most mutations missenseMost mutations missense

Most family specific Most family specific

Mutation hotspots rareMutation hotspots rare

Up to 5% patients > 1 pathogenic variant Up to 5% patients > 1 pathogenic variant

Mutation DistributionMutation Distribution GeneGene LocusLocus % of cases% of cases

MYH7MYH7 14q1214q12 4444

MYBPC3MYBPC3 11p11.211p11.2 3535

TNNT2TNNT2 1q321q32 77

TNNI3TNNI3 19q13.419q13.4 55

TPM1TPM1 15q22.115q22.1 2.52.5

MYL2 MYL2 12q24.312q24.3 22

MYL3MYL3 3p213p21 11

ACTC1ACTC1 15q1415q14 11

TTNTTN 2q312q31 <1<1

CSPR3CSPR3 11p15.111p15.1 <1<1

TCAPTCAP 17q2117q21 <1<1

MYOZ2MYOZ2 4q264q26 <1<1

VCL VCL 10q22.110q22.1 <1<1

MYBPC3

MYH7

Project Aims Project Aims

Aim: to provide a screening service for 6 of Aim: to provide a screening service for 6 of the commonly associated HCM genes the commonly associated HCM genes

MYH7MYH7,, MYBPC3 MYBPC3,, TNNT2 TNNT2,, TNNI3 TNNI3,, TPM1 TPM1 && MYL2 MYL2 (coding 112 exons)(coding 112 exons)

Mutation DistributionMutation Distribution GeneGene LocusLocus % of cases% of cases

MYH7MYH7 14q1214q12 4444

MYBPC3MYBPC3 11p11.211p11.2 3535

TNNT2TNNT2 1q321q32 77

TNNI3TNNI3 19q13.419q13.4 55

TPM1TPM1 15q22.115q22.1 2.52.5

MYL2 MYL2 12q24.312q24.3 22

MYL3MYL3 3p213p21 11

ACTC1ACTC1 15q1415q14 11

TTNTTN 2q312q31 <1<1

CSPR3CSPR3 11p15.111p15.1 <1<1

TCAPTCAP 17q2117q21 <1<1

MYOZ2MYOZ2 4q264q26 <1<1

VCL VCL 10q22.110q22.1 <1<1

Project Aims Project Aims

Aim: to provide a screening service for 6 of Aim: to provide a screening service for 6 of the commonly associated HCM genes the commonly associated HCM genes

MYH7MYH7,, MYBPC3 MYBPC3,, TNNT2 TNNT2,, TNNI3 TNNI3,, TPM1 TPM1 && MYL2 MYL2 (coding 112 exons)(coding 112 exons)

Introduce high throughput sequencing Introduce high throughput sequencing Miniaturise reaction volumesMiniaturise reaction volumes

Evaluate Biomek NX robotEvaluate Biomek NX robot

Evaluate magnetic bead cleanup Evaluate magnetic bead cleanup

Primer designPrimer design

Initial design with MutScreener program Initial design with MutScreener program 5 possible primer pairs5 possible primer pairs

Redesign if SNP under primerRedesign if SNP under primer

All primers checkedAll primers checkedNGRL Manchester SNP checkNGRL Manchester SNP check

Primer placementPrimer placement

BLAST & BLAT BLAST & BLAT

RepeatMasker RepeatMasker

28 29 30 31MYBPC3MYBPC3 exons 28-31 exons 28-31

50µl50µl reaction reaction

Commercial buffer

1µlµl of DNA

Standardised PCR conditions

28 29 30 31MYBPC3 MYBPC3 exons 28-31 exons 28-31

12µl12µl reaction reaction

40ng of DNA40ng of DNA

Standardised PCR conditions Standardised PCR conditions

shows primer-dimer (PD)shows primer-dimer (PD)

Reducing primer conc. removed PDReducing primer conc. removed PD

384 plate sequencing (5384 plate sequencing (5µl)µl)

2µlµl PCR product

25 cycles

1.5µl PCR product µl PCR product

35 cycles35 cycles

384 well sequencing (5384 well sequencing (5µl)µl)

2µlµl PCR product

25 cycles

QV20: 156 - 298

CRL: 151 - 305

1.5µl PCR product µl PCR product

35 cycles35 cycles

QV20: 297 - 357QV20: 297 - 357

CRL: 305 - 353 CRL: 305 - 353

Variants identifiedVariants identified10 variants detected in 18 patients10 variants detected in 18 patients

MYH7 MYH7 – 2 missense variants (1 of them reported as pathogenic) 2 missense variants (1 of them reported as pathogenic) – 1 splice variant1 splice variant– 1 deletion1 deletion– 1 silent variant 1 silent variant

MYBPC3MYBPC3– 2 missense variants2 missense variants– 1 nonsense (reported as pathogenic) 1 nonsense (reported as pathogenic) – 1 splice variant 1 splice variant

MYL2MYL2– 1 missense variant 1 missense variant

Patient 45755 – Patient 45755 – MYL2MYL2 exon 3 exon 3

Missense variant

c.141C>A, p.Asn47Lys

Associated with rapidly progressing late onset mid-ventricular hypertrophy

A/C

Normal

Variant

C

Costing breakdown (per patient) Costing breakdown (per patient)

Consumables Consumables £111.95£111.95

Use of 3730 (at MRC) Use of 3730 (at MRC) £122.63£122.63

Staffing (1.5x clinical scientist & 1 MTO) Staffing (1.5x clinical scientist & 1 MTO) £631.40£631.40

Repeat rate (10%)Repeat rate (10%) £86.60£86.60

Cost estimate per patient = £952.58Cost estimate per patient = £952.58

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 DesignFuture Service Design

Future Work Future Work

Referral criteria Referral criteria Screening all 6 genesScreening all 6 genes

Reporting guidelinesReporting guidelines Minimising repeats Minimising repeats

Implement pre-PCR roboticsImplement pre-PCR roboticsUnclassified variantsUnclassified variantsData management Data management Backlog (Backlog (TPM1TPM1 & & MYL2MYL2) ) GLEAM – other genes? GLEAM – other genes?

AcknowledgementsAcknowledgements

Austin Diamond, Judith Pagan, Jon Austin Diamond, Judith Pagan, Jon Warner, Paul Westwood & Nicola WilliamsWarner, Paul Westwood & Nicola Williams

Dr Vicky Murday (Glasgow) for patient Dr Vicky Murday (Glasgow) for patient samplessamples

Stewart McKay – MRC HGU Edinburgh Stewart McKay – MRC HGU Edinburgh

Everyone else in the molecular labEveryone else in the molecular lab

Edinburgh clinical genetics Edinburgh clinical genetics