Transcript Sequencing of the Hypertophic Cardiomyopathy genes using

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
Clinical features: LVH,
heart failure, cardiac
arrhythmias and SCD
Annual mortality rate
of ~1%
Image from: www.bestsyndication.com
Structure of the Cardiac Sarcomere
Image from: http://gilead.org.il/hcm/sarcomere.jpg
Genetics of HCM
16 different sarcomere and myofilamentrelated genes
>450 mutations described
Most mutations missense
Most family specific
Mutation hotspots rare
Up to 5% patients > 1 pathogenic variant
Mutation Distribution
Gene
Locus
% of cases
MYH7
14q12
44
MYBPC3
11p11.2
35
TNNT2
1q32
7
TNNI3
19q13.4
5
TPM1
15q22.1
2.5
MYL2
12q24.3
2
MYL3
3p21
1
ACTC1
15q14
1
TTN
2q31
<1
CSPR3
11p15.1
<1
TCAP
17q21
<1
MYOZ2
4q26
<1
VCL
10q22.1
<1
MYH7
MYBPC3
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)
Mutation Distribution
Gene
Locus
% of cases
MYH7
14q12
44
MYBPC3
11p11.2
35
TNNT2
1q32
7
TNNI3
19q13.4
5
TPM1
15q22.1
2.5
MYL2
12q24.3
2
MYL3
3p21
1
ACTC1
15q14
1
TTN
2q31
<1
CSPR3
11p15.1
<1
TCAP
17q21
<1
MYOZ2
4q26
<1
VCL
10q22.1
<1
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
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
MYBPC3 exons 28-31
28
29
30
28
29
30
31
 50µl reaction
 Commercial buffer
 1µl of DNA
 Standardised PCR conditions
MYBPC3 exons 28-31
 12µl reaction
 40ng of DNA
 Standardised PCR conditions

shows primer-dimer (PD)
 Reducing primer conc. removed PD
31
384 plate sequencing (5µl)
 2µl PCR product
 25 cycles
 1.5µl PCR product
 35 cycles
384 well sequencing (5µl)
 2µl PCR product
 25 cycles
 QV20: 156 - 298
 CRL: 151 - 305
 1.5µl PCR product
 35 cycles
 QV20: 297 - 357
 CRL: 305 - 353
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
Patient 45755 – MYL2 exon 3
Normal
C
Variant
A/C
 Missense variant
 c.141C>A, p.Asn47Lys
 Associated with rapidly progressing late onset mid-ventricular
hypertrophy
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
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
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?
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