Sequencing of the Hypertrophic Cardiomyopathy (HCM) genes using an automated high throughput strategy

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Sequencing of the Hypertrophic Cardiomyopathy
(HCM) genes using an automated high throughput
strategy

• Aisha Ansari
• Edinburgh Molecular Genetics

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

• 16 different sarcomere and myofilament-related
  genes
• gt450 mutations described
• Most mutations missense
• Most family specific
• Mutation hotspots rare
• Up to 5 patients gt 1 pathogenic variant

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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 lt1
CSPR3 11p15.1 lt1
TCAP 17q21 lt1
MYOZ2 4q26 lt1
VCL 10q22.1 lt1
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(No Transcript)
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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)

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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 lt1
CSPR3 11p15.1 lt1
TCAP 17q21 lt1
MYOZ2 4q26 lt1
VCL 10q22.1 lt1
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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

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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

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• MYBPC3 exons 28-31
• 50µl reaction
• Commercial buffer
• 1µl of DNA
• Standardised PCR conditions

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384 plate sequencing (5µl)

• 2µl PCR product
• 25 cycles

• 1.5µl PCR product
• 35 cycles

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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

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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

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Patient 45755 MYL2 exon 3

• Missense variant
• c.141CgtA, p.Asn47Lys
• Associated with rapidly progressing late onset
  mid-ventricular hypertrophy

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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

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Future Service Design

• 7 patients/plate zero
• Full screen 9.4x96 well plates/7patients
• Combined into 384 plates for sequencing
• 4.7x384 plates
• gt1500 sequencing reactions

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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?

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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