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INTRODUCTION

A MUTATION SCREENING SERVICE FOR HYPERTROPHIC CARDIOMYOPATHY (HOCM) Shalaka Samant Dept. of Medical Genetics Aberdeen. INTRODUCTION. Disease of the cardiac muscle Prevalence of 1 in 500 Autosomal dominant Variable penetrance, age of onset, severity of the disease

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INTRODUCTION

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  1. A MUTATION SCREENING SERVICE FOR HYPERTROPHIC CARDIOMYOPATHY (HOCM)Shalaka SamantDept. of Medical GeneticsAberdeen

  2. INTRODUCTION • Disease of the cardiac muscle • Prevalence of 1 in 500 • Autosomal dominant • Variable penetrance, age of onset, severity of the disease • Most common cause of Sudden Cardiac death (SCD) in young at exercise Fig.1 Myocyte disarray in HOCM

  3. TABLE 1: CRITERIA FOR DIAGNOSIS OF HOCM[According to the World Health Organisation (WHO) Guidelines]

  4. FIG 1. NORMAL AND HYPERTROPHIC HEART Taken from www.cardiomyopathy.org

  5. TABLE 2: GENES ASSOCIATED WITH HOCM

  6. INTRODUCTION (CONTD.) • Marked genotype phenotype variation MYH7 Severe hypertrophy and low risk of SCD TNNT2 Mild hypertrophy, high risk of SCD, early onset of hypertrophy MYBPC3 Onset of hypertrophy in elderly

  7. AIMS OF THE PROJECT • To develop and optimise methods for mutation screening of MYH7 and TNNT2 • To estimate the frequency of MYH7 and TNNT2 mutations in Grampian patients with HOCM, particularly where there has been SCD

  8. SAMPLE SELECTION • 84 patients with suspected HOCM and/or SCD were screened (Males: Females 65:18) • 58 SCD • 32 Post-Mortem (PM)/Family History (FH) evidence of HOCM • 26 PM/FH information not available • 26 living ? HOCM affected cases based on FH/Clinical information • Majority were tissue samples (51) • 2 genes, MYH7 (Ex 3-23) and TNNT2 (Ex 2-16) were screened

  9. ß myosin heavy chain (MYH7) Locus 14q1 ~ 25 kb genome, 40 exons Most mutations localised within exons 3-23 Myosin is the principal component of thick filaments and directs energy from ATP hydrolysis into movement of sliding filaments Cardiac troponin T (TNNT2) Locus 1q32.1 15 coding exons Different isoforms due to alternative splicing Most mutations localised within within exons 8, 9,11, and 14-16 Troponin T links the troponin complex to tropomyosin in the sarcomere

  10. METHODOLOGY Extraction of genomic DNA from blood and tissue samples of our patients Primers sequences checked for binding site polymorphisms PCR optimisation, amplification and gel electrophoresis Determination of specific melting curves for each PCR fragment based on optimal temperature for heteroduplex separation WAVE fragment analysis of amplified fragments by dHPLC Bi-directional DNA sequencing of samples showing aberrant elution profile on WAVE Confirmation of sequence changes using fresh dilutions Assessment of pathogenecity of the sequence changes detected (dbSNP, Align GVD, SIFT, Polyphen, Fruit fly, etc)

  11. RESULTS • 34 sequence variants were identified • 2 pathogenic missense mutations • 4 unclassified variants (unknown clinical significance) • 28 non-pathogenic sequence changes (6 novel, 22 previously reported)

  12. TABLE 3: LIST OF SEQUENCE CHANGES IN MYH7

  13. TABLE 4: LIST OF SEQUENCE CHANGES IN TNNT2

  14. PATHOGENIC SEQUENCE CHANGES Seq change c.1954A>G; p.Arg652Gly in MYH7 Asymptomatic at age 60. His son was diagnosed following syncope after vigorous exercise at age 36. Father died young (WW2), however no other clinical details available Seq change c.832 C>T; p.Arg278Cys in TNNT2 Breathlessness on exertion, palpitations at age 53. Echocardiogram showed asymmetric LV hypertrophy. Maternal family history of HOCM. No additional family members available

  15. CASE STUDY (Variant c.426T>G in TNNT2) c.426T>G; p. Asp142Lys seq variant I-4 I -1 I -2 I -3 II-2 II-1 Dx age 23, Heart transplant age 44 III-4 III-1 III-2 III-3 Dx age 20, chest pains IV-1 IV-2

  16. CONCLUSION • Our study had a combined pick-up rate of 2.2% • TNNT2 mutations found in 1-20% and MYH7 mutations in 15-35% of HOCM cases (Wu Heng-fang et al 2004, Richard et al 2003). This may have been due to: • Selection criteria • Phenocopies of hypertrophy can occur as a part of Noonan’s syndrome, Friedreich’s ataxia, Fabry’s disease, hypertension ordue to mutations in the mitochondrial genome • SCD may occur due to Coronary artery disease (CAD), Long QT, Brugada syndrome

  17. CONCLUSIONS (CONTD.) • General correlations between causative gene and clinical severity are not universal • Proposed service • Further clinical information is needed before screening is undertaken • Triage protocol required to decide which cases should be tested • Screening should include MYH7, MYBPC3 and TNNT2 in all cases

  18. SUMMARY • HOCM is a disease of the cardiac muscle • Characterised by left ventricular hypertrophy and myocyte disarray • SCD is often the first initial manifestation, hence the importance of genetic testing • 84 samples were screened for mutations in TNNT2 and MYH7 (Ex 3-23) • A screening strategy was successfully developed for the screening of the two genes • 2 pathogenic sequence changes, 4 unclassified variants and 28 non-pathogenic sequence changes were identified • Mutations in MYH7 and TNNT2 in our patient group appear to be relatively uncommon

  19. ACKNOWLEDGEMENTS • Dr. John Dean • Dr. Kevin Kelly • Caroline Clark • Dr. Christine Bell • Dawn O’Sullivan • Prof. Jamie Grieves • Members of the DNA laboratory (Aberdeen)

  20. THANK YOU

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