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A MUTATION SCREENING SERVICE FOR HYPERTROPHIC CARDIOMYOPATHY (HOCM) Shalaka Samant Dept. of Medical Genetics Aberdeen.

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Presentation on theme: "A MUTATION SCREENING SERVICE FOR HYPERTROPHIC CARDIOMYOPATHY (HOCM) Shalaka Samant Dept. of Medical Genetics Aberdeen."— Presentation transcript:

1 A MUTATION SCREENING SERVICE FOR HYPERTROPHIC CARDIOMYOPATHY (HOCM) Shalaka Samant Dept. of Medical Genetics Aberdeen

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] Major criteria Minor criteria Echocardiography - Left ventricular wall thickness ≥13 mm in the anterior septum or posterior wall, or ≥15 mm in posterior septum or free wall - Severe systolic anterior motion (SAM) - Left ventricular wall thickness of 12 mm in anterior septum or posterior wall, or 14 mm in posterior septum or free wall - Moderate SAM Electrocardiography - Left ventricular hypertrophy (LVH) + repolarisation changes - T wave inversion in leads I and aVL - Abnormal Q wave (> 40 ms or >25% R wave) - Complete bundle branch block (BBB) or interventricular conduction defect (in LV leads) - Minor repolarisation changes in LV leads - Deep S wave in V2 (>25 mm) Clinical features - Unexplained chest pain, dyspnea, syncope, fatigue, angina Diagnostic criteria 1 major criterion or 2 minor echocardiographic criteria or 1 minor echocardiographic +1 electrocardiographic criteria

4 FIG 1. NORMAL AND HYPERTROPHIC HEART
Taken from

5 TABLE 2: GENES ASSOCIATED WITH HOCM
Gene symbol Locus Gene Name Frequency MYH7 14q11.2-q12 β-myosin heavy chain 15-35% MYBPC3 11p11.2 Myosin heavy binding protein C TNNT2 1q32 Cardiac troponin T 1-20% TNNI3 19p13.2-q13.2 Cardiac troponin I <5% TNNC1 3p21.3-p21.2 Cardiac troponin C <2% TPM1 15q22.1 α-tropomyosin MYL3 3p21.2-p21.2 Ventricular essential myosin light chain <1% MYL2 12q23-q24.3 Ventricular regulatory myosin light chain ACTC 15q14 α-cardiac actin Rare TTN 2q24.1 Titin MYH6 14q12 α-myosin heavy chain KCNQ4 1p34 Voltage gated K channel ??? MTTI Mitochondria Isoleucine tRNA, glycine tRNA PRKAG2 7q3 Protein kinase A

6 INTRODUCTION (CONTD.) Marked genotype phenotype variation
MYH Severe hypertrophy and low risk of SCD TNNT Mild hypertrophy, high risk of SCD, early onset of hypertrophy MYBPC 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) Cardiac troponin T (TNNT2)
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
Sequence region Sequence change dbSNP ID Freq Reported heterozygosity values Type of change Ex 17 c.1954A>G; p.Arg652Gly 0.012 Pathogenic Int 8 c G>T Not reported ------ May affect splicing Int15 c C>T Int 10 c G>A 0.024 Does not affect splicing Int 15 c _26delAG Int 17 c insT 5’ UTR c.1-33G>T rs 0.06 0.079 Non-pathogenic Int 3 189C>T; p.Thr63Thr rs 0.58 0.50 Ex 7 c.597G>A; p.Ala199Ala rs 0.005 Ex 8 c.732C>T; p.Phe244Phe rs 0.20 0.411 Ex 9 c.975C>T; p.Asp325Asp rs 0.014 c T>C rs 0.13 0.276 Ex 12 c.1062C>T; p.Gly354Gly rs735712 0.046 c.1095G>A; p.Lys365Lys rs735711 0.230 0.074 c.1128C>T; p.Asp376Asp rs 0.07 0.396 Int 19 c A>G rs 0.19 c A>G rs 0.18 0.237 Ex 21 c.2334C>T; p.Asp778Asp rs

13 TABLE 4: LIST OF SEQUENCE CHANGES IN TNNT2
Sequence region Sequence change dbSNP ID Freq Reported heterozygosity values Type of change Ex 16 c.832C>T; p.Arg278Cys 0.012 Pathogenic Ex 10 c.426T>G;p.Asn142Lys Not reported ------ Unclassified variant 3’ UTR 848+66G>A May affect splicing Int 2 c.42-58A>G rs868407 0.43 Non-pathogenic Int 3 c.52+48G>A 0.048 c.53-11insCTTCT 0.53 Int 4 c C>T rs 0.22 0.105 Int 5 c C>T rs 0.30 0.308 c G>A rs 0.483 Int 8 c.207G>A; p.Ser69Ser rs 0.07 0.11 Int 9 c delC c.318C>T; p.Ile106Ile rs 0.46 Int 11 c C>A rs Ex 14 c.785A>G; p.Lys253Arg rs 0.024 0.17 Int 14 c C>G rs 0.25 0.33 c C>T rs 0.405 Int 15 c C>T rs 0.18

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-1 II-2 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 or due 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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