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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 Disease of the cardiac muscle Prevalence of 1 in 500 Prevalence of 1 in 500 Autosomal dominant Autosomal dominant Variable penetrance, age of onset, severity of the disease Variable penetrance, age of onset, severity of the disease Most common cause of Sudden Cardiac death (SCD) in young at exercise 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 Echocardiography - Left ventricular wall thickness 13 mm in - Left ventricular wall thickness 13 mm in the anterior septum or posterior wall, or the anterior septum or posterior wall, or 15 mm in posterior septum or free wall 15 mm in posterior septum or free wall - Severe systolic anterior motion (SAM) - Severe systolic anterior motion (SAM) - Left ventricular wall thickness of 12 mm in - Left ventricular wall thickness of 12 mm in anterior septum or posterior wall, or 14 anterior septum or posterior wall, or 14 mm in posterior septum or free wall mm in posterior septum or free wall - Moderate SAM - Moderate SAM Electrocardiography Electrocardiography - Left ventricular hypertrophy (LVH) + - Left ventricular hypertrophy (LVH) + repolarisation changes repolarisation changes - T wave inversion in leads I and aVL - T wave inversion in leads I and aVL - Abnormal Q wave (> 40 ms or >25% R - Abnormal Q wave (> 40 ms or >25% R wave) wave) - Complete bundle branch block (BBB) or - Complete bundle branch block (BBB) or interventricular conduction defect (in LV interventricular conduction defect (in LV leads) leads) - Minor repolarisation changes in LV leads - Minor repolarisation changes in LV leads - Deep S wave in V2 (>25 mm) - Deep S wave in V2 (>25 mm) Clinical features Clinical features - Unexplained chest pain, dyspnea, - Unexplained chest pain, dyspnea, syncope, fatigue, angina syncope, fatigue, angina Diagnostic criteria Diagnostic criteria 1 major criterion or 1 major criterion or 2 minor echocardiographic criteria or 2 minor echocardiographic criteria or 1 minor echocardiographic +1 electrocardiographic criteria 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 MYH714q11.2-q12 β-myosin heavy chain 15-35% MYBPC311p11.2 Myosin heavy binding protein C 15-35% TNNT21q32 Cardiac troponin T 1-20% TNNI319p13.2-q13.2 Cardiac troponin I <5% TNNC13p21.3-p21.2 Cardiac troponin C <2% TPM115q22.1 α-tropomyosin <2% MYL33p21.2-p21.2 Ventricular essential myosin light chain <1% MYL212q23-q24.3 Ventricular regulatory myosin light chain <1% ACTC15q14 α-cardiac actin Rare TTN2q24.1TitinRare MYH614q12 α-myosin heavy chain Rare KCNQ41p34 Voltage gated K channel ??? MTTIMitochondria Isoleucine tRNA, glycine tRNA ??? PRKAG27q3 Protein kinase A ???

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

7 AIMS OF THE PROJECT To develop and optimise methods for mutation screening of MYH7 and TNNT2 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 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) 84 patients with suspected HOCM and/or SCD were screened (Males: Females 65:18) 58 SCD 58 SCD 32 Post-Mortem (PM)/Family History (FH) evidence of HOCM 32 Post-Mortem (PM)/Family History (FH) evidence of HOCM 26 PM/FH information not available 26 PM/FH information not available 26 living ? HOCM affected cases based on FH/Clinical information 26 living ? HOCM affected cases based on FH/Clinical information Majority were tissue samples (51) Majority were tissue samples (51) 2 genes, MYH7 (Ex 3-23) and TNNT2 (Ex 2-16) were screened 2 genes, MYH7 (Ex 3-23) and TNNT2 (Ex 2-16) were screened

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

10 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) METHODOLOGY

11 RESULTS 34 sequence variants were identified 34 sequence variants were identified 2 pathogenic missense mutations 2 pathogenic missense mutations 4 unclassified variants 4 unclassified variants (unknown clinical significance) 28 non-pathogenic sequence changes 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 0.012Pathogenic Int 8 c G>T Not reported May affect splicing Int15c C>T Not reported May affect splicing Int 10 c G>A Not reported Does not affect splicing Int 15 c _26delAG Not reported Does not affect splicing Int 17 c insT Not reported Does not affect splicing 5 UTR c.1-33G>Trs Non-pathogenic Int 3 189C>T; p.Thr63Thr rs Non-pathogenic Ex 7 c.597G>A; p.Ala199Ala rs Non-pathogenic Ex 8 c.732C>T; p.Phe244Phe rs Non-pathogenic Ex 9 c.975C>T; p.Asp325Asp rs Non-pathogenic Int 10 c T>Crs Non-pathogenic Ex 12 c.1062C>T; p.Gly354Gly rs Non-pathogenic Ex 12 c.1095G>A; p.Lys365Lys rs Non-pathogenic Ex 12 c.1128C>T; p.Asp376Asp rs Non-pathogenic Int 19 c A>Grs Not reported Non-pathogenic Int 19 c A>Grs Non-pathogenic Ex 21 c.2334C>T; p.Asp778Asp rs Not reported Non-pathogenic

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 0.012Pathogenic Ex 10 c.426T>G;p.Asn142Lys Not reported Unclassified variant 3 UTR G>A Not reported May affect splicing Int 2 c.42-58A>Grs Non-pathogenic Int 3 c.52+48G>A Not reported Non-pathogenic Int 3 c.53-11insCTTCT Not reported Non-pathogenic Int 4 c C>Trs Non-pathogenic Int 5 c C>Trs Non-pathogenic c G>Ars Non-pathogenic Int 8 c.207G>A; p.Ser69Ser rs Non-pathogenic Int 9 c delC Not reported Non-pathogenic Int 9 c.318C>T; p.Ile106Ile rs Non-pathogenic Int 11 c C>Ars Non-pathogenic Ex 14 c.785A>G; p.Lys253Arg rs Non-pathogenic Int 14 c C>Grs Non-pathogenic c C>Trs Non-pathogenic Int 15 c C>Trs Non-pathogenic

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 -1I -2 I-4 I -3 II-1 II-2 III-1 III-2 III-3 III-4 IV-1 IV-2 Dx age 20, chest pains Dx age 23, Heart transplant age 44

16 CONCLUSION Our study had a combined pick-up rate of 2.2% Our study had a combined pick-up rate of 2.2% TNNT2 mutations found in 1-20% and MYH7 mutations in % of HOCM cases (Wu Heng-fang et al 2004, Richard et al 2003). This may have been due to: TNNT2 mutations found in 1-20% and MYH7 mutations in % of HOCM cases (Wu Heng-fang et al 2004, Richard et al 2003). This may have been due to: Selection criteria Selection criteria Phenocopies of hypertrophy can occur as a part of Noonans Phenocopies of hypertrophy can occur as a part of Noonans syndrome, Friedreichs ataxia, Fabrys disease, hypertension or due to mutations in the mitochondrial genome syndrome, Friedreichs ataxia, Fabrys disease, hypertension or due to mutations in the mitochondrial genome SCD may occur due to Coronary artery disease (CAD), Long QT, Brugada syndrome 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 General correlations between causative gene and clinical severity are not universal Proposed service Proposed service Further clinical information is needed before screening is undertaken Further clinical information is needed before screening is undertaken Triage protocol required to decide which cases should be tested Triage protocol required to decide which cases should be tested Screening should include MYH7, MYBPC3 and TNNT2 in all cases Screening should include MYH7, MYBPC3 and TNNT2 in all cases

18 SUMMARY HOCM is a disease of the cardiac muscle HOCM is a disease of the cardiac muscle Characterised by left ventricular hypertrophy and myocyte disarray Characterised by left ventricular hypertrophy and myocyte disarray SCD is often the first initial manifestation, hence the importance of genetic testing 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) 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 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 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 Mutations in MYH7 and TNNT2 in our patient group appear to be relatively uncommon

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

20 THANK YOU


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