A MUTATION SCREENING A MUTATION SCREENING SERVICE FOR SERVICE FOR

HYPERTROPHIC HYPERTROPHIC CARDIOMYOPATHY CARDIOMYOPATHY

(HOCM)(HOCM)

Shalaka SamantShalaka SamantDept. of Medical GeneticsDept. of Medical Genetics

AberdeenAberdeen

INTRODUCTIONINTRODUCTION Disease of the cardiac muscleDisease of the cardiac muscle

Prevalence of 1 in 500 Prevalence of 1 in 500

Autosomal dominantAutosomal dominant

Variable penetrance, age of onset, severity of Variable penetrance, age of onset, severity of the diseasethe disease

Most common cause of Sudden Cardiac death Most common cause of Sudden Cardiac death (SCD) in young at exercise(SCD) in young at exercise

Fig.1 Myocyte disarray in HOCM

TABLE 1: CRITERIA FOR DIAGNOSIS TABLE 1: CRITERIA FOR DIAGNOSIS OF HOCMOF HOCM

[According to the World Health Organisation (WHO) Guidelines][According to the World Health Organisation (WHO) Guidelines]

Major criteriaMajor criteria Minor criteriaMinor criteria EchocardiographyEchocardiography

- 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 wall15 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 wallmm in posterior septum or free wall

- Moderate SAM- Moderate SAM

ElectrocardiographyElectrocardiography

- Left ventricular hypertrophy (LVH) + - Left ventricular hypertrophy (LVH) +

repolarisation changesrepolarisation 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 featuresClinical features

- Unexplained chest pain, dyspnea, - Unexplained chest pain, dyspnea,

syncope, fatigue, anginasyncope, fatigue, angina

Diagnostic criteriaDiagnostic criteria

1 major criterion 1 major criterion or or

2 minor echocardiographic criteria 2 minor echocardiographic criteria or or

1 minor echocardiographic +1 electrocardiographic criteria1 minor echocardiographic +1 electrocardiographic criteria

FIG 1. NORMAL AND FIG 1. NORMAL AND HYPERTROPHIC HEARTHYPERTROPHIC HEART

Taken from www.cardiomyopathy.org

TABLE 2: GENES ASSOCIATED WITH TABLE 2: GENES ASSOCIATED WITH HOCMHOCM

Gene Gene symbolsymbol

LocusLocus Gene NameGene Name Frequency Frequency

MYH7MYH7 14q11.2-q1214q11.2-q12 ββ-myosin heavy chain-myosin heavy chain 15-35%15-35%

MYBPC3MYBPC3 11p11.211p11.2 Myosin heavy binding protein CMyosin heavy binding protein C 15-35%15-35%

TNNT2TNNT2 1q321q32 Cardiac troponin TCardiac troponin T 1-20%1-20%

TNNI3TNNI3 19p13.2-q13.219p13.2-q13.2 Cardiac troponin ICardiac troponin I <5%<5%

TNNC1TNNC1 3p21.3-p21.23p21.3-p21.2 Cardiac troponin CCardiac troponin C <2%<2%

TPM1TPM1 15q22.115q22.1 αα-tropomyosin-tropomyosin <2%<2%

MYL3MYL3 3p21.2-p21.23p21.2-p21.2 Ventricular essential myosin light chainVentricular essential myosin light chain <1%<1%

MYL2MYL2 12q23-q24.312q23-q24.3 Ventricular regulatory myosin light chainVentricular regulatory myosin light chain <1%<1%

ACTCACTC 15q1415q14 αα-cardiac actin-cardiac actin RareRare

TTNTTN 2q24.12q24.1 TitinTitin RareRare

MYH6MYH6 14q1214q12 αα-myosin heavy chain-myosin heavy chain RareRare

KCNQ4KCNQ4 1p341p34 Voltage gated K channelVoltage gated K channel ??????

MTTIMTTI MitochondriaMitochondria Isoleucine tRNA, glycine tRNAIsoleucine tRNA, glycine tRNA ??????

PRKAG2PRKAG2 7q37q3 Protein kinase AProtein kinase A ??????

INTRODUCTION (CONTD.)INTRODUCTION (CONTD.)

Marked genotype phenotype variationMarked genotype phenotype variation

MYH7MYH7 Severe hypertrophy and low Severe hypertrophy and low risk of SCDrisk of SCD

TNNT2TNNT2 Mild hypertrophy, high risk of Mild hypertrophy, high risk of

SCD, earlySCD, early onset of hypertrophyonset of hypertrophy MYBPC3MYBPC3 Onset of hypertrophy in elderly Onset of hypertrophy in elderly

AIMS OF THE PROJECTAIMS OF THE PROJECT

To develop and optimise methods for To develop and optimise methods for mutation screening of mutation screening of MYH7 MYH7 and and TNNT2TNNT2

To estimate the frequency of To estimate the frequency of MYH7MYH7 and and TNNT2TNNT2 mutations in Grampian patients mutations in Grampian patients with HOCM, particularly where there with HOCM, particularly where there has been SCD has been SCD

SAMPLE SELECTION SAMPLE SELECTION

84 patients with suspected HOCM and/or SCD 84 patients with suspected HOCM and/or SCD were screened (Males: Females 65:18)were screened (Males: Females 65:18)

58 SCD58 SCD 32 Post-Mortem (PM)/Family History (FH) 32 Post-Mortem (PM)/Family History (FH)

evidence of HOCMevidence of HOCM 26 PM/FH information not available26 PM/FH information not available

26 living ? HOCM affected cases based on 26 living ? HOCM affected cases based on FH/Clinical information FH/Clinical information

Majority were tissue samples (51)Majority were tissue samples (51)

2 genes, 2 genes, MYH7MYH7 (Ex 3-23) and (Ex 3-23) and TNNT2TNNT2 (Ex 2-16) (Ex 2-16) were screenedwere screened

ß myosin heavy ß myosin heavy chain (chain (MYH7MYH7))

Locus 14q1Locus 14q1 ~ 25 kb genome, 40 ~ 25 kb genome, 40

exonsexons Most mutations Most mutations

localised within localised within exons 3-23 exons 3-23

Myosin is the Myosin is the principal component principal component of thick filaments and of thick filaments and directs energy from directs energy from ATP hydrolysis into ATP hydrolysis into movement of sliding movement of sliding filamentsfilaments

Cardiac troponin Cardiac troponin T (T (TNNT2TNNT2))

Locus 1q32.1Locus 1q32.1 15 coding exons15 coding exons Different isoforms due Different isoforms due

to alternative splicingto alternative splicing Most mutations Most mutations

localised within within localised within within exons 8, 9,11, and 14-exons 8, 9,11, and 14-1616

Troponin T links the Troponin T links the troponin complex to troponin complex to tropomyosin in the tropomyosin in the sarcomeresarcomere

Extraction of genomic DNA from blood and tissue samples of our Extraction of genomic DNA from blood and tissue samples of our patientspatients

Primers sequences checked for binding site polymorphismsPrimers sequences checked for binding site polymorphisms

PCR optimisation, amplification and gel electrophoresisPCR optimisation, amplification and gel electrophoresis

Determination of specific melting curves for each PCR fragment Determination of specific melting curves for each PCR fragment based on optimal temperature for heteroduplex separationbased on optimal temperature for heteroduplex separation

WAVE fragment analysis of amplified fragments by dHPLCWAVE fragment analysis of amplified fragments by dHPLC

Bi-directional DNA sequencing of samples showing aberrant elution Bi-directional DNA sequencing of samples showing aberrant elution profile on WAVEprofile on WAVE

Confirmation of sequence changes using fresh dilutionsConfirmation of sequence changes using fresh dilutions

Assessment of pathogenecity of the sequence changes detected Assessment of pathogenecity of the sequence changes detected

(dbSNP, Align GVD, SIFT, Polyphen, Fruit fly, etc)(dbSNP, Align GVD, SIFT, Polyphen, Fruit fly, etc)

METHODOLOGYMETHODOLOGY

RESULTSRESULTS

34 sequence variants were identified 34 sequence variants were identified

2 pathogenic missense mutations2 pathogenic missense mutations 4 unclassified variants 4 unclassified variants

(unknown clinical significance)(unknown clinical significance) 28 non-pathogenic sequence changes28 non-pathogenic sequence changes

(6 novel, 22 previously reported) (6 novel, 22 previously reported)

TABLE 3: LIST OF SEQUENCE CHANGES TABLE 3: LIST OF SEQUENCE CHANGES IN IN MYH7MYH7

Sequence Sequence regionregion

Sequence changeSequence change dbSNP IDdbSNP ID FreqFreq Reported Reported heterozygosityheterozygosityvaluesvalues

Type of changeType of change

Ex 17Ex 17 c.1954A>G; p.Arg652Glyc.1954A>G; p.Arg652Gly 0.0120.012 PathogenicPathogenic

Int 8Int 8 c.640-32G>Tc.640-32G>T Not reportedNot reported 0.0120.012 ------------ May affect splicingMay affect splicing

Int15Int15 c.1579-47C>Tc.1579-47C>T Not reportedNot reported 0.0120.012 ------------ May affect splicingMay affect splicing

Int 10Int 10 c.895+17G>Ac.895+17G>A Not reportedNot reported 0.0240.024 ------------ Does not affect splicingDoes not affect splicing

Int 15Int 15 c.1579-25_26delAGc.1579-25_26delAG Not reportedNot reported 0.0120.012 ------------ Does not affect splicingDoes not affect splicing

Int 17Int 17 c.1956+173insTc.1956+173insT Not reportedNot reported 0.0120.012 ------------ Does not affect splicing Does not affect splicing

5’ UTR5’ UTR c.1-33G>Tc.1-33G>T rs3729992rs3729992 0.060.06 0.0790.079 Non-pathogenicNon-pathogenic

Int 3Int 3 189C>T; p.Thr63Thr189C>T; p.Thr63Thr rs2069540rs2069540 0.580.58 0.500.50 Non-pathogenicNon-pathogenic

Ex 7Ex 7 c.597G>A; p.Ala199Alac.597G>A; p.Ala199Ala rs2069541rs2069541 0.0240.024 0.0050.005 Non-pathogenicNon-pathogenic

Ex 8Ex 8 c.732C>T; p.Phe244Phec.732C>T; p.Phe244Phe rs2069542rs2069542 0.200.20 0.4110.411 Non-pathogenicNon-pathogenic

Ex 9Ex 9 c.975C>T; p.Asp325Aspc.975C>T; p.Asp325Asp rs2231124rs2231124 0.060.06 0.0140.014 Non-pathogenicNon-pathogenic

Int 10Int 10 c.999-44T>Cc.999-44T>C rs3729810rs3729810 0.130.13 0.2760.276 Non-pathogenicNon-pathogenic

Ex 12Ex 12 c.1062C>T; p.Gly354Glyc.1062C>T; p.Gly354Gly rs735712rs735712 0.0120.012 0.0460.046 Non-pathogenicNon-pathogenic

Ex 12Ex 12 c.1095G>A; p.Lys365Lysc.1095G>A; p.Lys365Lys rs735711rs735711 0.2300.230 0.0740.074 Non-pathogenicNon-pathogenic

Ex 12Ex 12 c.1128C>T; p.Asp376Aspc.1128C>T; p.Asp376Asp rs2231126rs2231126 0.070.07 0.3960.396 Non-pathogenicNon-pathogenic

Int 19Int 19 c.2162+89A>Gc.2162+89A>G rs2851746rs2851746 0.190.19 Not reportedNot reported Non-pathogenicNon-pathogenic

Int 19Int 19 c.2163-56A>Gc.2163-56A>G rs3729818rs3729818 0.180.18 0.2370.237 Non-pathogenicNon-pathogenic

Ex 21Ex 21 c.2334C>T; p.Asp778Aspc.2334C>T; p.Asp778Asp rs2069544rs2069544 0.0240.024 Not reportedNot reported Non-pathogenicNon-pathogenic

TABLE 4: LIST OF SEQUENCE CHANGES IN TABLE 4: LIST OF SEQUENCE CHANGES IN TNNT2TNNT2

Sequence Sequence regionregion

Sequence changeSequence change dbSNP IDdbSNP ID FreqFreq Reported Reported heterozygosity heterozygosity valuesvalues

Type of changeType of change

Ex 16Ex 16 c.832C>T; p.Arg278Cysc.832C>T; p.Arg278Cys 0.0120.012 PathogenicPathogenic

Ex 10Ex 10 c.426T>G;p.Asn142Lysc.426T>G;p.Asn142Lys Not reportedNot reported 0.0120.012 ------------ Unclassified variantUnclassified variant

3’ UTR3’ UTR 848+66G>A848+66G>A Not reportedNot reported 0.0120.012 ------------ May affect splicingMay affect splicing

Int 2Int 2 c.42-58A>Gc.42-58A>G rs868407rs868407 0.430.43 0.430.43 Non-pathogenicNon-pathogenic

Int 3Int 3 c.52+48G>Ac.52+48G>A Not reportedNot reported 0.0480.048 ------------ Non-pathogenicNon-pathogenic

Int 3Int 3 c.53-11insCTTCTc.53-11insCTTCT Not reportedNot reported 0.530.53 ------------ Non-pathogenicNon-pathogenic

Int 4Int 4 c.68-131C>Tc.68-131C>T rs3753999rs3753999 0.220.22 0.1050.105 Non-pathogenicNon-pathogenic

Int 5Int 5 c.133+121C>Tc.133+121C>T rs3722984rs3722984 0.300.30 0.3080.308 Non-pathogenicNon-pathogenic

Int 5Int 5 c.134-50G>Ac.134-50G>A rs3729843rs3729843 0.530.53 0.4830.483 Non-pathogenicNon-pathogenic

Int 8Int 8 c.207G>A; p.Ser69Serc.207G>A; p.Ser69Ser rs3729845rs3729845 0.070.07 0.110.11 Non-pathogenicNon-pathogenic

Int 9Int 9 c.382-93delCc.382-93delC Not reportedNot reported 0.0120.012 ------------ Non-pathogenicNon-pathogenic

Int 9Int 9 c.318C>T; p.Ile106Ilec.318C>T; p.Ile106Ile rs3729547rs3729547 0.460.46 0.460.46 Non-pathogenicNon-pathogenic

Int 11Int 11 c.571-32C>Ac.571-32C>A rs1104859rs1104859 0.430.43 0.4830.483 Non-pathogenicNon-pathogenic

Ex 14Ex 14 c.785A>G; p.Lys253Argc.785A>G; p.Lys253Arg rs3730238rs3730238 0.0240.024 0.170.17 Non-pathogenicNon-pathogenic

Int 14Int 14 c.781-122C>Gc.781-122C>G rs3762584rs3762584 0.250.25 0.330.33 Non-pathogenicNon-pathogenic

Int 14Int 14 c.781-33C>Tc.781-33C>T rs2275863rs2275863 0.250.25 0.4050.405 Non-pathogenicNon-pathogenic

Int 15Int 15 c.821+46C>Tc.821+46C>T rs2758864rs2758864 0.250.25 0.180.18 Non-pathogenicNon-pathogenic

PATHOGENIC SEQUENCE PATHOGENIC SEQUENCE CHANGESCHANGES

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

CASE STUDY CASE STUDY (Variant c.426T>G in (Variant c.426T>G in TNNT2TNNT2))

c.426T>G; p. Asp142Lys

seq variant

I -1 I -2 I-4I -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, Hearttransplant age 44

CONCLUSIONCONCLUSION Our study had a combined pick-up rate of Our study had a combined pick-up rate of 2.2%2.2%

TNNT2TNNT2 mutations found in 1-20% and mutations found in 1-20% and MYH7 MYH7 mutations in 15-35% of HOCM cases (mutations in 15-35% of HOCM cases (Wu Heng-fang Wu Heng-fang et al 2004, Richard et al 2003et al 2004, Richard et al 2003). This may have been ). This may have been due to:due to:

Selection criteriaSelection criteria

Phenocopies of hypertrophy can occur as a part of Phenocopies of hypertrophy can occur as a part of Noonan’sNoonan’s

syndrome, Friedreich’s ataxia, Fabry’s disease, syndrome, Friedreich’s ataxia, Fabry’s disease, hypertension orhypertension or due to mutations in the due to mutations in the mitochondrial genomemitochondrial genome

SCD may occur due to Coronary artery disease SCD may occur due to Coronary artery disease

(CAD), Long QT, Brugada syndrome(CAD), Long QT, Brugada syndrome

CONCLUSIONS (CONTD.)CONCLUSIONS (CONTD.)

General correlations between causative General correlations between causative gene and clinical severity are not gene and clinical severity are not universaluniversal

Proposed serviceProposed service Further clinical information is needed Further clinical information is needed

before screening is undertakenbefore screening is undertaken Triage protocol required to decide which Triage protocol required to decide which

cases should be testedcases should be tested Screening should include Screening should include MYH7, MYH7,

MYBPC3 MYBPC3 and and TNNT2TNNT2 in all cases in all cases

SUMMARYSUMMARY HOCM is a disease of the cardiac muscleHOCM is a disease of the cardiac muscle

Characterised by left ventricular hypertrophy and myocyte Characterised by left ventricular hypertrophy and myocyte disarraydisarray

SCD is often the first initial manifestation, hence the SCD is often the first initial manifestation, hence the importance of genetic testingimportance of genetic testing

84 samples were screened for mutations in 84 samples were screened for mutations in TNNT2TNNT2 and and MYH7MYH7 (Ex 3-23) (Ex 3-23)

A screening strategy was successfully developed for the A screening strategy was successfully developed for the screening of the two genesscreening of the two genes

2 pathogenic sequence changes, 4 unclassified variants 2 pathogenic sequence changes, 4 unclassified variants and 28 non-pathogenic sequence changes were identifiedand 28 non-pathogenic sequence changes were identified

Mutations in Mutations in MYH7MYH7 and and TNNT2TNNT2 in our patient group in our patient group appear to be relatively uncommonappear to be relatively uncommon

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Dr. John DeanDr. John Dean Dr. Kevin KellyDr. Kevin Kelly Caroline ClarkCaroline Clark Dr. Christine BellDr. Christine Bell Dawn O’SullivanDawn O’Sullivan Prof. Jamie GrievesProf. Jamie Grieves Members of the DNA laboratory (Aberdeen)Members of the DNA laboratory (Aberdeen)

THANK YOUTHANK YOU