Transcript A Mutation screening service for Hypertrophic

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
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Autosomal dominant
Fig.1 Myocyte disarray in HOCM
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Variable penetrance, age of onset, severity of the disease
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Most common cause of Sudden Cardiac death (SCD) in young
at exercise
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
FIG 1. NORMAL AND HYPERTROPHIC
HEART
Taken from www.cardiomyopathy.org
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
15-35%
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
<2%
MYL3
3p21.2-p21.2
Ventricular essential myosin light chain
<1%
MYL2
12q23-q24.3
Ventricular regulatory myosin light chain
<1%
ACTC
15q14
α-cardiac actin
Rare
TTN
2q24.1
Titin
Rare
MYH6
14q12
α-myosin heavy chain
Rare
KCNQ4
1p34
Voltage gated K channel
???
MTTI
Mitochondria
Isoleucine tRNA, glycine tRNA
???
PRKAG2
7q3
Protein kinase A
???
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
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
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)
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2 genes, MYH7 (Ex 3-23) and TNNT2 (Ex 2-16) were screened
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ß myosin heavy chain
(MYH7)
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Cardiac troponin T
(TNNT2)
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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
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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
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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)
RESULTS
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34 sequence variants were identified
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2 pathogenic missense mutations
4 unclassified variants
(unknown clinical significance)
28 non-pathogenic sequence changes
(6 novel, 22 previously reported)
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TABLE 3: LIST OF SEQUENCE CHANGES IN MYH7
Sequence
region
Sequence change
dbSNP ID
Freq
Reported
heterozygosity
values
Ex 17
c.1954A>G; p.Arg652Gly
Int 8
c.640-32G>T
Not reported
0.012
------
May affect splicing
Int15
c.1579-47C>T
Not reported
0.012
------
May affect splicing
Int 10
c.895+17G>A
Not reported
0.024
------
Does not affect splicing
Int 15
c.1579-25_26delAG
Not reported
0.012
------
Does not affect splicing
Int 17
c.1956+173insT
Not reported
0.012
------
Does not affect splicing
5’ UTR
c.1-33G>T
rs3729992
0.06
0.079
Non-pathogenic
Int 3
189C>T; p.Thr63Thr
rs2069540
0.58
0.50
Non-pathogenic
Ex 7
c.597G>A; p.Ala199Ala
rs2069541
0.024
0.005
Non-pathogenic
Ex 8
c.732C>T; p.Phe244Phe
rs2069542
0.20
0.411
Non-pathogenic
Ex 9
c.975C>T; p.Asp325Asp
rs2231124
0.06
0.014
Non-pathogenic
Int 10
c.999-44T>C
rs3729810
0.13
0.276
Non-pathogenic
Ex 12
c.1062C>T; p.Gly354Gly
rs735712
0.012
0.046
Non-pathogenic
Ex 12
c.1095G>A; p.Lys365Lys
rs735711
0.230
0.074
Non-pathogenic
Ex 12
c.1128C>T; p.Asp376Asp
rs2231126
0.07
0.396
Non-pathogenic
Int 19
c.2162+89A>G
rs2851746
0.19
Not reported
Non-pathogenic
Int 19
c.2163-56A>G
rs3729818
0.18
0.237
Non-pathogenic
Ex 21
c.2334C>T; p.Asp778Asp
rs2069544
0.024
Not reported
Non-pathogenic
0.012
Type of change
Pathogenic
TABLE 4: LIST OF SEQUENCE CHANGES IN TNNT2
Sequence
region
Sequence change
dbSNP ID
Freq
Reported
heterozygosity
values
Ex 16
c.832C>T; p.Arg278Cys
Ex 10
c.426T>G;p.Asn142Lys
Not reported
0.012
------
Unclassified variant
3’ UTR
848+66G>A
Not reported
0.012
------
May affect splicing
Int 2
c.42-58A>G
rs868407
0.43
0.43
Non-pathogenic
Int 3
c.52+48G>A
Not reported
0.048
------
Non-pathogenic
Int 3
c.53-11insCTTCT
Not reported
0.53
------
Non-pathogenic
Int 4
c.68-131C>T
rs3753999
0.22
0.105
Non-pathogenic
Int 5
c.133+121C>T
rs3722984
0.30
0.308
Non-pathogenic
Int 5
c.134-50G>A
rs3729843
0.53
0.483
Non-pathogenic
Int 8
c.207G>A; p.Ser69Ser
rs3729845
0.07
0.11
Non-pathogenic
Int 9
c.382-93delC
Not reported
0.012
------
Non-pathogenic
Int 9
c.318C>T; p.Ile106Ile
rs3729547
0.46
0.46
Non-pathogenic
Int 11
c.571-32C>A
rs1104859
0.43
0.483
Non-pathogenic
Ex 14
c.785A>G; p.Lys253Arg
rs3730238
0.024
0.17
Non-pathogenic
Int 14
c.781-122C>G
rs3762584
0.25
0.33
Non-pathogenic
Int 14
c.781-33C>T
rs2275863
0.25
0.405
Non-pathogenic
Int 15
c.821+46C>T
rs2758864
0.25
0.18
Non-pathogenic
0.012
Type of change
Pathogenic
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
CASE STUDY (Variant c.426T>G in TNNT2)
c.426T>G; p. Asp142Lys
seq variant
I -1
I -2
I-4
I -3
II-2
II-1
Dx age 23, Heart
transplant age 44
III-4
III-1
III-2
IV-1
III-3
Dx age 20, chest pains
IV-2
CONCLUSION

Our study had a combined pick-up rate of 2.2%
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TNNT2 mutations found in 1-20% and MYH7 mutations in 1535% of HOCM cases (Wu Heng-fang et al 2004, Richard et al
2003). This may have been due to:
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Selection criteria
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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
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SCD may occur due to Coronary artery disease (CAD), Long
QT, Brugada syndrome
CONCLUSIONS (CONTD.)

General correlations between causative gene and
clinical severity are not universal
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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
SUMMARY

HOCM is a disease of the cardiac muscle
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Characterised by left ventricular hypertrophy and myocyte disarray
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SCD is often the first initial manifestation, hence the importance of
genetic testing
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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
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2 pathogenic sequence changes, 4 unclassified variants and 28 nonpathogenic sequence changes were identified

Mutations in MYH7 and TNNT2 in our patient group appear to be
relatively uncommon
ACKNOWLEDGEMENTS
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Dr. John Dean
Dr. Kevin Kelly
Caroline Clark
Dr. Christine Bell
Dawn O’Sullivan
Prof. Jamie Grieves
Members of the DNA laboratory (Aberdeen)
THANK YOU