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High frequency of multiple mutations found by array-based resequencing of 16 genes implicated
in hypertrophic cardiomyopathy (HCM)
Purpose> To shed new light on the
complexity of genotypes associated
with hypertrophic cardiomyopathy
(HCM) and to determine the use of
exhaustive screening of sixteen disease
genes.
Methods> DNA samples of 20 patients
presenting with suspected familial HCM
were subjected to array-based
resequencing using commercially
available mutation detection assays
HCM1 and HCM2 (CorTAG, Dortmund,
Germany).
Results> At least one possibly disease
causing mutation was identified in 14
out of 20 samples (70%). Solely the
major or the minor disease genes were
affected in 8 and 2 patients (40% and
10%, resp.). As to the major disease
genes addressed by the CorTAG™
HCM_1 assay, double mutations were
found in two samples (10%). As to the
minor disease genes addressed by the
HCM_2 assay, a single patient (5%)
was found to carry two mutations. In
addition, 4 individuals (20%) showed
complex genotypes made up of at least
one mutation in the major and one
mutation in the minor disease genes.
Thus, as much as 7 out of 20 patients
(35%) were found to carry more than
one mutation possibly implicated in the
development of HCM.
Conclusions> Exhaustive resequencing using microarrays reveals multiple
mutations which should all be
considered upon counseling of patients
and family members at risk.
S. Waldmüller1, T. Lawrenz2, J. Haremza3, M. Müller3, K. Rackebrandt3, J. Davies1, J. Friedrich3, P. Binner3, T. Scheffold1
Genes analysed by array-based resequencing
3
Flow-Chart of Array-Based Resequencing
Expected
Mutation
Frequency
[%]
CorTAG™
mutation
detection
assay
Required
Number
of PCR
Reactions
Gene Name
Protein Name
MYH7
cardiac β-myosin heavy chain
20
HCM1
3
MYBPC3
myosin binding protein C
30
HCM1
4
TNNT2
TCAP
cardiac troponin T
telethonin/titin-cap
5
4
HCM1
HCM2
2
1
TNNI3
troponin I
3
HCM2
1
MYH6
α-myosin heavy chain
3
HCM2
3
GLA
α-galactosidase
3
HCM2
1
TPM1
α-tropomyosin
2
HCM2
3
MYL2
regulatory myosin light chain
2
HCM2
1
CRYAB
α-B crystallin
2
HCM2
1
CSRP3/CLP
muscle LIM protein
1
HCM2
2
ACTC1
cardiac α-actin
1
HCM2
1
MYL3
essential myosin light chain
1
HCM2
1
TNNC1
troponin C
<1
HCM2
1
PRKAG2
AMP-activated protein kinase,γ-subunit
<1
HCM2
8
CAV3
caveolin-3
<1
HCM2
2
TTN*
titin
<1
HCM2
1
marker
fragm. pool
2
1
genomic
DNA
11.0 kb
long-PCR
day 1
0.3 kb
pooling
[bp]
250
fragmentation
50
day 2
day 3
labeling
hybridisation
CorTAG™
HCM2
data analysis
*note: the design comprises probes for only the kinase domain of titin
Results of the first series of 10 consecutive samples
Results of the second series of 10 consecutive samples
Mutations Detected using CorTAG™ mutation detection assays
Sample
HCM_1 ("major disease genes")
HCM_2 ("minor disease genes")
1
-
-
2
-
3
Mutations Detected using CorTAG™ mutation detection assays
HCM_1 ("major disease genes")
HCM_2 ("minor disease genes")
11
-
-
-
12
MYBPC3: c.2308G>A; p.D770N
-
-
-
13
-
GLA: c.937G>T; p.D313Y
4
MYH7: c.2761G>A, p.E921K
TCAP: c.316C>T; p.R106C
14
MYH7: c.1988G>A; p.R663H
MYH6: c.3010G>T; p.A1004S
5
MYBPC3: c. 3580G>A, p.A1194T
-
15
MYH7: c.4159G>A; p.E1387K
-
5
MYBPC3: c.977G>A; p.R326Q
-
16
MYH7: c.2348G>A; p.R783H
-
6
MYH7: c.2156G>GA; p.R719Q
-
17
-
-
7
MYH7: c.428G>A, p.R143Q
-
18
MYBPC3: c.236_237insA; p.Y79fsX1
-
8
-
TNNI3: c.146T>A; p.L49Q
19
MYH7: c.1987C>T; p.R663C
-
8
-
CAV3: c.233C>T; p.T78M
19
MYBPC3: c.2125G>A; p.D709N
-
9
MYBPC3: c.1484G>A; p.R495Q
MYH6: c.3346C>A, p.R1116S
20
MYBPC3: c.977G>A; p.R326Q
PRKAG2: c.247C>T; p.P83S
10
-
-
20
MYBPC3: c.3697C>T; p.Q1233X
-
Mutations highlighted in red are "novel" (i.e. have not previously been described). The mutation
highlighted in purple has previously been associated with Long QT syndrome and elevation of serum
creatine kinase (i.e. Hyper-CKemia).
This work was supported by the Dr. Adolf Schilling Stiftung, Münster, Germany
Sample
Mutations highlighted in red are "novel" (i.e. have not previously been described). The mutation
highlighted in green has been linked to Fabry disease while that marked in blue is suspected of
causing pathological glycogen storage.
[email protected]