American Journal of Medical Genetics 86:325–327 (1999)
Mutational Analysis of the Cardiac Actin Gene in
Familial and Sporadic Dilated Cardiomyopathy
Eiji Takai,1 Hozuka Akita,1* Nobuyuki Shiga,1 Kenji Kanazawa,1 Shinichiro Yamada,1
Masahiro Terashima,1 Yasuaki Matsuda,1 Chikao Iwai,1 Keisuke Kawai,1 Yoshiyuki Yokota,2 and
Mitsuhiro Yokoyama1
1
First Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan
2
Faculty of Health Science, Kobe University School of Medicine, Kobe, Japan
Dilated cardiomyopathy (DCM) results in
part from genetic disorders. Recently, mis-
sense mutations of the cardiac actin gene
have been reported to cause DCM. We stud-
ied 136 Japanese DCM cases to elucidate
how frequently the gene mutations are in-
volved in its pathogenesis. Genomic DNA
samples were obtained from 136 DCM cases
(107 males, 29 females), containing 30 famil-
ial DCM (5 confirmed and 25 suspected). All
six exons of the cardiac actin gene were ana-
lyzed by polymerase chain reaction, single-
strand conformation polymorphism, and se-
quencing. We detected no mutations of the
disease causation previously reported
(G867A or A1014G) but two silent mutations
(G979C and C1018T) in exon 6 and one point
mutation (T1080A) in the 3ⴕ-untranslated re-
gion. As a result of screening 128 healthy
subjects, these novel silent mutations were
found to be mere genetic polymorphisms,
not responsible for the disease. Although
some genetic polymorphisms exist in the
cardiac actin gene, mutations of the gene
are rarely responsible for DCM, at least in
the Japanese patients. Am. J. Med. Genet.
86:325–327, 1999. © 1999 Wiley-Liss, Inc.
KEY WORDS: genetics; mutation; cytoskel-
etal protein; heart failure
INTRODUCTION
Dilated cardiomyopathy (DCM) is a primary myocar-
dial disease characterized by ventricular dilation and
depressed myocardial contractility, resulting in conges-
tive heart failure (CHF), arrhythmia, and sudden
*Correspondence to: Dr. Hozuka Akita, First Department of
Internal Medicine, Kobe University School of Medicine, Kusu-
noki-Cho 7-5-2, Chuo-Ku, Kobe 650-0017, Japan. E-mail:
ahozu@med.kobe-u.ac.jp
Received 14 January 1999; Accepted 3 June 1999
© 1999 Wiley-Liss, Inc.
death. Recent progress of the treatments of CHF, such
as those with angiotensin converting enzyme inhibitors
and ␤-blockers, has remarkably improved the progno-
sis of DCM [Cleland et al., 1998]. However, the 5-year
mortality rate remains as high as approximately 20%
[Dec and Fuster, 1994]. This is primarily due to un-
known etiology of DCM and, therefore, its elucidation
is a research priority. To date, some mechanisms for
the pathogenesis of DCM have been proposed: viral in-
fections, autoimmune responses, and genetic factors.
Familial occurrence is observed in approximately 35%
of DCM cases [Grunig et al., 1998], suggesting that
genetic factors play an important role in its pathogen-
esis. Although autosomal dominant inheritance is the
most common form in DCM and six chromosomal loci
have been determined by linkage analysis [Messina et
al, 1997], none of the responsible genes has been iden-
tified. In contrast, X-linked DCM (XLDCM) was found
to be caused by mutations of the dystrophin gene en-
coding a cytoskeletal protein present at sarcolemma
[Muntoni et al., 1993], but we showed that no mutation
was detected in the critical regions of the gene in 92
DCM patients examined [Shiga et al., 1998]. In animal
models abnormalities of some cytoskeletal proteins, in-
cluding muscle LIM protein, delta-sarcoglycan, and
tropomodulin, reveal a DCM-like phenotype [Arber et
al., 1997; Sakamoto et al., 1997; Sussman et al., 1998].
This evidence, along with the findings in human
XLDCM, strongly suggests that incompleteness of cy-
toskeletal proteins is closely related to the pathogen-
esis of DCM.
Actin is a highly conserved cytoskeletal protein,
which not only is a main component of cytoskeleton but
also forms thin filaments of the sarcomeres essential to
muscle contractions. Six isoforms are known to exist
including cardiac, skeletal, vascular, and enteric
muscle types and two non-muscle types. The cardiac
muscle type of actin is predominantly expressed in the
human adult heart [Vandekerckhove et al., 1986]. Re-
cently, Olson et al. [1998] showed that two missense
mutations of the cardiac actin gene caused DCM in two
small families. However, it remains unclear how fre-
quently actin gene mutations occur in the whole DCM
population. The purpose of this study is to research the
incidence of mutations of the cardiac actin gene in a
326 Takai et al.

relatively large population of both familial and spo- TABLE I. Characteristics of DCM Cases*
radic DCM. Familial DCM
Sporadic
DCM Confirmed Suspected
MATERIALS AND METHODS
Patients
Subjects Number 106 5 25
We studied data of 136 Japanese patients with DCM Age of patients
(years) 50.1 ± 12.8 43.4 ± 12.5 52.2 ± 13.9
(107 males and 29 females) collected between June Sex (male/female) 82/24 5/0 20/5
1995 and June 1998. DCM was confirmed according to Echocardiogram
previously reported criteria, that is, left ventricular LVDd (mm) 63.9 ± 7.1 64.6 ± 9.7 63.2 ± 7.1
(LV) diastolic diameter >27 mm/m2, and LV ejection LVDd/BSA
fraction <45% or fractional shortening <25% [Grunig et (mm/m2) 37.6 ± 5.8 36.6 ± 6.0 37.5 ± 6.7
al., 1998; Manolio et al., 1992]. Of the 136 patients, 132 FS (%) 14.6 ± 4.9 17.2 ± 5.2 16.1 ± 4.1
Left ventriculogram
were also examined by cardiac catheterization, and 123 Number 102 5 25
by endomyocardial biopsy to exclude coronary artery LVEDVI (ml/m2) 151.3 ± 44.6 153.0 ± 49.6 159.3 ± 45.8
disease (ⱖ50% obstruction in a major coronary vessel) EF (%) 28.7 ± 8.4 27.5 ± 11.0 28.2 ± 8.2
and specific cardiomyopathies, such as myocarditis,
*Data are expressed as mean ± SD. Ages are not current, but at diagnosis.
cardiac amyloidosis, sarcoidosis, and metabolic heart LVDd, left ventricular end-diastolic dimension; BSA, body surface area;
diseases. In addition, other specific cardiomyopathies, FS, fractional shortening; LVEDVI, left ventricular end-diastolic volume
including general systemic disease, neuromuscular dis- index; EF, ejection fraction.
orders, and toxic reactions, were excluded. A detailed
family history was obtained by interviewing all cases. patients (3.7%) from four families and was suspected in
The methods of family evaluation have been already 25 (18.4%). There was no significant difference of char-
published elsewhere [Honda et al., 1995]. Familial acteristics among three groups. Both echocardiograph-
DCM was confirmed when at least one member of first- ic and left ventriculographic parameters were similar
degree relatives had DCM that was documented by ei- among these groups.
ther echocardiogram or catheterization. Familial DCM All six exons of the cardiac actin gene were examined
was suspected when at least one first-degree relative by PCR and SSCP. There was no abnormal finding in
died suddenly or died of heart failure or when heart exons 1 to 5. However, in exon 6, three abnormal pat-
failure was suspected by the interviewer. Healthy sub- terns of migration were detected by SSCP and were
jects were recruited from company employees and no found to correspond to three different point mutations
abnormalities were demonstrated by physical work-up, (G979C, C1018T, and T1080A), respectively, by se-
ECG, and chest X ray. The study design was approved quencing (on the nucleotide numbers, see reference
by the Institutional Committee on Human Research at [Gunning et al., 1983]). The G979C was detected in 2
Kobe University Hospital. All patients and healthy patients, C1018T in 1, and T1080A in 3 without over-
participants gave written informed consent. lapping, and all mutations were heterozygous. Al-
Gene Analysis though G979C and C1018T are located in the coding
region, they do not change encoded amino acids, that is,
Genomic DNA was extracted from peripheral whole silent mutations. The T1080A is located at 20 nucleo-
blood using Genomix Kit (Tarent, Trieste, Italy). All six tides downstream of the termination codon in the 3⬘-
exons of the cardiac actin gene were amplified by poly- untranslated regions (Fig. 1). The two missense muta-
merase chain reaction (PCR) and screened for small tions (G867A and A1014G) reported to be causative for
mutations by single-strand conformation polymor- DCM [Olson et al., 1998] were not detected in the pre-
phism (SSCP) analysis as described previously [Nishi sent study, although these two mutants generated by
et al., 1992; Olson et al., 1998]. PCR fragments showing us could be detected by SSCP analysis.
abnormal patterns of migration by SSCP were sub- All patients possessing these novel mutations were
cloned into pT7 Blue vector (Novagen, Madison, WI) sporadic. To elucidate whether the mutations are re-
and sequenced using ABI PRISM Dye Terminator sponsible for DCM, we examined 128 unrelated,
Cycle Sequencing FS Ready Reaction Kit and ABI healthy participants (mean age 47.2 ± 9.3 years, all
PRISM 377 DNA sequencer (Perkinn-Elmer, Norwalk, males). The G979C was detected in 7 persons, C1018T
CT). Two mutants (G867A and A1014G) were gener- in 1, and T1080A in 1. We therefore conclude that these
ated by a PCR-based method as described previously to mutations are mere genetic polymorphisms and are not
check the ability of the SSCP method to detect the mu- responsible for the disease.
tations reported [Maruta et al., 1991]. Resulting data
were expressed as mean ± standard deviation, and DISCUSSION
compared by Scheffé’s F test. Sex distribution was ana-
lyzed by chi-square test or Fisher’s exact probability This is the first study to examine on all six exons of
test. the cardiac actin gene in a relatively large DCM popu-
lation. Olson et al. [1998] identified two mutations
RESULTS (G867A and A1014G) in two DCM families of German
or Swedish–Norwegian ancestry. However, we did not
Clinical characteristics of 136 DCM patients are detect these mutations in our patients. Although the
shown in Table I. Familial DCM was confirmed in 5 SSCP patterns of given nucleotide changes are variable
 Cardiac Actin Gene and Dilated Cardiomyopathy
depending on experimental conditions, we could detect
the two generated mutations corresponding to the mu-
tations reported by this SSCP method. Therefore, we
concluded that these two mutations do not exist in our
study population. It is likely that genetic differences
between ethnic groups influence the results. This is
supported by the fact that the Japanese population (pa-
tients and normal controls) we used have three poly-
morphisms in exon 6, whereas none was detected in
435 control subjects in the study by Olson et al. [1998].
It is unclear how the mutant actin leads to DCM.
Dysfunction of actin was suggested to reduce the trans-
mission of the force generated in sarcomeres to adja-
cent sarcomeres and myocytes and to the extracellular
matrix [Olson et al., 1998]. In contrast, hypertrophic
cardiomyopathy (HCM) is thought to be caused by com-
pensatory hypertrophy of the cardiac muscle, which is
induced by chronic reduction of force generation due to
dysfunction of sarcomere proteins [Kimura et al.,
1997]. The two hypotheses seem controversial because
in cardiomyopathic hamsters the abnormality of delta-
sarcoglycan, which is thought to transmit the forces to
extracellular matrix, leads to both phenotypes DCM
(TO-2) and HCM (BIO14.6) [Sakamoto et al., 1997],
suggesting that other factors for dilation or hypertro-
phy are involved. Thus, further studies on the mecha-
nism of cardiac dilation and hypertrophy are absolutely
necessary to clarify the pathogenesis of DCM and de-
velop direct treatments for the disease.
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