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American Journal of Medical Genetics 79:226–227 (1998)
Letter to the Editor
Study on Mutations Affecting the Muscle
Promoter/First Exon of the Dystrophin Gene in 92
Japanese Dilated Cardiomyopathy Patients
To the Editor:
Dilated cardiomyopathy (DCM), the most common
form of primary myocardial disease, is characterized by
ventricular dilatation and impaired systolic function.
The cause of DCM is unknown and the causal and
pathogenic mechanisms underlining DCM are a research priority. The only inherited DCM in which the
disease gene is known is X-linked DCM (XLDCM), a
rapidly progressive DCM generally manifested in
young males as congestive heart failure in the absence
of clinical signs of skeletal myopathy.
The identification of deletions affecting the muscle
promoter and first exon of the dystrophin gene in two
XLDCM families [Muntoni et al., 1993; Yoshida et al.,
1993] suggested a critical role of this region of the dystrophin gene for developing dilated cardiomyopathy. A
G-to-T substitution that altered the consensus sequence of the splice site at the muscle first exon-intron
junction was found in another XLDCM family [Milasin
et al., 1996]. Three further reports describing mutation
of the dystrophin gene in DCM were published in 1997.
A missense mutation in exon 9 and duplication of exons
2–7 of the dystrophin gene were identified in different
families of XLDCM [Bies et al., 1997; Ortiz-Lopez et al.,
1997]. Deletion mutations in the central region of the
dystrophin gene were identified in two patients with
idiopathic DCM by Muntoni et al. [1997]. These accumulating data suggest that some cases of DCM have a
mutation of the dystrophin gene, especially in the
muscle promoter/ first exon region.
In order to clarify the incidence of mutations in the
muscle promoter/first exon region of the dystrophin
gene in Japanese DCM, we analyzed this region in
DCM patients by polymerase chain reaction (PCR) amplification and direct sequencing. Ninety-two Japanese
idiopathic DCM patients (73 males, 19 females) from
90 families who had been admitted to Kobe University
Hospital [Honda et al., 1995] were enrolled in this
*Correspondence to: Masafumi Matsuo, Division of Genetics,
International Center for Medical Research, Kobe University
School of Medicine, 7-5-1 Kusunokicho, Chuo, Kobe 650, Japan.
E-mail: matsuo@kobe-u.ac.jp
Received 8 April 1998; Accepted 28 May 1998
© 1998 Wiley-Liss, Inc.
study. All DCM patients were diagnosed on the basis of
the results of echocardiographic analysis, with left ventricular end-diastolic dimension of >55 mm and fractional shortening <25%. All patients were examined by
catheterization and biopsy, and the possible existence
of coronary heart diseases or specific heart muscle diseases was excluded. Of the 90 families, 4 were confirmed as familial DCM, since at least one other relative was diagnosed as DCM by echocardiographic examination. A further 18 cases were suspected to be
familial, because interview information suggested the
presence of DCM patients in their families [Honda et
al., 1995]. However, none of the families could be classified as having XLDCM.
Genomic DNA was extracted from the patients’ peripheral blood by the standard phenol/chloroform
method. A region comprising 709 bp of muscle-specific
promoter/first exon and 50 bp of intron 1 was amplified
by PCR with M13 sequence-tailed primers (Fig. 1) under the conditions described previously [Milasin et al.,
1996]. Direct sequencing was performed using a dye
primer cycle sequencing kit (Perkin Elmer, Foster City,
CA), Cy5-labeled M13 primers (Pharmacia Biotech,
Uppsala, Sweden) and an ALFred DNA sequencer
(Pharmacia Biotech). From 92 DCM patients the amplified product was obtained, and the amplified product
was always exactly the same size as that of the control,
indicating the absence of major deletions or insertions.
Furthermore, complete sequencing of the amplified
fragments failed to show the existence of a nucleotide
change in any of the 92 DCM patients. However, C
residue at nucleotide −168 [Klamut et al., 1990] was
not observed in all samples as reported before [Fracasso and Patarnello, 1998]. Thus, this nucleotide deletion was found to be not pathogenic.
Our results indicated that mutation of the muscle
promoter/first exon region is not common among Japanese DCM cases, even though one example of a deletion
mutation has been reported in a Japanese XLDCM
case [Yoshida et al., 1993]. This is because none of our
90 families examined exhibited classical XLDCM,
whereas most of the 22 families with potential familial
occurrence show autosomal dominant inheritance
[Honda et al., 1995].
Among the 92 DCM cases examined here, 4 had high
serum creatine kinase level ranging from 182 to 738
Letter to the Editor
227
bell KP (1997): A 5⬘ dystrophin duplication mutation causes membrane
deficiency of a-dystroglycan in a family with X-linked cardiomyopathy.
J Mol Cell Cardiol 29:3175–3188.
Fracasso C, Patarnello T (1998): Evolution of the dystrophin muscular
promoter and 5⬘ flanking region in primates. J Mol Evol 46:168–179.
Honda Y, Yokota Y, Yokoyama M (1995): Familial aggregation of dilated
cardiomyopathy evaluation of clinical characteristics and prognosis.
Jpn Circ J 37:260–269.
Klamut HJ, Gangopadhyay SB, Worton RG, Ray PN (1990): Molecular and
functional analysis of the muscle specific promoter region of the Duchenne muscular dystrophy gene. Mol Cell Biol 10:193–205.
Michels VV, Pastores GM, Moll PP, Driscoll DJ, Miller FA, Burnett JC,
Rodeheffer RJ, Tajik JA, Beggs AH, Kunkel LM, Thibodeau SN (1993):
Dystrophin analysis in idiopathic dilated cardiomyopathy. J Med
Genet 30:955–957.
Fig. 1. a: Scheme of the examined region of the dystrophin gene. The
muscle promoter/first muscle exon region is shown schematically. The box
represents the first muscle exon. Shaded boxes upstream and downstream
of the exon indicate promoter and intron sequences, respectively. Vertical
lines show protein binding sites in the promoter region, named as indicated
above the line. Horizontal arrows show the locations and directions of
primers that were used for PCR amplification and direct sequencing. Bold
lines with hatched ends and a vertical arrow over the boxes indicate two
deletion mutations and one point mutation, respectively, that have been
reported previously. Brackets indicate size of the examined fragment. b:
Sequence of primers.
(normal: less than 150 IU/liter). Although these cases
were strong candidates for a mutation in the dystrophin gene, we could not identify a mutation in this
crucial region. These data suggest that abnormality of
this region is a rare cause of DCM. Other parts of the
dystrophin gene have been designated as candidate loci
responsible for DCM [Towbin et al., 1993]. One study
conducted in the United States failed to detect a deletion mutation in the dystrophin gene by using either
PCR amplification or Southern blot analysis in 27 idiopathic DCM patients [Michels et al., 1993], but the
existence of point mutations in the muscle promoter/
first exon region was not examined in their study. Recently, mutations outside the muscle promoter/first
exon region of the dystrophin gene have been identified
in XLDCM [Bies et al., 1997; Ortiz-Lopez et al., 1997]
or idiopathic DCM [Muntoni et al., 1997]. Therefore,
other regions of the dystrophin gene in the DCM patients studied here will now be examined for mutations.
REFERENCES
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Nobuyuki Shiga
Masafumi Matsuo
Division of Genetics
International Center for Medical Research
Kobe University School of Medicine
Kobe, Japan
Mitsuhiro Yokoyama
First Department of Internal Medicine
Kobe University School of Medicine
Kobe, Japan
Yoshiyuki Yokota
Faculty of Health Science
Kobe University School of Medicine
Kobe, Japan
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