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Anovel mutation in the GNE gene and a linkage disequilibrium in Japanese pedigrees.

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A Novel Mutation in
the GNE Gene and a
Linkage Disequilibrium in
Japanese Pedigrees
Aki Arai, MD,1 Keiko Tanaka, MD,1
Takeshi Ikeuchi, MD,1 Shuichi Igarashi, MD,1
Hisashi Kobayashi, MD,1 Tomoya Asaka, MD,2
Hidetoshi Date, MS,1 Masaaki Saito, MD,1
Hajime Tanaka, MD,1 Sari Kawasaki, MD,1
Eiichiro Uyama, MD,3 Hidehiro Mizusawa, MD,4
Nobuyoshi Fukuhara, MD,5 and Shoji Tsuji, MD1
Distal myopathy with rimmed vacuoles (DMRV) is an autosomal recessive muscular disorder characterized by weakness of the anterior compartment of the lower limbs with
onset in early adulthood and sparing of the quadricep muscles. The UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene was recently identified as
the causative gene for hereditary inclusion body myopathy
(HIBM). To investigate whether DMRV and HIBM are allelic diseases, we conducted mutational analysis of the GNE
gene of six Japanese DMRV pedigrees and found that all the
pedigrees share a homozygous mutation (V572L) associated
with a strong linkage disequilibrium, suggesting a strong
founder effect in Japanese DMRV pedigrees.
Ann Neurol 2002;52:516 –519
Distal myopathy with rimmed vacuoles (DMRV), also
known as Nonaka distal myopathy (OMIM 605820), is
an autosomal recessive muscular disorder.3 The disease
develops in early adulthood characterized by weakness of
the anterior compartment of the lower limbs and
hamstring muscles and sparing the quadricep muscles
until its late stage. This disease usually progresses
slowly, although sometimes rather rapidly. No laboratory findings such as mildly elevated serum creatine
kinase levels or mixed characteristics of myopathic
and neuropathic patterns in electromyograms are spe-
From the 1Department of Neurology, Brain Research Institute, Niigata University, Niigata; 2Department of Neurology, School of
Medicine, Kanazawa University, Kanazawa; 3Department of Neurology, School of Medicine, Kumamoto University, Kumamoto;
Department of Neurology, Tokyo Medical and Dental University,
Tokyo; and 5Department of Neurology, National Saigata Hospital,
Niigata, Japan.
Received Jan 3, 2002, and in revised form Jun 11. Accepted for
publication Jun 21, 2002
Published online Aug 25, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10341
Address correspondence to Dr Tsuji, Department of Neurology,
Brain Research Institute, Niigata University, 1 Asahimachi, Niigata
951-8585, Japan. E-mail:
Published 2002 by Wiley-Liss, Inc.
cific for DMRV.4 – 8 Argov and Yarom reported a
similar disease with autosomal recessive inheritance
among Iranian Jews, which is now designated as hereditary inclusion body myopathy (HIBM; OMIM
600737).9 The clinical manifestations of HIBM, including the characteristic distribution of affected muscles and its clinical course, are very similar to those of
DMRV. Both DMRV and HIBM are characterized
by the presence of numerous “rimmed vacuoles”
without inflammatory changes in muscle specimens,
as shown by modified Gomori trichrome staining.
Numerous proteins, including amyloid ␤ protein,
apolipoprotein E, ubiquitin, and hyperphosphorylated
␶ protein seen in brains of patients with Alzheimer’s
disease, have been demonstrated to be associated with
rimmed vacuoles.10 –13 Segregated heterogeneous materials derived from cytoplasmic organelles or cell
membranes in the muscle fibers and increased
lysosome-related proteins are seen in rimmed vacuoles.14 Despite these findings, the pathophysiological
mechanisms underlying rimmed vacuole formation remain to be elucidated.
Mitrani-Rosenbaum and colleagues performed linkage analyses of nine Persian Jewish families affected by
HIBM and demonstrated that HIBM is linked to 9p1q1.15 After this report, we conducted a linkage analysis
of six Japanese DMRV pedigrees and confirmed that
DMRV also was linked to 9p1-q1,1 raising the possibility that HIBM and DMRV are allelic diseases. Asaka
and colleagues further conducted a detailed linkage
analysis of Japanese DMRV pedigrees and found a
strong linkage disequilibrium that allows narrowing of
the DMRV critical region.16 Mirabella and colleagues17 also have reported an Italian family with autosomal recessive quadricep-sparing inclusion body myopathy linked to chromosome 9p1.
Eisenberg and colleagues have applied a positional
cloning approach to narrow the candidate gene region18,19 and recently have identified that the UDP-Nacetylglucosamine-2-epimerase/N-acetylmannosamine
kinase (GNE) gene is responsible for HIBM.2,19 GNE
is known as a bifunctional enzyme that has an epimerase domain and a kinase domain. It plays an important role in the sialic acid biosynthetic pathway. To
address the issue of whether HIBM and DMRV are
allelic diseases, we conducted a mutational analysis of
the GNE gene. We found that all the Japanese DMRV
pedigrees examined share a common mutation in the
homozygous state associated with a strong linkage disequilibrium in the flanking regions, suggesting a strong
founder effect in Japanese DMRV pedigrees.
Subject and Methods
Six consanguineous Japanese families (Pedigrees 262, 484,
544, 753, 1548, and 2181) including seven patients with
DMRV were analyzed (Fig). Blood samples were obtained
from 21 subjects (including 7 affected subjects: 6 male and 1
female patients) with their informed consent. All of the affected subjects exhibited very similar clinical presentations as
follows: (1) distal muscle weakness particularly in the anterior compartment with early adulthood onset, (2) sparing
quadricep muscles until its late stage, (3) mild increase in the
serum creatine kinase level (less than five times the normal
level), (4) rimmed vacuole formation in their muscle fibers
without inflammatory infiltration, and (5) autosomal recessive inheritance.
Mutational Analysis
High-molecular-weight genomic DNA was extracted from peripheral blood cells according to standard procedures. Eleven
coding exons of the GNE gene (exon 2 to exon 12) were amplified by polymerase chain reaction using exon-specific primer
sets. Supplementary information for primer sequences is available upon request. The polymerase chain reaction products
were subjected to direct nucleotide sequence analysis using an
automated DNA sequencer (ABI 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA).
Linkage Disequilibrium Analysis
In addition to the previously described microsatellite markers
(D9S1878, D9S1791, and D9S1874), the following microsatellite markers were developed based on the BAC sequences
recently released by the Sanger Institute (http://www.sanger., which include the following microsatellite
loci: 553CA12, 270CA20, 274GT21, 834CA10, and
792GT15. The primer pairs for these new polymorphic loci
were as follows: 553CA12 (forward primer, 5⬘-ATTTCTCACCACCACCACCA-3⬘; reverse primer, 5⬘- TTGTTCTTGCCCCATTGCCA-3⬘); 270CA20 (forward primer, 5⬘TAGGAGTTTGAGACCAGCCT-3⬘; reverse primer, 5⬘CCTGGCTAATTTGTGGTGTG-3⬘); 274GT21 (forward
primer, 5⬘-AATGCTACCTTGACCTCTGG-3⬘; reverse
primer, 5⬘-TTCTGATCCTCTAGCCCATG-3⬘); 834CA10
(forward primer, 5⬘-CATAGACTTGAGAGGGAATG-3⬘; reverse primer, 5⬘-AGTCTGGATACACAAGCTCT-3⬘); and
792GT15 (forward primer, 5⬘-GCTTATTGGTCATTCGTGTG-3⬘; reverse primer, 5⬘- AACAAAATTAGCTGGGCGTG-3⬘). The physical maps of these microsatellite loci
and the GNE gene are shown in the Fig. The polymorphism
of each marker was confirmed by analyzing 30 unrelated Japanese subjects. These microsatellite loci were analyzed using an
automated DNA sequencer (ABI 310 Genetic Analyzer; Applied Biosystems).
Mutational Analysis
By screening the 11 coding exons (from exon 2 to exon
12) of the GNE gene, we identified a novel missense
mutation among all the six unrelated Japanese families
investigated. The mutation was a G3 C transversion
in exon 10, substituting leucine for valine at codon
572. This mutation was not found in 50 healthy Japanese subjects. Other nucleotide changes were not
identified in the entire coding sequences.
Arai et al: A Novel Mutation in the GNE Gene
Fig. Physical maps of distal myopathy with rimmed vacuole (DMRV) locus. Seven microsatellite loci including the previously described microsatellite markers (D9S1878, D9S1791, and D9S1874) and the newly developed microsatellite markers (553CA12,
270CA20, 274GT21, 834CA10, and 792GT15) are shown along with the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene and the BAC clones. The DMRV critical region is defined by recombination events in the affected subjects, which are identified in this study.
Haplotype Analyses
As shown in the Table, we found that these families
share a common haplotype. Four Japanese families (Pedigrees 484, 544, 753, and 1548) shared the 7-2-4-3-9
haplotype at 270CA20, 274GT21, 834CA10,
792GT15, and D9S1791 in the homozygous state. Furthermore, all the pedigrees carry common alleles at
834CA10 and 792GT15, consistent with the location of
the GNE gene between 834CA10 and 792GT15 (see
Fig). These results further indicate a strong founder effect in these Japanese pedigrees, which is consistent with
the findings that all the unrelated Japanese DMRV pedigrees analyzed carry the novel mutation.
In this study, we found a novel mutation (V572L) in
the GNE gene in homozygous state, which is interestingly shared by all the six Japanese DMRV families. As
it has been strongly suggested by previous linkage analyses,16,19 this study confirmed that DMRV and HIBM
are allelic diseases caused by mutations in the GNE
gene. The mutation identified in the six Japanese pedigrees is a novel one that has not been described in
patients of other ethnic populations. The V572L mutation is located in the kinase domain, which is conserved among mammalian species as the other three
missense mutations.2 To date, several mutations in the
Table. Haplotypes Cosegregating with DMRV 1
DMRV ⫽ distal myopathy with rimmed vacuoles.
Haplotypes cosegregating with DMRV.
Annals of Neurology
Vol 52
No 4
October 2002
GNE gene have been identified in HIBM pedigrees,
which include G576E, A631T, V696M, C303X,
R246Q, D225N, and M712T in Middle Eastern Jewish pedigrees of typical HIBM.2
In this study, we found that a single mutation in the
kinase domain is shared by all the DMRV pedigrees
examined, which is consistent with the strong linkage
disequilibrium in the Japanese DMRV pedigrees (see
Table). In our patients carrying V572L, the mean age
at onset of the disease is 23.4 years old. Most of our
cases became nonambulant in their third or fourth decade within 10 years from the onset of the disease.
Moreover, two cases became nonambulant 6 years after
the onset, and the other two cases became bedridden in
their early fourth decade. Nonaka and colleagues also
showed that the patients with DMRV become nonambulant within an average of 12 years by analyzing 22
patients.8 As described above, most of the DMRV patients appear to develop severer consequences compared with those of typical HIBM patients who remain
ambulant even at the advanced stage. Note, however,
that one patient with DMRV in this study can still
walk with use of short leg braces and gave birth to a
healthy baby normally 6 years after the onset. Thus,
there appears to be heterogeneities in the severity of the
clinical presentations even in the patients with the
V572L. One patient of HIBM of non-Jewish origin in
India, who has compound heterozygous mutations in
epimerarse and kinase domains, showed more diffuse
muscle involvement and less tendency of quadricep sparing compared with typical cases of HIBM in Middle
Eastern Jews.20 Although several patients with HIBM/
DMRV have been described, only limited data are available as to the mutational analyses of these cases. Therefore, to discuss the geneotype–phenotype correlation,
and furthermore the effect of mutations on the kinase or
epimerase domain functions, accumulation of many
HIBM/DMRV patients with identified mutations will
be required. Functional analyses of the wild-type and
mutant GNE will also be important. Given the variety
of mutations involving the GNE gene, elucidation of the
pathophysiological mechanisms underlying HIBM and
DMRV, particularly those involved in vacuole formation
in muscles, is also highly expected.
This work was supported in part by a grant for the Research for the
Future Program from the Japan Society for the Promotion of Science,
and the Research Grant (11B-1) for Nervous and Mental Disorders
from the Ministry of Health, Labour and Welfare, Japan (K.T.).
We thank Dr I. Nonaka for his valuable advice.
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Arai et al: A Novel Mutation in the GNE Gene
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