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Unequal Crossing-over in Unique PABP2 Mutations in Japanese Patients
A Possible Cause of Oculopharyngeal Muscular Dystrophy
Mika Nakamoto, MD, PhD;
Satoshi Nakano, MD, PhD;
Shingo Kawashima, MD, PhD;
Masafumi Ihara, MD;
Yo Nishimura, MD;
Akiyo Shinde, MD;
Akira Kakizuka, MD, PhD
Arch Neurol. 2002;59:474-477.
ABSTRACT
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Background Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset autosomal
dominant muscle disease with a worldwide distribution. Recent findings reveal
the genetic basis of this disease to be mutations in the polyA binding–protein
2 (PABP2) gene that involve short expansions of the
GCG trinucleotide repeat encoding a polyalanine tract. The underlying mechanism
causing the triplet-expansion mutation in PABP2 remains
to be elucidated, although the DNA slippage model is thought to be a plausible
explanation of that.
Methods and Results We analyzed PABP2 using polymerase chain reaction
analysis and DNA sequencing in Japanese patients with pathologically confirmed
OPMD, and found mutated (GCG)6GCA(GCG)3(GCA)3GCG
and (GCG)6(GCA)3(GCG)2(GCA)3GCG
alleles instead of the normal (GCG)6(GCA)3GCG allele.
These mutated alleles could be explained by the insertions or duplications
of (GCG)3GCA and (GCG)2(GCA)3, respectively,
but not by the simple expansion of GCG repeats. The clinical features of our
patients were compatible with those of other Japanese patients carrying PABP2 that encodes a polyalanine tract of the same length,
but were not compatible with those of Italian patients.
Conclusions The mutated alleles identified in our Japanese patients with OPMD were
most likely due to duplications of (GCG)3GCA and (GCG)2(GCA)3 but not simple expansions of the GCG repeats. Therefore, unequal crossing-over
of 2 PABP2 alleles, rather than DNA slippage, is
probably the causative mechanism of OPMD mutations. All mutations that have
been reported in patients with OPMD so far can be explained with the mechanism
of unequal crossing-over. On the other hand, comparison of the clinical features
of our patients with those of other patients in previous reports suggests
that specific clinical features cannot be attributed to the length of the
polyalanine tract per se.
INTRODUCTION
OCULOPHARYNGEAL muscular dystrophy (OPMD) is a distinct clinicopathologic
category of late-onset autosomal dominant muscle disorders. The disease is
characterized by progressive dysphagia, ptosis, and limb muscle weakness.1 The hallmark of OPMD is the accumulation of 8.5-nm
tubulofilamentous inclusions within the nuclei of skeletal muscle fibers.2
Genetically, short (GCG)8-13 expansions of a (GCG)6 repeat located in exon 1 of the polyA binding–protein 2 (PABP2) gene were determined to be the genetic basis of
this disease in 1998.3 This gene encodes PABP2,
a nuclear protein known to bind to polyA tails of messenger RNAs and to control
their length.4-5 In the normal
allele, (GCG)6 is followed by a (GCA)3GCG nucleotide
sequence. Since the GCG and GCA codons are translated into alanine residues,
the normal sequence is translated into a stretch of 10 alanine residues. In
the diseased allele, (GCG)6 is expanded to (GCG)8-13,
resulting in an elongation of the length of the polyalanine tract to 12 to
17 tandem repeats of alanine in the PABP2 protein. It has been suggested that
these polyalanine expansions induce aggregation of the PABP2 protein into
insoluble inclusions. Recent studies have shown that the 8.5-nm tubulofilamentous
nuclear inclusions found in OPMD are composed of the PABP2 protein.6 The disease is thought to be a consequence of the
toxic effects of the aggregates or of the dysfunction of the protein.6
Our report serves 2 purposes. First, the underlying mechanism causing
the triplet-expansion mutation in PABP2 remains to
be elucidated, although the DNA slippage model is thought to be a plausible
explanation. We analyzed PABP2 in patients from 2
unrelated Japanese families with OPMD who had OPMD-specific nuclear inclusions
in their skeletal muscle fibers, and found 2 unique mutations. Our findings
show that the arrangements of the nucleotide sequences detected in our patients
indicated that unequal crossing-over could be a mechanism causing the triplet-repeat
expansion in OPMD.
Second, in many other triplet-repeat disorders, a gene-dosage effect
and an inverse relationship between the age of onset as well as clinical severity
of the disease and the length of the pathologically expanded repeat have been
reported.7 With regard to a gene-dosage effect
in OPMD, studies in the 2 largest clusters (French Canadians and Bukhara Jews)
showed that patients who were homozygous for the (GCG)9(GCA)3GCG allele exhibited more severe clinical features than those who were
heterozygous for the mutation.3, 8
However, it is unclear whether such an inverse relationship exists in OPMD.
This uncertainty is partially due to the 2 largest clusters of OPMD, which
are almost homogeneous for the mutation, probably resulting from a founder
effect.3, 8-9 We investigated
the relationship between genotype and phenotype in our patients.
SUBJECTS AND METHODS
REPORT OF CASES
The first patient (patient A) was a 65-year-old man born in Hyogo Prefecture,
Japan. At 55 years of age, a swallowing disturbance developed. At 60 years
of age, he noticed ptosis on both sides, which gradually worsened. Results
of an examination showed moderate ptosis and mild weakness of the facial and
pharyngeal muscles. Extraocular muscles showed no weakness. He exhibited moderate
symmetrical weakness of the proximal lower limb muscles. Results of laboratory
examination showed a 6-fold increase in serum creatine kinase (CK) level (843
U/L; reference value, <141 U/L). His father had had dysphagia and dysarthria
since the sixth decade of life. A sister of patient A had had dysphagia since
her sixth decade of life, whereas a brother had no neuromuscular symptoms.
The second patient (patient B) was a 69-year-old woman born in Shiga
Prefecture, Japan. She had no neuromuscular symptoms until 61 years of age,
when she noticed ptosis on both sides. The symptoms progressed gradually,
and she underwent an operation for it at 65 years of age. At 66 years of age,
easy fatigability on walking, swallowing difficulty, and speech disturbance
developed. Results of an examination disclosed ptosis that completely covered
the pupils. Medium grade of ophthalmoparesis was noted in all directions.
Eye and mouth closures were moderately impaired. She had moderate symmetrical
muscle weakness of the proximal limb and neck muscles. Her serum CK level
was 300 U/L. She was separated from her parents in early childhood, which
hindered confirmation of parental symptoms. She had a sister with ptosis and
a child without neuromuscular symptoms.
In patients A and B, results of muscle biopsy revealed the occasional
occurrence of muscle fibers containing rimmed vacuoles. Results of electron
microscopic studies showed intranuclear tubulofilamentous inclusions with
a diameter of 8.5 nm, which is a characteristic of OPMD.
METHODS
We analyzed PABP2 in both index patients and
the 2 siblings of patient A after obtaining informed consent.
Biopsy specimens of muscle tissue from patients A and B were stored
at -70°C and were then cut into 5- to 10-mg sections using a razor.
The sections of muscle tissue were incubated in a solution containing 50mM
Tris hydrochloride (pH, 8.0), 10mM EDTA, 100mM sodium chloride, 0.5% sodium
dodecyl sulfate, and 1-mg/mL proteinase K (Boehringer Mannheim, Mannheim,
Germany) for 5 hours at 50°C. After phenol extraction, genomic DNA was
precipitated using 99% ethanol, then dissolved in a solution consisting of
10mM Tris hydrochloride (pH, 7.5) and 0.1mM EDTA. Peripheral blood samples
were obtained from the 2 siblings of patient A and Japanese control subjects.
Genomic DNA was extracted from the samples with the use of a blood kit (QIAamp;
QIAGEN, Hilden, Germany) according to the manufacturer's instructions.
Polymerase chain reaction analysis was performed in 50-µL volumes
containing 100 ng of genomic DNA, 250µM of each dNTP (ie, doxyadenosine
triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and
deoxythymidine triphosphate), 1µM of the forward 5'-CGCAGTGCCCCGCCTTAGAGGTG-3'
and backward 5'-ACAAGATGGCGCCGCCGCCCCGGC-3' primers, 1.25 U of Taq DNA polymerase (TaKaRa LA Taq;
TaKaRa, Kyoto, Japan) and its specific reaction buffer (GC Buffer II; TaKaRa).
After initial denaturation at 95°C for 5 minutes, amplification was performed
in 30 cycles consisting of denaturation at 95°C for 18 seconds, annealing
at 70°C for 30 seconds, and extension at 74°C for 40 seconds. The
final extension proceeded at 74°C for 10 minutes. Products were separated
on a 5% nondenaturing polyacrylamide gel. After electrophoresis, the gels
were stained using ethidium bromide. Allele-specific bands were excised from
the gels, eluted, and subcloned into a plasmid vector (pGEM-T Easy Vector;
Promega, Madison, Wis). Sequencing of the normal and mutated fragments was
performed bidirectionally using a dideoxy sequencing kit (Amplicycle; Applied
Biosystems, Foster City, Calif) on at least 6 clones for every allele, for
validation. The sequences obtained were compared with the genomic sequence
of the human PABP2 gene (accession number AF026029;
GenBank, Bethesda, Md; available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi).
RESULTS
Sequence analyses disclosed a 12–base pair (bp) elongation in
patient A and his affected sister and a 15-bp elongation in patient B (Figure 1) within the (GCG)6(GCA)3 GCG normal sequence in exon 1 of PABP2. The
mutated sequence detected in patient A and his sister was (GCG)6GCA(GCG)3(GCA)3GCG, and that in patient B was (GCG)6 (GCA)3(GCG)2(GCA)3GCG. Both sequences could be explained
by the insertions or duplications of (GCG)3GCA and (GCG)2(GCA)3, respectively, into the normal sequence. The normal
sequence in PABP2 is translated into a series of
10 alanine residues. The mutations in our patients increase the number of
alanine residues encoded from 10 to 14 in patient A and his sister, and to
15 in patient B. All of the patients were heterozygous for the mutated and
the normal alleles. The unaffected brother of patient A was homozygous for
the normal allele.
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Figure 1. Mutant sequences found in Japanese
patients with oculopharyngeal muscular dystrophy. Electropherograms of alleles
of the polyA binding–protein 2 gene show normal allele, mutant allele
detected in patient A (mutation A), and that detected in patient B (mutation
B). Open circles represent GCG triplets; filled circles, GCA triplets.
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In addition to the duplications, we observed in all patients and 10
Japanese controls a CG-to-GGGC change in position 1146, a G deletion in position
1215, a GG insertion in position 1229, and an ATC-to-CAT change in position
1250 (Figure 2). The latter 2 changes
have previously been described in an Italian population.10
All 4 changes are located in the 5'-untranslated region of PABP2 and were present in all healthy and affected Japanese subjects
studied. This indicates that they may be polymorphisms; the former two are
characteristic to date of the Japanese population and the latter two are shared
to date by the Japanese and Italian populations.10
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Figure 2. Schematic representation of the
exon 1 structure of the polyA binding–protein 2 gene. The horizontal
line and the box indicate the 5'-untranslated region and the coding
region of the exon 1, respectively. Positions of the 4 putative polymorphisms
identified in the Japanese population are represented by vertical lines in
the 5'-untranslated region. The filled portion of the box indicates
the location of the sequence depicted in Figure 1; arrows, the positions of
the polymerase chain reaction primers used in this study.
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COMMENT
MECHANISM OF REPEAT EXPANSIONS IN OPMD
Except for the mutation found in Cajun patients who had a (GCG)6GCA (GCG)2(GCA)3GCG mutated allele,11
all of the PABP2 mutations reported so far in patients
with OPMD were expansions of the (GCG)6 repeat. These expansions
make a (GCG)8-13(GCA)3GCG allele from the (GCG)6(GCA)3GCG normal sequence.3
We found 2 novel mutations, (GCG)6GCA(GCG)3(GCA)3GCG and (GCG)6(GCA)3(GCG)2(GCA)3GCG, in Japanese patients with OPMD.
The molecular mechanism causing the repeat expansion in PABP2 has not been determined. In general, the following 2 types of
mechanisms leading to the generation of longer DNA sequences have been proposed:
replication- and recombination-associated expansion.12
Repeat expansions that involve the process of DNA replication may originate
from slipped mispairing between repeated sequences, as has been described
for the slippage model.13 It has been shown
recently that expanded triplet repeats are responsible for a number of hereditary
neuromuscular diseases.7, 14-15
These pathologic repeat expansions can be explained by the slippage model.
However, it has been proposed that tracts of approximately 25 to 35 perfect
trinucleotide repeats are required for instability and expansion via slippage.16 The heterogeneous sequences of the mutated alleles
of PABP2 detected in the Cajun patients and our Japanese
patients and the fact that even the longest perfect repeat reported (13 repeats)
is less than 25 repeats argue against the slippage model.
Recombination-associated repeat expansion may result from homologous
recombination, which occurs in germ cells during meiosis and sometimes during
mitosis.12 The mutations that we found suggest
that the molecular mechanism resulting in generation of longer DNA sequences
in PABP2 is unequal crossing-over (Figure 3), which is a kind of homologous recombination. The (GCG)6(GCA)3GCG normal allele was reported to be found in 98%
and 99% of French Canadian and Japanese control chromosomes, respectively,
whereas the rest of both populations carried a (GCG)7(GCA)3GCG polymorphism (Figure 3A).3, 17 Unequal pairing with variable degrees
of overlap can generate each of the (GCG)8-12(GCA)3GCG
mutant alleles by crossing-over of the 2 (GCG)6(GCA)3
GCG normal alleles (Figure 3B).
At most, a (GCG)13(GCA)3GCG allele can be derived by
unequal crossing-over of the (GCG)6(GCA)3GCG normal
allele and the (GCG)7(GCA)3GCG polymorphic allele. This
mechanism can also explain the (GCG)6GCA(GCG)2(GCA)3 GCG mutation reported in Cajun patients (Figure 3C)11 and the (GCG)6GCA(GCG)3(GCA)3GCG (Figure 3D) and (GCG)6(GCA)3(GCG)2(GCA)3GCG mutations found in our patients (Figure 3E). Since the (GCG)8-10(GCA)3 GCG
pathologic expansions in PABP2 are reported to be
stable with no variation among family members and between such different tissues
as blood and skeletal muscle in the same individual,10
we suspect that unequal crossing-over occurred once at meiosis in an ancestor
of each patient with OPMD. Similar expansions of cryptic repeats composed
of mixed synonymous codons causing a polyalanine expansion in the homeobox
D13 (HOXD13) protein has been found to cause synpolydactyly.18
The HOXD13 mutation has been explained by unequal
crossing-over.19
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Figure 3. Schematic representation of GCG
and GCA repeats found in the polyA binding–protein 2 (PABP2) gene. Open circles represent GCG triplets; filled circles, GCA
triplets; Xs, possible points of the DNA crossing-over; and underlines, duplicated
nucleotides. A, Normal and polymorphic alleles of PABP2 found in healthy individuals consist of alanine-encoding 10- and 11-triplet
repeats, respectively. B-E, All of the reported mutant PABP2 alleles can be generated by unequal crossing-over.
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GENOTYPE/PHENOTYPE RELATIONSHIP IN OPMD
The muscle degeneration seen in OPMD may be a consequence of the toxic
effects of the aggregates caused by the expanded polyalanine tracts, or of
the altered properties of the mutated PABP2 protein.6
In both cases, the length of the polyalanine tract may be a key determinant
of the effect. The (GCG)6(GCA)3GCG sequence in the normal PABP2 gene is translated into 10 alanine residues in the
protein. The mutated alleles in our patients encode 14 and 15 alanine residues
instead of 10. In that case, the number of alanine residues in our patients
are equivalent to those reported to be generated by the (GCG)10(GCA)3GCG and (GCG)11(GCA)3GCG expanded alleles, respectively.
All of our patients were heterozygous for the mutations. To our knowledge,
only 1 Japanese family and 2 Italian families heterozygous for the (GCG)10(GCA)3GCG allele and 2 Japanese families heterozygous for
the (GCG)11(GCA)3GCG allele have been described clinically
and genetically.10, 20-21
According to the reports, dysphagia with moderately increased CK levels and
proximal myopathy of the lower legs initially developed in the Japanese patients
heterozygous for the (GCG)10(GCA)3GCG allele.20 Ptosis first developed in the 2 Japanese families
heterozygous for the (GCG)11(GCA)3GCG allele, whereas
their CK levels were generally within normal limits or only mildly increased.20-21 These clinicogenetic correlations
are compatible with those of our patients, especially with respect to the
initial symptoms and CK levels. However, both Italian families heterozygous
for the (GCG)10(GCA)3GCG allele presented with ptosis
as the first symptom, with later development of dysphagia and severe weakness
of limb and pelvic girdle muscles.10 Therefore,
the specific clinical feature perhaps cannot be attributed to the length of
the polyalanine tract of PABP2 per se. Although these
observations argue that the pathologic characteristics of OPMD may be the
result of nucleotide configuration, intrapopulational similarities of phenotype
among the Japanese and among the Italian patients are more likely to be due
to the genetic background of each race. Further case accumulation is needed
to clarify the relationship between genotype and phenotype in OPMD.
AUTHOR INFORMATION
Accepted for publication August 9, 2001.
Author contributions: Study
concept and design, analysis and interpretation of data, and obtained funding (Drs Nakamoto and Kakizuka); acquisition of data (Drs Nakamoto, Nakano, Kawashima, Ihara, Nishimura, and Shinde); drafting of the manuscript (Dr Nakamoto); critical revision of the manuscript for important intellectual content
(Drs Nakamoto, Nakano, Kawashima, Ihara, Nishimura, Shinde, and Kakizuka);
and administrative, technical, and material support and
study supervision (Dr Kakizuka).
This work was supported by research fellowships from the Japan Society
for the Promotion of Science for Young Scientists, Tokyo (Dr Nakamoto), and
by CREST, Japan Science and Technology Corporation, Saitama.
We thank Akiko H. Popiel for the proofreading.
Corresponding author and reprints: Akira Kakizuka, MD, PhD, Laboratory
of Functional Biology, Graduate School of Biostudies, Kyoto University, Yoshida
Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan (e-mail: kakizuka{at}lif.kyoto-u.ac.jp).
From the Fourth Department, Osaka Bioscience Institute, Osaka (Drs
Nakamoto and Kakizuka), and the Departments of Neurology, Kyoto University
Faculty of Medicine, Kyoto (Drs Nakano, Kawashima, Ihara, and Shinde), and
Nishikobe Medical Center, Hyogo (Dr Nishimura), Japan. Dr Nakamoto is now
with the Department of Neurology, Emory University Graduate School of Arts
and Sciences, Atlanta, Ga; Dr Nakano, the Department of Neurology, Kansai
Medical University, Osaka, Japan; and Dr Kakizuka, Laboratory of Functional
Biology, Graduate School of Biostudies, Kyoto University.
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