| Research Abstract |
Oculopharyngeal muscular dystrophy (OPMD), an autosomal dominant disorder characterized by late-onset ptosis and dysphagia, and the presence of intranuclear tubulofilamentous inclusions (ITFI) of 8.5 nm outer diameter. The gene locus has recently mapped to chromosome 14q11.2-13q in French Canadian families, whose common ancestor emigrated from France to Quebec in 1634.
Thus far morphologically-confirmed OPMD families have been documented in more than 15 white communities around the world. We has been continued cloning the OPMD-gene with research groups in Canada, France, and other 12 contries as a collaborate study. In 1998, the poly(A) binding protein 2 gene (PABP2) from a 217-kb candidate interval on chromosome 14q11. A (GCG)6 repeat encoding a polyalanine tract located at the N terminus of the protein was expanded to (GCG)8-13 in the 144 OPMD families including our Japanese families.
In 1996, we have identified two unrelated Japanese families of oculopharyngeal muscular dystrophy (OPM
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D), who live in areas distant from each other : Family 1 in Kumamoto and Family 2 in Shizuoka (Neurology 46 : 773).
To clarify the phenotype-genotye relationship in this disease, we perfollned clinico-genetic investigations for 7 unrelated Japanese families including 45 affected individuals of OPMD.Genomic DNA was isolated from blood samples under informed consent. We analysed PCR amplified products for PABP2 in each case, and in 91 Japanese control individuals. Those were sequenced and the number of the (GCG)n repeats were also counted using a Fragment Manager. A11 cases were diagnosed as having OPMD on clinical grounds and 5 of 7 families were confirmed on EM findings. We identified the genotype in each affected family as following : one family in Tokyo =(GCG)6/(GCG)8, two unrelated families in Oita and Miyagi =(GCG)6/(GCG)9, one family in Shizuoka=(GCG)6/(GCG)10, and two families in Kumamoto and one family in Oita=(GCG)6/(GCG) 11.Although there were several minor differences on clinical aspects among affected families originated unrelated ancestors, we failed to find tight phenotype/genotype relationship.
The genotype of 90 Japanese control individual (98.9% ) was (GCG)6/(GCG)6, and another one (1.1%) was (GCG)6/(GCG)7 as similar to those of French-Canadian populations. Thus, the results collaborate our previous hypothesis that the affected Japanese individuals maybe caused by indipendent mutations of PABP2. The ship between OPMD phenotype and (GCG)n expansions of PABP2 remains uncertain at least in the range from (GCG) 8to (GCG) 11.
To elucidate the molecular mechanism, we raised an antiserum against a synthetic peptide fragment predicted from PABP2 cDNA.The peptide corresponded to amino acids 271-291 where a cluster of post-translational arginine methylation occurrs. We examined the subcellular localization of PABP2 in muscle specimens from 5 patients with OPMD, 14 patients with various neuromuscular disorders, and 3 normal controls. All Japanese OPMD patients revealed expanded (GCG)_<8, 9 or 11> mutations in PABP2, as well as intranuclear tubulofilamentous inclusions (ITFI) of 8.5 nm. Positive immunostaining for polyclonal PABP2 was confined to the intranuclear aggregates of muscle fibers exclusively in OPMD patients, with a frequency of 2%. In contrast, nuclear immunostaining was not detected in any samples from normal or disease controls. The results suggest the presence of molecular modification of the product of expanded PABP2, since the synthetic antigen peptide did not recognize a highly dimethylated cluster of arginine residues of the native PABP2, but did recognize the mutated form. Nuclear accumulation ofexpanded PABP2 product implies a causative role for ITFI. Less
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