Oculopharyngeal muscular dystrophy : studies on molecular pathology and molecular biology

眼咽肌营养不良症（OPMD）是一种常染色体显性遗传疾病，其特征为迟发性上睑下垂和吞咽困难，以及存在外径为8.5 nm的核内管状丝状包涵体（ITFI）。该基因定位于1634年从法国移民到魁北克的法裔加拿大人的染色体14 q11.2 - 13 q上。迄今为止，在世界上已有15个以上的白色群体中有形态学证实的OPMD家系。我们与加拿大、法国等12个国家的研究小组合作，继续克隆OPMD基因。在1998年，多聚腺苷酸结合蛋白2基因（PABP 2）从染色体14 q11上的一个217 kb的候选区间。1996年，我们在包括日本家系在内的144个OPMD家系中发现了两个不相关的日本人眼咽型肌营养不良症（OPM）家系 ...更多信息 为了阐明该病的表型-基因型关系，我们对7个无关的日本家族进行了临床遗传学调查，其中包括45名患有OPMD的个体。我们分析了PCR扩增产物PABP 2在每种情况下，并在91个日本对照个体。对这些进行测序，并使用片段管理器对（GCG）n重复的数量进行计数。11例经临床诊断为OPMD，7个家系中有5个经电镜检查确诊。我们确定了每个受影响家庭的基因型如下：东京1个家系=（GCG）6/（GCG）8，大分和宫城2个无关家系=（GCG）6/（GCG）9，静冈1个家系=（GCG）6/（GCG）10，熊本2个家系和大分1个家系=（GCG）6/（GCG）11。90例日本对照个体（98.9%）的基因型为（GCG）6/（GCG）6，另1例（1.1%）的基因型为（GCG）6/（GCG）7。因此，结果支持我们先前的假设，即受影响的日本个体可能是由PABP 2的独立突变引起的。PABP 2的（GCG）n扩增与OPMD表型之间的关系至少在（GCG）8 ~（GCG）11范围内还不清楚。我们研究了5例OPMD患者，14例各种神经肌肉疾病患者和3例正常对照的肌肉标本中PABP 2的亚细胞定位。所有日本OPMD患者均显示PABP 2中的扩展（GCG）_<8、9或11>突变，以及8.5nm的核内管状丝状包涵体（ITFI）。多克隆PABP 2的阳性免疫染色仅限于OPMD患者肌纤维的核内聚集体，频率为2%。相反，在正常或疾病对照的任何样品中均未检测到核免疫染色。结果表明存在扩增PABP 2产物的分子修饰，因为合成抗原肽不识别天然PABP 2的精氨酸残基的高度二甲基化簇，但识别突变形式。PABP 2扩增产物的核积累暗示ITFI的致病作用。少

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 … More 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