Oculopharyngeal muscular dystrophy : studies on molecular pathology and molecular biology

眼咽肌营养不良症：分子病理学和分子生物学研究

• 批准号: 10670594
• 负责人: UYAMA Eiichiro
• 金额: $ 2.24万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670594/
• 关键词: oculo har n eal muscular d stro h; OPMD; poly (A) binding protein; PABP2; GCG repeats; intranuclear inclusion body; aniti-PABP2; polyclonal antibody; OPMD; PABP2; ポリクローナル抗体; GCG リピート; 表現型; 遺伝子型; 眼咽頭筋ジスロフィー; 14g11; リピート病; GCG異状伸長; ハプロタイプ解

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

眼咽肌营养不良症(OPMD)是一种常染色体显性遗传性疾病，以晚发的上睑下垂和吞咽困难为特征，并存在外径8.5 nm的核内管丝状包涵体(ITFI)。该基因座最近被定位在染色体14q11.2-13q的法裔加拿大家庭，他们的共同祖先于1634年从法国移民到魁北克。到目前为止，在世界上15个以上的白人社区中已经记录了OPMD家族的形态。作为一项合作研究，我们一直在与加拿大、法国和其他12个国家的研究小组继续克隆OPMD-基因。1998年，从染色体14q11上217kb的候选区间克隆了聚(A)结合蛋白2基因(PABP2)。在包括日本人在内的144个OPMD家系中，我们发现了两个不相关的眼咽肌营养不良症(OPM…)日本家系更多的D)，他们生活在相距较远的地区：熊本的1号家庭和静冈的2号家庭(神经病学46：773)。为了阐明这种疾病的表型-基因关系，我们对7个无关的日本家庭进行了临床遗传学调查，其中包括45名OPMD患者。我们分析了每个病例和91名日本对照个体的PABP2扩增产物。对这些序列进行测序，并使用片段管理器计算(GCG)n重复的数量。11例患者均经临床诊断为OPMD，7个家系中有5个家系经EM确诊。每个家系的基因分型如下：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=(GCG)6/(GCG)11个家系。虽然这些家系的临床表现略有不同，但我们没有发现紧密的表型/基因关系。90名日本对照个体(98.9%)的基因为(GCG)6/(GCG)6，另1个(1.1%)为(GCG)6/(GCG)7，与法裔加拿大人相似。因此，这些结果支持了我们之前的假设，即受影响的日本人可能是由PABP2的独立突变引起的。至少在(GCG)8到(GCG)11的范围内，PABP2的OPMD表型和(GCG)n扩展之间的联系仍然不确定。为了阐明其分子机制，我们提出了一种针对PABP2 cDNA合成多肽片段的抗血清，该多肽对应于271-291氨基酸，其中发生了一系列翻译后精氨酸甲基化。我们检测了5例OPMD患者、14例各种神经肌肉疾病患者和3例正常对照肌肉标本中PABP2的亚细胞定位。所有日本OPMD患者均显示PABP2基因扩增的(GCG)_lt；8、9或11&gt；突变，以及8.5 nm的核内小管丝状包涵体(ITFI)。多克隆PABP2免疫阳性染色仅见于OPMD患者的肌纤维核内集合体，阳性率为2%。相比之下，在正常或疾病对照组的任何样本中都没有检测到核免疫染色。结果表明，由于合成的抗原肽不识别天然PABP2的高度二甲基化的精氨酸残基，但识别突变形式，因此扩增的PABP2产物存在分子修饰。扩增的PABP2产物的核聚集暗示了ITFI的致病作用。较少

项目成果

Uyama E, et al.: "Autosomal recessive oculopharyngodistal myopathy in light of distal myopathy with rimmed vacuole and OPMD"Neuromusc Disord. 8. 119-125 (1998)

Uyama E 等人：“常染色体隐性遗传性眼咽远端肌病，伴有边缘空泡和 OPMD 的远端肌病”神经肌肉疾病。

Uyama E at al: "Oculopharyngeal muscular dystrophy (in Japanese)"Clinical Neuroscience. 78. 1152-1154 (1999)

Uyama E 等人：“眼咽肌营养不良症（日语）”临床神经科学。

Tkahashi T et.al: "A family with oculopharyngeal muscular dystrophy with (GCG) 9 expansion in which a sister had neck as well as proximal and her brother proximal lower limb muscle weakness"ClinicNeurol. 40. 911-914 (2000)

Tkahashi T 等人：“一个患有眼咽肌营养不良症 (GCG) 9 扩展的家庭，其中一个姐妹有颈部和近端肌肉无力，而她的兄弟则有下肢近端肌肉无力”ClinicNeurol。

Brais B, et al.: "Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy"Nature Genet. 18. 164-167 (1998)

Brais B 等人：“PABP2 基因中的短 GCG 扩展导致眼咽肌营养不良”Nature Genet。

宇山英一郎: "眼咽頭筋ジスロフィー(OPMD)/眼咽頭筋ミオパチー(印刷中)"日本臨床「骨格筋症候群」. (2001)

Eiichiro Uyama：“眼咽肌营养不良症（OPMD）/眼咽肌肌病（印刷中）”日本临床“骨骼肌综合征”（2001）。