BACKGROUND As genome-wide approaches prove difficult with genetically heterogeneous orphan diseases, we developed a new approach to identify candidate genes. We applied this to Emery-Dreifuss muscular dystrophy (EDMD), characterised by early onset contractures, slowly progressive muscular wasting, and life-threatening heart conduction disturbances with wide intra- and inter-familial clinical variability. Roughly half of EDMD patients are linked to six genes encoding nuclear envelope proteins, but the disease mechanism remains unclear because the affected proteins function in both cell mechanics and genome regulation. METHODS A primer library was generated to test for mutations in 301 genes from four categories: (I) all known EDMD-linked genes; (II) genes mutated in related muscular dystrophies; (III) candidates generated by exome sequencing in five families; (IV) functional candidates - other muscle nuclear envelope proteins functioning in mechanical/genome processes affected in EDMD. This was used to sequence 56 unlinked patients with EDMD-like phenotype. FINDINGS Twenty-one patients could be clearly assigned: 18 with mutations in genes of similar muscular dystrophies; 3 with previously missed mutations in EDMD-linked genes. The other categories yielded novel candidate genes, most encoding nuclear envelope proteins with functions in gene regulation. INTERPRETATION Our multi-pronged approach identified new disease alleles and many new candidate EDMD genes. Their known functions strongly argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction due to connectivity of candidates from the nuclear envelope to the plasma membrane. This approach highlights the value of testing for related diseases using primer libraries and may be applied for other genetically heterogeneous orphan diseases. FUNDING The Wellcome Trust, Muscular Dystrophy UK, Medical Research Council, European Community's Seventh Framework Programme "Integrated European -omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)".

中文翻译：

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多阶段测序策略确定了 Emery-Dreifuss 肌营养不良症的新候选等位基因，并支持将基因失调作为其发病机制。

背景由于全基因组方法证明难以处理遗传异质性孤儿疾病，我们开发了一种新方法来鉴定候选基因。我们将此应用于 Emery-Dreifuss 肌营养不良症 (EDMD)，其特征是早发性挛缩、缓慢进行性肌肉萎缩和危及生命的心脏传导障碍，具有广泛的家族内和家族间临床变异性。大约一半的 EDMD 患者与编码核膜蛋白的六个基因有关，但疾病机制仍不清楚，因为受影响的蛋白质在细胞力学和基因组调控中都起作用。方法 生成引物文库以测试来自四类的 301 个基因的突变：（I）所有已知的 EDMD 连锁基因；(II) 相关肌营养不良的基因突变；（三）5个家族的外显子组测序产生的候选者；(IV) 功能候选者——在受 EDMD 影响的机械/基因组过程中起作用的其他肌肉核包膜蛋白。这用于对 56 名具有 EDMD 样表型的未关联患者进行测序。发现 21 名患者可以明确归类： 18 名具有类似肌营养不良症的基因突变；3 之前遗漏了 EDMD 相关基因的突变。其他类别产生了新的候选基因，大多数编码具有基因调控功能的核膜蛋​​白。解释 我们的多管齐下的方法确定了新的疾病等位基因和许多新的候选 EDMD 基因。他们已知的功能强烈认为 EDMD 病理机制是由于候选者从核包膜到质膜的连通性而改变的基因调控和机械转导。这种方法突出了使用引物库检测相关疾病的价值，并可应用于其他遗传异质性孤儿疾病。资助 Wellcome Trust、英国肌肉萎缩症、医学研究委员会、欧洲共同体第七框架计划“用于诊断和治疗罕见神经肌肉和神经退行性疾病 (NEUROMICS) 的欧洲综合组学研究项目”。