Use of Whole-Exome Sequencing for Diagnosis of Limb-Girdle Muscular Dystrophy Outcomes and Lessons Learned
Use of Whole-Exome Sequencing for Diagnosis of Limb-Girdle Muscular Dystrophy Outcomes and Lessons Learned
复制标题
利用全基因组测序诊断肢腰肌营养不良症的结果和经验教训
DOI：
10.1001/jamaneurol.2015.2274
复制
发表时间：
2015-12-01
期刊：
影响因子：
29
通讯作者：
Clarke, Nigel F.
中科院分区：
文献类型：
--
作者：
Ghaoui, Roula;Cooper, Sandra T.;Clarke, Nigel F.
据我们所知，下一代测序从研究环境转移到神经肌肉诊所的功效从未得到评估。目的将全外显子组测序 (WES) 转化为临床实践，对一大群肢带型肌营养不良症 (LGMD) 患者进行基因诊断，基于蛋白质的分析和靶向桑格测序未能确定其疾病的遗传原因。设计、设置和参与者我们对 60 个家庭进行了 WES与 LGMD（100 个外显子组）。数据分析于2014年1月6日至12月19日期间使用x Browse生物信息学界面（Broad Institute）进行。 LGMD 患者是通过神经科学研究所和肌肉研究生物样本库在 2006 年至 2014 年间进行回顾性确定的。纳入的患者已通过蛋白质研究和候选基因测序进行了广泛的研究，但仍未得到诊断。患者出现超过 2 年的肌肉无力，并且肌肉活检标本中出现营养不良或肌病性变化。 主要结果和措施 澳大利亚 LGMD 的诊断率以及不同 LGMD 亚型的相对频率。我们的中心目标是改进 LGMD 的基因诊断，调查 WES 平台是否充分覆盖已知的 LGMD 相关基因，并识别新的 LGMD 相关基因。 结果 通过 WES，我们在 60 个家族中的 27 个家族中识别出了已知肌病基因中可能的致病突变。 12 个家族的已知 LGMD 相关基因发生突变。然而，15 个家族存在通常与 LGMD 无关的疾病相关基因变异，这凸显了 LGMD 与其他肌病之间的临床重叠。表型重叠的常见原因是先天性肌营养不良症相关基因（4个家族）和胶原蛋白肌病相关基因（4个家族）的突变。不太常见的肌病包括代谢性肌病（2 个家族）、先天性肌无力综合征 (DOK7)、先天性肌病 (ACTA1)、管状聚集性肌病 (STIM1)、肌原纤维肌病 (FLNC) 和 CHD7 突变（通常与 CHARGE 综合征相关）。家庭成员的纳入提高了 WES 的诊断效率，“三人组”（父母双方均受影响的先证者）的诊断率为 60%，而单身先证者的诊断率为 40%。对目标神经肌肉疾病相关基因组未确诊的患者进行后续筛查并没有提高我们的诊断率。结论和相关性通过 WES，我们在难以诊断的 LGMD 患者队列中实现了 45.0% 的诊断成功率。我们扩展了与已知肌病基因相关的临床表型，并强调准确的临床检查和组织病理学结果对于解释 WES 的重要性，许多诊断需要对活检标本或血清样本进行后续审查和辅助研究。
IMPORTANCE To our knowledge, the efficacy of transferring next-generation sequencing from a research setting to neuromuscular clinics has never been evaluated.OBJECTIVE To translate whole-exome sequencing (WES) to clinical practice for the genetic diagnosis of a large cohort of patients with limb-girdle muscular dystrophy (LGMD) for whom protein-based analyses and targeted Sanger sequencing failed to identify the genetic cause of their disorder.DESIGN, SETTING, AND PARTICIPANTS We performed WES on 60 families with LGMDs (100 exomes). Data analysis was performed between January 6 and December 19, 2014, using the x Browse bioinformatics interface (Broad Institute). Patients with LGMD were ascertained retrospectively through the Institute for Neuroscience and Muscle Research Biospecimen Bank between 2006 and 2014. Enrolled patients had been extensively investigated via protein studies and candidate gene sequencing and remained undiagnosed. Patients presented with more than 2 years of muscle weakness and with dystrophic or myopathic changes present in muscle biopsy specimens.MAIN OUTCOMES AND MEASURES The diagnostic rate of LGMD in Australia and the relative frequencies of the different LGMD subtypes. Our central goals were to improve the genetic diagnosis of LGMD, investigate whether the WES platform provides adequate coverage of known LGMD-related genes, and identify new LGMD-related genes.RESULTS With WES, we identified likely pathogenic mutations in known myopathy genes for 27 of 60 families. Twelve families had mutations in known LGMD-related genes. However, 15 families had variants in disease-related genes not typically associated with LGMD, highlighting the clinical overlap between LGMD and other myopathies. Common causes of phenotypic overlap were due to mutations in congenital muscular dystrophy-related genes (4 families) and collagen myopathy-related genes (4 families). Less common myopathies included metabolic myopathy (2 families), congenital myasthenic syndrome (DOK7), congenital myopathy (ACTA1), tubular aggregate myopathy (STIM1), myofibrillar myopathy (FLNC), and mutation of CHD7, usually associated with the CHARGE syndrome. Inclusion of family members increased the diagnostic efficacy of WES, with a diagnostic rate of 60% for "trios" (an affected proband with both parents) vs 40% for single probands. A follow-up screening of patients whose conditions were undiagnosed on a targeted neuromuscular disease-related gene panel did not improve our diagnostic yield.CONCLUSIONS AND RELEVANCE With WES, we achieved a diagnostic success rate of 45.0% in our difficult-to-diagnose cohort of patients with LGMD. We expand the clinical phenotypes associated with known myopathy genes, and we stress the importance of accurate clinical examination and histopathological results for interpretation of WES, with many diagnoses requiring follow-up review and ancillary investigations of biopsy specimens or serum samples.