Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston.
JAMA Neurol. 2015 Dec;72(12):1424-32. doi: 10.1001/jamaneurol.2015.2274.
To our knowledge, the efficacy of transferring next-generation sequencing from a research setting to neuromuscular clinics has never been evaluated.
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 xBrowse 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.
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.
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.
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.
据我们所知，将下一代测序从研究环境转移到神经肌肉诊所的功效尚未得到评估。
将外显子组测序（WES）转化为临床实践，用于对一组患有肢带型肌营养不良症（LGMD）的患者进行基因诊断，这些患者的蛋白质分析和靶向 Sanger 测序未能确定其疾病的遗传原因。
设计、地点和参与者：我们对 60 个 LGMD 家族（100 个外显子）进行了 WES。数据分析于 2014 年 1 月 6 日至 12 月 19 日进行，使用 xBrowse 生物信息学接口（Broad Institute）。LGMD 患者通过 2006 年至 2014 年之间的神经科学和肌肉研究生物样本库进行回顾性确定。入组患者通过蛋白质研究和候选基因测序进行了广泛的调查，但仍未确诊。患者表现出超过 2 年的肌肉无力，肌肉活检显示出营养不良或肌病变化。
澳大利亚 LGMD 的诊断率和不同 LGMD 亚型的相对频率。我们的主要目标是提高 LGMD 的基因诊断水平，研究 WES 平台是否为已知的 LGMD 相关基因提供了足够的覆盖范围，并确定新的 LGMD 相关基因。
通过 WES，我们在 60 个家庭中的 27 个家庭中发现了已知肌病基因的可能致病性突变。12 个家庭存在已知 LGMD 相关基因的突变。然而，15 个家庭的疾病相关基因中存在变体，这些基因通常与 LGMD 无关，突出了 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 个家庭）和胶原肌病相关基因（4 个家庭）的突变。不太常见的肌病包括代谢性肌病（2 个家庭）、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 综合征有关。纳入家庭成员提高了 WES 的诊断效果，“三联体”（受影响的先证者和父母双方）的诊断率为 60%，而单个先证者的诊断率为 40%。对靶向神经肌肉疾病相关基因面板未确诊的患者进行后续筛查并未提高我们的诊断率。
通过 WES，我们在难以诊断的 LGMD 患者队列中达到了 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，并强调了准确的临床检查和组织病理学结果对 WES 解释的重要性，许多诊断需要随访审查和对活检标本或血清样本的辅助检查。
