Gene identification and functional analyses of genetically unsolved patients with neuromuscular disorders

在约45%的非5 q-脊髓性肌萎缩症（SMA）/下运动神经元疾病患者中，已确定了致病基因。在最近完成的EU-FP 7 Neuromics项目（2012-2017）中，我们招募了214名新的非5 q-SMA/LMND患者和216名家庭成员并进行了基因检测。我们根据HPO条款建立了严格的流程，并将临床数据存储到PhenoTips中。我们开发了一个NGS基因面板，包括479个已知的神经肌肉疾病（NMD）基因，并测试了91个索引病例。在40/91例（44%）病例中鉴定出致病性或可能致病性变异。24例未解决的病例以及52例新招募的患者使用短读NGS技术进一步进行外显子组或基因组测序，作为单例或与其他家族成员作为三人组或四人组。其中，我们解决了27/76（35%）。所有测序数据均可通过电子方式获得并存储在EGA数据库中。除了阐明已知致病基因中的变体外，我们还鉴定、功能表征并发表了六种新的NMD致病基因（BICD 2、VAMP 1、PIEZO 2、CHP 1、PRUNE和MCM 3AP）。在这个项目中，我们的目标是使用最终的测序策略，并进行trio/quattro WGS分析（N= 129）和/或RNAseq（N=16，一式三份），以确定43个未解决的病例的致病基因，其中NMD面板/WES失败。变异分析将在WGGC使用VarBank和新开发的基因组和RNA分析算法进行。在我们的常规诊断实验室中，我们不断使用NMD面板对新患者进行基因检测。此外，我们与土耳其和伊朗的神经科医生建立了良好的国际合作，他们不断为我们推荐非常有趣的、主要是具有NMD表型的近亲家庭。到2019年底，我们的目标是进一步纳入40个未解决的病例作为WES分析的三重/四重（Sure Select V7试剂盒），同时这仍然是识别蛋白质编码基因突变的最便宜和最快的方法。我们将测试这一策略的效力，如果成功，最终可能也会被纳入我们的常规诊断中。正如过去成功展示的那样，我们将验证候选变异，在我们自己的大数据集和全球数据集中搜索更多的家族。我们将对这些新的候选基因以及从神经组学项目中获得的两个有希望的候选基因GBF 1和CAPRIN 1进行功能分析。我们将进行生物化学，分子和细胞研究，并将使用IPSC，斑马鱼苍蝇和/或小鼠模型。这是一个简单，全面和医学高度相关的项目。每一个新的疾病基因都将加深我们对神经通路和回路的了解，并可能为罕见但也可能为复杂和年龄相关的神经病变开辟全新的治疗可能性，正如我们的出版记录所有力证明的那样。

In about 45% of patients with non-5q-spinal muscular atrophy (SMA)/lower motor neuron disorders, the disease-causing gene has been identified. Within the recently completed EU-FP7 NeurOmics project (2012-2017), we recruited and genetically tested 214 new patients with non-5q-SMA/LMND and 216 family members. We established a stringent pipeline based on HPO terms and stored the clinical data into PhenoTips. We developed an NGS gene panel including 479 known neuromuscular disease (NMD) genes and tested 91 index cases. Pathogenic or likely pathogenic variants were identified in 40/91 (44%) cases. 24 unsolved cases together with 52 newly recruited patients were further subjected to exome or genome sequencing using short-read NGS technology either as singletons or with further family members as trios or quattros. Of these, we solved 27/76 (35%). All sequencing data is publically available and stored within the EGA database. In addition to the elucidation of variants in known disease-causing genes, we identified, functionally characterized and published six novel NMD-causing genes (BICD2, VAMP1, PIEZO2, CHP1, PRUNE and MCM3AP). Two further strong candidates are in the pipeline to be characterized within this project.Here we aim to use the ultimate sequencing strategies and perform trio/quattro WGS analysis (N= 129) and/or RNAseq (N=16 in triplicates) to identify the causative gene for 43 unsolved cases in which either NMD panel/WES failed. Variant analysis will be carried out with the VarBank and newly developed algorithms for genome and RNA analysis at the WGGC.In our routine diagnostic lab, we are constantly genetically testing new patients using our NMD panel. Moreover, we established excellent international collaborations with neurologist in Turkey and Iran, who are constantly referring us very interesting, mainly consanguineous families with NMD phenotype. Until the end of 2019, we aim to further include 40 unsolved cases as trios/quattros for a WES analysis (Sure Select V7 kit), which meanwhile remains as the cheapest and fastest way to identify mutations in protein coding genes. We will test the power of this strategy, which - if successful- might ultimately be also included in our routine diagnosis.As successfully shown in past, we will validate the candidate variants, search for further families in our own sizable dataset and world-wide data sets. We will functionally analyze the novel candidate genes together with two promising candidate genes, GBF1 and CAPRIN1, obtained from the NeurOmics project. We will perform biochemical, molecular and cellular studies and will use IPSCs, zebrafish fly and/or mouse models.This is a straightforward, comprehensive and medically highly relevant project. Every new disease gene will deepen our knowledge on neuronal pathways and circuits and might open up completely new therapeutic possibilities for rare but maybe also for complex and age-related neuropathies, as strongly proven by our publication record.