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

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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.