RNF170 - a novel disease gene causing Hereditary Spastic Paraplegia

Abstract

Hereditary spastic paraplegias are a clinically and genetically extremely heterogenous group of monogenic neurodegenerative diseases. Their most prominent symptom is a slowly progressing lower limb spasticity and weakness caused by length dependent axonopathy of the upper motor neurons. Clinical classification distinguishes between pure and complicated forms of HSP. The latter variably includes additional symptoms like optic atrophy, cognitive impairment, ataxia and peripheral neuropathy. Mutations in more than 80 genes are known to cause autosomal dominant, recessive, X-linked and mitochondrial forms of the disease. Nevertheless, about one third of HSP cases remain unexplained by mutations in these known genes. In this study we aimed at the identification of novel genes causing HSP in order to solve undiagnosed families and improve our understanding of the functional networks leading to axon degeneration. Thus we used WGS and WES data from a cohort of 142 individuals suffering from HSP without a molecular diagnosis. We used the GENESIS platform to apply a stepwise variant filtering approach on these NGS datasets and consecutively performed segregation analysis of top-ranked candidate variants. This approach led to identification of a novel candidate disease gene (RNF170) in an autosomal recessive family with two affected siblings. To confirm the functional effect of the c.396+3A>G variant, which was predicted to affect splicing, we performed RNA studies on patient peripheral blood cells and fibroblasts which demonstrated an aberrant transcript lacking exon 5 resulting in absent or a truncated protein. Accordingly, qRT-PCR showed significantly reduced RNF170 expression levels in both cell types and western blot in patient fibroblasts showed no residual RNF170 protein. As RNF170 works as an E3 ubiquitin ligase which targets IP3Rs for proteasomal degradation we performed additional western blots for IP3R3 in patient fibroblasts to gain more functional evidence about the consequences of RNF170 deficiency. Thereby we could demonstrate that loss of RNF170 impairs both basal and stimulation-dependent turnover of IP3Rs in mutant fibroblasts, resulting in significant IP3R accumulation and indicating a possible effect of RNF170 deficiency on IP3R-dependent Ca2+ signaling. To provide additional genetic proof for the pathogenetic relevance of the newly discovered gene we performed genetic matchmaking via the GeneMatcher collaboration platform and identified three more families with seven affected patients carrying biallelic loss of function mutations in RNF170. Due to the growing number of publications which associated HSP as well as ADSA with RNF170 mutations, this gene should be included in diagnostic panels for both ataxia and HSP. If the functional impact seen on IP3R signaling in fibroblasts effectively transfers to Ca2+ homeostasis as well has yet to be examined. Moreover, further functional experiments in a suitable neuronal cell model, for example patient-derived iPSCs which can be differentiated to post mitotic-neuronal cells, are required. Taken together the growing number of neurodegenerative diseases in which dysregulation of IP3R-dependent ER calcium release play an essential role emphasize that IP3R-dependent Ca2+ signaling may be a promising pathway for future targeted therapies in HSP and other diseases.