High-throughput functional characterization of variants of uncertain significance and systematic analysis of tandem repeat expansions to reveal hidden genetic factors in neurodegenerative disorders

Neurodegenerative disorders (NDDs) are characterized by the selective loss of neurons in motor, sensory, or cognitive systems. Familial cases suggest a genetic basis for NDDs, but a significant portion of heritability remains unexplained. This study investigates novel genetic variants and repeat expansions underlying NDD pathogenesis. Variants of uncertain significance (VUS) identified from whole genome sequencing (WGS) of 140 patients, were functionally assessed using two high-throughput in vitro assays: Massively Parallel Reporter Assay (MPRA) and MNeuassively Parallel Splicing Assay (MaPSy). MPRA analyzed, in parallel, a list of putative regulatory variants mapped in a panel of known NDD genes, identifying five genes (ELOVL5, GIGYF2, OMA1, CWF19L1, and NEK1) that significantly affected gene expression in our model. MaPSy, applied after SpliceAI in silico predictions, validated 14/17 predicted splicing variants and uncovered 15 additional aberrant splicing events, emphasizing the importance of functional validation for VUS. To investigate tandem repeat polymorphisms (TRPs), GangSTR was applied to genotype 777,099 loci across ALS (n=1,122) and NDD (n=76) cohorts, filtered using an “R score” approach. Predicted small expansions (R≥1.5were experimentally validated in HTT, ATXN1, ATXN3, and RPL14 genes, while large expansions (R≥5) lacked validation. Moreover, where BAM files were available, a second tool was applied: ExpansionHunter. It predicted pathogenic expansions in ATXN1, HTT, and FGF14 genes (then experimentally confirmed), causing spinocerebellar ataxia, Huntington’s disease, and late-onset cerebellar ataxia. By combining high-throughput functional assays and comprehensive TRP analysis, this study highlights the critical roles of non-coding regions, splicing mechanisms, and repeat expansions in NDD pathogenesis. These findings provide new insights into the missing heritability of NDDs and pave the way for improved genetic diagnostics and therapeutic strategie.