Massively parallel reporter assays discover de novo exonic splicing mutants in paralogs of Autism genes

Author summaryAutism Spectrum Disorders (ASD) is the most common neurodevelopmental disorder that presents with both phenotypic and genetic heterogeneity. Before genetically defined therapeutic approaches can be applied to the treatment of ASD, it is crucial to understand the genetic basis of the disorder. Tremendous effort by consortiums, such as the Simon Foundation for Autism Research Initiative, has led to the identification of variants and, in turn, genes associated with ASD. However, the effect of ASD variants have on the process of splicing is often not fully considered when assessing ASD risk genes. Using a high-throughput splicing assay, MaPSy, ~6.3% of ASD-associated variants affected splicing, highlighting a substantial contribution of defective splicing to ASD. This analysis led to identification of genes, such as TNRC6C, that warrant further investigation with respect to ASD risk. To determine the contribution of defective splicing in Autism Spectrum Disorders (ASD), the most common neurodevelopmental disorder, a high throughput Massively Parallel Splicing Assay (MaPSY) was employed and identified 42 exonic splicing mutants out of 725 coding de novo variants discovered in the sequencing of ASD families. A redesign of the minigene constructs in MaPSY revealed that upstream exons with strong 5' splice sites increase the magnitude of skipping phenotypes observed in downstream exons. Select hits were validated by RT-PCR and amplicon sequencing in patient cell lines. Exonic splicing mutants were enriched in probands relative to unaffected siblings -especially synonymous variants (7.5% vs 3.5%, respectively). Of the 26 genes disrupted by exonic splicing mutations, 6 were in known ASD genes and 3 were in paralogs of known ASD genes. Of particular interest was a synonymous variant in TNRC6C - an ASD gene paralog with interactions with other ASD genes. Clinical records of 3 ASD patients with TNRC6C variant revealed respiratory issues consistent with phenotypes observed in TNRC6 depleted mice. Overall, this study highlights the need for splicing analysis in determining variant pathogenicity, especially as it relates to ASD.