Abstract
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.
Highlights
The remarkable advent of generation sequencing technologies in the past decade has led to the discovery that each individual carries millions of genetic variants, including more than ~10,000 peptide-altering variants [1,2]
Before genetically defined therapeutic approaches can be applied to the treatment of Autism Spectrum Disorders (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
Summary
The remarkable advent of generation sequencing technologies in the past decade has led to the discovery that each individual carries millions of genetic variants, including more than ~10,000 peptide-altering variants [1,2]. Classical variant interpretation methods rely heavily on the variant’s impact on the peptide sequence and its evolutionary constraint [3,4,5]. Nonsense, frameshift, and splice site mutations may lead to loss-of-function of the target gene and are expected to be deleterious ( known as likely gene disrupting). These methods cannot determine whether an exonic variant residing outside the canonical splice site may impact splicing thereby having a more deleterious effect than expected. We have developed a high-throughput reporter assay, MaPSy, to screen ~5,000 disease-associated variants as a functional approach to detect exonic splice altering variants on a high-throughput scale [6]. MaPSy revealed ~10% of exonic disease-causing variants disrupted splicing, highlighting the relevance of splicing in disease
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