Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing

Abstract

Purpose: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains around 50%, suggesting some clinically relevant rare variants may be missed by standard analyses. Here we analyze poison exons (PEs) which, while often absent from standard gene annotations, are alternative exons whose inclusion results in a premature termination codon. Variants that alter PE inclusion can lead to loss-of-function and may be highly penetrant contributors to disease. Methods: We curated published RNA-seq data from developing mouse cortex to define 1,937 PE regions conserved between humans and mice and potentially relevant to NDDs. We then analyzed variants found by genome sequencing in multiple NDD cohorts. Results: Across 2,999 probands, we found six clinically relevant variants in PE regions that were previously overlooked. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), associated with epilepsies. One variant is in SNRPB, associated with Cerebrocostomandibular Syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and were observed in probands with features consistent with those reported for the associated gene. Conclusion: With only a minimal increase in variant analysis burden (most probands had zero or one candidate PE variants in a known NDD gene, with an average of 0.77 per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Solving the unsolved genetic epilepsies: Current and future perspectives

“…Additional benefit lies in the interpretation of synonymous variants within coding exons that can influence mRNA splicing, and the increased notice that intronic variants creating altered splice acceptor and donor sites (often far away from the nearest exon) can result in the increased inclusion of poison exons with a premature termination codon in the mature mRNA transcript of an expressed gene, targeting the transcript for nonsense‐mediated decay and thus leading to gene dysfunction. Such poison exons have been found to affect the expression of SCN1A , SCN2A , and SCN8A , causing epilepsy in affected individuals 65,66 …”

“…Such poison exons have been found to affect the expression of SCN1A, SCN2A, and SCN8A, causing epilepsy in affected individuals. 65,66 Whereas in most current studies, RNA derived from peripheral blood was investigated, sampling other clinically accessible tissues such as fibroblasts might be further beneficial, given that these tissues express up to 70% of all OMIM morbid genes (including the majority of known epilepsy-related genes) at sufficient coverage, and not all genes are expressed in blood cells. 63,64 In the future, innovations from the field of cell reprogramming such as fibroblast to induced-neuron transdifferentiation might further advance the utility of RNA-seq to specifically analyze genes with a neuronal restricted gene expression pattern that can only be analyzed in neurons but not in patient derived fibroblasts.…”

Long-read genome sequencing and variant reanalysis increase diagnostic yield in neurodevelopmental disorders

“…We note that in 7/9 cases, variants were identified in an unaffected or mildly affected parent, which was somewhat unexpected in these cases due to suspicion of high penetrance (also see Supplemental Case Reports). Lastly, we identified a variant of interest in SCN1A that resulted from a targeted analysis of candidate "poison exon" variants (Proband 16, Felker et al 2023).…”

Treatability of the KMT2-Associated Neurodevelopmental Disorders Using Antisense Oligonucleotide-Based Treatments

“…Human Mutation putative targets for generalized treatment approaches (Table 3). This is the case for KMT2A, KMT2B, KMT2C, KMT2E, and ASH1L, where poison exons have recently been reported [89,90,92]. Not all of the PEs have been reported consistently across different publications; thus, it is necessary to confirm the presence and abundance of these alternative events in the affected tissues, which in the case of the KMT2-associated disorders is the central nervous system.…”

“…: alternative splicing; PE: poison exon; UTR: untranslated region; NATs: naturally occurring antisense transcripts; GoF: gain of function. The additional symbols indicate in which publications the features have been reported: ǂ, Felker et al[89]; * , Lim et al[90]; ⊢, Liu et al[91]; and ¶, Mittal et al[92].…”