Contribution of Previously Unrecognized RNA Splice-Altering Variants to Congenital Heart Disease

Abstract

BACKGROUND: Known genetic causes of congenital heart disease (CHD) explain <40% of CHD cases, and interpreting the clinical significance of variants with uncertain functional impact remains challenging. We aim to improve diagnostic classification of variants in patients with CHD by assessing the impact of noncanonical splice region variants on RNA splicing. METHODS: We tested de novo variants from trio studies of 2649 CHD probands and their parents, as well as rare (allele frequency, <2×10 − 6 ) variants from 4472 CHD probands in the Pediatric Cardiac Genetics Consortium through a combined computational and in vitro approach. RESULTS: We identified 53 de novo and 74 rare variants in CHD cases that alter splicing and thus are loss of function. Of these, 77 variants are in known dominant, recessive, and candidate CHD genes, including KMT2D and RBFOX2 . In 1 case, we confirmed the variant’s predicted impact on RNA splicing in RNA transcripts from the proband’s cardiac tissue. Two probands were found to have 2 loss-of-function variants for recessive CHD genes HECTD1 and DYNC2H1 . In addition, SpliceAI—a predictive algorithm for altered RNA splicing—has a positive predictive value of ≈93% in our cohort. CONCLUSIONS: Through assessment of RNA splicing, we identified a new loss-of-function variant within a CHD gene in 78 probands, of whom 69 (1.5%; n=4472) did not have a previously established genetic explanation for CHD. Identification of splice-altering variants improves diagnostic classification and genetic diagnoses for CHD. REGISTRATION: URL: https://clinicaltrials.gov ; Unique identifier: NCT01196182.

Therapeutic potential of alternative splicing in cardiovascular diseases

“… 30 This finding has been replicated across multiple studies-with several different de novo loss-of-function mutations in RBFOX2 identified in probands with HLHS. 30 , 31 , 32 Although no published studies have identified molecular mechanisms in genetic animal models with human pathogenic mutations identical to those in humans, numerous animal experiments have demonstrated the necessity of RBFOX2 for heart development. Specifically, Rbfox2 loss in zebrafish resulted in HLHS-like phenotypes, characterised by a reduced ventricle size, abnormal myocardial segments, and mitochondrial defects.…”

Noncanonical Splice-Altering Variants: Hidden Culprits of Congenital Heart Disease

“…10 Further, contributions from common variants toward the development of certain CHD subtypes, including transposition of the great arteries and other complex disease, have been highlighted. 11,12 In this edition of Circulation: Genomics and Precision Medicine, Jang et al 13 evaluate noncoding variants near canonical splice sites and their potential to disrupt RNA processing resulting in CHD. Using a combination of computational and in vitro (minigene) assays, they identified 53 de novo variants and 74 rare variants resulting in aberrant splicing in a cohort of 4474 CHD probands from the Pediatric Cardiac Genomics Consortium.…”

“…Over the last decade, following significant advances in genomic technology, we are beginning to piece together the complex puzzle of CHD etiology (Figure ), with the study by Jang et al 13 contributing another important piece. A significant portion of the remaining unsolved cases are hypothesized to result from complex interactions between multiple genetic/epigenetic changes and factors affecting the fetal-placental-maternal environment.…”

“…19 However, there is still much work to be done in understanding the dark matter of the genome, that is, the noncoding regions, and the likely contribution of these hidden culprits to CHD development. The recent studies by Jang et al 13 and Richter et al, 10 focussing on noncanonical splice variants and noncoding enhancers and RNA-binding proteins, respectively, are beginning to address this important area. Further, how the genome, encompassing both the coding and noncoding regions, interacts with the fetal-placental-maternal environment, including the timing of the likely environmental insult and the effect on the (epi)genome and vice versa, should constitute an important research focus in the coming years.…”

“…Percentages displayed in the diagram are based on the following. 2,3,4,10,13,20 Created with BioRender.com. CHD indicates congenital heart disease.…”