Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies

Abstract

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies.

Performance Evaluation of SpliceAI for the Prediction of Splicing of NF1 Variants

“…These results were similar with those of the present study, showing 94.5% sensitivity and 94.3% specificity with a cut-off value of >0.22. The present study might be highlighted in that a large number of variants of NF1 were evaluated, since previous studies using SpliceAI evaluated mainly variants of BRCA1/BRCA2 , CFTR , FBN1 , and PLCγ1 genes [ 22 , 23 , 24 ].…”

Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

“…Firstly, many +2T>C variants in human disease genes that have been capable of generating some wild-type transcripts are likely to have gone largely unreported; this represents a significant deficiency in terms of our understanding of genotype-phenotype relationships and tailored treatment options given that even the minor retention of wild-type transcripts derived from a variant allele might significantly impact disease expression and severity (Ramalho et al, 2002;Den Uijl et al, 2011;Raraigh et al, 2018;Lin et al, 2019;Scalet et al, 2019;Joynt et al, 2020). In this regard, it is pertinent to mention that CFTR c.3873+2T>C and c.4242+2T>C transitions (Joynt et al, 2020) and SRP68 c.184+2T>C (Schmaltz-Panneau et al, 2021) are among the most recently reported examples of disease-causing +2T>C variants that generated some wild-type transcripts. Secondly, +2T>C variants in human disease genes may not invariably be pathogenic, a notion that has received support from at least two recent publications, which reclassified BRCA2 c.8331+2T>C (Nix et al, 2021) and BAP1 c.783+2T>C (Goldberg et al, 2021) as variants of unknown significance.…”

Clotting factor genes are associated with preeclampsia in high altitude pregnant women in the Peruvian Andes

“…The copyright holder for this preprint this version posted May 21, 2021. ; https://doi.org/10.1101/2021.05.20.21257549 doi: medRxiv preprint research could evaluate its pathogenic potential in Peruvian populations (Joynt et al, 2020, Lin et al, 2019.…”

“…However, intronic variants are known to have functional impacts on RNA splicing patterns (Cooper, 2010). To elucidate the functional significance of this variant, future research could evaluate its pathogenic potential in Peruvian populations (Joynt et al, 2020, Lin et al, 2019).…”