Evidence-based recommendations for application of construct-based splicing data in clinical variant classification

Abstract

Minigene RT-PCR assays are widely used to assess variant impact on splicing, with increasing reports of massively parallel splicing assays (MPSAs) demonstrating potential to upscale diagnostic use of construct-based data. This study conducted a comprehensive evaluation of > 41,000 variants from construct-based splicing assays, to build evidence-based recommendations to support the consistent application of such assays in clinical variant interpretation. Seven MPSAs were reviewed for design limitations, and their discriminatory performance evaluated by assessing: assay score distribution; consistency of splice-impact thresholds with expectations based on SpliceAI predictions. A traditional minigene RT-PCR dataset comprising 673 variants from 14 studies was analysed to: assess potential for SpliceAI score to predict level of aberration; demonstrate performance of SpliceAI-10k calculator (SAI-10k-calc) to accurately predict variant-induced splicing events; calibrate evidence strength towards or against spliceogenicity based on SpliceAI score. Traditional minigene results were compared to patient-derived RNA results, and calibrated for evidence strength towards or against pathogenicity using assertions from ClinVar. MPSA datasets lacked specific information on variant-induced transcripts and had design limitations preventing detection of some aberrant splice events. Assay scores generated by five of seven MPSAs were unable to differentiate aberrant from natural splicing events. Traditional minigene results showed high predictive agreement between predicted and observed variant-induced events: SpliceAI score of 0.285 showed 90% sensitivity and 90% specificity for separating no/low versus intermediate-to-complete aberration, and agreement was 87% for aberration type using SAI-10k-calc. Minigene and patient-derived RNA results showed 89% (81/91) complete or high concordance for predominant transcripts. Clinical calibration of minigene results showed that high (≥ 80%) or low (≤ 20%) expression of variant-induced loss of function (LOF) transcripts provide strong evidence towards or against pathogenicity, respectively. Evidence-based recommendations for design and critique of construct-based assays were built based on these and other findings. MPSA screens require review of design limitations and performance evaluation before considering their suitability for clinical variant interpretation. Well-designed multi-exon minigene assays can provide quantitative RNA results to supplement patient-derived RNA findings, and clinical calibration justifies their use in the diagnostic setting. Altogether, these findings support more consistent application of RNA evidence in clinical practice.