ParSE- seq: A Calibrated Multiplexed Assay for Clinical Interpretation of Putative Splice- Altering Variants

Background: Up to 10% of disease-associated variants are predicted to affect RNA-splicing. Interpreting the clinical significance of putative splice-altering variants outside the 2-base pair canonical splice sites remains difficult without functional studies. Goal: To develop a calibrated, high-throughput assay to adjudicate variant effects on RNA splicing. Methods: We established Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We used the tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select curated benign and pathogenic controls in SCN5A , which encodes the major voltage-gated cardiac sodium channel. We transfected 271 barcoded minigene constructs into HEK cells or induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and calibrated to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data correlated to SpliceAI predictions. We studied selected variants by automated patch clamp (APC) in HEK cells and by assessing splicing and channel function directly in CRISPR-edited iPSC-CMs. Results: ParSE-seq revealed the splicing effect of 245 SCN5A variants in iPSC-CMs. The assay had concordant scores for 43/45 benign and pathogenic control variants, achieving both PS3- and BS3-strong levels of evidence after calibration. 41/114 exonic variants and 30/66 intronic variants with determinate scores disrupted splicing. 9 of 42 Variants of Uncertain Significance were reclassified, and 30 of 34 variants with conflicting interpretations were reinterpreted using the functional data. Predictions from SpliceAI performed well on experimental data (AUC = 0.96). We identified 3 clinically relevant splice-altering missense variants that had normal function when tested with a cDNA-based APC assay. A splice-altering intronic variant, c.1891-5C>G also disrupted splicing and sodium channel electrophysiology when introduced into iPSC-CMs by CRISPR-editing. Conclusions: ParSE-seq is an efficient, calibrated, high-throughput assay to facilitate splice-altering variant interpretation.