Functional and in-silico interrogation of rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders

PurposeTo develop a comprehensive analysis framework to identify pre-messenger RNA splicing mutations in the context of rare disease.\n\nMethodsWe assessed variants of uncertain significance through six in-silico prioritization strategies. Firstly, through comparison to functional analyses, we determined the precise effect on splicing of variants identified through clinical multi-disciplinary meetings. Next, we calculated the sensitivity of in-silico prioritization strategies to distinguish known splicing mutations from common variation (>2% in allele frequency in gnomAD) within relevant disease genes. These approaches defined an accurate in-silico strategy for variant prioritization, which we retrospectively applied to a large cohort of 2783 individuals who had previously received genomic testing for rare genomic disorders. We assessed the clinical impact of such prioritization strategies alongside routine diagnostic testing strategies.\n\nResultsWe identified 21 variants that potentially impacted splicing, and used cell based splicing assays to identify those variants which disrupted normal splicing. These findings underpinned new molecular diagnoses for 14 individuals. This process established that the use of pre-defined thresholds from a machine learning splice prediction algorithm, SpliceAI, was the most efficient method for variant prioritization, with a positive predictive value of 86%. We analysed 1,346,744 variants identified through diagnostic testing for 2783 individuals and observed that splicing variant prioritization strategies would improve clarity in clinical analysis for 15% of the individuals surveyed. Prioritized variants could provide new molecular diagnoses or provide additional support for molecular diagnosis for up to 81 individuals within our cohort.\n\nConclusionWe present an in-silico and functional analysis framework for the assessment of variants impacting pre-messenger RNA splicing which is applicable across monogenic disorders. Incorporation of these strategies improves clarity in diagnostic reporting, increases diagnostic yield and, with the advent of targeted treatment strategies, can directly alter patient clinical management.\n\nKey HighlightsO_LIWe establish an in-silico and functional analysis framework for the incorporation of splice variant assessment into diagnostic testing that is applicable across monogenic disorders.\nC_LIO_LIAfter assessment of six distinct variant prioritization strategies, we concluded that SpliceAI was the best method to accurately identify genomic variation disrupting normal pre-mRNA splicing. We determined this through (i) functional assessment of novel variants of uncertain significance described in this study, and (ii) calculation of sensitivity and specificity for prioritization strategies to distinguish known splicing mutations from common variants in the general population.\nC_LIO_LIWe describe novel disease-causing variants with support from cell based functional assays which underpin autosomal recessive, autosomal dominant and X-linked Mendelian disorders. This includes variants which are deeply intronic, within the nearby splice region of canonical splice sites and variants which activate cryptic splice sites within the protein-coding regions of genes.\nC_LIO_LIWe integrated the best performing variant prioritization strategy alongside clinical diagnostic testing for 2783 individuals referred to a well-established targeted gene panel test available through the UK National Health Service. We show that integration of such strategies will increase accuracy and clarity of diagnostic reporting, including the identification of variants which could provide new diagnoses and new carrier findings for referred individuals.\nC_LIO_LIFunctional assessment is essential for accurate clinical assessment of variants disrupting pre-mRNA splicing. We show through cell based functional assessments that variants impacting splicing may have complex impacts on pre-mRNA splicing, which may cause multiple interpretable consequences according to ACMG guidelines.\nC_LI