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Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of β-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMDintron 5 of 46 nt as the minimal productive D-BP length, demonstrating spliceosome assembly constraint persists at D-BP lengths of 47-56 nt. We exploit the common HBBexon 1 β-thalassemia variant that strengthens a cryptic donor (NM_000518.5(HBB):c.79G > A) to provide a simple barometer for the earliest signs of space constraint, via cryptic donor activation. For clinical evaluation of intronic deletions, we assert D-BP lengths > 60 nt present low mis-splicing risk while space constraint increases exponentially with D-BP lengths < 55 nt, with critical risk and profound splicing abnormalities with D-BP lengths < 50 nt.

Even for essential splice-site variants that are almost guaranteed to alter mRNA splicing, no current method can reliably predict whether exon-skipping, cryptic activation or multiple events will result, greatly complicating clinical interpretation of pathogenicity. Strikingly, ranking the four most common unannotated splicing events across 335,663 reference RNA-sequencing (RNA-seq) samples (300K-RNA Top-4) predicts the nature of variant-associated mis-splicing with 92% sensitivity. The 300K-RNA Top-4 events correctly identify 96% of exon-skipping events and 86% of cryptic splice sites for 140 clinical cases subject to RNA testing, showing higher sensitivity and positive predictive value than SpliceAI. Notably, RNA re-analyses showed we had missed 300K-RNA Top-4 events for several clinical cases tested before the development of this empirical predictive method. Simply, mis-splicing events that happen around a splice site in RNA-seq data are those most likely to be activated by a splice-site variant. The SpliceVault web portal allows users easy access to 300K-RNA for informed splice-site variant interpretation and classification.

Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy).

X-linked myotubular myopathy (XLMTM) is a severe congenital myopathy characterised by generalised weakness and respiratory insufficiency. XLMTM is associated with pathogenic variants in MTM1; a gene encoding the lipid phosphatase myotubularin. Whole genome sequencing (WGS) of an exome-negative male proband with severe hypotonia, respiratory insufficiency and centralised nuclei on muscle biopsy identified a deep intronic MTM1variant NG_008199.1(NM_000252.2):c.1468-577A>G, which strengthened a cryptic 5' splice site (A>G substitution at the +5 position). Muscle RNA sequencing was non-diagnostic due to low read depth. Reverse transcription PCR (RT-PCR) of muscle RNA confirmed the c.1468-577A>G variant activates inclusion of a pseudo-exon encoding a premature stop codon into all detected MTM1transcripts. Western blot analysis establishes deficiency of myotubularin protein, consistent with the severe XLMTM phenotype. We expand the genotypic spectrum of XLMTM and highlight benefits of screening non-coding regions of MTM1in male probands with phenotypically concordant XLMTM who remain undiagnosed following exome sequencing.

We establish autosomal recessive DESvariants p.(Leu190Pro) and a deep intronic splice variant causing inclusion of a frameshift-inducing artificial exon/intronic fragment, as the likely cause of myopathy with cardiac involvement in female siblings. Both sisters presented in their twenties with slowly progressive limb girdle weakness, severe systolic dysfunction, and progressive, severe respiratory weakness. Desmin is an intermediate filament protein typically associated with autosomal dominant myofibrillar myopathy with cardiac involvement. However a few rare cases of autosomal recessive desminopathy are reported. In this family, a paternal missense p.(Leu190Pro) variant was viewed unlikely to be causative of autosomal dominant desminopathy, as the father and brothers carrying this variant were clinically unaffected. Clinical fit with a DES-related myopathy encouraged closer scrutiny of all DESvariants, identifying a maternal deep intronic variant within intron-7, predicted to create a cryptic splice site, which segregated with disease. RNA sequencing and studies of muscle cDNA confirmed the deep intronic variant caused aberrant splicing of an artificial exon/intronic fragment into maternal DESmRNA transcripts, encoding a premature termination codon, and potently activating nonsense-mediate decay (92% paternal DEStranscripts, 8% maternal). Western blot showed 60–75% reduction in desmin levels, likely comprised only of missense p.(Leu190Pro) desmin. Biopsy showed fibre size variation with increased central nuclei. Electron microscopy showed extensive myofibrillar disarray, duplication of the basal lamina, but no inclusions or aggregates. This study expands the phenotypic spectrum of recessive DEScardio/myopathy, and emphasizes the continuing importance of muscle biopsy for functional genomics pursuit of ‘tricky’ variants in neuromuscular conditions.