RT‐PCR analysis of mRNA revealed the splice‐altering effect of rare intronic variants in monogenic disorders

Background Variants perturbing the normal splicing of pre‐mRNA can lead to human diseases. The splice‐altering effect and eventual consequence on gene function was sometimes uncertain and hinders a definitive molecular diagnosis. Methods The impact of four rare intronic variants on splicing was analyzed through reverse transcription ‐ polymerase chain reaction (RT‐PCR) analysis of mRNA derived from the peripheral blood of patients. The results were compared with in‐silico prediction. Potential implication on molecular diagnosis was discussed. Results Four rare intronic variants of SLC9A6, DLG3, GAA, and OCRL were identified in patients with suspected disorders, respectively. Although these four variants were all predicted to alter splicing by in‐silico tools, RT‐PCR analysis of mRNA derived from peripheral blood showed these variants affected splicing in different ways: c.899+3_899+6del of SLC9A6 resulted in one‐exon skipping and an out‐of‐frame transcript; c.905‐2A > G of DLG3 resulted in a mix of in‐frame transcripts; c.1195‐11T > A of GAA resulted in the in‐frame insertion of nine nucleotides; c.723‐2A > C of OCRL resulted in one‐exon skipping and in‐frame deletion of 102 nucleotides. The consequence revealed by mRNA analysis is essential for accurate interpretation of pathogenicity. Conclusion Four intronic variants all caused aberrant mRNA splicing. For intronic variants with uncertain impact on splicing, mRNA analysis is helpful for ascertainment of alternative splicing and accurate interpretation of pathogenicity.