Improving the accuracy of variant interpretation during diagnostic sequencing is a major goal for genomic medicine. To explore an often‐overlooked splicing effect of missense variants, we developed the functional assay (“minigene”) for the majority of exons of CAPN3, the gene responsible for limb girdle muscular dystrophy. By systematically screening 21 missense variants distributed along the gene, we found that eight clinically relevant missense variants located at a certain distance from the exon–intron borders (deep exonic missense variants) disrupted normal splicing of CAPN3 exons. Several recent machine learning‐based computational tools failed to predict splicing impact for the majority of these deep exonic missense variants, highlighting the importance of including variants of this type in the training sets during the future algorithm development. Overall, 24 variants in CAPN3 gene were explored, leading to the change in the American College of Medical Genetics and Genomics classification of seven of them when results of the “minigene” functional assay were considered. Our findings reveal previously unknown splicing impact of several clinically important variants in CAPN3 and draw attention to the existence of deep exonic variants with a disruptive effect on gene splicing that could be overlooked by the current approaches in clinical genetics.
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