Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay

Abstract

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.

Molecular landscape of DYSF mutations in dysferlinopathy: From a Chinese multicenter analysis to a worldwide perspective

“…In the Chinese dysferlinopathy cohort, several standards of the ACMG/AMP guideline were modified according to the actual situations of the cohort and previous similar studies (Charnay et al, 2021; Dionnet et al, 2020): (1) PVS1 score was assigned in accordance with ClinGen Sequence Variant Interpretation (SVI) recommendations for interpreting the loss of function variants (Abou Tayoun et al, 2018); (2) Since functional studies of the RNA or protein activity were not performed, PS3 score was not applicable; (3) PP3 score was assigned if CADD PHRED score above 25; (4) PM3 score was assigned referring to the SVI Recommendation for in trans Criterion PM3 (Version 1.0); (5) PP1 score was assigned according to the recommendations on recessive disorders by the Hearing Loss ClinGen Working group (Oza et al, 2018); (6) Though protein expression assay was performed in only few patients (Table ), PP4_strong was assigned if Dysferlin was absent in muscle tissues (detected by western blot or immunohistochemistry) in at least two patients either homozygous for the variant or compound heterozygous in trans with a pathogenic variant (PP4_moderate if absent in one patient, PP4_supporting if decreased in one patient); and (7) the criteria for the allele frequency were set as following: PM2: 0.02%, BS1: 0.5%, BA1: 5%.…”

“…Variant ACMG/AMP classifications for the Chinese dysferlinopathy cohortIn the Chinese dysferlinopathy cohort, several standards of the ACMG/ AMP guideline were modified according to the actual situations of the cohort and previous similar studies(Charnay et al, 2021;Dionnet et al, 2020): (1) PVS1 score was assigned in accordance with ClinGen Sequence Variant Interpretation (SVI) recommendations for interpreting the loss of function variants (Abou Tayoun et al, 2018); (2) Since functional studies of the RNA or protein activity were not performed, PS3 score was not applicable; (3) PP3 score was assigned if CADD PHRED score above 25; (4) PM3 score was assigned referring to the SVI Recommendation for in trans Criterion PM3 (Version 1.0); (5) PP1 score was assigned according to the recommendations on recessive disorders by the Hearing Loss ClinGen Working group (Oza et al, 2018); (6) Though protein expression assay was performed in only few patients (Table…”

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency

“…However, for exonic variants, particularly those farther from exon junctions, splicing defects may be more challenging to identify bioinformatically. [73][74][75] Efforts to interpret these variants will need to account for the functional impacts of changing the encoded protein sequence as well as its splicing. Finally, as our results illustrate, different variants in a single gene may lead to distinct splicing outcomes with diverse consequences ranging from the straightforward loss of function to dominant-negative effects.…”

High-throughput splicing assays identify missense and silent splice-disruptivePOU1F1variants underlying pituitary hormone deficiency

“…Variants at or near canonical spice sites are readily recognized as pathogenic 67 , and these can be identified predicted with high accuracy by algorithms such as SpliceAI. However, for exonic variants, particularly those farther from exon junctions, splicing defects may be more challenging to identify bioinformatically [68][69][70] . Efforts to interpret these variants will need to account for the functional impacts of changing the encoded protein sequence as well as its splicing.…”