Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene predominantly expressed in hepatocytes. Different types of mutations have been identified in ABCC6 including canonical splice site mutations affecting pre-mRNA splicing. Non-canonical splicing variants near or within exon-intron boundary in ABCC6 are under-recognized due to the lack of liver biopsies from patients carrying such variants, and consequently, inability to determine their consequences on splicing by transcriptional analysis. The interpretation of such variants has primarily relied on the prediction by in silico algorithms; often such programs do not provide consensus predictions. Among seven non-canonical splicing variants identified in ABCC6 in patients with PXE, five variants were found to affect splicing in in vitro minigene splicing assays in transfected HepG2 cells. Four of the variants, c.1868-5T>G, c.2070+5G>A, c.3735G>A (p.E1245=), and c.3883-46A>G, caused skipping of the respective adjacent exon. One variant, c.3883-6G>A, created a new splicing donor site resulting in a 4-bp retention from the intronic region. The results provide experimental evidence that these non-canonical variants disrupt splicing, suggesting the presence of pathogenic mutations beyond the canonical splice sites, including silent variants in the coding region.

Assessment of Non-Canonical Splicing Variants in ABCC6 and Comparison with Bioinformatics Predictions