406 An in vitro splicing assay reveals the pathogenicity of intronic variants in ABCC6, the gene at fault in pseudoxanthoma elasticum

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. Splicing variants in ABCC6 are often overlooked due to the unavailability of liver biopsies from patients with such variants to determine their consequences on splicing by transcriptional analysis. Thus, the interpretation of intronic variants purely relies on computational algorithms to predict their effects on splicing. Very often the prediction programs do not provide a consensus as to the effect of variants for disrupting normal splicing. Our recent ABCC6 mutation analysis of over 400 patients with PXE shows that this continues to be a problem: among 7 intronic variants in ABCC6 with conflicting results by current splice site prediction algorithms, 3 variants were found to affect splicing. These three intronic variants were analyzed in an in vitro functional splicing assay using expression of minigene constructs in transfected HepG2 cells. Total RNA was extracted and RT-PCR was performed to analyze the splice pattern followed by Sanger sequencing. Two of the variants, c.3883-46A>G and c.2070+5G>A, caused skipping of the adjacent exon, predicted to cause out-of-frame translation and premature stop of translation. One variant, c.3883-6G>A, created a new splice acceptor site resulting in a 4-bp retention from the intronic region and causing frame-shift of translation. The results provided evidence that intronic variants away from the canonical splice sites disrupted splicing, clearly demonstrating the presence of mutations beyond the canonical splice sites.