Splicing Analysis of MYO5B Noncanonical Variants in Patients with Low Gamma-Glutamyltransferase Cholestasis

Figure 2

Splicing effects of MYO5B variants based on minigene splicing assays. (a) Schematic of minigene system. AV-E1 and AV-E2 are adenovirus exon 1 and exon 2, embedded in the pcDNA3.1 vector; amplicons of interest (exons of interest and 500~1000 bp of flanking intronic sequences in this study) were inserted into the pcDNA3.1 vector. SeqF and SeqR are primers for cDNA amplifying and the Sanger sequencing. (b) The splicing products were amplified by PCR and underwent 2% agarose gel electrophoresis. Gray boxes indicate two adenovirus exons; white boxes are MYO5B exons and are marked with an exon number individually; the arrangement of boxes is consistent with the schematic of minigene system. c.3538-1G>A and c.839-1G>A were selected as positive controls for the assay. Δ, deletion; ins, insertion; nt, nucleotides; bp, base pairs. (c) qPCR for relative quantity of the normal splicing in the mutant minigenes with correctly spliced transcript identified. Compared to the wild-type minigenes, c.1322+5G>A, c.2349A>G, c.1669-35A>C, c.3045+3A>T, and c.4221G>A have only 20%, 35%, 17%, 15%, and 30% of normal splicing, respectively. The first variant in each pair represents the common allele, while the second variant represents the rare allele.

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