Two novel intronic mutations in ABCB4 gene: clinical manifestations and molecular mechanisms and literatures review

Abstract Background and Aims: ABCB4 gene is closely related to bile transport and excretion. There are few studies on intronic mutations. In this study, we aimed to explore the pathogenicity of 2 newly discovered intronic mutations of the ABCB4 gene. Methods: This research enrolled 2 patients with novel ABCB4 intronic mutations and we analyzed their clinical characteristics. The pathogenicity of 2 variants was predicted and analyzed in silicon. Minigene analysis was made to investigate their effects on splicing patterns. And the effects of mutations on the expression of ABCB4 mRNA and MDR3 were further explored. Additionally, we also conducted a literature review on ABCB4 intronic mutations. Results: The main clinical manifestation of 2 patients was cholestasis. Liver pathology showed that 2 patients had different degrees of bile duct damage, inflammation and fibrosis. The results of the three pathogenicity prediction tools were similar, and it suggested that 2 intronic mutations might interfere with normal splicing. Moreover, minigene experiments demonstrated that the c.537-32G > T create a new alternative intron splicing receptor, resulting in the retention of 16-bp intronic sequence. And the c.833 + 2T > C created a new alternative intron splicing donor, resulting in the retention of 67-bp intronic sequence. The mRNA expression of c.537-32G > T was up-regulated compared with the wild type, while the mRNA expression of c.833 + 2T > C was down-regulated. Both of these 2 mutations led to the decrease of MDR3 expression. The review of ABCB4 intronic variants reported so far indicates that Conclusions: The mutation of c.537-32G > T and c.833 + 2T > C of ABCB4 gene are splicing mutations, and they tempered with 3' splicing site and 5' splicing site respectively. Clinical data, bioinformatics prediction, and in vitro experiments all indicated that the 2 mutations are pathogenic.