The Role of ARF6 in Biliary Atresia

BACKGROUND & AIMS: Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA). METHODS: To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6). RESULTS: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10(-7), OR 2.66; 0.286 vs. 0.13, P = 5.57x10(-7), OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10(-2), OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10(-7)), ERK/MAPK and CREB canonical pathways (p<1 x10(-34)), and functional networks for cellular development and proliferation (p<1 x10(-45)), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA. CONCLUSIONS: The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.