The Role of ARF6 in Biliary Atresia

Abstract

Background & AimsAltered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).MethodsTo 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).ResultsAmong 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.ConclusionsThe 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.

Role of ErbB/HER family of receptor tyrosine kinases in cholangiocyte biology

“…The role of the EGFR/Notch1 pathway should be confirmed in human liver as well as its applicability during embryonic liver development. Recently, the EGFR/guanine nucleotide exchange factor 100 (GEF100)/ADP‐ribosylation factor 6 (Arf6) signaling pathway has been envisaged as a regulator of intrahepatic biliary morphogenesis in a zebrafish model . Arf6 knockdown or pharmacological inhibition of EGFR in zebrafish embryo resulted in poor bile duct development.…”

Making It New Again

“…As hepatocytes seem unaffected in snapc4 mutants, indicating that the function of Snapc4 is specific to biliary cells. In many of these studies, live imaging of GFP-expressing biliary cells in cultured livers or in embryos provided invaluable insights into cell behaviors driving this dynamic morphogenetic process (Lorent et al, 2004; Ningappa et al, 2015). …”

“…A recent study in zebrafish found that only depletion of add3 caused biliary devects similar to BA, prioritizing ADD3 as a BA susceptibility gene over XPNPEP1 (Tang et al, 2016). Another study of Caucasian BA cases identified a susceptibility locus on human chromosome 14q21.3, including the ARF6 gene (Ningappa et al, 2015). The two zebrafish homologs, arf6a and arf6b are expressed in the liver and knock down experiments showed they are required for early biliary development.…”

Using Zebrafish to Model Liver Diseases-Where Do We Stand?

“…Excessive Hedgehog activation has also been observed in children with BA [30], suggesting that Hedgehog signaling has evolutionarily conserved roles in cholangiocyte development. The Sindhi group knocked down a different candidate gene, ADP-ribosylation factor 6 ( arf6 ), which encodes a GTP-binding protein [31]. They observed similar intrahepatic biliary defects to those seen in gpc1 and add3 knockdown larvae.…”

“…The main challenge for the follow-up studies of patient GWAS is to determine which variants are indeed causative. Four groups have used zebrafish to validate GWAS variants in BA patients, as well as in patients with elevated liver enzyme levels [28, 98, 31, 29]. Furthermore, CRISPR/Cas9-mediated targeted mutagenesis can now be applied in zebrafish not only to generate null mutations, but also to precisely recapitulate the same mutations found in patients.…”