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The role of ARF6 in biliary atresia

Ningappa, M and So, J and Glessner, J and Ashokkumar, C and Ranganathan, S and Min, J and Higgs, BW and Sun, Q and Haberman, K and Schmitt, L and Vilarinho, S and Mistry, PK and Vockley, G and Dhawan, A and Gittes, GK and Hakonarson, H and Jaffe, R and Subramaniam, S and Shin, D and Sindhi, R (2015) The role of ARF6 in biliary atresia. PLoS ONE, 10 (9).

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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 = 5.94 x 10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57 x 10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58 x 10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x 10-7), ERK/MAPK and CREB canonical pathways (p<1 x 10-34), and functional networks for cellular development and proliferation (p<1 x 10-45), 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.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Ningappa, M
So, Jjuhoon@pitt.eduJUHOON
Glessner, J
Ashokkumar, Ccsa3@pitt.eduCSA3
Ranganathan, Ssar1@pitt.eduSAR10000-0001-6214-9167
Min, J
Higgs, BW
Sun, Qqingsun@pitt.eduQINGSUN
Haberman, K
Schmitt, L
Vilarinho, S
Mistry, PK
Vockley, Ggev1@pitt.eduGEV10000-0002-8180-6457
Dhawan, A
Gittes, GKgkg2@pitt.eduGKG2
Hakonarson, H
Jaffe, R
Subramaniam, S
Shin, Ddonghuns@pitt.eduDONGHUNS
Sindhi, R
ContributionContributors NameEmailPitt UsernameORCID
Centers: Other Centers, Institutes, Offices, or Units > McGowan Institute for Regenerative Medicine
Date: 17 September 2015
Date Type: Publication
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Journal or Publication Title: PLoS ONE
Volume: 10
Number: 9
DOI or Unique Handle: 10.1371/journal.pone.0138381
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Pathology
School of Medicine > Pediatrics
Refereed: Yes
Date Deposited: 23 Aug 2016 14:38
Last Modified: 22 Jun 2021 10:55


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