title: The Role of ARF6 in Biliary Atresia.
abstract: OBJECTIVE
  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.
authors.affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.
authors.forename: Mylarappa
authors.initials: M
authors.lastname: Ningappa
authors.affiliation: Department of Developmental Biology and McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, 15261, United States of America.
authors.forename: Juhoon
authors.initials: J
authors.lastname: So
authors.affiliation: Center for Applied Genomics of the Children's Hospital of Philadelphia, Philadelphia, PA, 19104, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Glessner
authors.affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.
authors.forename: Chethan
authors.initials: C
authors.lastname: Ashokkumar
authors.affiliation: Department of Pathology, Division of Pediatric Pathology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, 15224, United States of America.
authors.forename: Sarangarajan
authors.initials: S
authors.lastname: Ranganathan
authors.affiliation: Department of Bioengineering, University of California San Diego, La Jolla, CA, 92013, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Min
authors.affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.
authors.forename: Brandon W
authors.initials: BW
authors.lastname: Higgs
authors.affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.
authors.forename: Qing
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Haberman
authors.affiliation: Histology Core Laboratory, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, 15224, United States of America.
authors.forename: Lori
authors.initials: L
authors.lastname: Schmitt
authors.affiliation: Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, 06510, United States of America.
authors.forename: Silvia
authors.initials: S
authors.lastname: Vilarinho
authors.affiliation: Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, 06510, United States of America.
authors.forename: Pramod K
authors.initials: PK
authors.lastname: Mistry
authors.affiliation: Department of Pediatrics and Human Genetics, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, 15224, United States of America.
authors.forename: Gerard
authors.initials: G
authors.lastname: Vockley
authors.affiliation: Paediatric Liver, GI, and Nutrition, King's College Hospital, London, WC2R 2LS, England.
authors.forename: Anil
authors.initials: A
authors.lastname: Dhawan
authors.affiliation: Pediatric General and Thoracic Surgery, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, 15224, United States of America.
authors.forename: George K
authors.initials: GK
authors.lastname: Gittes
authors.affiliation: Center for Applied Genomics of the Children's Hospital of Philadelphia, Philadelphia, PA, 19104, United States of America.
authors.forename: Hakon
authors.initials: H
authors.lastname: Hakonarson
authors.affiliation: Histology Core Laboratory, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, 15224, United States of America.
authors.forename: Ronald
authors.initials: R
authors.lastname: Jaffe
authors.affiliation: Department of Bioengineering, University of California San Diego, La Jolla, CA, 92013, United States of America.
authors.forename: Shankar
authors.initials: S
authors.lastname: Subramaniam
authors.affiliation: Department of Developmental Biology and McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, 15261, United States of America.
authors.forename: Donghun
authors.initials: D
authors.lastname: Shin
authors.affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Sindhi
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics*; D000818:Animals; D001656:Biliary Atresia / Q000235:genetics*; D016022:Case-Control Studies; D049109:Cell Proliferation / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics; D005787:Gene Frequency / Q000235:genetics; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005838:Genotype; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D015027:Zebrafish / Q000235:genetics
chemical_list: D000089982:ADP-Ribosylation Factor 6; C512478:EGFR protein, human; D066246:ErbB Receptors; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human