title: Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity.
abstract: OBJECTIVE
  Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a potentially lethal autosomal recessive liver disease associated with mutations in ABCB4, the gene encoding the canalicular translocator of phosphatidylcholine MDR3. While some affected children benefit from ursodeoxycholic acid (UDCA) therapy, others evolve to end-stage liver disease. We aimed to evaluate whether these different outcomes are related to the impact of ABCB4 mutations.
  
  
  METHODS
  Six children with PFIC3 were investigated by sequencing of ABCB4 exons and flanking intron-exon boundaries and by immunohistochemistry. ABCB4 missense mutations were phenotyped in vitro by assessing their effects on MDR3 expression, subcellular localisation, and phosphatidylcholine-translocating activity. The resulting data were contrasted with the clinical outcomes.
  
  
  RESULTS
  Eight distinct ABCB4 mutations were identified: one nonsense, one splicing and six missense mutations, four of which (G68R, T201M, P479L, D459H) affected MDR3 expression level. G68R and D459H also led to retention of the protein in endoplasmic reticulum. Phosphatidylcholine efflux assays indicated that T201M, P479L, S978P and E1118K mutations impaired MDR3 activity to variable degrees. Three children with mutations that caused a total loss of MDR3 expression/function manifested progressive liver disease refractory to UDCA treatment. This was also the case in a patient carrying two different mutations that, in combination, resulted in a 90% reduction in total MDR3 activity. A favourable response to UDCA was achieved in two patients with estimated MDR3 activities of 50% and 33%, respectively.
  
  
  CONCLUSIONS
  These data provide experimental evidence of the correlation between the degree of MDR3 floppase activity and the clinical outcomes of PFIC3.
authors.affiliation: La Paz University Hospital Health Research Institute-IdiPAZ, Madrid, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Gordo-Gilart
authors.affiliation: La Paz University Hospital Health Research Institute-IdiPAZ, Madrid, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Andueza
authors.affiliation: La Paz University Hospital Health Research Institute-IdiPAZ, Madrid, Spain Pediatric Liver Service, La Paz Children's University Hospital, Madrid, Spain.
authors.forename: Loreto
authors.initials: L
authors.lastname: Hierro
authors.affiliation: La Paz University Hospital Health Research Institute-IdiPAZ, Madrid, Spain Institute of Medical and Molecular Genetics (INGEMM), La Paz University Hospital, Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Martínez-Fernández
authors.affiliation: Paediatric Gastroenterology-Hepatology Division, Liver-Intestinal Transplantation Center, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Daniel
authors.initials: D
authors.lastname: D'Agostino
authors.affiliation: La Paz University Hospital Health Research Institute-IdiPAZ, Madrid, Spain Pediatric Liver Service, La Paz Children's University Hospital, Madrid, Spain.
authors.forename: Paloma
authors.initials: P
authors.lastname: Jara
authors.affiliation: La Paz University Hospital Health Research Institute-IdiPAZ, Madrid, Spain.
authors.forename: Luis
authors.identifier: http://orcid.org/0000-0003-4821-0640
authors.initials: L
authors.lastname: Alvarez
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000172:deficiency* / Q000235:genetics / Q000502:physiology; D002648:Child; D002780:Cholestasis, Intrahepatic / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D020125:Mutation, Missense; D016896:Treatment Outcome; D014580:Ursodeoxycholic Acid / Q000627:therapeutic use*
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D014580:Ursodeoxycholic Acid; C095122:multidrug resistance protein 3
keywords: Cholestatic Liver Diseases; Gene Mutation; Inherited Metabolic Disease; Liver Transplantation; Paediatric Liver Disease