Journal of Gastroenterology

, Volume 53, Issue 8, pp 945–958 | Cite as

Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and TJP2 gene variants analysis by high-throughput sequencing

  • Giovanni Vitale
  • Stefano Gitto
  • Francesco Raimondi
  • Alessandro Mattiaccio
  • Vilma Mantovani
  • Ranka Vukotic
  • Antonietta D’Errico
  • Marco Seri
  • Robert B. Russell
  • Pietro AndreoneEmail author
Original Article—Liver, Pancreas, and Biliary Tract



Mutations in ATP-transporters ATPB81, ABCB11, and ABCB4 are responsible for progressive familial intrahepatic cholestasis (PFIC) 1, 2 and 3, and recently the gene for tight junction protein-2 (TJP2) has been linked to PFIC4.


As these four genes have been poorly studied in young people and adults, we investigated them in this context here.


In patients with cryptogenic cholestasis, we analyzed the presence of mutations by high-throughput sequencing. Bioinformatics analyses were performed for mechanistic and functional predictions of their consequences on biomolecular interaction interfaces.


Of 108 patients, 48 whose cause of cholestasis was not established were submitted to molecular analysis. Pathogenic/likely pathogenic mutations were found in ten (21%) probands for 13 mutations: two in ATP8B 1, six in ABCB11, two in ABCB4, three in TJP2. We also identified seven variants of uncertain significance: two in ATP8B1, one in ABCB11, two in ABCB4 and two in TJP2. Finally, we identified 11 benign/likely benign variants. Patients with pathogenic/likely pathogenic mutations had higher levels of liver stiffness (measured by FibroScan®) and bile acids, as well as higher rates of cholestatic histological features, compared to the patients without at least likely pathogenic mutations. The multivariate analysis showed that itching was the only independent factor associated with disease-causing mutations (OR 5.801, 95% CI 1.244–27.060, p = 0.025).


Mutations in the genes responsible for PFIC may be involved in both young and adults with cryptogenic cholestasis in a considerable number of cases, including in heterozygous status. Diagnosis should always be suspected, particularly in the presence of itching.


Progressive familial intrahepatic cholestasis Cryptogenic disease Pathogenic mutations Genetic variants Bioinformatics analysis 



Progressive familial intrahepatic cholestasis


Tight junction protein-2


Familial intrahepatic cholestasis 1


Bile salt export pump


Multidrug resistance P-glycoprotein 3




Alkaline phosphatase


Benign intrahepatic cholestasis


Low-phospholipid-associated cholelithiasis


Intrahepatic cholestasis of pregnancy


Drug-induced cholestasis


High-throughput sequencing


Next-generation sequencing


Primary sclerosing cholangitis


Bile acids


Minor allele frequency


Sorting Intolerant From Tolerant


Human Gene Mutation Database


American College of Medical Genetics and Genomics




Likely pathogenic


Variants of uncertain significance


Likely benign




Standard deviation


Confidence interval


Single-nucleotide polymorphism


Alanine aminotransferase


Odds ratio


Author contributions

GV and PA designed the study and collected data. AM, VM, and MS performed the DNA sequencing and applied prediction tools; AD supervised the histological evaluations, FR and RBR performed protein modeling by Mechismo; SG, AM, VM, GV, RV, and PA analyzed the patients’ data. GV wrote the manuscript; all authors critically revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Financial support

No grants and other financial support were received.

Supplementary material

535_2017_1423_MOESM1_ESM.pdf (6 kb)
Supplementary material 1 (PDF 5 kb)
535_2017_1423_MOESM2_ESM.docx (25 kb)
Supplementary material 2 (DOCX 25 kb)
535_2017_1423_MOESM3_ESM.xlsx (20 kb)
Supplementary material 3 (XLSX 20 kb)
535_2017_1423_MOESM4_ESM.doc (52 kb)
Supplementary material 4 (DOC 51 kb)


  1. 1.
    Hori T, Nguyen JH, Uemoto S. Progressive familial intrahepatic cholestasis. Hepatobiliary Pancreat Dis Int. 2010;9:570–8.PubMedGoogle Scholar
  2. 2.
    Paulusma CC, Elferink RP, Jansen PL. Progressive familial intrahepatic cholestasis type 1. Semin Liver Dis. 2010;30:117–24.CrossRefPubMedGoogle Scholar
  3. 3.
    Lam P, Soroka CJ, Boyer JL. The bile salt export pump: clinical and experimental aspects of genetic and acquired cholestatic liver disease. Semin Liver Dis. 2010;30:125–33.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Smit JJ, Schinkel AH, Oude Elferink RP, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell. 1993;75:451–62.CrossRefPubMedGoogle Scholar
  5. 5.
    Davit-Spraul A, Gonzales E, Baussan C, et al. The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects. Semin Liver Dis. 2010;30:134–46.CrossRefPubMedGoogle Scholar
  6. 6.
    Sambrotta M, Strautnieks S, Papouli E, et al. Mutations in TJP2 cause progressive cholestatic liver disease. Nat Genet. 2014;46:326–8.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pauli-Magnus C, Meier PJ, Stieger B. Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy. Semin Liver Dis. 2010;30:147–59.CrossRefPubMedGoogle Scholar
  8. 8.
    Poupon R, Rosmorduc O, Boëlle PY, et al. Genotype-phenotype relationships in the low-phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients. Hepatology. 2013;58:1105–10.CrossRefPubMedGoogle Scholar
  9. 9.
    Gordo-Gilart R, Hierro L, Andueza S, et al. Heterozygous ABCB4 mutations in children with cholestatic liver disease. Liver Int. 2016;36:258–67.CrossRefPubMedGoogle Scholar
  10. 10.
    Colombo C, Vajro P, Degiorgio D, et al. SIGENP Study Group for Genetic Cholestasis. Clinical features and genotype-phenotype correlations in children with progressive familial intrahepatic cholestasis type 3 related to ABCB4 mutations. J Pediatr Gastroenterol Nutr. 2011;52:73–83.CrossRefPubMedGoogle Scholar
  11. 11.
    Dröge C, Bonus M, Baumann U, et al. Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants. J Hepatol. 2017;S0168–8278:32147–55.Google Scholar
  12. 12.
    Xuan J, Yu Y, Qing T, et al. Next-generation sequencing in the clinic: promises and challenges. Cancer Lett. 2013;340:284–95.CrossRefPubMedGoogle Scholar
  13. 13.
    Herbst SM, Schirmer S, Posovszky C, et al. Taking the next step forward—diagnosing inherited infantile cholestatic disorders with next generation sequencing. Mol Cell Probes. 2015;29:291–8.CrossRefPubMedGoogle Scholar
  14. 14.
    Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Schwarz JM, Cooper DN, Schuelke M, et al. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods. 2014;11:361–2.CrossRefPubMedGoogle Scholar
  16. 16.
    Richards S, Aziz N, Bale S, et al. ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Betts MJ, Lu Q, Jiang Y, et al. Mechismo: predicting the mechanistic impact of mutations and modifications on molecular interactions. Nucleic Acids Res. 2015;43:e10.CrossRefPubMedGoogle Scholar
  18. 18.
    Pieper U, Webb BM, Dong GQ, et al. ModBase, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res. 2014;42(Database issue):D336–46.CrossRefPubMedGoogle Scholar
  19. 19.
    Hornbeck PV, Chabra I, Kornhauser JM, et al. PhosphoSite: a bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics. 2004;4:1551–61.CrossRefPubMedGoogle Scholar
  20. 20.
    Jay JJ, Brouwer C. Lollipops in the CLINIC: information dense mutation plots for precision medicine. PLoS ONE. 2016;11:e0160519.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Painter JN, Savander M, Ropponen A, et al. Sequence variation in the ATP8B1 gene and intrahepatic cholestasis of pregnancy. Eur J Hum Genet. 2005;13:435–9.CrossRefPubMedGoogle Scholar
  22. 22.
    Vitale G, Pirillo M, Mantovani V, et al. Bile salt export pump deficiency disease: two novel, late onset, ABCB11 mutations identified by next generation sequencing. Ann Hepatol. 2016;15:795–800.PubMedGoogle Scholar
  23. 23.
    Anzivino C, Odoardi MR, Meschiari E, et al. ABCB4 and ABCB11 mutations in intrahepatic cholestasis of pregnancy in an Italian population. Dig Liver Dis. 2013;45:226–32.CrossRefPubMedGoogle Scholar
  24. 24.
    Lang C, Meier Y, Stieger B, et al. Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury. Pharmacogenet Genomics. 2007;17:47–60.CrossRefPubMedGoogle Scholar
  25. 25.
    Jin MS, Oldham ML, Zhang Q, et al. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans. Nature. 2012;490:566–9.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Degiorgio D, Colombo C, Seia M, et al. Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3). Eur J Hum Genet. 2007;15:1230–8.CrossRefPubMedGoogle Scholar
  27. 27.
    Müllenbach R, Linton KJ, Wiltshire S, et al. ABCB4 gene sequence variation in women with intrahepatic cholestasis of pregnancy. J Med Genet. 2003;40:e70.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Liu C, Aronow BJ, Jegga AG, et al. Novel resequencing chip customized to diagnose mutations in patients with inherited syndromes of intrahepatic cholestasis. Gastroenterology. 2007;132:119–26.CrossRefPubMedGoogle Scholar
  29. 29.
    Fanning AS, Anderson JM. Zonula occludens-1 and -2 are cytosolic scaffolds that regulate the assembly of cellular junctions. Ann NY Acad Sci. 2009;1165:113–20.CrossRefPubMedGoogle Scholar
  30. 30.
    Szklarczyk D, Franceschini A, Wyder S, et al. STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015;43:D447–52.CrossRefPubMedGoogle Scholar
  31. 31.
    van Mil SW, Houwen RH, Klomp LW. Genetics of familial intrahepatic cholestasis syndromes. J Med Genet. 2005;42:449–63.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. J Pediatr Gastroenterol Nutr. 2008;46:241–52.CrossRefPubMedGoogle Scholar
  33. 33.
    Zhou S, Hertel PM, Finegold MJ, et al. Hepatocellular carcinoma associated with tight-junction protein 2 deficiency. Hepatology. 2015;62:1914–6.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Degiorgio D, Crosignani A, Colombo C, et al. ABCB4 mutations in adult patients with cholestatic liver disease: impact and phenotypic expression. J Gastroenterol. 2016;51:271–80.CrossRefPubMedGoogle Scholar
  35. 35.
    Gotthardt D, Runz H, Keitel V, et al. A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults. Hepatology. 2008;48:1157–66.CrossRefPubMedGoogle Scholar
  36. 36.
    Ziol M, Barbu V, Rosmorduc O, et al. ABCB4 heterozygous gene mutations associated with fibrosing cholestatic liver disease in adults. Gastroenterology. 2008;135:131–41.CrossRefPubMedGoogle Scholar
  37. 37.
    Van Ooteghem NA, Klomp LW, Van Berge-Henegouwen GP, et al. Benign recurrent intrahepatic cholestasis progressing to progressive familial intrahepatic cholestasis: low GGT cholestasis is a clinical continuum. J Hepatol. 2002;36:439–43.CrossRefPubMedGoogle Scholar
  38. 38.
    Padda MS, Sanchez M, Akhtar AJ, et al. Drug-induced cholestasis. Hepatology. 2011;53:1377–87.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Meier Y, Zodan T, Lang C, et al. Increased susceptibility for Intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the 1331T>C polymorphism in the bile salt export pump. World J Gastroenterol. 2008;14:38–45.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Pauli-Magnus C, Lang T, Meier Y, et al. Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy. Pharmacogenetics. 2004;14:91–102.CrossRefPubMedGoogle Scholar
  41. 41.
    Dixon PH, van Mil SW, Chambers J, et al. Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy. Gut. 2009;58:537–44.CrossRefPubMedGoogle Scholar
  42. 42.
    Ulzurrun E, Stephens C, Crespo E, et al. Role of chemical structures and the 1331T>C bile salt export pump polymorphism in idiosyncratic drug-induced liver injury. Liver Int. 2013;33:1378–85.CrossRefPubMedGoogle Scholar
  43. 43.
    Gudbjartsson DF, Helgason H, Gudjonsson SA, et al. Large-scale whole-genome sequencing of the Icelandic population. Nat Genet. 2015;47:435–44.CrossRefPubMedGoogle Scholar
  44. 44.
    Boldt K, van Reeuwijk J, Lu Q, et al. UK10K Rare Diseases Group. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms. Nat Commun. 2016;7:11491. Scholar
  45. 45.
    Raimondi F, Singh G, Betts MJ, et al. Insights into cancer severity from biomolecular interaction mechanisms. Sci Rep. 2016;6:34490. Scholar
  46. 46.
    Wang NL, Lu YL, Zhang P, et al. Specially designed multi-gene panel facilitates genetic diagnosis in children with intrahepatic cholestasis: simultaneous test of known large insertions/deletions. PLoS ONE. 2016;11:e0164058.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Togawa T, Sugiura T, Ito K, et al. Molecular genetic dissection and neonatal/infantile intrahepatic cholestasis using targeted next-generation sequencing. J Pediatr. 2016;171:171–4.CrossRefPubMedGoogle Scholar
  48. 48.
    Gomez-Ospina N, Potter CJ, Xiao R, et al. Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis. Nat Commun. 2016;18(7):10713.CrossRefGoogle Scholar
  49. 49.
    Qiu YL, Gong JY, Feng JY, et al. Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis. Hepatology. 2017;65:1655–69.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Gonzales E, Taylor SA, Davit-Spraul A, et al. MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease. Hepatology. 2017;65:164–73.CrossRefPubMedGoogle Scholar

Copyright information

© Japanese Society of Gastroenterology 2017

Authors and Affiliations

  • Giovanni Vitale
    • 1
  • Stefano Gitto
    • 1
  • Francesco Raimondi
    • 3
    • 4
  • Alessandro Mattiaccio
    • 2
  • Vilma Mantovani
    • 2
  • Ranka Vukotic
    • 1
  • Antonietta D’Errico
    • 5
  • Marco Seri
    • 1
  • Robert B. Russell
    • 3
    • 4
  • Pietro Andreone
    • 1
    • 6
    Email author
  1. 1.Department of Medical and Surgical SciencesUniversity of BolognaBolognaItaly
  2. 2.Center for Applied Biomedical Research (CRBA)University HospitalBolognaItaly
  3. 3.CellNetworks, BioquantHeidelberg UniversityHeidelbergGermany
  4. 4.Bioochemie Zentrum Heidelberg (BZH)Heidelberg UniversityHeidelbergGermany
  5. 5.Addari Institute of Oncology and Transplant Pathology, Policlinico S. Orsola-MalpighiUniversity of BolognaBolognaItaly
  6. 6.Department of Medical and Surgical Sciences and Research Center for the Study of HepatitisUniversity of Bologna, ItalyBolognaItaly

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