ABCMdb

ABCB4 p.Ser320Phe

ClinVar:	c.959C>T , p.Ser320Phe ? , Conflicting interpretations of pathogenicity
Predicted by SNAP2:	A: N (66%), C: D (66%), D: D (71%), E: D (80%), F: D (80%), G: N (53%), H: D (66%), I: N (57%), K: D (80%), L: D (80%), M: D (66%), N: N (78%), P: D (85%), Q: D (75%), R: D (80%), T: N (97%), V: N (61%), W: D (85%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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[hide] Molecular characterization and structural implicat... Eur J Hum Genet. 2007 Dec;15(12):1230-8. Epub 2007 Aug 29. Degiorgio D, Colombo C, Seia M, Porcaro L, Costantino L, Zazzeron L, Bordo D, Coviello DA
Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3).
Eur J Hum Genet. 2007 Dec;15(12):1230-8. Epub 2007 Aug 29., [PMID:17726488]

Abstract [show]
Progressive familial intrahepatic cholestasis type 3 (PFIC3) is an autosomal-recessive disorder due to mutations in the ATP-binding cassette, subfamily B, member 4 gene (ABCB4). ABCB4 is the liver-specific membrane transporter of phosphatidylcholine, a major and exclusive component of mammalian bile. The disease is characterized by early onset of cholestasis with high serum gamma-glutamyltranspeptidase activity, which progresses into cirrhosis and liver failure before adulthood. Presently, about 20 distinct ABCB4 mutations associated to PFIC3 have been described. We report the molecular characterization of 68 PFIC3 index cases enrolled in a multicenter study, which represents the largest cohort of PFIC3 patients screened for ABCB4 mutations to date. We observed 31 mutated ABCB4 alleles in 18 index cases with 29 distinct mutations, 25 of which are novel. Despite the lack of structural information on the ABCB4 protein, the elucidation of the three-dimensional structure of bacterial homolog allows the three-dimensional model of ABCB4 to be built by homology modeling and the position of the mutated amino-acids in the protein tertiary structure to be located. In a significant fraction of the cases reported in this study, the mutation should result in substantial impairment of ABCB4 floppase activity. The results of this study provide evidence of the broad allelic heterogeneity of the disease, with causative mutations spread along 14 of the 27 coding exons, but with higher prevalence on exon 17 that, as recently shown for the closely related paralogous ABCB1 gene, could contain an evolutionary marker for mammalian ABCB4 genes in the seventh transmembrane segment.

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18 The two TMDs contain specific sites for substrate binding and translocation, whereas the two NBDs, which display a high degree of sequence similarity with the equivalent domain of ABC transporters, couple the energy obtained from ATP hydrolysis to substrate transport.8 The ICDs are deemed to be involved in mediating the coupling between NBD conformational changes and the reorientation of TM helices concomitant with substrate extrusion.9 The ABCB1 gene, one of the most extensively studied ABC transporters, is responsible for the human multidrug resistance phenotype that is a rapidly growing obstacle to the treatment of numerous infectious diseases, including human immunodeficiency10 and malaria.11 The properties of this transporter are also exploited in cancer pharmacological therapy where ABCB1 translocates the chemotherapeutic drugs and other molecules with a broad but defined specificity.12 A gene duplication of ABCB1 and additional mutations selected as advantageous have created in mammals the T715I G723E L724AfsX744 A737V G954S G762X T775M G126E S320F A840D OUT IN Linker region F357L L701P A364V NBD-NH2 terminal NBD-COOH terminal A1193T NH2 COOH 1 2 54 6 7 8 129 11 10 EC2EC1 ICD2 A250P Y279X A286V ICD1 R159X T175A ICD3 EC3 EC4 EC6EC5 ICD4 ICD6 ICD5 E888X Y403H V475A A511T E558K R590Q T593A M630V 3 S379KfsX413 P726T Figure 1 (a) Localization of the 29 mutations identified in this study in the ABCB4 protein, schematically represented in its domains. Login to comment
77 Notably, two mutations already described were found in members of different families that are not part of a genetic isolate: p.T175A and p.S320F were identified in unrelated Caucasian patients (three and two cases, respectively). Login to comment
84 There are no PFIC3 epidemiologic data available to date; however, knowing that the number of newborns in Italy has been on average 500 000/year in the last 14 years (http://demo.istat.it/), since we observed 18 patients with ABCB4-mutated alleles born within a 14-year period (with Table 2 Mutations identified in ABCB4 Type of mutationb Exons cDNA locusa Missense Frameshift or nonsense ABCB4-predicted domain GenBank accession numberc Exon 6 c.377G4A G126E TM2 DQ861346 Exon 6 c.523A4G T175A ICD1 Exon 6 c.475C4T R159X ICD1 DQ861347 Exon 8 c.748G4C A250P ICD2 DQ861349 Exon 9 c.837T4A Y279X ICD2 DQ861348 Exon 9 c.857C4T A286V ICD2 DQ861350 Exon 9 c.959C4T S320F TM5 Exon 10 c.1069T4C F357L ICD3 DQ861351 Exon 10 c.1091C4T A364V ICD3 DQ861352 Exon 11 c.1135_1136insAA S379KfsX413 ICD3 DQ861353 Exon 11 c.1207T4C Y403H NBD-NH2 A-loop EF035007 Exon 13 c.1424T4C V475A NBD-NH2 ter DQ861354 Exon 13 c.1531G4A A511T NBD-NH2 ter DQ861355 Exon 14 c.1672G4A E558K NBD-NH2 ter DQ861356 Exon 15 c.1769G4A R590Q NBD-NH2 ter Exon 15 c.1777A4G T593A NBD-NH2 ter DQ861357 Exon 15 c.1888A4G M630V NBD-NH2 ter DQ861358 Exon 17 c.2102T4C L701P Linker region DQ861359 Exon 17 c.2144C4T T715I TM7 DQ861360 Exon 17 c.2168G4A G723E TM7 DQ861361 Exon 17 c.2169_2170insG L724AfsX744 TM7 DQ861362 Exon 17 c.2176C4A P726T TM7 DQ861363 Exon 17 c.2210C4T A737V EC4 DQ861364 Exon 18 c.2284G4T G762X TM8 DQ861365 Exon 19 c.2324C4T T775M TM8 Exon 21 c.2519C4A A840D TM9 DQ861366 Exon 21 c.2662G4T E888X ICD5 DQ861367 Exon 23 c.2860G4A G954S TM11 DQ861368 Exon 27 c.3577G4A A1193T NBD-COOH ter DQ861369 a cDNA sequence is based on reference sequence GenBank NM_018849. Login to comment

[hide] Sequence analysis of bile salt export pump (ABCB11... Pharmacogenetics. 2004 Feb;14(2):91-102. Pauli-Magnus C, Lang T, Meier Y, Zodan-Marin T, Jung D, Breymann C, Zimmermann R, Kenngott S, Beuers U, Reichel C, Kerb R, Penger A, Meier PJ, Kullak-Ublick GA
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 Feb;14(2):91-102., [PMID:15077010]

Abstract [show]
Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder associated with increased risk of intrauterine fetal death and prematurity. There is increasing evidence that genetically determined dysfunction in the canalicular ABC transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) might be risk factors for ICP development. This study aimed to (i). describe the extent of genetic variability in BSEP and MDR3 in ICP and (ii). identify new disease-causing mutations. Twenty-one women with ICP and 40 women with uneventful pregnancies were recruited between April 2001 and April 2003. Sequencing of BSEP and MDR3 spanned 8-10 kb per gene and comprised the promoter region and 100-350 bp of the flanking intronic region around each exon. DNA sequencing of polymerase chain reaction fragments was performed on an ABI3700 capillary sequencer. MDR3 promoter activity of promoter constructs carrying different ICP-specific mutations was studied using reporter assays. A total of 37 and 51 variant sites were detected in BSEP and MDR3, respectively. Three non-synonymous sites in codons for evolutionarily conserved amino acids were specific for the ICP collective (BSEP, N591S; MDR3, S320F and G762E). Furthermore, four ICP-specific splicing mutations were detected in MDR3 [intron 21, G(+1)A; intron 25, G(+5)C and C(-3)G; and intron 26, T(+2)A]. Activity of the mutated MDR3 promoter was similar to that observed for the wild-type promoter. Our data further support an involvement of MDR3 genetic variation in the pathogenesis of ICP, whereas analysis of BSEP sequence variation indicates that this gene is probably less important for the development of pregnancy-associated cholestasis.

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8 Three nonsynonymous sites in codons for evolutionarily conserved amino acids were specific for the ICP collective (BSEP, N591S; MDR3, S320F and G762E). Login to comment
136 1 , 1 Splicing 2 , 1 N591S 3 NA 4 3.7 Splicing 5 NA 6 9.0 7 , 1 8 , 1 9 , 1 10 1.7 G762E 11 , 1 12 2.6 13 2.8 14 4.5 15 2.9 Splicing 16 , 1 17 4.2 18 2.5 Splicing 19 2.3 20 1.4 S320F 21 , 1 ICP-specific variants are indicated with their respective variant number (Tables 1 and 2). Login to comment
109 Non-synonymous changes newly observed as singletons or doubletons in our sample set coded for the following amino acid changes: S320F, E528D, G762E and T775M. Login to comment
116 The ICP-specific variants included two non-synonymous sites (heterozygous : exon 18, G762E; homozygous : exon 19, S320F) and one synonymous site (exon 6, T459C). Login to comment
142 Q R I K R I Q I D F H G I E H D T T E L H S I D D T L K I S E G I G D K R Q R K F F H A I L R G A V A G V T R I G G Q H L E K A Q K H L L G A E I F E YA R R T L I E G L S H A F K A H R D F H S H D Q D K H S T G A L A Q V Q G A T G T R L R S R H G A L L K R E I A E T A T S L T Q E R V Y H S E F L P Y R H S V Q K K I K D E L E A A G H E L A K A Y D A T Q G K K V Q E P I L I E A I S C Q Q R I A I E V V Q G L S L P A K L S H KL H A D T A L R T A K K A I H V V K E Y G K K F D A R V K Q Q R I L A P V F Q G G A V Q H T G H E Q H L K A A S C S S G V L L A Q L G I R H D G Y A G Q L S G G P R A H V P F G R S TT A D L L L I E I H A D L I Q I K Q A L L G V V H S F Y S L Q R E F L Q V T S K G K L H V Q H I D Q S V V E R V G D K H V V F H Y P V K E TE E D L A S T T I H R L S T A E S Y S D I D F K E L L L D G Q E A A K A A A V E L P K K Y F G H I T F E Y L A TE E V V K E S Q E R T C I V G D A L K R A H H I P I F A P L G V T K G L P K D Y F H D L E R H A T G H K P T S E L G I S S K F D G V T K T K K R K H I F R Y S D H Q L T L D K A A Q K A E G V F V D K G A T H F S S L S F H V H L L P K K H T R E E E F P I H A Y Y Y Y H T D S R G H K A I I E S K Q Q K C H I F VG P G D D A F R F C G H R F R D V G A HY L I V H G Y G H Q V F G I GE L H H P S G L G I A H A T T H G H S LFL G A G L A Y I A T L V L S P I I I G HT T V F H S I L F G A F I V G Q S A P C A F S LI L F L F I I S G F T F F L Q G F T F G F A T F V G H F V Y G S H L A F A A S Y Y L L I F I FV A F S P G L Q G I A H A T V C G A H L I A L I L S I S F H I Y A T L L L V F I A IS F G A V A LV H AG S S F G I T Q A F H Y F S Y A L L L V V A I I P V S A I V G A Q H G T G I I I S F I F V V YG I A H F Q Q F A G F I V G F I A K A Y E K D L S F A S L A E A R E A H E K G I K I K A ISAH I E E T D L D H R V T E H K K E S V K F Y V G D K A A T V A H R L S A L L EI K H P A H G H Q K S L D C L A Q V E A D F G A I V V Q G S H S S T Q H H S E F E L H R G R T T I A E E G EE H F L R S K R K L K H S Q H S T QQ QG ID A IVK K G G S L L E A T S Q D R I A I R Q P K I L L A H A V L A V E R G A G P K F D T L Q L E V A K K R I E Y A H A F H H T K D E V H C Y G R G I E E S V V G Q T S F L V P T I H L R E I Y D R H F H V I Q P Y L R Q D I I T G E D H D S T V Q L T G S G C G K S V K H L G K L V Q G S Q V T A R H V K I A V F S Y P S H D P H G E K L G K I S D H E KAGLHF QIIILS F S D I K P H H D I I D V P P V H A E L G D T E V F V I I A A G R A H A F A D P K V L K F H K T E H L R R S L Q T G A Q H IAL V I T S H A I G L L A A S H A V L V V V S Q H T F A A L S K E Y I K T175A E528D T775M R652G S Cytoplasm R T G762E S320F Extracellular K Fig. 2 Secondary structure of multidrug resistance protein 3 with non-synonymous coding region genetic variants. Login to comment
176 Out of the coding region changes, two were newly identified non-synonymous mutations located in highly conserved regions of the protein (S320F and G762E). Login to comment

[hide] Adult progressive intrahepatic cholestasis associa... Hepatol Res. 2009 Jun;39(6):625-31. Epub 2009 Feb 24. Hsu YC, Chen HL, Wu MZ, Liu YJ, Lee PH, Sheu JC, Chen CH
Adult progressive intrahepatic cholestasis associated with genetic variations in ATP8B1 and ABCB11.
Hepatol Res. 2009 Jun;39(6):625-31. Epub 2009 Feb 24., [PMID:19260995]

Abstract [show]
Severe intrahepatic cholestasis with low serum gamma-glutamyltranspeptidase (gamma-GT) activity is exceptionally rare in adult patients, and its association with multi-genetic alterations of bile salt transporters has not been reported. We investigated a 25-year-old man presenting with a four-year history of jaundice. Laboratory and radiographic examinations revealed clinical pictures of progressive intrahepatic cholestasis with low gamma-GT. Serial liver histopathology demonstrated cirrhosis resulting from progressive persistent cholestatic injury. Genetic sequencing studies for the entire coding exons of ATP8B1 and ABCB11 uncovered a heterozygous missense mutation 1798 C->T (R600W) in ATP8B1, and a homozygous nucleotide substitution 1331 T->C (V444A) in ABCB11. In conclusion, this is a rare case of adult onset progressive intrahepatic cholestasis with low gamma-GT associated with heterozygous ATP8B1 mutation and homozygous ABCB11 polymorphism. Further studies are necessary to investigate the impact of heterozygous R600W mutation and whether other cholestatic disorders are multi-genetic.

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86 In contrast, homozygous V444A of ABCB11 was originally considered as a frequent polymorphism in the western population, which might be associated with intrahepatic cholestasis of pregnancy.15 Keitel et al. investigated an unusual case of severe intrahepatic cholestasis of pregnancy and demonstrated that a homozygous V444A in combination with a homozygous MDR3 mutation (S320F) was associated with diminished canalicular BSEP.16 Kubitz et al. reported compound heterozygosity of V444A and E186G in a BRIC-2 patient with decreased canalicular BSEP.12 Lang et al. described a significant association of V444A with drug-related cholestasis and concluded that V444A was susceptible for cholestasis under certain challenges, based on their in vitro study of BSEP expression and its transporter activity.17 It is possible that the multi-genetic alterations in this patient contributed to an inherited susceptibility for secondary insults, which caused the phenotype with disease onset at a later age than usual genetic diseases. Login to comment

[hide] Combined functional variants of hepatobiliary tran... Liver Int. 2009 Sep;29(8):1286-8. Zimmer V, Mullenbach R, Simon E, Bartz C, Matern S, Lammert F
Combined functional variants of hepatobiliary transporters and FXR aggravate intrahepatic cholestasis of pregnancy.
Liver Int. 2009 Sep;29(8):1286-8., [PMID:19490418]

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32 Genetic analysis Sequencing of all exons and exon-intron boundaries of the ABCB4 and ABCB11 genes was performed, revealing the rare ABCB4 mutation c.959C 4 T (p.S320F) and the common ABCB11 variant c.1331T 4C (p.V444A). Login to comment

[hide] Combined features of low phospholipid-associated c... Liver Int. 2010 Feb;30(2):327-31. Epub 2009 Oct 19. Poupon R, Barbu V, Chamouard P, Wendum D, Rosmorduc O, Housset C
Combined features of low phospholipid-associated cholelithiasis and progressive familial intrahepatic cholestasis 3.
Liver Int. 2010 Feb;30(2):327-31. Epub 2009 Oct 19., [PMID:19840255]

Abstract [show]
Adenosine triphosphate-binding cassette, subfamily B, member 4 (ABCB4) gene alterations can cause two distinct clinical entities: progressive familial intrahepatic cholestasis type 3 (PFIC3) and low phospholipid-associated cholelithiasis (LPAC). Based on the findings in two siblings and a review of the literature, we aimed to identify determinants of disease phenotypic traits associated with ABCB4 gene alterations. Two siblings presented, before the age of 30 years, recurrent symptomatic cholelithiasis and extensive biliary fibrosis that progressed towards portal hypertension and liver failure necessitating liver transplantation. We analysed the sequence of the ABCB4 gene and immunolocalization of the protein in the liver. Sequence analysis of ABCB11, potentially involved in similar symptoms, was also performed. Two heterozygous non-synonymous variants of ABCB4 were found in both siblings. One of them (c.959C>T; p.Ser320Phe) was previously implicated in LPAC and the second one (c.2858C>A; p.Ala953Asp) in PFIC3. Both patients were also heterozygous for the ABCB11 variant Val444Ala, which predisposes to cholestatic disorders. ABCB4 was normally detected at the canalicular membrane of hepatocytes. The review of ABCB4 gene variants reported so far shows that the vast majority of variants causing PFIC3 and LPAC are distinct. Also as a general rule, homozygous variants cause PFIC3 while heterozygous variants lead to LPAC. Combined PFIC3 and LPAC phenotype is a rare clinical event, which may be determined by the coexistence of ABCB4 variants related to both phenotypes and also potentially to the ABCB11 variant. Thus, most of the patients presenting with LPAC are not at a particular risk of developing PFIC3 features in adulthood.

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7 One of them (c.959C 4 T; p.Ser320Phe) was previously implicated in LPAC and the second one (c.2858C 4 A; p.Ala953Asp) in PFIC3. Login to comment
89 Sequence ABCB4 gene analysis showed the presence of composite heterozygous double alterations affecting exon 9 (c.959C 4 T; p.Ser320Phe) and exon 23 (c.2858C4 A; p.Ala953Asp) in both siblings (Fig. 1, arrows). Login to comment
110 One was located in exon 9, and resulted in a substitution of serine for phenylalanine (p.Ser320Phe). Login to comment
135 (Ala953Asp), previously reported in a homozygous state in PFIC3 (3) and one heterozygous missense mutation (Ser320Phe) previously reported in LPAC (5). Login to comment
136 The Ser320Phe has never been described in PFIC3 (1-3) and the Ala953Asp has never been found in LPAC [(4, 5) and unpublished personal data]. Login to comment

[hide] Genetic determinants of drug-induced cholestasis a... Semin Liver Dis. 2010 May;30(2):147-59. Epub 2010 Apr 26. Pauli-Magnus C, Meier PJ, Stieger B
Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy.
Semin Liver Dis. 2010 May;30(2):147-59. Epub 2010 Apr 26., [PMID:20422497]

Abstract [show]
Intrahepatic cholestasis of pregnancy and drug-induced cholestasis are two clinically important forms of acquired cholestatic liver disease. The understanding of the underlying mechanisms of acquired cholestasis has recently made considerable progress by the identification of canalicular ATP-binding cassette (ABC) transporters as likely targets for these forms of cholestasis. Cholestasis of pregnancy is linked to estrogen and progesterone metabolites. These metabolites have been shown to impair the bile salt export pump (BSEP) function by an indirect mechanism. In addition, genetic variants (as well as mutants) of the genes coding for the phosphatidylcholine translocator MDR3 and BSEP and for the farnesoid X receptor, which is critical in the transcriptional activation of MDR3 ( ABCB4) and BSEP ( ABCB11) have been associated with intrahepatic cholestasis of pregnancy. The pathogenesis of drug-induced liver injury encompasses a wide spectrum of mechanisms, some of which are still poorly understood. BSEP is now known to be subject to drug inhibition in susceptible patients. Information on genetic factors rendering individuals susceptible to inhibition of BSEP by drugs or their metabolites is still scarce. Besides rare mutations that have been linked to drug-induced cholestasis, the common p.V444A polymorphism of BSEP has been identified as a potential risk factor. In this review, the authors summarize key concepts of physiology of bile formation, diagnostic principles to indentify these forms of acquired cholestasis, as well as pathogenetic mechanisms leading to intrahepatic cholestasis of pregnancy or drug-induced cholestasis. In addition, they review the current knowledge on genetic susceptibility factors for these two forms of cholestasis.

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82 In line with this, a recent report of an ICP patient suffering from alterations in three genes: p.S320F in ABCB4 (previously described in a patient with ICP113 ), p.A444V in ABCB11, and c.-1G> T in FXR,114 again highlights the role of FXR as a factor associated with ICP. Login to comment

[hide] Aspects of liver pathology in adult patients with ... Virchows Arch. 2012 Mar;460(3):291-8. Epub 2012 Feb 14. Wendum D, Barbu V, Rosmorduc O, Arrive L, Flejou JF, Poupon R
Aspects of liver pathology in adult patients with MDR3/ABCB4 gene mutations.
Virchows Arch. 2012 Mar;460(3):291-8. Epub 2012 Feb 14., [PMID:22331132]

Abstract [show]
The aims of this study were to describe the histological liver lesions in adult patients with MDR3/ ABCB4 mutation and to study the usefulness of MDR3 immunostaining as a diagnostic tool. All adult patients from our institution with an MDR3/ABCB4 mutation and a liver histology were included (n = 13). Eleven patients had a single heterozygous gene mutation and two patients had two heterozygous mutations. Two patients had no liver lesions. Eight patients had a mild ductular reaction and portal fibrosis. One patient had a few fibrous septa and two patients had biliary cirrhosis. In three cases intraductal lipid crystals were identified. Two patients had biliary fibroobliterative lesions with no sclerosing cholangitis on cholangiography. Biliary dysplasia was identified in hepatectomy specimens from two patients, one of whom developed an intrahepatic cholangiocarcinoma. One patient with biliary cirrhosis developed a hepatocellular carcinoma. MDR3 immunostainings performed on formalin-fixed paraffin-embedded sections showed a strong canalicular staining in all patients except in one. To conclude, the predominant histological features were ductular reaction with no or mild fibrosis without cholangitis. Liver lesions previously unreported in association with MDR3/ABCB4 gene mutations (biliary dysplasia, cholangiocarcinoma, small duct sclerosing cholangitis) were also found. Lipid crystals in bile ducts may be suggestive of MDR3/ABCB4 mutation. MDR3 immunostaining on formalin-fixed paraffin-embedded sections does not seem to be sensitive for the diagnosis of heterozygous MDR3/ABCB4 mutations.

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107 Location and nucleotide change Effect on protein Status of variant Mutation category 1 c.1328dup p.Arg444Glu fsX4, truncating Heterozygous Insertion 2 c.1584 G > C p.Glu528Asp Heterozygous Missense 3 c.101 C > T p.Thr34Met Heterozygous Missense 4 c.1553delT p.Leu518Tyr fsX16, truncating Heterozygous Deletion 5 c.139 C > G p.Arg 47 Gly Heterozygous Missense 6 c.1217 G > A p.Arg 406 Gln Heterozygous Missense c.140 G > A p.Arg47Gln Heterozygous, compound Missense 7 c.857 C > T p.Ala 286 Val Heterozygous Missense 8 c.2324 C > T p.Thr775Met Heterozygous Missense c.2836 G > A p.Ala946Thr Heterozygous Missense 9 c.523A > G p.Thr175Ala Heterozygous Missense 10 c.1005 + 5 G > A p.? Login to comment
108 Heterozygous Splicing 11 c.523A > G p.Thr175Ala Heterozygous Missense 12 c.959 C > T Ser 320 Phe Heterozygous Missense 13 c.959 C > T Ser 320 Phe Heterozygous Missense Fig. 1 Liver lesions in adult patients with MDR3 deficiency (MDR3/ABCB4 mutation). Login to comment

[hide] Clinical features and genotype-phenotype correlati... J Pediatr Gastroenterol Nutr. 2011 Jan;52(1):73-83. Colombo C, Vajro P, Degiorgio D, Coviello DA, Costantino L, Tornillo L, Motta V, Consonni D, Maggiore G
Clinical features and genotype-phenotype correlations in children with progressive familial intrahepatic cholestasis type 3 related to ABCB4 mutations.
J Pediatr Gastroenterol Nutr. 2011 Jan;52(1):73-83., [PMID:21119540]

Abstract [show]
OBJECTIVES: The aim of the study was to estimate the frequency of ABCB4 mutations among children with chronic intrahepatic cholestasis with elevated gamma-glutamyl-transpeptidase (gamma-GT) activity and to characterize the genotypes with respect to severity of symptoms, response to ursodeoxycholic acid therapy, and outcome. PATIENTS AND METHODS: Molecular analysis of ABCB4 in 133 Italian children was performed, and ABCB4 mutations were classified as disease-causing mutations or benign substitutions according to the prediction algorithm PolyPhen. RESULTS: : Twenty-eight patients were identified carrying 31 mutations (20 disease causing). Twenty patients carried 2 mutated alleles and 8 only 1. At presentation (1-204 months), 20 children were symptomatic with jaundice and/or pruritus, whereas in 8 biochemical cholestasis was a fortuitous finding. Cirrhosis developed in 15 and 6 progressed to terminal liver failure. Disease-causing mutations on both alleles were found to be associated with reduced liver expression of ABCB4 protein, lack of response to ursodeoxycholic acid therapy, and progression to cirrhosis and end-stage liver disease, whereas mild genotypes, including single heterozygous mutations, were generally associated with less severe disease and, often, absence of symptoms. CONCLUSIONS: ABCB4 mutations are responsible for a chronic liver disease in more than one-third of patients with chronic intrahepatic cholestasis and elevated gamma-GT activity. In patients with severe ABCB4 genotype, the disease is often progressive with risk of developing cirrhosis and liver failure during the first 2 decades of life. Patients with mild genotypes, including single heterozygous mutations, have variable expressions of liver disease that may be influenced by comorbidity factors and modulated by still unknown genetic modifiers.

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107 Nucleotidechange (effectonprotein) Predictionscoresby PolyPhenanalysis Nucleotidechange (effectonprotein) Predictionscoresby PolyPhenanalysis Referencefor eachgenotype 1[1-I]c.475C>T(p.R159X)XUnknownUnknown20 1[1-II]c.475C>T(p.R159X)XUnknownUnknownThisstudy 2[2-I]c.523A>G(p.T175A)0.774c.1069T>C(p.F357L)þc.2324C>T(p.T775M)1.079þ0.59720 3[3-I]c.1135_1136insAA(p.S379KfsX413)Xc.2102T>C(p.L701P)2.22620 4[4-I]c.2662G>T(p.E888X)Xc.748G>C(p.A250P)Rc.1888A>G(p.M630V)1.871R1.67720 5[5-I]c.959C>T(p.S320F)1.287c.857C>T(p.A286V)1.40820 6[6-I]c.377G>A(p.G126E)1.998c.1531G>A(p.A511T)2.1720 6[6-II]c.377G>A(p.G126E)1.998c.1531G>A(p.A511T)2.1720 7[7-I]c.2176C>A(p.P726T)2.086c.1769G>A(p.R590Q)þc.2284G>T(p.G762X)2.623RX20 8[8-1]c.1091C>T(p.A364V)1.343c.2210C>T(p.A737V)0.21720 9[9-I]c.1777A>G(p.T593A)2.044UnknownUnknown20 10[10-I]c.2144C>T(p.T715I)0.383UnknownUnknown20 11[11-I]c.2519C>A(p.A840D)1.803c.1424T>C(p.V475A)2.60320 12[12-I]c.1672G>A(p.E558K)2.486c.2168G>A(p.G723E)Rc.3577G>A(p.A1193T)1.548þ2.34120 12[12-II]c.1672G>A(p.E558K)2.486c.2168G>A(p.G723E)Rc.3577G>A(p.A1193T)1.548þ2.34120 13[13-I]c.2860G>A(p.G954S)0.245c.2860G>A(p.G954S)0.24520 14[14-I]c.523A>G(p.T175A)0.774UnknownUnknown20 15[15-I]c.959C>T(p.S320F)1.287c.837T>A(p.Y279X)X20 16[16-I]c.523A>G(p.T175A)0.774UnknownUnknown20 17[17-I]c.2169_2170insG(p.L724AfsX744)Xc.2169_2170insG(p.L724AfsX744)X20 17[17-II]c.2169_2170insG(p.L724AfsX744)Xc.2169_2170insG(p.L724AfsX744)X20 18[18-I]c.1207T>C(p.Y403H)2.798c.1207T>C(p.Y403H)2.79820 19[19-I]c.208G>C(p.G70R)þc.1769G>A(p.R590Q)1.497þ2.623c.959C>T(p.S320F)1.287Thisstudy 19[19-II]c.208G>C(p.G70R)þc.1769G>A(p.R590Q)1.497þ2.623c.959C>T(p.S320F)1.287Thisstudy 19[19-III]c.208G>C(p.G70R)þc.1769G>A(p.R590Q)1.497þ2.623c.959C>T(p.S320F)1.287Thisstudy 20[20-I]c.217C>G(p.L73V)0.489UnknownUnknown22,thisstudy 21[21-I]c.959C>T(p.S320F)1.287c.959C>T(p.S320F)1.28716,thisstudy 22[22-I]c.1207T>C(p.Y403H)2.798UnknownUnknownThisstudy Xidentifiesmutationsthatpredictprematureterminationoftranslation.PolyPhenpredictionwithPSICscoredifferencesbelow1.5definebenignsubstitutions;PSICscoredifferencesencompassing between1.5and2.0(bold)definesubstitutionspossiblydamaging,whereasabove2.0(underlined)definesubstitutionsprobablydamaging. Login to comment
108 substitution p.S320F in 3 cases. Login to comment

[hide] ABCB4 gene mutations and single-nucleotide polymor... J Med Genet. 2009 Oct;46(10):711-5. Epub 2009 Jul 6. Bacq Y, Gendrot C, Perrotin F, Lefrou L, Chretien S, Vie-Buret V, Brechot MC, Andres CR
ABCB4 gene mutations and single-nucleotide polymorphisms in women with intrahepatic cholestasis of pregnancy.
J Med Genet. 2009 Oct;46(10):711-5. Epub 2009 Jul 6., [PMID:19584064]

Abstract [show]
AIM: To evaluate the nature and frequency of ATP-binding cassette subfamily B member 4 (ABCB4) gene variants in a series of French patients with intrahepatic cholestasis of pregnancy (ICP). METHODS: In this prospective study, the entire ABCB4 gene coding sequence was analysed by DNA sequencing in 50 unrelated women with ICP defined by pruritus and raised serum alanine aminotransferase activity or bile acid concentration, with recovery after delivery. Genomic variants detected in patients with ICP were sought in 107 control pregnant women. Patients with ICP and controls were of Caucasian origin. RESULTS: Eight genomic variants were observed. One nonsense mutation (p.Arg144Stop) and two missense mutations (p.Ser320Phe and p.Thr775Met) were revealed each in one heterozygous patient. A third missense mutation (p.Arg590Gln) was detected in three heterozygous patients and in two homozygous patients also homozygous for a particular haplotype of three single-nucleotide polymorphisms (c.175C>T, c.504T>C, c.711A>T). The chromosomal frequency of the p.Arg590Gln variant was significantly different between the ICP and control group (7.0% vs 0.5%; p = 0.0017; OR 16.03, 95% CI 1.94 to 132.16). An association was also found between allele T of the c.504T>C silent nucleotide polymorphism and ICP (68.0% vs 53.7%; p = 0.017; OR 1.83, 95% CI 1.08 to 3.11). The chromosomal frequency of the p.Arg652Gly variant did not differ between the ICP and control group (p = 0.40). CONCLUSIONS: This study shows that 16% of Caucasian patients with ICP bear ABCB4 gene mutations, and confirms the significant involvement of this gene in the pathogenesis of this complex disorder.

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5 One nonsense mutation (p.Arg144Stop) and two missense mutations (p.Ser320Phe and p.Thr775Met) were revealed each in one heterozygous patient. A third missense mutation (p.Arg590Gln) was detected in three heterozygous patients and in two homozygous patients also homozygous for a particular haplotype of three single-nucleotide polymorphisms (c.175C.T, c.504T.C, c.711A.T). Login to comment
48 A nonsense mutation (nucleotide position from ATG, c.462C.T; amino acid position, p.Arg144Stop) was detected in exon 6 in one heterozygous patient.14 A missense mutation (c.959C.T, p.Ser320Phe) was detected in exon 9 in one heterozygous patient. A missense mutation (c.1769G.A, p.Arg590Gln) was detected in exon 15 in two homozygous and three heterozygous patients with ICP and in one heterozygous control. Login to comment
66 The heterozygous nonsense mutation (p.Arg144Stop) detected in one patient has previously been reported by our group.14 Three missense mutations (p.Ser320Phe, p.Arg590Gln, p.Thr775Met) were detected in this study. Login to comment
68 The p.Ser320Phe mutation has been described in homozygous patients with ICP16 21 and was absent in our control group. Login to comment
74 Furthermore, Arg590 is well conserved during evolution (fig 1), and the missense mutation p.Arg590Gln is localised in the first ATP-binding domain of the ABCB4 protein, where it substitutes a basic amino acid for a Table 3 Allele frequencies of ABCB4 genomic variants in 50 patients with intrahepatic cholestasis of pregnancy (100 chromosomes) and 107 controls (214 chromosomes) Genomic variant Exon Allele identification ICP (n (%)) Controls (n (%)) p Value Deduced effect Protein domain c.175 C.T 4 C 86 (86) 180 (84.1) 0.66 Silent T 14 (14) 34 (15.9) c.462 C.T 6 C 99 (99) 214 (100) 0.14 p.Arg144Stop (nonsense) Intracytoplasmic first loopT 1 (1) 0 (0) c.504 T.C 6 T 68 (68) 115 (53.7) 0.017 Silent C 32 (32) 99 (46.3) c.711 A.T 8 A 86 (86) 176 (82.2) 0.40 Silent T 14 (14) 38 (17.8) c.959 C.T 9 C 99 (99) 214 (100) 0.14 p.Ser320Phe (missense) TM5 T 1 (1) 0 (0) c.1769 G.A 15 G 93 (93) 213 (99.5) 0.0017 p.Arg590Gln (missense) NBD1 A 7 (7) 1 (0.5) c.1954 A.G 16 A 97 (97) 202 (94.4) 0.40 p.Arg652Gly (missense) NBD1 G 3 (3) 12 (5.6) c.2324 C.T 19 C 99 (99) 214 (100) 0.32 p.Thr775Met (missense) TM8 T 1 (1) 0 (0) ICP, intrahepatic cholestasis of pregnancy; NBD, nucleotide-binding domain; TM, transmembrane domain; n, number of chromosomes. Login to comment
94 Indeed, 82% of Table 4 Characteristics of eight patients with intrahepatic cholestasis of pregnancy (ICP) exhibiting ABCB4 mutations Nature of mutation (exon) Age Onset of pruritus (weeks of gestation) Total bilirubin* (N = 17 mmol/l) ALT* (N = 35 U/l) Total bile acids* (N = 6 mmol/l) GGT* (N = 15 U/l) Biliary lithiasis p.Arg144Stop:R/X (6) 23 26 36 1280 120.9 63 No p.Ser320Phe:S/F (9) 37 30 22 437 128.7 16 No p.Arg590Gln:R/Q (15) 39 30 8 73 17.7 13 No p.Arg590Gln:R/Q (15) 28 34 21 134 17.7 119 No p.Arg590Gln:Q/Q (15) 33 35 33 137 26.0 37 No p.Arg590Gln:Q/Q (15) 37 30 15 204 6.1 19 No p.Arg590Gln:R/Q (15) 24 30 14 173 78.0 20 No p.Thr775Met:T/M (19) 35 36 17 144 36.2 26 No *Maximum values during one occurrence of ICP. Login to comment

[hide] Hepatobiliary phospholipid transporter ABCB4, MDR3... Dig Liver Dis. 2008 May;40(5):366-70. Epub 2007 Dec 20. Floreani A, Carderi I, Paternoster D, Soardo G, Azzaroli F, Esposito W, Montagnani M, Marchesoni D, Variola A, Rosa Rizzotto E, Braghin C, Mazzella G
Hepatobiliary phospholipid transporter ABCB4, MDR3 gene variants in a large cohort of Italian women with intrahepatic cholestasis of pregnancy.
Dig Liver Dis. 2008 May;40(5):366-70. Epub 2007 Dec 20., [PMID:18083082]

Abstract [show]
BACKGROUND: Intrahepatic cholestasis of pregnancy is a multifactorial disorder of pregnancy associated with a genetic background. AIM: To evaluate the genetic contribution of ABCB4, MDR3 gene in the development of intrahepatic cholestasis of pregnancy in a large cohort of Italian subjects. METHODS: This study represents an extension of a previous multicentre-prospective study including three Italian referral centres. In all, we enrolled 96 women at the 3rd trimester of pregnancy. Genomic DNA was extracted from peripheral venous blood leucocytes by standard procedures. Polymerase chain reaction was used to amplify exon 14, 15 and 16 of MDR3 gene. RESULTS: We found 3 non-synonymous heterozygous mutations in exon 14 (E528D, R549H, G536A), 1 in exon 15 (R590Q) and 2 in exon 16 (R652G, T6671). MDR3 gene variants in exons 14, 15 and 16 occurred in 7/96 of pregnant mothers with intrahepatic cholestasis of pregnancy (7.2%), and in none of 96 pregnant controls matched for age and parity. All seven patients had normal gamma-glutamyl transpeptidase, normal bilirubin, but high levels of both alanine transferase and serum bile acids. One had cholesterol biliary lithiasis. The outcome of pregnancy was normal in four cases (with vaginal delivery), while there was one fetal distress. CONCLUSIONS: MDR3 mutations are responsible for the development of intrahepatic cholestasis of pregnancy in only a small percentage of Italian women. Further genetic studies are warranted, however, to clarify the role of different mutations in intrahepatic cholestasis of pregnancy.

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90 In a recent reported case of severe ICP in a woman of Moroccan descent [21], gene sequencing revealed a homozygous MDR3 mutation (S320F). Login to comment
91 The patient was also homozygous for the common BSEP polymorphism V444A. Login to comment

[hide] Low phospholipid associated cholelithiasis: associ... Orphanet J Rare Dis. 2007 Jun 11;2:29. Rosmorduc O, Poupon R
Low phospholipid associated cholelithiasis: association with mutation in the MDR3/ABCB4 gene.
Orphanet J Rare Dis. 2007 Jun 11;2:29., [PMID:17562004]

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Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of ABCB4 mutations and low biliary phospholipid concentration with symptomatic and recurring cholelithiasis. This syndrome is infrequent and corresponds to a peculiar small subgroup of patients with symptomatic gallstone disease. The patients with the LPAC syndrome present typically with the following main features: age less than 40 years at onset of symptoms, recurrence of biliary symptoms after cholecystectomy, intrahepatic hyperechoic foci or sludge or microlithiasis along the biliary tree. Defect in ABCB4 function causes the production of bile with low phospholipid content, increased lithogenicity and high detergent properties leading to bile duct luminal membrane injuries and resulting in cholestasis with increased serum gamma-glutamyltransferase (GGT) activity. Intrahepatic gallstones may be evidenced by ultrasonography (US), computing tomography (CT) abdominal scan or magnetic resonance cholangiopancreatography, intrahepatic hyperechogenic foci along the biliary tree may be evidenced by US, and hepatic bile composition (phospholipids) may be determined by duodenoscopy. In all cases where the ABCB4 genotyping confirms the diagnosis of LPAC syndrome in young adults, long-term curative or prophylactic therapy with ursodeoxycholic acid (UDCA) should be initiated early to prevent the occurrence or recurrence of the syndrome and its complications. Cholecystectomy is indicated in the case of symptomatic gallstones. Biliary drainage or partial hepatectomy may be indicated in the case of symptomatic intrahepatic bile duct dilatations filled with gallstones. Patients with end-stage liver disease may be candidates for liver transplantation.

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55 The mutation S320F has also been recently identified in a patient with intrahepatic cholestasis of pregnancy and cholelithiasis [7,11]. Login to comment
64 In addition, LPAC syndrome was observed in patients exhibiting the same or similar nonsense or missense mutations: in a mother and her elder son with an identical heterozygous nonsense mutation (1327insA); in independent patients from non-consanguineous families with the same homozygous missense mutation (Ser320Phe or Ala934Thr), while their heterozygous parents were asymptomatic; in patients with a similar nonsense mutation (1006-1016insT and 1006-1016delT) and in unrelated patients with the same missense mutations (Pro1161Ser). Login to comment

[hide] Function and pathophysiological importance of ABCB... Pflugers Arch. 2007 Feb;453(5):601-10. Epub 2006 Apr 19. Oude Elferink RP, Paulusma CC
Function and pathophysiological importance of ABCB4 (MDR3 P-glycoprotein).
Pflugers Arch. 2007 Feb;453(5):601-10. Epub 2006 Apr 19., [PMID:16622704]

Abstract [show]
Like several other ATP-binding cassette (ABC) transporters, ABCB4 is a lipid translocator. It translocates phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane of the hepatocyte. Its function is quite crucial as evidenced by a severe liver disease, progressive familial intrahepatic cholestasis type 3, which develops in persons with ABCB4 deficiency. Translocation of PC makes the phospholipid available for extraction into the canalicular lumen by bile salts. The primary function of biliary phospholipid excretion is to protect the membranes of cells facing the biliary tree against these bile salts: the uptake of PC in bile salt micelles reduces the detergent activity of these micelles. In this review, we will discuss the functional aspects of ABCB4 and the regulation of its expression. Furthermore, we will describe the clinical and biochemical consequences of complete and partial deficiency of ABCB4 function.

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141 Canalicular lipid transport defects can cause gallstone formation Cholesterol supersaturation of bile, which occurs in a large proportion of humans, leads to the formation of cholesterol Walker B; L556R 571del Truncation PFIC3 LPAC ICP 27 splice Truncation 132 del Truncation TM 2; W138R TM 12; 981 del Truncation Linker; Q636X Truncation TM 11; R957X Truncation TM 6; S346I E395G Walker B; I541F TM 12; G983S Walker A; V425M Walker A; T424A Walker B; D564G TM 7; F711S 180 del truncation 336 delT truncation Exon 22-23 del truncation F165I T175A TM 5; M301T TM 5; S320F 336 insT truncation Walker A; 432 insA truncation E528D L591Q W658stop 757 insT R788E A934T P1161S TM 5; S320F TM 8; G762ER144X Walker B; A546D Walker B; G535AALL 96 del Truncation Walker B; L556R 571del Truncation PFIC3 LPAC ICP 27 splice Truncation 132 del Truncation TM 2; W138R TM 12; 981 del Truncation Linker; Q636X Truncation TM 11; R957X Truncation TM 6; S346I E395G Walker B; I541F TM 12; G983S Walker A; V425M Walker A; T424A Walker B; D564G TM 7; F711S 180 del truncation 336 delT truncation Exon 22-23 del truncation F165I T175A TM 5; M301T TM 5; S320F 336 insT truncation Walker A; 432 insA truncation E528D L591Q W658stop 757 insT R788E A934T P1161S TM 5; S320F TM 8; G762ER144X Walker B; A546D Walker B; G535AALL 96 del Truncation Fig. 3 Summary of the known mutations and their localization in the protein, as identified in patients with PFIC type 3, LPAC syndrome (intrahepatic gallstone formation), and intrahepatic cholestasis of pregnancy (ICP). Login to comment

[hide] Combined mutations of canalicular transporter prot... Gastroenterology. 2006 Aug;131(2):624-9. Keitel V, Vogt C, Haussinger D, Kubitz R
Combined mutations of canalicular transporter proteins cause severe intrahepatic cholestasis of pregnancy.
Gastroenterology. 2006 Aug;131(2):624-9., [PMID:16890614]

Abstract [show]
Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic disorder that usually develops in the third trimester of pregnancy and persists until delivery. The cause of ICP remains elusive, but there is evidence that mutations in the canalicular ABC transporter phospholipid flippase (MDR3) and in the bile salt export pump (BSEP) can predispose for the development of ICP. MDR3 and BSEP were investigated by gene sequencing and immunofluorescence microscopy in a patient with severe ICP of early onset. ICP was diagnosed in a patient in the first trimester of pregnancy with severe pruritus, elevated levels of bile salts, and 48-fold elevation of transaminase levels. A liver biopsy specimen showed diminished canalicular expression of the bile salt export pump BSEP, while the expression and localization of the phospholipid flippase MDR3 was normal. Gene sequencing revealed a homozygous MDR3 gene mutation (S320F). The patient was also homozygous for the common BSEP polymorphism V444A. Treatment with ursodeoxycholate normalized transaminase levels but could not prevent further elevation of bile salt levels and preterm delivery. The combined homozygous alterations of the canalicular transporters may explain the early onset and severity of ICP in this patient. The common BSEP polymorphism V444A accounts for the reduced canalicular BSEP expression. Reduced bile salt secretion through BSEP may explain the persistence of elevated bile salt levels and incomplete efficacy of ursodeoxycholate treatment.

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5 Gene sequencing revealed a homozygous MDR3 gene mutation (S320F). Login to comment
32 A single nucleotide exchange from cytidine to thymidine at position 959 in exon 9 of MDR3 (959C¡T) led to a change from serine to phenylalanine (S320F) (Figure 2A), which has been described to be ICP specific.6 The second homozygous polymorphism was detected in the BSEP gene at the complementary DNA position 1331 with a thymidine replaced by a cytidine (1331T¡C), leading to an exchange of valine to alanine (V444A) (Figure 2B). Login to comment
35 The patient`s mother and aunt (the mother`s sister) were heterozygous for the MDR3 mutation S320F. Login to comment
58 Two ICP-specific nonsynonymous SNPs were detected in the MDR3 gene (S320F and G762E).6 In the BSEP gene, one ICP-specific SNP was found (N591S).6 Another nucleotide exchange from T to C at position Figure 2. Login to comment
63 The mutations found in this patient are within the fifth transmembrane domain (MDR3, S320F) and in the nucleotide binding fold (BSEP, V444A). Login to comment
81 The MDR3 mutation S320F has been shown to be specific for ICP.6 Strikingly, S320F does not result in an obvious decrease in MDR3 immunoreactivity as compared with BSEP. Login to comment
82 This is in line with recent observations that at least 4 of the 18 published severe, PFIC-3-associated, MDR3 mutations do not alter MDR3 localization.10,21,22 In conclusion, the ICP-relevant mutation S320F probably alters the activity but not the localization of MDR3. Login to comment
92 Treatment options for patients with ICP include cholestyramine and ursodeoxycholate.24 To date, ursodeoxycholate seems to represent the most effective therapy, which has a beneficial effect on pruritus and liver enzyme levels.24-26 Ursodeoxycholate may also improve fetal outcome by reducing the risk of preterm delivery.26 Typically, the total amount of serum bile salts (including ursodeoxycholate) is not significantly affected by ursodeoxycholate treatment.24 However, ursodeoxycholate replaces endogenous bile salts (by about 45%), thereby reducing the absolute (and relative) amount of endogenous bile salts by almost 60%.24 In patients with PFIC-3 with severe MDR3 mutations, residual MDR3 activity was shown to be essential for a response to ursodeoxycholate treatment.22,27 The remaining activity of MDR3 in our patient with the homozygous mutation S320F may account in part for the normalization of liver enzyme levels that was induced by UDCA. Login to comment
96 Both the mother and aunt of our patient, who were heterozygous for the MDR3 mutation S320F, had a milder course of ICP, pointing to a genotype-phenotype relationship. Login to comment
97 Homozygosity for both the S320F MDR3 mutation and the V444A BSEP mutation may account for the severity and early onset of ICP in our patient. Login to comment

[hide] Intrahepatic cholestasis of pregnancy: three novel... Aliment Pharmacol Ther. 2006 Jun 1;23(11):1649-53. Floreani A, Carderi I, Paternoster D, Soardo G, Azzaroli F, Esposito W, Variola A, Tommasi AM, Marchesoni D, Braghin C, Mazzella G
Intrahepatic cholestasis of pregnancy: three novel MDR3 gene mutations.
Aliment Pharmacol Ther. 2006 Jun 1;23(11):1649-53., [PMID:16696816]

Abstract [show]
BACKGROUND: The aetiology of intrahepatic cholestasis of pregnancy is unknown, but more than 10 different MDR3 gene mutations have recently been identified. AIM: To evaluate the genetic contribution of the MDR3 gene in the pathogenesis of intrahepatic cholestasis of pregnancy in Italian subjects. METHODS: We performed a multicentre prospective case-control study, enrolling 80 women with intrahepatic cholestasis of pregnancy at the third trimester of pregnancy and 80 pregnant women without intrahepatic cholestasis of pregnancy. Genomic DNA was extracted from peripheral venous blood leucocytes using standard procedures. The polymerase chain reaction was used to amplify exon 14 of the MDR3 gene and the polymerase chain reaction products were sequenced using a Big Dye Terminator Cycle Sequencing kit. RESULTS: Three novel non-synonymous heterozygous mutations in exon 14 were found (4%; E528D, R549H, G536R) among the 80 intrahepatic cholestasis of pregnancy patients, whereas the pregnant controls were all negative for exon 14 polymorphisms. The three patients involved had normal GGT and bilirubin, but high levels of both ALT and serum bile acids. One had cholesterol bile stones. The outcome of pregnancy was normal for two (with vaginal delivery), while foetal distress was recorded in the third. CONCLUSIONS: These three novel mutations add further information on the involvement of the MDR3 gene in intrahepatic cholestasis of pregnancy. As in other studies, we found only heterozygous mutations that could cause an impaired transport protein function, not its absence (which is responsible for more severe liver disease). Different genetic backgrounds might justify the presence of novel MDR3 gene mutations.

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35 MDR3 mutations reported in the literature Mutation (codon) Exon Reference (R144X) 6 Gendrot et al.5 481G>A (R150K) 6 Mu¨llenbach et al.6 426-432del (132) 6 DeVree et al.13 959C>T (S320F) 9 Rosmordurc et al.,14 Pauli-Magnus et al.9 (G535D) 14 Lucena et al.7 1669 C>A (A546D) 14 Dixon et al.4 1712 del T (571) 14 Jacquemin et al.8, 15 2285 G>A (G762E) 18 Pauli-Magnus et al.9 2901 C>T (R957X) 23 DeVree et al.13 conditions included an initial denaturation step at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s. Login to comment

[hide] ABCB4 gene mutation-associated cholelithiasis in a... Gastroenterology. 2003 Aug;125(2):452-9. Rosmorduc O, Hermelin B, Boelle PY, Parc R, Taboury J, Poupon R
ABCB4 gene mutation-associated cholelithiasis in adults.
Gastroenterology. 2003 Aug;125(2):452-9., [PMID:12891548]

Abstract [show]
BACKGROUND & AIMS: We recently put forward arguments in favor of ABCB4 gene (adenosine triphosphate-binding cassette, subfamily B, member 4) defects as a risk factor for symptomatic cholelithiasis in adults. In this study, we characterized ABCB4 gene mutations in a series of patients with symptomatic cholelithiasis to determine the genetic basis and the clinical phenotype of ABCB4 gene mutation-associated cholelithiasis. METHODS: We analyzed the entire ABCB4 gene coding sequences in a first group of 32 patients who had a clinical history compatible with the syndrome previously described, in a second group of 28 patients who presented with a classic gallstone disease that justified a cholecystectomy, and in a third group of 33 patients without a history of cholelithiasis. RESULTS: We identified both heterozygous and homozygous ABCB4 gene point mutations in 18 of 32 (56%) patients who presented with clinical criteria of the syndrome, whereas no mutation was detected in the 2 other groups of patients (P < 0.001). Three independent clinical features were strongly associated with point mutations: recurrence of symptoms after cholecystectomy (odds ratio, 8.5); intrahepatic hyperechoic foci, intrahepatic sludge, or microlithiasis (odds ratio, 6.1); and age <40 years at the onset of symptoms (odds ratio, 3.0). ABCB4 gene point mutations were detected exclusively in the patients who showed 2 or 3 of these clinical features. CONCLUSIONS: Our results show that ABCB4 gene mutations represent a major genetic risk factor in a symptomatic and recurring form of cholelithiasis in young adults.

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68 ABCB4 Gene Mutations in Patients With LPAC Syndrome Gene position Location and nucleotide change Peptide change Protein domain Status 6 495T3A Phe165Ile First intracellular loop Heterozygous 523T3C Thr175Ala between TM2 and TM3 Heterozygous 9 902T3C Met301Thr TM5 Heterozygous 959C3T Ser320Phe TM5 Homozygous 10 1007-1015insT 355Stop TM6 Heterozygous 1007-1015delT 341Stop TM6 Heterozygous 12 1327insA 447Stop Close to NBD11 Heterozygous 14 1584G3C Glu528Asp Close to NBD11 Heterozygous 15 1772T3A Leu591Gln Third intracellular loop Homozygous 17 1973G3A Try658Stop Third intracellular loop linker domain Heterozygous 18 2270-2273insT 793Stop Fourth intracellular loop between TM8 and TM9 Heterozygous 19 2363G3T Arg788Glu Fourth intracellular loop between TM8 and TM9 Heterozygous 23 2800G3T Ala934Thr Fifth intracellular loop between TM10 and TM11 Homozygous 26 3481C3T Pro1161Ser Close to NBD2 Heterozygous NOTE. The A of ATG of the initiator Met codon was denoted as "nucleotide ϩ 1." Login to comment
105 In addition, the LPAC syndrome was observed in patients with similar nonsense or missense mutations: in a mother and her elder son with an identical heterozygous nonsense mutation (1327insA); in patients from nonconsanguineous families with the same homozygous missense mutation (Ser320Phe or Ala934Thr) with asymptomatic heterozygous parents; in patients with a similar nonsense mutation (1006-1016insT and 1006-1016delT); and in unrelated patients with the same missense mutations (Pro1161Ser). Login to comment
106 In addition, the S320F mutation was recently identified in a patient who presented with ICP and cholelithiasis.5 For these reasons, all these mutations could be considered as causing the LPAC syndrome. Login to comment

[hide] A second heterozygous MDR3 nonsense mutation assoc... J Med Genet. 2003 Mar;40(3):e32. Gendrot C, Bacq Y, Brechot MC, Lansac J, Andres C
A second heterozygous MDR3 nonsense mutation associated with intrahepatic cholestasis of pregnancy.
J Med Genet. 2003 Mar;40(3):e32., [PMID:12624161]

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131 Two of them, from independent and non-consanguineous families, had a homozygous missense mutation in exon 9 (S320F) and the third woman had a heterozygous 1 bp insertion in exon 12 (1327insA), with an early stop codon. Login to comment
169 By contrast, we did not find the six mutations of MDR3 already described in exons 6, 9, 12, 14, and 23 in patients suffering from ICP, PFIC, or cholestasis during pregnancy associated with chronic liver disease.9-13 The role of MDR3 is essential for the secretion of phosphatidylcholine into bile, as has been shown for its close murine homologue Mdr2.15 The Mdr2 knockout mouse is completely unable to secrete phosphatidylcholine into bile and develops mild liver disease, since the augmentation of free biliary bile acids alters the canalicular membrane of the hepatocyte.14 15 In patients with PFIC3, human bile salts are very hydrophobic and the absence of human phosphatidylcholine translocater coded by MDR3 results in serious liver disease.16 The apparent heterozygous status of our patient is in agreement with previous studies in consanguineous families.9 11 In these studies, women affected by ICP were heterozygous for the familial mutation, whereas patients affected by PFIC3 were homozygous.9 10 Moreover, a homozygous state for the missense mutation S320F was observed in two women suffering from chronic liver disease with clinical and biological features outside pregnancy.13 The discovery of these mutations in MDR3 opens the way for a better understanding of the pathogenesis of ICP. Login to comment

[hide] MDR3 gene defect in adults with symptomatic intrah... Gastroenterology. 2001 May;120(6):1459-67. Rosmorduc O, Hermelin B, Poupon R
MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis.
Gastroenterology. 2001 May;120(6):1459-67., [PMID:11313316]

Abstract [show]
BACKGROUND & AIMS: Many studies indicate that gallstone susceptibility has genetic components. MDR3 is the phosphatidylcholine translocator across the hepatocyte canalicular membrane. Because phospholipids are a carrier and a solvent of cholesterol in hepatic bile, we hypothesized that a defect in the MDR3 gene could be the genetic basis for peculiar forms of cholesterol gallstone disease, in particular those associated with symptoms and cholestasis without evident common bile duct stone. METHODS: We studied 6 adult patients with a peculiar form of cholelithiasis. MDR3 gene sequence was determined by reverse-transcription polymerase chain reaction amplification of mononuclear cell RNAs followed by direct sequencing. Hepatic bile was analyzed in 2 patients. RESULTS: All patients shared the following features: at least 1 episode of biliary colic, pancreatitis, or cholangitis; biochemical evidence of chronic cholestasis; recurrence of symptoms after cholecystectomy; presence of echogenic material in the intrahepatic bile ducts; and prevention of recurrence by ursodeoxycholic acid therapy. Hepatic bile composition showed a high cholesterol/phospholipid ratio and cholesterol crystals. In all patients, we found MDR3 gene mutations involving a conserved amino acid region. CONCLUSIONS: These preliminary observations suggest that MDR3 gene mutations represent a genetic factor involved in this peculiar form of cholesterol gallstone disease in adults. They require further studies to assess the prevalence of MDR3 gene defects in symptomatic and silent cholesterol gallstone disease.

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155 Sequencing of amplified genomic DNA confirmed the homozygosity of patients 1 and 4 for mutation S320F, the heterozygosity of patients 2 and 3 for mutation 1327insA, the heterozygosity of patient 5 for mutation T175V, and the homozygosity of patient 6 for mutation P1161S. Login to comment
157 Restriction endonuclease digestion also confirmed the presence of the S320F mutation in patients 1 (homozygous transition) and 1a and 1b (heterozygous transition) and the absence of mutation in healthy unrelated individuals (Figure 4). Login to comment
166 This amino acid has recently been shown to be included in a very conserved cluster of 4 amino acids at position 169-172 (TRLT) in the central portion of the intracellular loop of the protein required for adenosine triphosphatase activity.19 The S320F mutation (patients 1 and 4) was located in exon 9, at the Figure 3. Login to comment
186 Detection of the (S320F) point mutation in genomic DNA by restriction analysis for patients 1, 1a, and 1b. Login to comment
187 The mutation (S320F) (A) creates a new HinfI site in a 210-bp PCR fragment of exon 9 and (B) removes a BamHI site. Login to comment
190 Lane 1: amplified DNA fragment without enzymatic digestion; lane 2: S320F mutated homozygote (patient 1); lanes 3 and 4: S320F heterozygotes (patients 1a and 1b); lane 5: wild-type homozygote. Login to comment

[hide] ABCB4: Insights from pathobiology into therapy. Clin Res Hepatol Gastroenterol. 2014 Oct;38(5):557-63. doi: 10.1016/j.clinre.2014.03.001. Epub 2014 Jun 19. Falguieres T, Ait-Slimane T, Housset C, Maurice M
ABCB4: Insights from pathobiology into therapy.
Clin Res Hepatol Gastroenterol. 2014 Oct;38(5):557-63. doi: 10.1016/j.clinre.2014.03.001. Epub 2014 Jun 19., [PMID:24953525]

Abstract [show]
Adenosine triphosphate (ATP)-binding cassette, sub-family B, member 4 (ABCB4), also called multidrug resistance 3 (MDR3), is a member of the ATP-binding cassette transporter superfamily, which is localized at the canalicular membrane of hepatocytes, and mediates the translocation of phosphatidylcholine into bile. Phosphatidylcholine secretion is crucial to ensure solubilization of cholesterol into mixed micelles and to prevent bile acid toxicity towards hepatobiliary epithelia. Genetic defects of ABCB4 may cause progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare autosomic recessive disease occurring early in childhood that may be lethal in the absence of liver transplantation, and other cholestatic or cholelithiasic diseases in heterozygous adults. Development of therapies for these conditions requires understanding of the biology of this transporter and how gene variations may cause disease. This review focuses on our current knowledge on the regulation of ABCB4 expression, trafficking and function, and presents recent advances in fundamental research with promising therapeutic perspectives.

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108 The A286 V mutation was inactive, the S320F was fully active but its expression reduced to 50%, and the A953D mutation affected both the expression and the activity of ABCB4. Login to comment

[hide] Genetic variations of bile salt transporters. Drug Discov Today Technol. 2014 Jun;12:e55-67. doi: 10.1016/j.ddtec.2014.03.006. Kubitz R, Droge C, Kluge S, Stindt J, Haussinger D
Genetic variations of bile salt transporters.
Drug Discov Today Technol. 2014 Jun;12:e55-67. doi: 10.1016/j.ddtec.2014.03.006., [PMID:25027376]

Abstract [show]
Bile salt transporters directly or indirectly influence biological processes through physicochemical or signalling properties of bile salts. The coordinated action of uptake and efflux transporters in polarized epithelial cells of the liver, biliary tree, small intestine and kidney determine bile salt concentrations in different compartments of the body. Genetic variations of bile salt transporters lead to clinical relevant phenotypes of varying severity ranging from a predisposition for drug-induced liver injury to rapidly progressing end-stage liver disease. This review focuses on the impact of genetic variations of bile salt transporters including BSEP, NTCP, ASBT and OSTalpha/beta and discusses approaches for transporter analysis.

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115 A Vol. 12, 2014 Drug Discovery Today: Technologies | Transporter assays patient homozygous for p.V444A of BSEP and p.S320F of MDR3 (ABCB4), suffered from a severe form of ICP. Login to comment

[hide] Successful management of severe intrahepatic chole... BMC Gastroenterol. 2014 Sep 13;14:160. doi: 10.1186/1471-230X-14-160. Kamimura K, Abe H, Kamimura N, Yamaguchi M, Mamizu M, Ogi K, Takahashi Y, Mizuno K, Kamimura H, Kobayashi Y, Takeuchi M, Yoshida K, Yamada K, Enomoto T, Takakuwa K, Nomoto M, Obata M, Katsuragi Y, Mishima Y, Kominami R, Kamimura T, Aoyagi Y
Successful management of severe intrahepatic cholestasis of pregnancy: report of a first Japanese case.
BMC Gastroenterol. 2014 Sep 13;14:160. doi: 10.1186/1471-230X-14-160., [PMID:25218883]

Abstract [show]
BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is a cholestasis condition caused by elevated levels of serum bile acids that mainly occurs in the third trimester of pregnancy. Maternal symptoms include pruritus; elevation of transaminases, biliary enzymes, and bilirubin levels; and abnormal liver function tests. Fetal symptoms include spontaneous preterm labor, fetal distress, and intrauterine death. It is more prevalent in the Caucasians and is rarely found in Asian countries, including Japan. The etiology of ICP has been reported as involving various factors such as, environmental factors, hormone balance, and genetic components. The genetic factors include single-nucleotide polymorphisms (SNPs) in the genes of canalicular transporters, including ABCB4 and ABCB11. It has also been reported that the combination of these SNPs induces severe cholestasis and liver dysfunction. CASE PRESENTATION: Here, we report for the first time a 24-year Japanese case of severe ICP diagnosed by typical symptoms, serum biochemical analysis, and treated with the administration of ursodeoxycholic acid which improved cholestasis and liver injury and prevented fetal death. The sequence analysis showed SNPs reported their association with ICP in the ABCB11 (rs2287622, V444A) and ABCB4 (rs1202283, N168N) loci. CONCLUSION: The risk of ICP has been reported to be population-specific, and it is rare in the Japanese population. Our case was successfully treated with ursodeoxycholic acid and the genetic sequence analysis has supported the diagnosis. Because genetic variation in ABCB4 and ABCB11 has also been reported in the Japanese population, we need to be aware of potential ICP cases in pregnant Japanese women although further studies are necessary.

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23 For example, the combination of homozygous polymorphisms in ABCB11 at the complementary DNA position 1331 with a thymine replaced by a cytosine (1331 T > C, rs2287622), leading to an exchange from valine to alanine (V444A), and in ABCB4 at position 959 in exon 9 with a cytosine replaced by a thymine (959 C > T), leading to an exchange of serine to phenylalanine (S320F), and some other synonymous SNPs are considered to be related to the etiology of severe type of ICP [15]. Login to comment

[hide] Functional Characterization of ABCB4 Mutations Fou... Korean J Physiol Pharmacol. 2013 Dec;17(6):525-30. doi: 10.4196/kjpp.2013.17.6.525. Epub 2013 Dec 16. Kim TH, Park HJ, Choi JH
Functional Characterization of ABCB4 Mutations Found in Low Phospholipid-Associated Cholelithiasis (LPAC).
Korean J Physiol Pharmacol. 2013 Dec;17(6):525-30. doi: 10.4196/kjpp.2013.17.6.525. Epub 2013 Dec 16., [PMID:24381502]

Abstract [show]
Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations.

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8 Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Login to comment
26 In this study, we selected 3 novel missense mutations of ABCB4 that were first reported by Rosmorduc et al. [3] and F165I 5`-AGG AAA TAG GAT GGA TTG ACA TCA ATG ACA-3` M301T 5`-GCA AAC ATT TCC ACG GGT ATT GCC TTS CTG-3` S320F 5`-AGG ACA CAA ATC AGA CAG CAT CAA AGG GAA-3` The SNP sites were marked by bold-faced letters with underlines. Login to comment
56 RESULTS Mutations of ABCB4 examined in this study To perform a molecular characterization of ABCB4 muta- cDNA position Amino acid substitution Predicted domain a c.495T&#ff1e;A F165I ICD1 c.902T&#ff1e;C M301T TM5 c.959C&#ff1e;T S320F TM5 Position of each mutant was based upon the translational start site. Login to comment
80 The ABCB4 mutant, F165I might be located in the ICD1, while other two mutants, M301T and S320F are located in the TM5 [3]. Login to comment
84 To exclude ATPase activity by other endogenous ABC transporters including MDR1, values for transport activity were obtained by subtracting the uptake in empty vector-transfected cells from that in cells transfected with ABCB4 reference or mutant-bearing vectors, at each corresponding paclitaxel concentration. The uptake of paclitaxel Vmax (nmol mg &#ff0d;1 per min) Km (mM) Vmax/Km ratio (nmol mg&#ff0d;1 min &#ff0d;1 per mM) Reference 28.98&#b1;1.565 1.114&#b1;0.1391 27.13&#b1;5.232 F165I 16.82&#b1;1.565* 1.028&#b1;0.1189 15.56&#b1;4.658* M301T 23.23&#b1;0.8641 1.206&#b1;0.2875 20.60&#b1;5.628 S320F 18.55&#b1;2.726* 1.185&#b1;0.1064 15.99&#b1;3.736* Data (mean&#b1;SD) are from 5 separate experiments. Login to comment
92 was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Login to comment
95 We observed that the average values of Vmax/Km for F165I and S320F were significantly reduced compared to that of the ABCB4 reference. Login to comment
100 The 2 mutations, F165I and S320F that showed decreased transport activities as compared to the reference, had significantly decreased MDR3 expression as compared to reference. Login to comment
102 Then, we investigated MDR3 expression levels of these mutants on the plasma membrane by cell surface biotinylation and observed that those of F165I and S320F were significantly decreased by 31%, compared to that of the reference (Fig. 2B). Login to comment
103 The results from immunoblotting or cell surface biotinylation experiments could explain the reason of altered transport activities of ABCB4 mutants, F165I and S320F; the decreased transport activities of these mutants were due to the reduced expression of functional MDR3 on the plasma membrane. Login to comment
106 The subcellular expression of M301T was comparable with that of the reference while the co-localization of F165I and S320F with plasma membrane was decreased. Login to comment
115 Through a membrane vesicular transport assay using paclitaxel, we found that 2 mutants, F165I and S320F, showed significantly decreased transport activity compared to the reference (Fig. 1). Login to comment
116 F165I and S320F are located in the ICD1 and TM5 of MDR3, respectively, that might be involved in coupling the energy from ATP hydrolysis to substrate transport and the conformational change involved in substrate extrusion, respectively [3,15]. Login to comment
120 The reduced transport function of F165I and S320F mutants also might be due to the decreased expression of functional MDR3 on the plasma membrane (Fig. 2). Login to comment
125 2 mutants, F165I and S320F, examined in this study also showed decreased transport activity and protein expression, although the extent of reduction was less than that of I541F. Login to comment

[hide] ABCB4 mutations in adult patients with cholestatic... J Gastroenterol. 2015 Sep 1. Degiorgio D, Crosignani A, Colombo C, Bordo D, Zuin M, Vassallo E, Syren ML, Coviello DA, Battezzati PM
ABCB4 mutations in adult patients with cholestatic liver disease: impact and phenotypic expression.
J Gastroenterol. 2015 Sep 1., [PMID:26324191]

Abstract [show]
BACKGROUND: The ABCB4 gene encodes the MDR3 protein. Mutations of this gene cause progressive familial intrahepatic cholestasis type 3 (PFIC3) in children, but their clinical relevance in adults remains ill defined. The study of a well-characterized adult patient series may contribute to refining the genetic data regarding cholangiopathies of unknown origin. Our aim was to evaluate the impact of ABCB4 mutations on clinical expression of cholestasis in adult patients. METHODS: We consecutively evaluated 2602 subjects with hepatobiliary disease. Biochemical evidence of a chronic cholestatic profile (CCP) with elevated serum gamma-glutamyltransferase activity or diagnosis of intrahepatic cholestasis of pregnancy (ICP) and juvenile cholelithiasis (JC) were inclusion criteria. The personal/family history of additional cholestatic liver disease (PFH-CLD), which includes ICP, JC, or hormone-induced cholestasis, was investigated. Mutation screening of ABCB4 was carried out in 90 patients with idiopathic chronic cholestasis (ICC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), ICP, and JC. RESULTS: Eighty patients had CCP. PSC and ICC patients with PFH-CLD had earlier onset of disease than those without it (p = 0.003 and p = 0.023, respectively). The mutation frequency ranged from 50 % (ICP, JC) to 17.6 % (PBC). Among CCP patients, presence or absence of PFH-CLD was associated with ABCB4 mutations in 26.8 vs 5.1 % (p = 0.013), respectively; in the subset of ICC and PSC patients, the corresponding figures were 44.4 vs 0 % (p = 0.012) and 28.6 vs 8.7 % (p = 0.173). CONCLUSIONS: Cholangiopathies attributable to highly penetrant ABCB4 mutant alleles are identifiable in a substantial proportion of adults that generally have PFH-CLD. In PSC and ICC phenotypes, patients with MDR3 deficiency have early onset of disease.

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72 CCP chronic cholestatic profile, NCCP patients without chronic cholestatic profile, PBC primary biliary cirrhosis, PSC primary sclerosing cholangitis, ICP intrahepatic cholestasis of pregnancy, JC juvenile cholelithiasis, AIH autoimmune hepatitis, OS overlap syndrome between AIH and PBC or PSC, ICC idiopathic chronic cholestasis; a with (n = 14) or without (n = 23) other cholangiopathies in the personal/family history (see ''Methods``), b with (n = 9) or without (n = 15) other cholangiopathies in the personal/family history (see ''Methods``), c with (n = 2) or without (n = 2) other cholangiopathies in the personal/family history (see ''Methods``) Table 1 Heterozygous nucleotide changes within the ABCB4 gene identified in 18 adult patients with cholangiopathies and predicted impact on MDR3 Nucleotide changea Involved regions Type of mutation Mutant protein Location on the protein Degree of conservationb Reference genotypesc c.217C[G Exon 4 Missense p.(L73V) TM1 B 7, 21 c.475C[T Exon 6 Non-sense p.(R159X) ICD1 X 8, 14 c.523A[G Exon 6 Missense p.(T175A) ICD1 B 18, 21, 8, 25, 14 c.959C[T Exon 9 Missense p.(S320F) TM5 B 18, 8, 14 c.1529A[G Exon 13 Missense p.(N510S) N-ter NBD B 14 c.1531G[A Exon 13 Missense p.(A511T) N-ter NBD A 8, 14 c.1633C[T Exon 14 Missense p.(R545C) N-ter NBD A 21 c.1769G[A Exon 15 Missense p.(R590Q) N-ter NBD A 8, 13, 14, 25 c.1846G[A Exon 15 Missense p.(E616K) N-ter NBD A This study (JN392435) c.1901G[A Exon 16 Missense p.G634E Linker region B This study (JN392436) c.2431G[C Exon 20 Missense p.(G811R) ICD4 A This study (JN392437) c.2544_2548delATCAT Exon 21 Frameshift p.(S849YfsX24) TM9 X This study (JN392438) c.2576T[G Exon 21 Missense p.(L859W) TM10 B This study (JN392439) c.2844G[C Exon 23 Missense p.(M948I) TM11 B This study (JN392440) c.3541C[T Exon 27 Non-sense p.(Q1181X) C-ter NBD X This study (JN392441) TM transmembrane domain, ICD intracellular domain, N-ter NBD N-terminal nucleotide binding domain, C-ter NBD C-terminal nucleotide binding domain a The mutations were numbered according to GenBank NM_018849 and NP_061337. Login to comment
76 A second group of seven mutations is ascribed to type B [p.(L73V), p.(T175A), p.(N510S), p.(G634E), p.(L859W), and p.(M948I); and p.(S320F)] (Figs. 2, 3, residues in blue). Login to comment
81 L73V T175A S320F N510S A511T R590Q E616K R545C G634E G811R L859W M948I ...69_SGLPLMMIV_77... ...69_SGLPLMMIV_77... ...66_SGLPLMMIV_74... ...66_SGLPLMMIV_74... ...69_SGLPLMMIV_77... ...69_SGLPLMMIV_77... ...67_SGLPLMMIV_75..... ...63_AGLPLMMLV_71... ...92_LGFPIMTIL_100.. ...59_MSEPLMTVV_67... ...77_GFAMPALTI_85... ...79_ASFPIMSIL_87... ..107_LGMPLMSLV_115.. ...89_AGLPLMSIL_97... ...39_ASDTFMLSL_47... ...39_ASDTFMLSL_47... ...39_AADTYMISL_47... ...28_GIPMLIPLL_36... 171_TELNTRLTD_179... 171_TELNTRLTD_179... 168_TELNTRLTD_176... 168_TELNTRLTD_176... 171_TELNTRLTD_179... 171_TELNTRLTD_179... 169_TELNTRLTD_177... 169_GELNTRLTD_177... 180_GEVVGRMSG_188... 146_GEAASRISA_154.. 164_GEVVGRMSG_172.. 167_GEVVGRMSG_175.. 197_GEITTRITT_205.. 193_GTLATKLFD_201.. 122_GTLLSRITY_130... 122_GTLLSRITY_130... 122_GGLLSRITY_130... 118_GQVISRVIN_126... 586_VIAHRLSTV_594... 586_VIAHRLSTV_594... 583_VIAHRLSTI_591... 583_VIAHRLSTV_591... 586_VIAHRLSTI_594... 586_VIAHRLSTI_594... 584_VIAHRLSTI_592... 584_VIAHRLSTV_592... 595_VVAHRLSTV_603... 561_IVAHRLSTI_569... 579_VIAHRLTTI_587... 582_IVAHRLSTV_590... 621_VIAHRLSTI_629... 608_IIAHRLSTI_616... 534_VIAHRLSTI_542... 534_VIAHRLSTI_542... 534_VIAHRLSTI_542... 531_IVAHRLSTI_539... 316_FWYGSTLVI_324... 316_FWYGSTLVI_324... 313_FWYGSTLVI_321... 313_FWYGSTLVI_321... 316_FWYGSTLVI_324... 316_FWYGSTLVI_324... 314_FWYGSTLVI_322... 314_FWYGTTLVL_322... 325_VWYGGKMIL_333... 291_FWYGAKLVI_299... 309_LWYGSKLVL_317... 312_IWFGGKMIL_320... 342_FWEGGRLLH_350... 338_FYIGVGWVH_346... 267_LYAASFPSV_275... 267_LYAASFPSV_275... 267_LFLASVDSI_275... 263 IGVGAYLAI 271... 506_VKEANAYEFI_515... 506_VKEANAYEFI_515... 503_VKEANAYDFI_512... 503_VKEANAYDFI_512... 506_VKEANAYEFI_515... 504_VKEANAYEFI_513... 504_VKDANAYEFI_513... 504_VKEANAYDFI_513... 515_TELANASKFI_524... 481_AELANAANFI_490... 499_AYLANAARFI_508... 502_TELANAAKFI_511... 541_AKLANAYDFI_550... 528_CKMANAEKFI_537... 454_ARMAYAMDFI_463... 454_ARMAYAMDFI_463... 454_ARQAHAMEFI_463... 451_AKMANAHDFI_460... 541_IAIARALVR_549... 541_IAIARALVR_549... 538_IAIARALVR_546... 538_IAIARALVR_546... 541_IAIARALVR_549... 541_IAIARALVR_549... 539_IAIARALVR_547... 539_IAIARALVR_547... 550_IAVARAILK_558... 516_IAIARAILK_524... 534_VAIARAILK_542... 537_IAIARAILK_545... 576_IAIARAVIS_584... 563_IAIARALVR_571... 489_IAIARALLR_497... 489_IAIARALLR_497... 489_VAIARALLR_497... 486_LSIARIFLN_494... ...612_GSHSELMKK_620... ...612_GSHSELMKK_620... ...609_GSHSELMKK_617... ...609_GSHSELIKK_617... ...612_GSHGELMKK_620... ...612_GNHRELMKK_620... ...610_GSHNELMKK_618... ...610_GNHDELMKE_618... ...621_GSHSELLRD_629... ...587_GSHDELIKD_595... ...605_GTHFDLVQR_613... ...608_GSHSELLKD_616... ...647_GSHNELLDL_655... ...634_GDHRALMAQ_642... ...560_GTHNDLLEH_568... ...560_GTHSELLAQ_568... ...560_GRHADLLAQ_568... ...557_GTHRELIAK_565... 630_MQTSGSQIQ_638... 630_MQTSGSQIQ_638... 627_MQTAGSQIL_635... 627_MQTSGSQIL_635... 630_TQISGSQIQ_638... 630_MQTSGNQTQ_638... 628_MQTSGNQIQ_636... 628_MQTAGNEVE_636... 640_LQEDTKQTE_648... 620_SEVSTSRLK_628... 624_LQEMHQPPP_632... 627_LQEVNKESK_635... 666_QKLSGGEKD_674... 652_AQTFTDAVD_660... 578_MQFGQ----_582... 578_MQFGQ----_582... 578_IQFGE----_582... 575_IQNL-----_578... 807_KNSTGALST_815... 807_KNSTGALST_815... 804_KNSTGALST_812... 806_KNSTGALST_814... 804_KNSTGALST_812... 809_KNSTGALST_817... 806_KNSTGALST_814... 808_KNTTGALTT_816... 825_ENSSGAIGA_833... 797_SHSSGSLGA_805... 835_ENSSGALGA_843... 822_EHSSGAIGA_830... 891_ENTVGAITT_899... 849_QNASGKIST_857... 118_KQSTGTLLS_126... 118_KQSTGTLLS_126... 118_QESTGGLLS_126... 114_NNQVGQVIS_122... 855_QLTLLLLAV_863.... 855_QLTLLLLAV_863.... 852_QLTLLLLSV_860.... 854_QLTLLLLSV_862.... 852_QLTLLLLSV_860.... 857_QLTLLLLVV_865.... 854_QLTLLLLSV_862.... 856_QLTLLLLAI_864.... 873_QLAFIVLAM_881.... 845_KLTLTIMCP_853.... 883_QLALLVLAL_891.... 870_QLALVILVL_878.... 939_KLGLVTLST_947... 897_QMALLIIAI_905.... 166_QLSIILIVL_174.... 166_QLSIILVVL_174.... 166_QLSLVLIVV_174.... 162_KLTLAALFI_170.... 944_SQAFMYFSY_952... 944_SQAFMYFSY_952... 941_SQAFMYFSY_949... 943_SQAFMYFSY_951... 941_SQAFMYFSY_949... 946_SQAFMYFSY_954... 943_SQAFMYFSY_951... 945_TQAMMYFSY_953... 962_SFFVLFSSY_970... 940_SYLMVYLTY_948... 972_SNFVLFGSY_980... 959_SFFLLFSVY_967... 1028_AQGVTFLIN_1036.. 986_ASSVLYLLN_994... 255_IQLIASLAL_263... 255_IQLIASLAL_263... 255_IQMIASLAL_263... 251_INTVTDIGP_259... Hs_MDR3 Pt_MDR3 Mm_MDR3 Rn_MDR3 Bt_MDR3 Cf_MDR3 Md_MDR3 Hs_MDR1 At_MDR Os_MDR Sm_MDR Ptr_MDR Sp_MDR Ce_P-gly Esch-coli_Msba Salm-typh_Msba Vibrio-ch_Msba Staph-au_Sav1866 Hs_MDR3 Pt_MDR3 Mm_MDR3 Rn_MDR3 Bt_MDR3 Cf_MDR3 Md_MDR3 Hs_MDR1 At_MDR Os_MDR Sm_MDR Ptr_MDR Sp_MDR Ce_P-gly Esch-coli_Msba Salm-typh_Msba Vibrio-ch_Msba Staph-au_Sav1866 Fig. 2 Multiple sequence alignment of 18 ABC proteins concerning the amino acid sequences around the 12 ABCB4 missense mutations identified in this study. Login to comment
99 When the 19 patients affected by PBC, AIH, or OS, in whom PFH-CLD was among the enrollment criteria, were excluded from the analysis, the S320F L859W L73V M948I N-ter T175A G811R R545C A511T N510S R590Q E616K G634E C-ter Fig. 3 Ribbon representation of the three-dimensional structure of human MDR3. Login to comment
141 p.(S320F) Yes JC/HIC 16 F PSC 55 p.(R590Q) Yes 7 M ICC 16 p.(R545C) Yes JC Father of patient 72 18a,b M ICC 34 p.(S849YfsX24) Yes JC Family history of JC 49 F ICC 32 p.(G811R) Yes ICP/JC Family history of JC 72 F ICC 12 p.(R545C) Yes ICP/JC Daughter of patient 72 68 F PBC 31 p.(T175A) No ICP 73a F PBC 58 p.(R590Q) Yes JC 75 F PBC 64 p.(L73V) No JC 38 M JC 18 p.(S320F) No 55 M JC 26 p.(Q1181X) Yes 81 F JC 39 p.(R590Q) ? Login to comment
142 p.(M948I) Yes 48 F ICP 37 p.(L859W) No JC 88 F ICP 27 p.(S320F) No HIC PSC primary sclerosing cholangitis, ICC idiopathic chronic cholestasis, PBC primary biliary cirrhosis, JC juvenile cholelithiasis, ICP intrahepatic cholestasis of pregnancy, IBD inflammatory bowel disease, HIC hormone-induced cholestasis, OLT orthotopic liver transplantation a With cirrhosis b With major complications of portal hypertension c Radical mutation is considered highly deleterious for protein function (see ''Methods``) associated with cholesterol increase [11]. Login to comment