ABCMdb

ABCB4 p.Tyr403His

ClinVar:	c.1207T>C , p.Tyr403His D , Pathogenic
Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (91%), W: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: 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
76 Two patients were homozygous, one for the mutation p.G954S and the second for p.Y403H (Table 3). 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
102 Table 3) includes seven cases: p.Y403H, p.V475A, p.A511T, p.E558K, p.R590Q, p.T593A and p.A1193T. Login to comment
107 In particular, the two missense mutations p.Y403H and p.E558K are located in protein sites already extensively investigated in other ABC transporters. Login to comment
116 13_FRRLWPTIAPFKAGLIVAGVALILNAASDTFMLSLLKPLLDDG_55.. 13_FRRLWPTIAPFKAGLIVAGIALILNAASDTFMLSLLKPLLDDG_55.. 13_FKRLWTYIRLYKAGLVVSTIALVINAAADTYMISLLKPLLDEG_55.. 2_IKRYLQFVKPYKYRIFATIIVGIIKFGIPMLIPLLIKYAIDGV_44.. ...836_AQNIANLGT_844... ..1148_AQNIANLGT_1156.. ...833_AQNTANLGT_841... ...835_AQNTANLGT_843... ...987_AQNTANLGT_995... ...748_AQNTANLGT_756... ...837_TQNIANLGT_845... ...147_VREGASIIG_155... ...147_VREGASIIG_155... ...147_VREGASIIG_155... ..143_WLDCITIII_151... Hs_ABCB4 Pt_ABCB4 Mm_ABCB4 Rn_ABCB4 Bt_ABCB4 Md_ABCB4 Hs_ABCB1 Esch-coli_Msba Salm-typh_Msba Vibrio-ch_Msba Staph-au_Sav1866 950_FSYAGCFRF_958.... 1262_FSYAGCFRF_1270.... 947_FSYAGCFRF_955.... 949_FSYAGCFRF_957.... 1101_FSYAGCFRF_1109 862_FSYAGCFRF_870.... 951_FSYAGCFRF_959.... 261_LALAFVLYA_269.... 261_LALAFVLYA_269.... 261_LALFAVLFL_269.... 257_IGPIIVIGV_265.... 1189_RIAIARALI_1197.... 1494_RIAIARALI_1502.... 1179_RIAIARALI_1187.... 1181_RIAIARALI_1189.... --------- 1093_RIAIARALI_1101.... 1183_RIAIARALV_1191.... .488_RIAIARALL_496.... .488_RIAIARALL_496.... .488_RVAIARALL_496.... .485_RLSIARIFL_493.... Y403H T593AR590Q M630V L701P T715I P726TG723E A737V G954S A1193TA840D Figure 2 Multiple sequence alignment of ABCB4 protein sequences. 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
128 This may be the case for patient 18-I, who had moderate ABCB4 immunostaining (60% of the cells, Fig. 2) and was homozygous for the missense mutation p.Y403H. Login to comment

[hide] Two ABCB4 point mutations of strategic NBD-motifs ... Eur J Hum Genet. 2014 May;22(5):633-9. doi: 10.1038/ejhg.2013.214. Epub 2013 Sep 18. Degiorgio D, Corsetto PA, Rizzo AM, Colombo C, Seia M, Costantino L, Montorfano G, Tomaiuolo R, Bordo D, Sansanelli S, Li M, Tavian D, Rastaldi MP, Coviello DA
Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction.
Eur J Hum Genet. 2014 May;22(5):633-9. doi: 10.1038/ejhg.2013.214. Epub 2013 Sep 18., [PMID:24045840]

Abstract [show]
The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

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3 According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. Login to comment
7 On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile. Login to comment
31 The nucleotide change c.1207T4C that encodes the mutant protein characterized by p.(Y403H) was previously described at homozygous state in the child of family B and at heterozygous state in the other members of family B and C.14 To evaluate the expression of MDR3 in vitro, we used two plasmids: one containing the full-length cDNA of ABCB4 wild type on a pcDNA3.1/ Hygro( &#fe; ) backbone (pcDNA3.1/Hygro( &#fe; )/ABCB4) and the second containing the b-galactosidase gene (pcDNA3.1.V5/His-lacZ). Login to comment
33 The plasmid pcDNA3.1/Hygro(&#fe; )/ABCB4 was also used to obtain clones harboring the p.(Y403H) or the p.(L481R) mutation. Login to comment
80 Wild-type cells secreted more PC and Chol than the mutant ones; PC: 3.417 nmol/mg proteins versus 1.410 for p.L481R and 2.346 for p.Y403H and Chol: 4.476 nmol/mg proteins versus 3.193 for p.L481R and 3.811 for p.Y403H (Figures 4a and b; Supplementary Table 1). Login to comment
82 Although wild-type cells and p.Y403H mutant showed similar fold increases in PC excretion, p.L481R mutant showed higher sensitivity to NaTC reaching 11-fold increase in PC secretion at 3 mM compared with the absence of NaTC (Supplementary Table 1). Login to comment
85 In particular, p.Y403H displayed higher Chol excretion compared with wild-type cells at 3.0 mM NaTC: 9.04 versus 6.05 by referring at parameter 'fold increase` and 34.438 versus 27.074 (nmol/mg proteins) by referring at parameter 'absolute amount`. Login to comment
92 Lanes 5 and 6 show the stable expression of p.L481R-ABCB4 and p.Y403H, respectively. Login to comment
94 The PC/Chol ratio showed a similar trend in absence or with 1.0 and 3.0 mM of NaTC; it was reduced in both mutants, particularly in Y403H cells, whereas an inverted trend was shown after treatment with 0.5 mM NaTC. Login to comment
96 As reported in Figure 4c, in mutant Y403H cells low values of PC efflux are associated with high Chol into the culture medium. Login to comment
98 In this study, besides reporting the identification of a novel ABCB4 mutation (p.(L481R)) within the Q-loop in three siblings suffering from juvenile cholelithiasis, we demonstrate that this mutation, stably transfected in HUH28 cells, as well as the mutation p.(Y403H), previously described by our group,14 do not prevent protein targeting to the plasma membrane but induce an abnormal efflux of PC and Chol. Login to comment
102 The p.(L481R) mutation was identified in three out of seven affected siblings belonging to family A, whereas the already characterized p.(Y403H) mutation6,14 was identified in four affected members belonging to other two families. Login to comment
104 In particular, the infant bearing the homozygous p.(Y403H) mutation was diagnosed as PFIC-3 at the age of 3 months, and at the age of 3 years presented compensated cirrhosis with portal hypertension; (ii) a single mutant allele can be associated with hepatobiliary diseases with less significant and highly variable clinical features. Login to comment
111 Analysis of PC (a) and Chol (b) secretion, and PC-Chol correlation (c) in MDR3 wild-type, MDR3 p.Y403H and MDR3 p.L481R cells. Login to comment
118 As for biliary Chol, it is widely documented that in mice it is excreted into the canalicular lumen through different pathways; among them, a relevant rate of efflux is because of the direct activity of the specific ABC hemitransporters G5 and G8 (ABCG5 and ABCG8).27 Our data show that, in absence or low concentration of NaTC, mutant cells have less Chol efflux ability than wild-type cells, and this direct proportionality between reduced excretion of biliary PC and Chol was originally described in Mdr2/ mice.7 Instead, higher concentrations of NaTC seem to increase Chol efflux by mutant cells, with a maximum value obtained in presence of the p.Y403H mutation so that the ratio between PC and Chol is inverted. 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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106 By contrast, the Y403H and L481R mutations did not alter membrane targeting but decreased ABCB4 activity [61]. Login to comment