ABCMdb

PMID: 24530123

Naoi S, Hayashi H, Inoue T, Tanikawa K, Igarashi K, Nagasaka H, Kage M, Takikawa H, Sugiyama Y, Inui A, Nagai T, Kusuhara H
Improved liver function and relieved pruritus after 4-phenylbutyrate therapy in a patient with progressive familial intrahepatic cholestasis type 2.
J Pediatr. 2014 May;164(5):1219-1227.e3. doi: 10.1016/j.jpeds.2013.12.032. Epub 2014 Feb 13., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCB11 p.Arg1231Gln A homozygous c.3692G>A (p.R1231Q) mutation was identified in ABCB11. Login to comment 9 ABCB11 p.Arg1231Gln To test this hypothesis, we investigated the effects of 4PB therapy in a patient with PFIC2 carrying a homozygous c.3692G>A (p.R1231Q) mutation in ABCB11. Login to comment 22 ABCB11 p.Arg1231Gln She developed hepatocellular cholestasis and jaundice with normal serum gamma-glutamyl transferase (GGT) activity at the age of 2 months and was diagnosed with PFIC2 by the presence of the c.3692G>A (p.R1231Q) mutation in both alleles of ABCB11 and no detectable immunosignal for BSEP at the canalicular membrane of a liver section sample (Figure 1, A and B). Login to comment 28 ABCB11 p.Arg1231Gln ABCB11 p.Arg1231Gln ABCB11 p.Thr1210Pro ABCB11 p.Thr1210Pro In Vitro Studies pShuttle (Clontech, Palo Alto, California) containing complementary DNA (cDNA) of human BSEP with a hemagglutinin (HA) tag at the N-terminus (HA-BSEPwild type [WT] ) and that of HA-BSEPWT with the c.3692G>A (p.R1231Q) or p.T1210P mutation (HA-BSEPR1231Q and HA-BSEPT1210P ) were used for this study.8 The c.3692G>A (p.R1231Q) and p.T1210P mutations were introduced into pShuttle containing HA-BSEPWT cDNA by site-directed mutagenesis as described previously.9 HEK293T cells and McA-RH7777 cells transfected with pShuttle containing HA-BSEPWT , HA-BSEPR1231Q , or HA-BSEPT1210P cDNA, or empty vector (EV) (HA-BSEPWT , HA-BSEPR1231Q , HA-BSEPT1210P , or EV HEK293T cells and HA-BSEPWT , HA-BSEPR1231Q , HA-BSEPT1210P , or EV McA-RH7777 cells) were subjected to analysis of quantitative PCR (qPCR), cell surface biotinylation, immunofluorescence, and transport.Allinvitroexperiments were performed asdescribed previously,7,9 and a detailed description of the experiments is presented in the Appendix. Login to comment 48 ABCB11 p.Arg1231Gln The homozygous c.3692G>A (p.R1231Q) mutation in ABCB11 identified is shown by the arrowhead. Login to comment 56 ABCB11 p.Arg1231Gln HEK293T cells (top) and McA-RH7777 cells (bottom) expressing HA-BSEPWT (WT; left) or HA-BSEPR1231Q (R1231Q, right) were analyzed by confocal immunofluorescence microscopy as described in the Methods. Login to comment 64 ABCB11 p.Arg1231Gln Therefore, all encoding exons and flanking areas of both ATP8B1 and ABCB11 were sequenced, and a homozygous c.3692G>A (p.R1231Q) mutation in ABCB11 was identified, which has been reported previously in European white patients with PFIC2 (Figure 1, A).12 This result, combined with the immunosignal of ATP8B1, but not of BSEP at the canalicular membrane of liver sections (Figure 1, B), was the basis of the diagnosis of PFIC2. Login to comment 65 ABCB11 p.Arg1231Gln To characterize the effect of the c.3692G>A (p.R1231Q) mutation on BSEP, HA-BSEPWT and HA-BSEPR1231Q were expressed ectopically in HEK293T cells and McA-RH7777 cells, a rat hepatoma cell line that develops canalicular membranes through the formation of couplets as hepatocytes. Login to comment 79 ABCB11 p.Arg1231Gln These results suggest that 4PB treatment at a clinically relevant dosage for humans could increase BSEP expression at the canalicular membrane in patients with PFIC2 with the c.3692G>A (p.R1231Q) mutation in ABCB11 and, consequently, expand the capacity to secrete bile salt into bile. Login to comment 82 ABCB11 p.Arg1231Gln Therapeutic Effect of 4PB in the Patient with PFIC2 with the c.3692G>A (p.R1231Q) Mutation in ABCB11 Serum liver tests and the itching score did not improve during the period of 4PB treatment at the dosages of 200 and 350 mg/kg/day. However, the serum level of aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT) started to decrease when the dosage was increased to 500 mg/kg/day. Login to comment 97 ABCB11 p.Arg1231Gln HA-BSEPWT (WT) and HA-BSEPR1231Q (R1231Q) HEK293T cells were treated with or without 1 mM 4PB for 24 hour and subjected to A, qPCR and B, cell surface biotinylation and analyzed as described in Methods. Login to comment 99 ABCB11 p.Arg1231Gln McA-RH7777 cells expressing HA-BSEPWT (WT, left) or HA-BSEPR1231Q (R1231Q, right) were treated with or without 1 mM 4PB for 24 hours and then subjected to confocal immunofluorescence microscopy as described in Figure 1, E. Login to comment 113 ABCB11 p.Thr1210Pro Gonzales et al20 demonstrated that 4PB therapy improved liver tests in a patient with PFIC2 with a homozygous p.T1210P mutation in BSEP (BSEPT1210P ),20 which markedly reduced BSEP Figure 3. Login to comment 136 ABCB11 p.Arg1231Gln ABCB11 p.Arg1231Gln Analysis using an in vitro minigene system has suggested that c.3692G>A (p.R1231Q) in ABCB11 causes aberrant ABCB11 splicing.22 However, because BSEP was detected around 160 kDa, which is identical to the value in the control patients (Figure 4, C) and to the reported molecular weight,9 it is likely that the correct splicing occurred to some degree in our patient despite having c.3692G>A (p.R1231Q) in ABCB11 and its resultant protein product on the canalicular membrane was prevented from degradation by 4PB therapy. Login to comment