ABCMdb

PMID: 11477083

Mor-Cohen R, Zivelin A, Rosenberg N, Shani M, Muallem S, Seligsohn U
Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome.
J Biol Chem. 2001 Oct 5;276(40):36923-30. Epub 2001 Jul 26., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe One mutation, 3517A3T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G3A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Login to comment 7 ABCC2 p.Arg1150His Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. Login to comment 8 ABCC2 p.Ile1173Phe In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Login to comment 63 ABCC2 p.Ile1173Phe Incorporation of the mutations was verified by DNA sequencing. Two sets of primers were used to introduce the I1173F mutation:1) the forward primer (5Ј-CAGGTTT- GCCAGTTTTCCGTGCCTTTGAGC-3Ј) and the reverse primer (5Ј- GCTCAAAGGCACGGAAAACTGGCAAACCTG-3Ј); 2) the forward primer (5Ј-CCGTATCAGGTTTGCCAGTTTTCCGTGCCTTTGAGC-3Ј) and the reverse primer (5Ј-GCTCAAAGGCACGGAAAACTGGCAAAC- CTGATACGG-3Ј). Login to comment 65 ABCC2 p.Arg1150His The primers used to introduce the R1150H mutation are the forward primer (5Ј-GCCAGCTGAG- GCATCTGGACTCTGTCACCAG-3Ј) and the reverse primer (5Ј-CTG- GTGACAGAGTCCAGATGCCTCAGCTGGC-3Ј). Login to comment 70 ABCC2 p.Ile1173Phe Exon Fragment size Forward primer Reverse primer Name Sequence Name Sequence base pairs 1 404 1F 5Ј-TTGTTGGCCAGCTCTGTTG-3Ј 1R* 5Ј-ACTACCACTTGTTCTGAGTC-3Ј 2 310 2F* 5Ј-TGAAAGCAGTGGGATGTGC-3Ј 2R* 5Ј-CTCTACTGTGCAGCCAAGG-3Ј 3 295 3F* 5Ј-ATCTGAATCACTGCATACCG-3Ј 3R* 5Ј-TCACCTAGATGCCTATGGG-3Ј 4 251 4F* 5Ј-CTCAGTCCTCGGTTAGTGG-3Ј 4R* 5Ј-CTATGAGTTAGAGGTTGCCC-3Ј 5 266 5F* 5Ј-GCCATGTAGACTTCCTTTGG-3Ј 5R* 5Ј-ACCTTATTCTGGGCTTGTGG-3Ј 6 185 6F* 5Ј-TTAGAGTCCCATGAAGTTCC-3Ј 6R* 5Ј-AGTAAGGATACAGCCAATCC-3Ј 7 406 7F* 5Ј-TGGAGATAGCCTCTGACCC-3Ј 7R 5Ј-TGCACTGAGAAGTATGAAGTGC-3Ј 8 428 8F 5Ј-CCTGTACAGAGAAGGCCACG-3Ј 8R 5Ј-CGGTCTTCATGACACAATGC-3Ј 9 515 9F* 5Ј-GATAGTGTAGTCTAGCTGGC-3Ј 9R* 5Ј-TGAGCACCAGAACAGCTTGC-3Ј 10 435 10F* 5Ј-ACTCCCTAGTATCCTTGGC-3Ј 10R* 5Ј-GATGGTAGAAAGTCTTCCACCAGC-3Ј 11 348 11F 5Ј-ACAGTCAGGCAAGGGCTATG-3Ј 11R 5Ј-TCCTTACCCACAGAGAGCC-3Ј 12 389 12F* 5Ј-GGATCAGATACACCTGGTGC-3Ј 12R* 5Ј-ACGAAGGTGAAACTAGAGC-3Ј 13 512 13F* 5Ј-AAGGATTGGCTTAGGAGGC-3Ј 13R* 5Ј-AGTCATTCTGGACTCCAAGG-3Ј 14 256 14F* 5Ј-TTAGGAGATGCCAGCTGTGG-3Ј 14R* 5Ј-ATTCTGGCACCAGTACTGCG-3Ј 15 286 15F* 5Ј-GCACTTAGCAGAAACAATCC-3Ј 15R* 5Ј-ACCGAAGACATGCACATAGC-3Ј 16 343 16F* 5Ј-CCTGATACCAGACTTCATGG-3Ј 16R* 5Ј-GTCGGATGTCTCCAAGACC-3Ј 17 289 17F* 5Ј-CTTCAACCCTGCGTTTCTGG-3Ј 17R* 5Ј-CTCTTCAATATGCCTTCACCC-3Ј 18 ϩ 19 792 18F 5Ј-TCACAGGGTGACAAGCAAC-3Ј 19R 5Ј-TTTACCATTCCACCCATGGC-3Ј 20 352 20F 5Ј-GTGTCTCCCTAGTCCATGATGG-3Ј 20R 5Ј-TCACTCAGCTGGCATCAAAG-3Ј 21 330 21F* 5Ј-ATGCGCTTTGATGCCAGCTG-3Ј 21R* 5Ј-ATTGCTCCTGTAAGTATGCG-3Ј 22 417 22F* 5Ј-TTGGCATTCTAGGTGATTCC-3Ј 22R* 5Ј-CACCATGCACAGGAATCCC-3Ј 23 352 23F* 5Ј-CACAAGTCTTCAGGGATTCC-3Ј 23R* 5Ј-GGTACTCAAGAAACACTTGC-3Ј 24 307 24F* 5Ј-TTACATGAAGGAGTACTGGG-3Ј 24R* 5Ј-GGAAGGATGACTTAGCAATTTCC-3Ј 25 460 25F 5Ј-GGAGCCTCTCATCATTCTGC-3Ј 25R 5Ј-TTTCACACCACTAGCCATGC-3Ј 26 402 26F 5Ј-GAGGCATTGCCTAAGAGTGC-3Ј 26R 5Ј-AAAGATGGAGCCAGGGTTTG-3Ј 27 214 27F 5Ј-TTGGTTTCTGTGCCTATGATG-3Ј 27R* 5Ј-GCACTCTCGAAGGAGTTGC-3Ј 28 323 28F* 5Ј-TCTATGTCTCGAGTCCTGGG-3Ј 28R* 5Ј-CAAATGATGAAGGCTTAGGG-3Ј 29 285 29F* 5Ј-ATGGAGTAGCCAGTCACTGC-3Ј 29R* 5Ј-CCCGAGTAAGTTCTAGAGC-3Ј 30 321 30F* 5Ј-CAGGAATCCATCTCAGGCC-3Ј 30R* 5Ј-CACATCCTCTCATTGCCTGC-3Ј 31 282 31F* 5Ј-CTTTAGGAGCTAACACATGG-3Ј 31R* 5Ј-GAGCAAGGGTTAAGCCATCC-3Ј 32 192 32F* 5Ј-AATGCCTAGACTTGAGATGC-3Ј 32R 5Ј-CTGCTAGAATTTTGTGCTGTTCACATTC-3Ј three clones of each mutant were analyzed for activity, and all six clones of the I1173F mutant were analyzed for expression by Western blot. Login to comment 97 ABCC2 p.Ile1173Phe RESULTS Identification of Candidate Mutations in DJS Patients- Screening of all 32 exons of the MRP2 gene in an Iranian Jewish patient disclosed a 3517A3T transition in exon 25, predicting an I1173F substitution. Login to comment 105 ABCC2 p.Arg1150His The mutation predicts an R1150H substitution and results in loss of a BsaHI restriction site. Login to comment 120 ABCC2 p.Val417Ile ABCC2 p.Ser281Asn Identification of Novel Polymorphisms in the MRP2 Gene- Four dimorphisms in the MRP2 gene were identified: 1) -24C3T in the 5Ј-untranslated region, 2) 842G3A in exon 7 predicting a S281N substitution, 3) 1249G3A in exon 10 predicting a V417I substitution, and 4) IVS29-35G3A. Login to comment 157 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe Carboxyfluorescein Transport of Mutants MRP2-Transfected HEK-293 cells expressing the I1173F- and R1150H-MRP2 mutants were used to study the effect of the mutations on CF transport. Login to comment 160 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe Cells transfected with either I1173F-MRP2 (Fig. 5C) or R1150H-MRP2 (Fig. 5D) showed a slow efflux of CF, which was similar to that measured in control cells (Figs. Login to comment 164 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe The average rate constant of CF efflux (in s-1 ) by WT-MRP2 (562 Ϯ 56, n ϭ 4) was significantly higher than that measured in control cells (151 Ϯ 45, n ϭ 5), R1150H-MRP2 cells (90 Ϯ 40, n ϭ 5) and I1173F-MRP2 cells (104 Ϯ 66, n ϭ 3). Login to comment 165 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe No significant difference was found between the average rate constant of control, R1150H-MRP2, and I1173F-MRP2 cells. These data indicate TABLE III Frequency of haplotypes in Iranian Jewish and Moroccan Jewish controls and DJS patients Haplotype 5Ј-Untranslated region -24C3T Exon 7 842G3A Exon 10 1249G3A IVS29-35 G3A Frequency in Iranian Jewsa Frequency in Moroccan Jewsa Controls (n ϭ 144) Patients (n ϭ 26) Controls (n ϭ 118) Patients (n ϭ 8) % % 1 C G A A 0 100 0 0 2 C A G G 0.7 0 1.7 100 3 C G G G 63.2 0 61 0 4 C G A G 23.6 0 14.4 0 5 T G G G 11.1 0 18.6 0 Seven additional haplotypes 2.1 0 4.3 0 a n indicates number of informative alleles of unrelated individuals. Login to comment 178 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe Panel E shows the irreversible inhibition of efflux by cyclosporine A. that both I1173F and R1150H mutations impair the transport activity of MRP2. Login to comment 186 ABCC2 p.Arg1150His The R1150H-MRP2 behaved similarly to WT-MRP2, showing a very intense band at 190 kDa and a very weak band at 175 kDa when 20 ␮g of protein was used, and no 175-kDa band when 13 ␮g of protein was used. Login to comment 187 ABCC2 p.Ile1173Phe By contrast, the amount of protein and the ratio between the two bands was different for I1173F-MRP2. Login to comment 188 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe At 20 ␮g of I1173F-MRP2 protein, staining was much weaker than that observed with either WT or R1150H-MRP2. Login to comment 189 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe When 55 ␮g of protein from cells expressing I1173F-MRP2 was used, the overall amount of protein was comparable to that measured with 13 ␮g of WT or R1150H-MRP2. Login to comment 190 ABCC2 p.Ile1173Phe Notably, the 175-kDa band was more intense than the 190-kDa band for I1173F-MRP2. Login to comment 191 ABCC2 p.Ile1173Phe Cellular Localization of WT and Mutants MRP2-The significant amount of the 175-kDa band found in cells expressing I1173F-MRP2 suggests that processing of MRP2 was affected by this mutation. Login to comment 194 ABCC2 p.Arg1150His In cells transfected with WT-MRP2 and in R1150H-MRP2, the protein was localized at the plasma membrane (Fig. 7, A and B) and a Golgi-like structure. Login to comment 195 ABCC2 p.Arg1150His This suggested that processing and targeting of the R1150H-MRP2 mutant was normal. Login to comment 196 ABCC2 p.Ile1173Phe In contrast, I1173F-MRP2 showed largely a reticular pattern of staining (Fig. 7C). Login to comment 199 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe Both mutations are located in exon 25 of the MRP2 gene, with 3517A3T predicting I1173F substitution found in a cluster of Iranian Jewish DJS patients, and 3449G3A predicting R1150H substitution found in a cluster of Moroccan Jewish patients. Login to comment 204 ABCC2 p.Val417Ile ABCC2 p.Ser281Asn Two were in non-coding regions of the gene, -24C3T and IVS29-35G3A, and two were in coding regions of the gene, 842G3A and 1249G3A predicting S281N and V417I substitutions, respectively. Login to comment 218 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe Cells were transfected with GFP only (control, A), WT-MRP2 (B), I1173F-MRP2 (C), or R1150H-MRP2 (D). Login to comment 223 ABCC2 p.Gln1382Arg ABCC2 p.Arg768Tyr ABCC2 p.Arg1066* These mutations include two missense mutations (R768Y, Q1382R), a nonsense mutation (R1066stop), three splice site mutations (1815ϩ2, T3A; 1967ϩ2, T3C; 2439ϩ2, T3C) and a deletion mutation (4175del6). Login to comment 233 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe With this assay we showed that both I1173F and R1150H mutations impaired the MRP2 activity (Fig. 5). Login to comment 237 ABCC2 p.Arg1150His The R1150H mutation had no apparent effect on the relative amount of each MRP2 forms. Login to comment 238 ABCC2 p.Ile1173Phe In contrast, the I1173F mutation gave rise to a markedly reduced relative amount of the 190-kDa band and an increased relative amount of the 175-kDa form. Login to comment 239 ABCC2 p.Ile1173Phe Furthermore, the total amount of I1173F-MRP2 that was expressed was substantially decreased (Fig. 6), which suggests enhanced intracellular degradation, due to mistargeting and misfolding of the mutant protein. Login to comment 240 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe Indeed, immunolocalization of the different constructs showed that WT and R1150H-MRP2 were properly targeted to the plasma membrane, whereas I1173F-MRP2 remained largely confined to the ER. Login to comment 242 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe Our expression studies suggest that the I1173F mutation causes impaired maturation of MRP2, mislocalization probably to the ER, and augmented degradation, whereas the R1150H mutation does not affect the maturation and localization, but impairs the MRP2 transport activity. Login to comment 243 ABCC2 p.Arg1257Ala ABCC2 p.Arg1210Ala Interestingly, two site-directed mutations, which replaced arginine by alanine in the predicted third transmembrane region of MRP2 (R1210A and R1257A), were shown to cause FIG. 6. Login to comment 252 ABCC2 p.Arg1150His ABCC2 p.Ile1173Phe HEK 293 cells transfected with WT-MRP2 (A), R1150H-MRP2 (B), or I1173F-MRP2 (C) were detected by the monoclonal antibody M2III-6 and were examined by confocal laser scanning microscopy. Login to comment