ABCMdb

PMID: 17502832

Choi JH, Ahn BM, Yi J, Lee JH, Lee JH, Nam SW, Chon CY, Han KH, Ahn SH, Jang IJ, Cho JY, Suh Y, Cho MO, Lee JE, Kim KH, Lee MG
MRP2 haplotypes confer differential susceptibility to toxic liver injury.
Pharmacogenet Genomics. 2007 Jun;17(6):403-15., [PubMed]

Sentences

No. Mutations Sentence Comment

93 ABCC2 p.Arg768Trp ABCC2 p.Val417Ile ABCC2 p.Val417Ile ABCC2 p.Val417Ile ABCC2 p.Thr486Ile ABCC2 p.Thr486Ile The pGL3 plasmids containing WT or mutant MRP2 promoters were Table 3 Oligonucleotide primers used in the genotyping and functional molecular studies Primer name Nucleotide sequence Primers for SNaPshot or SNaPIT PCR primers c.334-49C > T Sense 50 -CAT GGG TCC TGG AAA GGT T-30 Antisense 50 -CCC CAT GGT ACC TCC TCA T-30 c.1249G > A (p.V417I) Sense 50 -TTT GTC CAT GGG TCC TAA TTT-30 Antisense 50 -ATG AAG TTG GTC ACA TCC ATG-30 c.1457C > T (p.T486I) Sense 50 -ATG GTG CTT GTA ATC CCA ATT-30 Antisense 50 -TTG CCC AAA CTC CCA TTA-30 c.2620 + 3A > G Sense 50 -AAA AAA GGA GAG TTT GCT AAG AAT C-30 Antisense 50 -ATA CTG AGC AGT TCA GGA ATT AGA TAT T-30 c.2934G > A (p.S978S) Sense 50 -AGT TCT ACT AAT ATT GAG GTG GGG A-30 Antisense 50 -AAT AAA AGC CAC AGA ATT CAT CA-30 c.3972C > T (p.I1324I) Sense 50 -TAC CGA CCT GAG CTG GAT C-30 Antisense 50 -CAT CCA GGC CTT CCT TCA-30 c.4147 - 35G > A Sense 50 -GTA GCC ACT CCG AGC CTT AG-30 Antisense 50 -GGA ATC AGA CCT GGA TGA AAA-30 c.4508 + 12G > A Sense 50 -CCC ACA GGC TGC ACA CCA T-30 Antisense 50 -TGT TAC TGT TGA GCA AGG GTT A-30 Genotyping primers c.334 - 49C > T 50 -TCT GAA GAA TAC TGC CAC TAA CCG A-30 c.1249G > A (p.V417I) 50 -GAC ATC AGG TTC ACT GTT TCT CCA A-30 c.1457C > T (p.T486I) 50 -GGG TGA CTT TTT CTT TAC CTG AAT G-30 c.2620 + 3A > G 50 -CTA TCT TGT CCC AAT CCT TCT TAC A-30 c.2934G > A (p.S978S) 50 -ACC TAC AAG CAA TAG GAT TGT TTT C-30 c.3972C > T (p.I1324I) 50 -CTC CAC CTA CCT TCT CCA TGC TAC C-30 c.4147 - 35G > A 50 -GAA CTC CGA GGT CCT TTT CTG GCA T-30 c.4508 + 12G > A 50 -AAG CCA TCC GTG TCA AGC CCT GTC C-30 Primers for the DNA sequencing of MRP2 promoter regions g. - 1774delG Sense 50 -GAT TCT CCA CCC TCT CTT TT-30 Antisense 50 -CAT TCA GTG TGG GAG AAA AT-30 g. - 1549G > A Sense 50 -CCC ACT TTT TAA TTT GTT AGT GTA-30 Antisense 50 -CTG GGA CTA CAG GCA CAT-30 g. - 24C > T and g. - 23G > A Sense 50 -TAG GCT CAC ACT GGA TAA GC-30 Antisense 50 -TGC ACA TCT AAC ATT TCT GG-30 Mutagenic primers - 24C-T 50 -CAA TCA TAT TAA TAG AAG AGT CTT CGT TCC AGA CGC AGT CCA GGA A-30 c.1249G > A (p.V417I) 50 -GGA AGG AGT ACA CCA TTG GAG AAA CAG TGA ACC TGA TGT C-30 c.2302C > T (p.R768W) 50 -AAA TCT TAG TGG GGG TCA GAA GCA GTG GAT CAG CCT GGC CAG-30 c.2934G > A (p.S978S) 50 -CTA CAA GCA ATA GGA TTG TTT TCA ATA TTC TTC ATC ATC-30 c.3972C > T (p.I1324I) 50 -GAG GGA TCA CTT GTG ACA TTG GTA GCA TGG AGA AGG TAG G-30 Primers for MRP2 promoter cloning First round PCR ( - 2314 to + 348) Sense 50 -AGA TTC ATG ACT TCC TGG CTC CTT-30 Antisense 50 -ACA ACA ATT CTC CTT CCT CAC ACG-30 Second round PCR ( - 2229 to -1) Sense (KpnI site) 50 -CGG GGT ACC TTG ATG AAC ATT TAG ATT CT-30 Antisense (NheI site) 50 -CTA GCT AGC GAT TCC TGG ACT GCG TCT GG-30 RT-PCR primers for exon 4 splicing Sense (exon 3) 50 -CGT GTA TAA ATC CAG GAC CAA GAG A-30 Antisense (exon 5) 50 -GGA GAT GAA GAA CAG GCA GGA GTA G-30 PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction. Login to comment 112 ABCC2 p.Val417Ile ABCC2 p.Thr486Ile Twelve genetic variations were found in the MRP2 gene after comprehensive gene scanning of a total of 100 samples derived from 50 healthy Table 4 Frequency of MRP2 genetic variations in control and toxic hepatitis patients Variant Control Hepatocellular Cholestatic or mixed n n P value n P value g. - 1774delG + / + 50 30 0.28 9 0.04* + / - 46 23 20 - / - 14 4 8 (0.34) (0.27) (0.49) g. - 1549G > A + / + 66 30 0.03* 24 0.11 + / - 42 22 10 - / - 2 5 3 (0.21) (0.28) (0.22) g. - 24C > T + / + 74 31 0.06 25 0.75 + / - 34 22 11 - / - 2 4 1 (0.17) (0.26) (0.18) g. - 23G > A + / + 105 55 37 + / - 5 2 0 (0.02) (0.02) (0.00) c.334 - 49C > T + / + 66 28 0.06 23 0.2 + / - 41 24 11 - / - 3 5 3 (0.21) (0.30) (0.23) c.1249G > A (p.V417I) 92 47 0.22 34 0.29 + / + 15 10 3 + / - 3 0 0 - / - (0.10) (0.09) (0.04) c.1457C > T (p.T486I) 107 56 36 + / + 3 1 1 + / - (0.01) (0.01) (0.01) c.2620 + 3A > G + / + 110 56 37 + / - 0 1 0 (0.00) (0.01) (0.00) c.2934G > A (p.S978S) + / + 100 52 0.52 35 0.35 + / - 9 5 2 - / - 1 0 0 (0.05) (0.04) (0.03) c.3972C > T (p.I1324I) + / + 61 29 0.02* 23 0.12 + / - 47 22 11 - / - 2 6 3 (0.23) (0.30) (0.23) c.4147-35G > A + / + 107 52 37 + / - 3 5 0 (0.01) (0.04) (0.00) c.4508 + 12G > A + / + 108 57 37 + / - 2 0 0 (0.01) (0.00) (0.00) + , major allele, - , minor allele. Login to comment 141 ABCC2 p.Arg768Trp The p.R768W mutation, which was known to cause the DJS by misfolding and premature degradation of MRP2 protein [14], was used as a positive control of MRP2 dysfunction. Login to comment 142 ABCC2 p.Arg768Trp ABCC2 p.Val417Ile None of the common coding region variants, p.V417I, c.2934G > A, or c.3972C > T, induced identifiable defects in protein expression, whereas p.R768W evoked a definite decrease (Fig. 2a). Login to comment 143 ABCC2 p.Arg768Trp Consistent with protein expression levels, MRP2-mediated CF efflux was decreased only in p.R768W, and none of the common variants in the coding regions showed significant changes in transport activity (Fig. 2b). Login to comment 149 ABCC2 p.Arg768Trp ABCC2 p.Arg768Trp ABCC2 p.Val417Ile ABCC2 p.Val417Ile Fig. 2 30 kDa 0 20 40 60 80 100 120 Transportactivity(%) 140 WT 190 kDa MRP2 Neomycin phosphotransferase II (a) (b) WT p.V417I c.2934G > A (Ser978) c.3972C > T (Ile1324) p.R768W Mock (c) 500 400 300 200 100 CC CT TT MRP2 exon 4 β-actin (1) (2) (3) (4) (5) (6) bp 300 p.V417I c.2934G > A (Ser978) c.3972C > T (Ile1324) p.R768W Functional studies of intragenic variations. Login to comment 152 ABCC2 p.Arg768Trp p.R768W was used as a positive control bearing defective protein expression. Login to comment