ABCMdb

PMID: 17352537

Jeong H, Herskowitz I, Kroetz DL, Rine J
Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay.
PLoS Genet. 2007 Mar 9;3(3):e39., 2007-03-09 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr ABCB1 p.Trp1108Arg The P-gp reference and nine variants carrying amino-acid-altering single nucleotide polymorphisms (SNPs) were tested on medium containing daunorubicin, doxorubicin, valinomycin, or actinomycin D, revealing SNPs that increased (M89T, L662R, R669C, and S1141T) or decreased (W1108R) drug resistance. Login to comment 4 ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg The R669C allele`s highly elevated resistance was compromised when in combination with W1108R. Login to comment 68 ABCB1 p.Gly185Val The cloned cDNA carried the G185V SNP of ABCB1, and therefore site-directed mutagenesis was used to restore it to the most common allele, referred to as the ABCB1 reference allele in the Pharmacogenetics of Membrane Transporters dataset (pJR2703) (http://pharmacogenetics.ucsf.edu or http://www. Login to comment 79 ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr ABCB1 p.Trp1108Arg We first focused on the five SNPs with highest Grantham values (.80): M89T, L662R, R669C, A893S, and W1108R. Login to comment 80 ABCB1 p.Met89Thr The M89T polymorphic site was not evolutionarily conserved, but the other four sites were highly conserved. Login to comment 81 ABCB1 p.Ser1141Thr ABCB1 p.Pro1051Ala In addition, the P1051A SNP was chosen because of its conservation despite a low Grantham value, and the S1141T SNP was included due to its relatively high allele frequency (11% in African Americans) and evolutionary conservation. Login to comment 82 ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys Although A893S, S1141T, and R669C SNPs are common variants (minor allele frequency !1% in at least one major ethnic group), the remaining four chosen variants are observed only once among 494 alleles from different populations. Login to comment 84 ABCB1 p.Arg669Cys The alignment and allele count of ABCB1 haplotypes based on the 14 nonsynonymous SNPs identified in the previous resequencing project are presented in Table S2. From the standpoint of functional impact, the R669C SNP was particularly interesting. Login to comment 87 ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg Third, the R669C SNP may be in phase with the W1108R variant. Login to comment 88 ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg ABCB1 p.Trp1108Arg One of the two R669C SNPs was detected in an individual whose ABCB1 gene also contained the W1108R variant, potentially resulting in haplotype R669C-W1108R. Login to comment 89 ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg This observation prompted us to test whether a R669C-W1108R allele had a unique phenotype relative to alleles carrying each individual SNP. Login to comment 92 ABCB1 p.Ser1141Thr ABCB1 p.Pro1051Ala ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr ABCB1 p.Trp1108Arg Features of the ABCB1 Variants Analyzed in This Study SNPa Evolutionary Conservationb Chemical Dissimilarity (Grantham Valuec ) Allele Count (out of 494 Alleles) M89T 0 81 1 L662R 3 102 1 R669C 2 180 2 A893S 2 99 151 P1051A 3 27 1 W1108R 3 101 1 S1141T 3 58 23 a Positions are relative to the transcription start site and based on the cDNA sequence from GenBank accession number M14758.1 with the change V185G, which is the most common haplotype in African Americans in the Pharmacogenetics of Membrane Transporters dataset. Login to comment 109 ABCB1 p.Ser1141Thr ABCB1 p.Pro1051Ala ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr ABCB1 p.Trp1108Arg ABCB1 p.Trp1108Arg Different P-gp variants displayed higher levels of resistance (A893S-M89T, L662R, and R669C) or lower levels of resistance (A893S, S1141T, A893S-R669C, A893S-P1051A, W1108R, and W1108R-R669C) relative to the P-gp reference (Figure 2A and 2B). Login to comment 111 ABCB1 p.Arg669Cys The replacement of Arg669 by Cys led to one of the most drastic gain-of-function effects on the ability of P-gp to confer drug resistance. Login to comment 112 ABCB1 p.Trp1108Arg This allele`s elevated resistance was compromised when in combination with W1108R. Login to comment 120 ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr ABCB1 p.Met89Thr In the liquid assay, three variants for daunorubicin (A893S-R669C, A893S-M89T, and R669C) and five variants for doxorubicin (A893S, S1141T, A893S-M89T, L662R, and R669C) exhibited statistically significant increases in EC50 values (p , 0.05) (Figure 2C; Table S4). Login to comment 129 ABCB1 p.Ser1141Thr ABCB1 p.Pro1051Ala ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr ABCB1 p.Trp1108Arg ABCB1 p.Trp1108Arg The average 6 standard deviation of three measurements of relative protein levels is 103 6 19 for A893S, 83 6 17 for S1141T, 94 6 16 for A893S-R669C, 115 6 20 for A893S-M89T, 93 6 19 for L662R, 70 6 22 for A893S-P1051A, 134 6 25 for R669C, 102 6 18 for W1108R, 148 6 24 for R669C-W1108R, and 53 6 13 for frame-shifted, relative to the average of reference P-gp set to 100. Login to comment 159 ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg ABCB1 p.Trp1108Arg Although some alleles showed similar trends of resistance for valinomycin and daunorubicin/doxorubicin, others (e.g., S1141T, W1108R, and W1108R-R669C) were qualitatively different in their resistances. Login to comment 165 ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr Five variants (S1141T, A893S-R669C, A893S-M89T, L662R, and R669C) exhibited a statistically significant increase in EC50 or EC30 values for two or more drugs. Login to comment 166 ABCB1 p.Pro1051Ala The A893S and A893S-P1051A variants caused an increase in resistance only for doxorubicin and valinomycin, respectively. Login to comment 167 ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg The compromising effect of W1108R on R669C was obvious in resistance for all four drugs (Figures 2 and 4A). Login to comment 174 ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Met89Thr ABCB1 p.Trp1108Arg The functional consequences of five ABCB1 polymorphisms were previously unknown: the M89T, L662R, R669C, and S1141T variants were associated with increased resistance to two or more drugs; and the W1108R variant strongly mitigated the impact of R669C on gain of P-gp function (Figures 2 and 4A). Login to comment 175 ABCB1 p.Ser1141Thr Due to its high allele frequency (11% in African Americans), the S1141T SNP in particular deserves further attention to define its clinical significance. Login to comment 176 ABCB1 p.Trp1108Arg As measured by plating efficiency in an acute exposure test, the difference between the reference and most sensitive (W1108R) alleles was approximately 30-fold. Login to comment 189 ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg ABCB1 p.Trp1108Arg The resistance profiles of the S1141T, W1108R, and W1108R-R669C variants showed the largest variation across substrates. Login to comment 192 ABCB1 p.Met89Thr In contrast, all seven SNPs for which functional consequences were determined in this study are located either in the extracellular region (M89T) or in the cytoplasmic region (the remaining six variants). Login to comment 194 ABCB1 p.Ser1141Thr ABCB1 p.Pro1051Ala ABCB1 p.Arg669Cys ABCB1 p.Leu662Arg ABCB1 p.Trp1108Arg Our data on functional consequences revealed that these predictions were sound: four functional SNPs (L662R, R669C, W1108R, and S1141T) scored highly on both criteria, while the two SNPs (A893S and P1051A) that showed no significant functional impact had lower scores on evolutionary conservation and chemical dissimilarity, respectively. Login to comment 195 ABCB1 p.Met89Thr One exception was the M89T variant that altered function despite being poorly conserved among mammals. Login to comment 199 ABCB1 p.Arg669Cys ABCB1 p.Trp1108Arg Indeed, it is striking that the strong impact of R669C on P-gp function diminished almost completely when combined with W1108R (Figures 2 and 4A). Login to comment 200 ABCB1 p.Trp1108Arg In contrast, the W1108R variant either alone or with A893S contributed no significant alterations in EC50 or EC30 values. Login to comment 207 ABCB1 p.Asn21Asp Third, only a few coding SNPs have been functionally tested, such as A893S and N21D, which our analysis predicted would have a weak functional impact [26]. Login to comment