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Human Molecular Genetics Advance Access originally published online on June 8, 2005
Human Molecular Genetics 2005 14(14):2075-2087; doi:10.1093/hmg/ddi212
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

A functional polymorphism within the MRP1 gene locus identified through its genomic signature of positive selection

Zihua Wang1,4, Baoshuang Wang5, Kun Tang1, Edmund J.D. Lee2, Samuel S. Chong3,6,7 and Caroline G.L. Lee1,5,*

1Department of Biochemistry, 2Department of Pharmacology, 3Department of Pediatrics and Obstetrics/Gynecology and 4Graduate Programme in Bioengineering, National University of Singapore, Singapore, 5Division of Medical Sciences, National Cancer Center, Singapore and 6Department of Pediatrics and 7Department of Gynecology and Obstetrics, McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

* To whom correspondence should be addressed at: Division of Medical Sciences, National Cancer Center, Level 6, Laboratory 5, 11 Hospital Drive, Singapore 169610, Singapore. Tel: +65 64368353; Fax: +65 62241778; Email: bchleec{at}nus.edu.sg

Received March 28, 2005; Revised May 4, 2005; Accepted June 5, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Searching for genomic evidence of positive selection has been hailed as an attractive strategy for identifying functional polymorphisms. Here, we demonstrate the feasibility of identifying functional polymorphism at the MRP1 gene locus using this strategy. The 190 kDa MRP1 protein is an efflux pump that regulates the accumulation of xenobiotics and drugs in cells. Functional sequence variations within this gene might account, in part, for inter-individual and population differences in drug response. To identify single nucleotide polymorphisms (SNPs) within the MRP1 gene with potentially important functional significance, we scanned for genomic signatures of recent positive selection at this locus in ~480 individuals sampled from the Chinese, Malay, Indian, European-American and African-American populations. The genetic profile of SNPs at this locus revealed high haplotype diversity and weak linkage disequilibrium (LD). Despite this weak LD, major allele G of SNP 5'FR/G-260C contained within a high frequency haplotype exhibited extended haplotype homozygosity across 135 kb in European-Americans. Using two independent genomic tests, long-range haplotype (LRH) test and the FST statistic, we found statistical evidence of positive selection for this allele in the European-American population. When this SNP was recapitulated in an in vitro MRP1 promoter–reporter assay, significantly lower activity was observed from the G-containing promoter when compared with the C-containing promoter in all four cell lines that we tested (P<0.01). These observations confirm the power of this strategy in identifying functionally different alleles of genes and suggest that the different alleles at this SNP locus in the MRP1 gene may account, in part, for inter-individual variations and population differences in drug response.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The human ATP-binding cassette (ABC) superfamily of transporters regulates the traffic of molecules across cell membranes. The 190 kDa multidrug resistance-associated protein-1 (MRP1/ABCC1) is a member of this superfamily of transporters and has been implicated in the resistance of various cancers to chemotherapy as it effluxes several drugs including doxorubicin, vincristine and colchicine from the cells (1Go). In addition to protecting cells within the body against drugs, environmental toxins and heavy metals, MRP1 has also been implicated to contribute to cellular antioxidative defense system and inflammation (2Go,3Go).

Polymorphisms within the gene of the MDR1 transporter, another closely related member of the ABC superfamily, have been correlated with differences in MDR1 expression/function, drug response and disease susceptibilities (4Go). Likewise, we hypothesize that genetic variation at the MRP1 gene may influence its expression and/or protein structure/function, resulting in differences in response to drugs and oxidative stress as well as susceptibilities to diseases in which environment plays an important role. The MRP1 gene contains 31 exons and spans at least 200 kb and encodes a membrane transport protein comprising 1531 amino acids (2Go,3Go,5Go). Numerous single nucleotide polymorphisms (SNPs) have been identified in the MRP1 gene (6Go–9Go). Attempts have been made to associate a few of these SNPs at the MRP1 gene locus with functional differences but no associations were found (6Go,10Go–12Go). A possible explanation for the failure to find association is that the SNPs selected were not functionally important or in linkage disequilibrium (LD) with those that are. Such studies which are based on only a few of more than 20 SNPs in potentially functional regions of the MRP1 gene, without knowledge of the functional SNP or its LD or haplotype profile in that population, may not have sufficient power to detect any association.

Searching for evidence of positive selection has been hailed as an attractive strategy for identifying functional polymorphisms because during the dispersal of mankind from Africa to other parts of the world, the different populations are exposed to different climate, pathogens and food and hence are challenged by different selective forces (13Go). Thus, phenotypic differences between individuals/groups could be due to these functional polymorphisms that facilitated survival in the ancestral human population (13Go). Several studies (reviewed in 13,14) reported that functionally important alleles in ‘adaptive’ genes, e.g. the well known G6PD-202A allele that protects individuals against malaria (15Go,16Go), show evidence of positive selection. Recently, we also demonstrated evidence of positive selection of SNP alleles within the MDR1 gene (17Go), which were previously reported to be associated with various functional differences (18Go). Here, we hypothesized that MRP1, like its superfamily member, MDR1, may also be acted upon by forces of natural selection in selected population(s), and genomic signatures of natural selection may thus be utilized to identify functionally important SNPs at this gene locus.

Our approach was to examine SNPs within the MRP1 gene locus in five different populations and utilize two different strategies to determine whether any of the polymorphisms within the MRP1 gene is under natural selection pressures. In the first strategy, we employed the modified long-range haplotype test (LRH) (16Go), which we previously utilized to demonstrate evidence of recent positive selection at SNP loci e21/G2677T/A and e26/C3435T of the MDR1 gene (17Go). The rationale underlying this strategy is that recent neutral polymorphism usually arises on an existing background haplotype characterized by complete LD between that new polymorphism and other linked polymorphisms. Hence, recent neutral polymorphism is usually low in frequency but high in LD. However, over time, additional polymorphisms and recombination reduce the LD of that neutral polymorphism. Hence, older neutral polymorphisms are usually high in frequency but low in LD. A polymorphism with an unusually long-range LD and high population frequency is thus indicative of being under recent positive selective pressures. In the second strategy, we utilized the statistic FST, which measures the variation in SNP allele frequencies between populations (19Go–21Go). Under neutral situations, when there are no selective pressures, FST is determined by genetic drift, which will affect all loci across the genome similarly and predictably. However, when there is natural selection at a particular locus, the FST value for that locus will deviate significantly from the FST values of other loci in the genome.

In this report, through the aforementioned two strategies, we demonstrated evidence of recent positive selection of SNP 5'FR/G-260C (SNP1), which resides in the promoter of the MRP1 gene. Although this SNP was found in the dbSNP database, it has not been examined in any association studies so far. Notably, through in vitro promoter–reporter assays, we demonstrated that cells carrying the positively selected G allele of SNP1 have significantly lower MRP1 promoter activity when compared with cells carrying the alternative C allele in all four cell lines that we examined. Our findings should facilitate future studies associating this gene with drug response/disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
LD extends for short distances at the MRP1 gene locus
Thirteen SNPs across the MRP1 genomic region (Table 1) were examined in five different populations, namely, Chinese, Malays, Indians, European-Americans and African-Americans. Similar to previous reports (17Go,22Go,23Go), allele frequency distribution at the MRP1 gene locus differed among the different populations. SNP frequencies in the African-American population were significantly different from the SNP frequencies in other non-African populations at most SNP loci (P<0.05). Although the SNP frequencies in the Chinese were similar to the SNP frequencies in the Malays at many SNP loci (10/13) of the MRP1 gene, no obvious trend could be observed between the SNP frequencies of the Indians and the European-Americans at this gene locus.


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  		Table 1. Allele frequency comparisons of the 13 SNPs at the MRP1/ABCC1 gene locus in five populations
 
Pair-wise LD between SNPs was evaluated using two statistical measures, |D'| and r2, which are useful for modeling recombination rates and association power, respectively (24Go). LD generally decreased with physical distance with R2 correlation coefficient of 0.549, 0.269, 0.334, 0.286 and 0.088 in Chinese, Malay, Indian, European-American and African-American, respectively (Fig. 1A). Half LD (|D'|0.5) was determined to be 26, 48, 35, 22 and 26 kb in the respective populations.



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  		Figure 1. Pairwise LD profiles. (A) Plot of |D'| between pairs of single SNPs against the physical distances in the five populations. (B) Plot of r2 between pairs of single SNPs against the physical distances in five populations.

 
When r2≥0.3 was utilized as a threshold to represent useful LD for association studies (25Go), useful LD extended only ~12 kb in the Chinese and European-Americans. No useful r2 LD could be identified in the Malays, Indians and African-Americans (Fig. 1B, Supplementary Material, Fig. S1).

High haplotype diversity at the MRP1 gene locus
A total of 227 haplotypes occurring in at least one population was observed at the MRP1 gene locus. Of these, 24, 22, 36, 26 and 55 haplotypes occurred only in the Chinese, Malays, Indians, European-Americans and African-Americans, respectively. These population-specific haplotypes accounted for 40.7, 44.9, 52.2, 40.6 and 70.5% of all the haplotypes in the respective populations.

Sixteen haplotypes occurring at >4% frequency in at least one population were labeled as major haplotypes (mh1–mh16) and shown in Figure 2. None of these haplotypes occurred at >20%. Curiously, in the Malay population, which has the longest |D'|0.5 (Fig. 1A), the sum of all their major haplotypes constituted >65% of all the observed haplotypes in that population. The sum of the major haplotypes in other populations constituted only between 35 and 50% of the total haplotypes. Three (Malays and Indians), four (Chinese and European-Americans) or five (African-Americans) different haplotypes accounted for only ~30% of all chromosomes in the respective population. Only three of these major haplotypes (mh4, 11 and 12) occurred in all five populations.



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  		Figure 2. Profile of MRP1 haplotypes whose frequencies occur at >4% in at least one population. Haplotype frequencies were inferred from genotype data. 59, 48, 69, 64 and 78 haplotypes were observed in the Chinese, Malay, Indian, European-American and African-American population, respectively. Sixteen major haplotypes (mh1–mh16) which occurs at >4% frequency in at least one population are shown in this figure. The frequencies of major haplotypes in five populations are presented in the bottom row. Cells highlighted in gray represent haplotype with frequencies>4%. Gray box with black outline represents the haplotype with the highest frequency in European-American, which is also the haplotype that shows the evidence of positive selection.

 
The aforementioned results suggest high haplotype diversity at the ABCC1 gene locus but no obvious geographical distribution in haplotype frequencies among the five populations.

SNP 5'FR/G-260C showed evidence of positive selection in European-Americans
An interesting observation arose from the detailed analyses of the haplotype profiles. In the European-Americans, the difference in haplotype frequency between the most common (mh10) and the second most common haplotype (mh12) (7.44%) was greater than that observed in the other populations (2.01–5.43%) (Fig. 2). Such predominance of a single major haplotype, despite the overall weak LD across the MRP1 gene, suggested the presence of strong selective forces acting to maintain this haplotype at high frequency in this population.

To identify whether there is evidence of positive selection occurring at any of the SNP loci within this gene, we plotted haplotype branching diagrams (HBDs) for all 13 SNPs. Figure 3 shows representative HBDs using SNP1, SNP11 and SNP13 as the tested loci in all five populations. Each SNP allele in the HBD is represented by a black dot and haplotypes are represented by lines/branches that connect the dots. The thickness of the lines and the size of the black dots correspond to the frequencies of the haplotype and SNP alleles, respectively. In general, thinner and greater number of branches can be observed as the distance from the root locus increases, suggesting LD decay. However, if an allele is under recent positive selection, the allele will increase in frequency rapidly and will have an unusually long LD and high haplotype frequency. Such alleles would be represented in the HBD as large dots (representing high allele frequency) with a predominant thick line (representing high haplotype frequency) that extended for a long distance (representing long LD). When HBDs of all the SNPs were examined, allele G of SNP1 in the European-Americans as well as allele T of SNP11 in the European-Americans and perhaps the Malays displayed large dots with a single distinctly predominant thick branch (representing haplotype mh10) that extended across at least 130 of the 195 kb MRP1 gene in the European-American population (Fig. 3). In contrast, the HBD of SNP13 did not display any single predominant thick branch extending over a considerable distance in any of the five populations examined. Hence, SNPs 1 and 11 represented SNPs that were potentially positively selected, whereas SNP13 was also portrayed to represent an SNP that is unlikely to be positively selected (Fig. 3).



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  		Figure 3. HBD for three selected SNPs at the MRP1 gene locus in five populations. Representative branching diagrams of three selected SNPs at the MRP1 gene locus. SNPs 1 and 11 represent SNPs that are potentially positively selected, whereas SNP13 represents SNP that is unlikely to be positively selected. Alleles of the test SNP (root locus) are labeled in the left of each row of diagrams. Each SNP allele is represented by a black dot and the position of each SNP locus along the x-axis is scaled to the physical distance between consecutive SNP alleles. Haplotypes are represented by lines joining the dots. The thickness of the lines and the size of the black dots correspond to the frequency of the haplotype and SNP allele, respectively. Thinner and greater number of branches can be observed as the distance from the root locus increase, suggesting LD decay.

 
To assess the significance of the aforementioned observation, the extended haplotype homozygosity (EHH) of SNPs 1 and 11 was then plotted against genomic distance for all the five populations. As shown in Figure 4 (column 1), EHH of the G allele of SNP1 (European-Americans, 0.218; Indians, 0.154; African-Americans, 0.062) decayed more slowly than its corresponding alternative C allele (European-Americans, 0.113; Indians, 0.176; African-Americans, 0.094) for 135 of 195 kb (from SNP1 to SNP10) at the MRP1 gene locus in European-Americans but not Indians or African-Americans. Likewise, EHH of the T allele of SNP11 (Chinese, 0.173; Malays, 0.317; Indians, 0.153; European-Americans, 0.316; African-Americans, 0.113) in several populations including the Chinese, Malay and European-American populations also decayed slower than the corresponding G allele (Chinese, 0.089; Malays, 0.108; Indians, 0.187; European-Americans, 0.111; African-Americans, 0.060) for 130 of 195 kb (from SNP11 to SNP2) (Fig. 4, column 2). Hence, selective forces may be acting on the G allele of SNP1 and T allele of SNP11.



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  		Figure 4. EHH and relative EHH analyses of SNPs 1 and 11. Left two panels: the EHH of the two alleles for SNPs 1 and 11 are plotted against distance between consecutive SNPs. Right two panels: for each allele of SNPs 1 and 11, relative EHH from SNP1 to SNP10 and SNP11 to SNP 1/2, respectively, are plotted against allele frequency (black diamonds). The grey dots represent simulated data under the constant size population model assuming recombination rate of 1.3 cM Mb–1. The 95th, 75th and 50th percentile lines of the distribution of the simulated alleles are also shown. Asterisk indicates that the EHH or relative EHH plots are not shown for SNP1 in the Chinese and Malay populations because the minor allele frequency of that SNP is either zero or too low in these populations and the LRH test is thus not applicable.

 
To evaluate whether selection on these alleles are statistically significant, relative EHH was plotted against their allele frequencies (Fig. 4). Relative EHH is the ratio of EHH of the tested allele relative to the EHH of the alternative/control allele(s) (16Go). The observed relative EHH was then compared against data simulated under four different population models and three recombination rates at the observed allele frequencies and P-values were generated (Fig. 4, right two columns; Table 2). Significantly, only the relative EHH value of SNP1G displayed statistically significant departure from selection neutrality when tested by coalescent simulation under all four population models simulated (constant size, expansion, bottleneck and structured) and all three recombination rate assumptions (0.65, 1.3 and 2.6 cM Mb–1) in the European-American population (Table 2, boxed). This allele in the Indian population showed significant evidence of positive selection under all tested population model and recombination rate assumptions except structured population model with recombination rate of 2.6 cM Mb–1. The T allele of SNP11 in Malays and European-Americans showed statistical evidence of positive selection under constant size, expansion and bottleneck population models but not the structured population model (Table 2). Hence, only the SNP1G allele in European-Americans showed evidence of positive selection under all tested population models and recombination rates.


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  		Table 2. LRH test: P-values computed through the comparison of the observed relative EHH of SNP1 and 11 with that of all of the simulated data points under the specified population history models assuming the specified recombination rates and critical value at level 0.05 of the maximum simulated rEHH distribution to account for Type I error
 
To account for Type I error in this LRH test, a simulation-based approach as described in the Materials and Methods was used. When SNP1G of the European-American was the anchor locus, the rEHH value was 1.865, which is greater than the critical value at level 0.05 of the maximum simulated rEHH distribution for all 12 models (Table 2), further strengthening the evidence that SNP1 is under recent positive selection in the European-American population.

To validate the evidence of recent positive selection at SNP1G in another population of European ancestry, genotype data of the MRP1 region from 60 unrelated Centre d'Etude du Polymorphisme Humain (CEPH) individuals were extracted from the HapMap database (http://www.hapmap.org/) and analyzed similarly. The CEPH samples were collected in 1980 from the United States residents with northern and western European ancestry by the CEPH. We found that SNP1 (SNP 5'FR/G-260C) in this additional population of European ancestry also showed statistical evidence of positive selection when tested by coalescent simulation under all four population models (constant size, expansion, bottleneck and structured population) and three recombination rates assumptions (0.65, 1.3 and 2.6 cM Mb–1) (Supplementary Material, Table S3).

Further signatures of positive or balancing selection at this SNP locus were obtained by assessing the FST values of the various MRP1 SNPs (Fig. 5). Significantly, SNP1 had the highest FST value of 0.628 when compared with the other SNPs (0.096–0.265) at the MRP1 gene locus (Fig. 5). This FST value was above the threshold of 0.45, which would correspond to an empirical significance level of {alpha}=0.026, on the basis of genome-wide distribution of FST values in autosomal loci (19Go). Hence, the MRP1 gene can be considered a high-FST candidate selection gene (19Go). To account for multiple testing, a simulation-based approach as described in the Materials and Methods was also employed. As different chromosomes were found to have different FST distributions (19Go), our simulation was performed on 13 random SNPs residing within 200 kb on chromosome 16, which is the chromosome that the MRP1 gene reside in. SNP1 has an FST value of 0.628, which is larger than the chromosome 16 critical FST value of 0.478 at 0.05 level, thus further strengthening our evidence of positive or balancing selection of SNPs at the MRP1 gene locus. Although SNP1 was found in the dbSNP database, it has not been examined in any association studies so far.



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  		Figure 5. Graphical representation and FST plot of the 13 SNPs at the MRP1 gene locus. Top: scaled representation of the 13 SNPs at the MRP1 gene locus. Bottom: plot of FST values of the 13 SNPs at the MRP1 gene locus. Dotted line represents an arbitrary threshold (FST=0.45) in which SNPs with FST values that are above that threshold are likely to be under selection pressures (19Go).

 
MRP1 promoter activity is lower in cells carrying the G allele at SNP 5'FR/G-260C
SNP1 resides in the reported core promoter region of the MRP1 gene (26Go) and is ~260 bp upstream of the transcription start site (TSS). The observed genomic signatures of positive selection for the SNP1G allele of this gene strongly suggested that this SNP may be functional. To evaluate the potential functional significance of SNP1, 635 bp of the proximal promoter (Fig. 6A) of MRP1 was cloned into a promoter–reporter construct (Fig. 6B) and its activity evaluated in four cell lines: KB3-1, MCF-7, Hep3B and HepG2. MRP1 promoter activity varied greatly between the different cell lines. However, for each cell line tested, expression from the promoter containing the SNP1G allele was significantly (P<0.01) lower when compared with the promoter containing the SNP1C allele (Fig. 6C). Further examination of the sequence flanking SNP1 was performed using three different web-based transcription factor binding site identification programs: AliBaba2.1 (http://www.gene-regulation.com/pub/programs/alibaba2/index.html) (27Go), Patch1.0 (or PatchSearch) (http://www.gene-regulation.com/cgi-bin/pub/programs/patch/bin/patch.cgi) (28Go) and TESS (http://www.cbil.upenn.edu/tess/) (29Go). The analysis suggested that the SNP1G allele may reside within a putative c-ETS-1 binding site, which will be abolished on the SNP1C allele.



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  		Figure 6. Effect of different alleles of SNP1 on MRP1 promoter activity. (A) Sequence of 635 bp of the MRP1 promoter region in which SNP1 is boxed. A putative c-Ets-1 binding site is predicted when allele of SNP1 is G but not C. The transcriptional start site, translational initiation codon (NM_004996 [GenBank] .2) and other putative binding sites within this region as predicted in silico are shown. (c-Ets-1, p54; cAMP REBP, cAMP-responsive element binding protein; Sp1, stimulating protein 1; AP-1,2, activator protein 1,2). (B) Schematic diagram of the construct used in MRP1 promoter assays. (C) MRP1 promoter activity in four cell lines transfected with constructs containing either G or C alleles at the position of SNP1. Asterisk denotes significant difference (P<0.01) between cells carrying the G and C alleles at SNP1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The MDR1 and MRP1 efflux pumps are members of the ABC superfamily of transporters and they represent the first and second multidrug transporters to be characterized (5Go,30Go). Both these transporters were found to confer on tumor cells resistance to similar spectrum of drugs including vincristine, colchicine, doxorubicin and daunorubicin (31Go), suggesting that they efflux some similar drugs. However, although the MDR1 pump transports unmodified drugs, the MRP1 pump primarily effluxes drugs conjugated or co-transported with glutathione and other anions (32Go). These two drug transporters may thus play important roles in determining an individual's response to various drugs.

Polymorphisms within the MDR1 gene have been variously associated with differences in drug response and disease susceptibilities (4Go). However, no associations were found between functional differences and SNPs that were examined at the MRP1 gene locus (6Go,10Go–12Go). It is possible that the failure to detect an association could be because those examined SNPs were not the causative SNPs or in LD with the causative SNPs. It may thus be important to identify the functional SNP or SNPs in LD with the functional SNP to facilitate association studies on this gene. An attractive strategy to identify functional SNP or SNP(s) in LD with the functional SNP is to identify SNPs that show evidence of recent positive selection (13Go).

In a previous study, we characterized the LD and haplotype profile of the MDR1 gene in five different populations and found evidence that SNPs 21/G2677T/A and 26/C3435T are under recent positive selection pressures (17Go). Here, we present the detailed characterization of the LD and haplotype profiles of the MRP1 gene in the same five populations and demonstrate evidence that an SNP within the core promoter region of the MRP1 gene has undergone recent positive selection. We further showed that this SNP is functional and modulates MRP1 promoter activity.

LD and haplotype profiles at the MRP1 locus are different from that at the MDR1 locus
We previously reported that at the MDR1 gene locus, although LD generally decreased with physical distance, there was great variation in the LD-distance relationship with R2 correlation coefficient ranging from 0.0006 in African-Americans to 0.1191 in Indians. Nonetheless, a reasonably strong LD was observed at this gene locus with |D'|0.5 ranging from 105 kb in the Malays to 150 kb in the European-Americans. Useful r2 LD at the MDR1 gene locus extended over distances of between 35 kb in the Chinese and Malays and 82 kb in the Indians (17Go). In contrast, there was better LD-distance relationship at the MRP1 gene locus with R2 correlation coefficient ranging from 0.088 in the African-Americans to 0.549 in the Chinese population (Fig. 1). However, LD extended only over very short distances at the MRP1 gene locus with |D'|0.5 ranging from 22 kb in the European-Americans to 48 kb in the Malays. Useful r2 LD was undetectable in the Malays, Indians and African-Americans at the MRP1 locus and extended only for 12 kb in the European-Americans and Chinese. LD profiles were found to vary greatly between different genomic regions and different populations (24,33–36Go). The |D'|0.5 LD at the MRP1 gene locus in all five populations (22–48 kb) are within the range of 6–155 kb but lower than the average of 60 kb observed at 19 randomly selected genomic regions in the European-American population (37Go). Interestingly, the Malays had the longest |D'|0.5 at the MRP1 gene locus (Fig. 1B) but the shortest |D'|0.5 at the MDR1 gene locus (17Go). The reverse was true for the European-American population.

Greater haplotype diversity was observed at the MRP1 gene locus (Fig. 2) when compared with the MDR1 gene locus (17Go). The number of different haplotypes occurring in at least one population at the MRP1 locus was 227, which was almost double that of the 118 haplotypes occurring at the MDR1 locus. Although three to six haplotypes could account for>60% of the total chromosomes at the MDR1 locus, three to five haplotypes could account for only ~30% of the total chromosomes at the MRP1 locus. Notably, although the most common haplotype at the MDR1 gene locus in the different populations accounted for between 25 and 50% of the total haplotypes in the different populations, the most common haplotype at the MRP1 gene locus accounted for only between 10 and 17% of the total haplotypes in the corresponding populations (Fig. 2).

The shorter LD and greater haplotype diversity at the MRP1 locus when compared with the MDR1 locus were also evident from analysis of the HBD. Thinner and greater number of branches were observed at the MRP1 locus in all five populations (Fig. 3) when compared with the MDR1 locus (17Go).

The observation of LD extending for much shorter distances at the MRP1 locus when compared with MDR1 locus has practical implications. Given the short LD at the MRP1 locus, association of any MRP1 SNP with functional difference may lead to easier identification of the causative SNP within a relatively smaller region of DNA. However, more SNPs need to be examined initially in order to establish an association with functional differences, and it may not be practical to identify tagging-SNPs for loci with short LD and great haplotype diversity. The short LD and great haplotype diversity of the MRP1 locus make it difficult to use surrogate SNPs to identify functional SNPs and could perhaps explain previous difficulties in associating random MRP1 SNPs with functional differences (6Go,10Go–12Go).

Evidence of positive selection at the MRP1 gene locus occurs in a different population from evidence of positive selection at the MDR1 locus
The G allele of SNP1 and T allele of SNP11 showed high relative EHH when compared with their alternative alleles in a few different populations. However, only SNP1G in European-Americans showed statistical evidence of positive selection when tested by coalescent simulation under all four population models (constant size, expansion, bottleneck and structured population) and three recombination rates assumptions (0.65, 1.3 and 2.6 cM Mb–1) (Table 2). The significantly higher FST value of SNP1 when compared with other MRP1 SNPs or SNPs in other genomic regions further strengthened the evidence of positive or balancing selection of SNP1 at the MRP1 gene locus.

We had previously demonstrated genomic evidence of recent positive selection at the MDR1 gene locus for the e21/2677T and e26/3435T alleles in the Chinese, e26/3435T allele in the Malay and e26/3435C in the African-American population (17Go). It is thus interesting that now genomic evidence of positive selection has been identified for an allele of the closely related and functionally similar MRP1 gene in the European-American population. These observations suggest that similar selective pressures may be acting on related and functionally similar genes in different populations.

Our finding of evidence of positive selection of the SNP1G allele at the MRP1 gene in European-Americans suggests that this SNP may be a functional SNP. SNP1G is ~260 bp from the TSS and hence resides in the reported core promoter region of the MRP1 gene (26Go). Significantly, using promoter–reporter assays, we demonstrated significant decrease (P<0.01) in MRP1 promoter activity in four different cell lines carrying the G allele when compared with those carrying the C allele, confirming the functionality of this SNP. Using in silico strategies, the SNP1G allele was found to reside within a putative c-ETS-1 binding site, which was abolished on the SNP1C allele. c-ETS-1 has been reported to be capable of repressing AP-1 induced transcriptional activity (38Go). AP-1 has been reported to function as a transcriptional enhancer of the MRP1 promoter (39Go), and two putative AP-1 binding sites reside within the 635 bp fragment of the MRP1 promoter that was tested (Fig. 6A). Hence, an attractive model to explain the observed differences in MRP1 promoter activity of the two different SNP1 alleles could be that the SNP1G allele, but not the SNP1C allele, allows c-ETS-1 binding onto the MRP1 promoter to inhibit AP-1 induction of transcription.

It is pertinent to note that in mice, loss of the Mrp1 gene results in increased resistance to Streptococcus pneumoniae induced pneumonia (40Go). It is tempting to speculate that increased survival against pneumonia may have been a positive selective force for the functionally weaker SNP1G allele of human MRP1 in populations or regions where community-acquired pneumonia is endemic.

Interestingly, significantly greater functional differences were observed between the SNP1G and SNP1C alleles in p53-negative Hep3B cells when compared with the related p53-positive HepG2 cells. The cellular p53 status has been found to affect the expression of MRP1 (41Go), and our present observations highlight the complexity of the role of SNPs in the regulation of MRP1 gene expression. They also imply that in p53-negative cells (e.g. in some cancer cells) with MRP1 genes containing the SNP1G allele, MRP1 expression levels would be expected to be very low, resulting in greater drug accumulation and thus increased cytotoxicity in these cells, whereas p53-negative cells with SNP1C containing MRP1 would be expected to show very high expression and thus greater drug resistance. Conversely, in p53-positive cells, the difference in expression levels between SNP1G- and SNP1C-containing MRP1 promoters is smaller and thus less likely to result in either increased cytotoxicity or drug resistance. It would thus be interesting to further examine the role of p53 in modulating MRP1 expression according to its promoter SNP1 allele status.

In summary, we have demonstrated a novel strategy for isolating functional SNPs within the MRP1 gene by first identifying genomic signatures of positive selection at this locus. We postulate that this positively selected functional SNP, which resides within the core promoter of the MRP1 gene, may account in part for inter-individual and population variation in response to various drugs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Study population
The five different populations examined in this study are the European-American, the African-American, the Chinese, the Malays and the Indians. The European-American population was chosen to represent the most commonly studied population in population genetics as well as association studies, whereas the African-American population represents the most ancient population that is publicly available. The three Asian populations in this study are the major ethnic populations living in Singapore. The Chinese and Indians of Singapore are predominantly descendants of migrants originating from Southern China and Southern India, respectively, whereas the Malays are indigenous to the Malay Archipelago. The Chinese-Singaporean population was chosen to represent the Han Chinese, which is the largest population in the world, while the Indian-Singaporean population was chosen to represent the Indians which is the second largest population in the world (http://www.cia.gov/cia/publications/factbook/rankorder/2119rank.html). The Malay-Singaporean population was selected to represent the indigenous population in Southeast Asia.

Genomic DNA of the European-American and African-American populations was obtained from the respective Human Variation Panels of the Coriell Cell Repositories (Camden, NJ, USA). Samples for the three main populations in Singapore (Chinese, Malay, Indians) were obtained from previously archived genomic DNAs extracted from unselected cord blood samples discarded after clinical newborn screening for glucose-6-phosphate dehydrogenase deficiency from one of the two major hospitals in the country. These samples have been anonymized, except for their ethnicity. Ethical approval for this study was obtained from National University of Singapore Institutional Review Board (NUS-IRB Reference Code: 04-126E). Approximately 100 samples from each population were examined to avoid biased estimates of population parameters (42Go).

SNP selection and genotyping
The MRP1 genomic DNA (NT_0101393.13) sequence was obtained from GenBank (http://www.ncbi.nlm.nih.gov) and used as the reference sequence. MRP1 SNPs were selected on the basis of two criteria: (1Go) SNPs should have relatively high minor allele frequencies (≥5%) to facilitate the characterization of the haplotype and LD profiles and (2Go) SNPs should be relatively dense and fairly evenly spaced.

Thirteen SNPs spanning 186.4 kb of the MRP1 gene were selected from two primary sources: published reports (6Go–9Go) and public databases including the National Center for Biotechnology Information SNP database (http://www.ncbi.nlm.nih.gov/SNP/), the SNP Consortium Ltd (http://snp.cshl.org/) and the Japanese SNP database (http://snp.ims.u-tokyo.ac.jp). The average distance between two consecutive SNPs in this panel of 13 SNPs was 15.5 kb (Supplementary Material, Table S1).

Three different mini-sequencing panels were set up to genotype the 13 SNPs at the MRP1 gene locus. Genotyping was carried out using multiplex PCR amplification and multiplex mini-sequencing as previously described (43Go). Information on the PCR and mini-sequencing primers, as well as the reaction conditions is tabulated in the Supplementary Material, Table S2. Genotyping accuracy was ascertained through dideoxy sequencing of random samples from each population.

Inference of haplotype and LD
Fisher's exact test was employed to evaluate the significance of deviation of each SNP in each population from Hardy–Weinberg equilibrium, as well as the significance of pair-wise comparisons of allele frequencies between two different groups.

Haplotype frequencies and LD between SNP pairs were estimated as described previously (17Go).

Signatures of positive or balancing selection
Signatures of positive or balancing selection were evaluated using two strategies. In the first strategy, evidence of recent positive selection was evaluated graphically using the HBD and assessed using a modified LRH test by plotting the EHH and relative EHH against genomic distance and allele frequencies, respectively, as described previously (17Go). Coalescent simulations were performed to validate the evidence of positive selection. Four different population models including constant population size model, expansion model, extreme bottleneck model and highly structured population model were simulated as described previously (17Go).

To correct for Type I error, a simulation approach was utilized to obtain the exact critical value for the LRH test. Similar coalescent simulations of the four population models with three recombination rates performed for the LRH test were also performed to correct for Type I error using each of the 13 SNPs as the anchor locus. For each model, a chromosome with a particular rEHH value at comparable distance as our experimental data was randomly selected from each of the 13 simulated pools and the maximum rEHH value was noted. This process was iterated 5000 times to obtain 5000 maximum rEHH values, which served as the reference null distribution of maximum simulated rEHH values (Table 2). The experimental rEHH values of the SNPs examined were then compared against the reference distribution of maximum simulated rEHH values.

In the second strategy, FST values for each SNP were calculated as described (19Go). FST≥0.45 in at least one SNP of an autosomal gene was considered a high-FST candidate selection gene. This threshold corresponds to an empirical significance level of 0.026 on the basis of the genome-wide distribution of FST (19Go).

To take into account the multiple tests, a simulation approach was also employed to adjust the exact critical value for the FST statistic. Different chromosomes were found to have different FST distributions (19Go), suggesting that the genome-wide distribution of FST might not be an appropriate reference distribution. As the 13 SNPs that we examined at the MRP1 gene locus reside within a region of ~200 kb on chromosome 16, we extracted FST values of 13 random SNPs on chromosome 16 that lies within a region of ~200 kb, from the FST database of 753 SNPs (19Go). The maximum FST value of the 13 random SNPs was noted. This process was iterated 1000 times to obtain 1000 maximum FST values, which served as the reference null distribution of the maximum FST values among the 13 SNPs on chromosome 16. The experimental FST values of the SNPs examined were then compared against the reference distribution of maximum chromosome 16 FST values.

In vitro analysis of promoter SNP5'FR/G-260C
A 635 bp promoter region of the MRP1 gene (NM_004996 [GenBank] .2) spanning from –441 to +194 (+1 denotes the TSS) (Fig. 6A) was amplified and inserted upstream of the ß-galactosidase (ß-gal) reporter gene in an expression vector, which also contains the enhanced green fluorescent protein (EGFP) reporter gene driven by the cytomegalovirus promoter (Fig. 6B) for normalization against differences in transfection efficiency. The C allele of the SNP 5'FR/G-260C was recapitulated via in vitro mutagenesis. The plasmid constructs were sequenced across the PCR amplified regions to exclude PCR-induced nucleotide mis-incorporations prior to use.

Promoter–reporter constructs were transfected into the HeLa subclone, KB-3-1, the MCF-7 breast cancer cell line or the HepG2 or Hep3B hepatocellular carcinoma cell lines using calcium phosphate co-precipitation as described previously (44Go). ß-galactosidase activity was quantitated in a kinetic assay using CPRG (chlorphenol red-ß-D-galactopyranoside) as substrate and measured at 1 min intervals over 60 min at 570 nm in a SpectraMAX PLUS microplate reader (Molecular Devices, Sunnyvale, CA, USA), while EGFP fluorescence was measured at 509 nm in a SpectraMAX Gemini microplate reader (Molecular Devices) after excitation at 488 nm. ß-galactosidase expression was normalized against EGFP activity.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We would also like to thank Dr Huihua LI from National Cancer Centre for biostatistical assistance and A/Professor Zehua Chen from the Department of Statistics and Applied Probability, National University of Singapore for expert biostatistical advice. We would also like to thank Mr Jingbo Wang for help with writing programs for the various simulations that we performed. This study is supported by a grant from the BioMedical Research Council (BMRC) (01/1/21/17/054), Singapore to C.G.L.L. and E.J.D.L.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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