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Am. J. Trop. Med. Hyg., 72(2), 2005, pp. 182-188
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

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ANALYSIS OF THE GENETIC DIVERSITY OF THE PLASMODIUM FALCIPARUM MULTIDRUG RESISTANCE GENE 5' UPSTREAM REGION

ALISSA MYRICK, OUSMANE SARR, THERESE DIENG, OMAR NDIR, SOULEYMANE MBOUP, AND DYANN F. WIRTH
Department of Medicine, San Francisco General Hospital, University of California at San Francisco, San Francisco, California; Laboratory of Bacteriology and Virology, Le Dantec Hospital, Dakar, Senegal; Laboratory of Parasitology, Faculty of Medicine and Pharmacy, Cheikh Anta Diop University, Dakar, Senegal; Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal. A comparison of six geographically diverse laboratory strains showed that the 1.94-kb 5'-untranslated region is highly monomorphic, with a total of four unique single nucleotide polymorphisms (SNPs) being identified. All of the mutations were localized to a 462-basepair region proximal to the transcription start point. Analysis of this region in field isolates shows the prevalence of one SNP throughout the entire population of parasites, irrespective of drug resistance status. The SNP frequency of the pfmdr1 upstream region is lower than that found in the noncoding region of other genes.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There has been renewed effort to discern the origins of the genetic diversity of Plasmodium falciparum.1–11 This research has been driven by the knowledge that defining the roots of genetic diversity and the population genetics of the parasite will enhance the ability of the scientific community to design strategies for the design of drugs targeting the malaria parasite. Much of this investigation has focused on the analysis of the level of genetic polymorphism in the parasite as a method to discern the age of its most recent common ancestor. However, data from this research has revealed unique characteristics of the P. falciparum genome.

To date, most studies of polymorphism in P. falciparum have analyzed the link between polymorphisms in the coding regions of selected genes and a phenotypic readout (e.g., levels of drug resistance). Further work needs to be done to determine whether single nucleotide polymorphisms (SNPs) can serve as markers for functionally important segments of genome.

In the case of the P. falciparum multidrug resistance 1 (pfmdr1) gene, amino acid changes at positions 86(N->Y) and 1246(D->Y) have been most strongly associated with resistance to chloroquine and other antimalarial drugs.12–17 These mutations have various levels of correlation depending upon the geographic location and the drug resistance profile of the isolate. However, mutations in pfmdr1 are most predictive of resistance when they occur alongside a mutant allele of the gene most tightly linked to chloroquine resistance, the P. falciparum chloroquine resistance transporter (pfcrt).18,19 Wootton and others analyzed the level of polymorphism in micro-satellite (MS) markers flanking pfcrt in a global panel of isolates with a range of chloroquine susceptibility levels.4 They found that there was a low level of MS diversity among all isolates in the 20 kb on either side of the pfcrt locus, and attributed this observation to a recent selective sweep due to drug pressure. These investigators also found regions of reduced diversity surrounding the pfmdr1 locus, but did not find a statistically significant association with a chloroquine resistance phenotype. Thus, prior to this work, evidence suggested that the region flanking pfmdr1 had a low level of genetic diversity. Assuming that pfmdr1 is a gene under selection pressure, we hypothesized that given the low frequency of background genetic diversity, any polymorphism found might have functional significance.

We used a polymerase chain reaction (PCR) and sequence-based approach to construct SNP maps of 3 kb of pfmdr1 upstream sequence from six geographically diverse laboratory strains with differing drug resistance profiles. Upon identification of a polymorphic hotspot in the upstream region, we mapped this portion of sequence in 22 field isolates collected from patients with uncomplicated P. falciparum malaria in Dakar, Senegal. They were part of a larger study testing the predictive value of pfmdr1 and pfcrt coding region polymorphisms as markers of chloroquine resistance.20,21 Therefore, data were available on both in vivo and in vitro chloroquine resistance. The results of the SNP mapping of the field isolates were examined for any link between the presence of the identified polymorphism and drug resistance.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of laboratory strains. To generate a comprehensive polymorphic profile of the 5' upstream region of pfmdr1, six geographically diverse laboratory strains with different chloroquine susceptibility levels were used for sequence analysis (Table 1Go). Strain 3D7, which was used as the reference strain, is chloroquine sensitive, and thought to originate in Africa. Strain D6 is a chloroquine sensitive west African strain. Strains 7G8 and HB3 are both from South America, with 7G8 being chloroquine resistant and HB3 chloroquine sensitive. Finally, two additional chloroquine-resistant strains were chosen: W2 from Thailand, and Muz12.4 from Papua New Guinea. It has been proposed that chloroquine resistance arose from two independent events, one in southeast Asia and one in South America. Thus, it was important to use isolates from different regions that might have undergone different types of selection pressure.4,10,22


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TABLE 1
Summary of SNPs and MS repeats in the 5' upstream region of the pfmdr1 gene in six laboratory strains*
 
Primer design and PCR. The intergenic sequence flanked by pfmdr1 and PFE1145w was obtained from the P. falciparum Genome Database.23 A set of primers spanning 2.9 kb of sequence upstream of the pfmdr1 start ATG was generated. Most primers were made in both the forward and reverse direction so that sequences could easily be formed into contigs for the detection of SNPs and MS sequences. This work was done in parallel to the mapping of the pfmdr1 5'-untranslated region (UTR) and transcription start point.24 Briefly, reverse transcriptase-PCR and 5'-rapid amplification of cDNA ends (RACE) analysis of the 5' upstream region showed that the 5' end of the transcript was located 1.94 kb upstream of the start methionine. Transient transfection assays demonstrated the presence of a putative promoter for pfmdr1 comprised of sequence 381 basepairs upstream of the transcription start point. Since this analysis was completed prior to the identification of the transcription start point, an extra 575 basepairs (from –953 to –381) that corresponds to the 3'-UTR of PFE1145w was also sequenced. Any nucleotide diversity discovered beyond the 5' end of the transcript was excluded from this analysis. Primers are labeled according to their distance from the transcription start site. The primers used for the laboratory strains were 121R 5'-CATTTAAACGGTAAAAAAAATG-3', -557F/R 5'-CATGGATATAGAAAGAAAAATGGAAG-3', -968F/R 5'-TTGGAGTACATGTTTATAACATAATATTC-3', 505F/R 5'-CTCAAATATCCTATAAATACCTTGAG-3', 288F/R 5'-GAACGATTTAAAAAAAAAAAAAC-AAAAGAACC-3', -381F/R 5'-CCTATATAAAAAAG-ATACCCCG-3', and -953F/R 5'-GAAAATATAAC-AATCTCTCAAGGG-3'.

The following conditions were used for the PCR: an initial denaturation at 93°C for 3 minutes, 30 cycles at 93°C for 1 minute, 50–55°C for 1.5 minutes, and 70°C for 3 minutes, and an additional extension at 70°C for 10 minutes. A modified 3:1 dNTP ratio for A-T rich genomes was used for all PCRs.25 At least three independent PCRs were performed for each primer pair.

Cloning and sequencing. The PCR products were visualized after electrophoresis on 1% agarose gels, and subsequently cloned into the pCR2.1Topo vector (Invitrogen, Carlsbad, CA). If multiple bands were seen on the gel, they were excised and gel purified using either the Freeze’ N Squeeze kit (Bio-Rad Laboratories, Hercules, CA) or the SNAPTM gel purification kit (Invitrogen). At least three positive clones were selected for each PCR product, and grown in 5-mL terrific broth (TB) (Teknova, Hollister, CA) cultures for plasmid recovery via the Wizard Plus Miniprep kit (Promega, Madison, WI) or the Plasmid 96vac Direct-bind miniprep system (Eppendorf, Hamburg, Germany). Eco R1 (New England Biolabs, Beverly, MA) restriction digests were performed to check for insert of the correct size, and candidate clones were then sequenced. Initial sequencing was done at Seqwright (Houston, TX), but the bulk of sequencing was performed at the Dana Farber Cancer Institute High Throughput Sequencing Center (Boston, MA). Inserts were sequenced with the M13F and M13R universal primers.

Sequence analysis. Sequence chromatograms were uploaded into the SeqMan sequence alignment program (DNAStar, Madison, WI). Sequences were edited, and a consensus sequence was generated. If a group of sequences contained a conflict that could not be resolved with 18 fold coverage, more clones were sequenced to clarify the problem. There is 16–18 fold coverage over 89.9% of all of the upstream regions for all of the laboratory strains. Some regions were so A-T rich and dominated by homopolymeric tracts that successful sequencing was extremely difficult. A total of 0.8% of the 5' upstream sequence for the laboratory isolates has less than four times coverage. Thus, there is a possibility that SNPs were missed in this portion of DNA. Regions containing polymorphism were sequenced to 18 fold coverage.

Once a consensus sequence was generated, it was entered into the MegAlign program (DNAStar) and compared with the 3D7 sequence using the ClustalW and Wilbur-Lipman alignment tools. If a putative SNP was identified in the consensus sequence, the original SeqMan file containing the 18 sequences from the 9 different clones was scrutinized to ensure that the polymorphism was found in a region of unquestionable sequence. If this proved to be the case, this was called a SNP. Microsatellites were defined according to the system of Volkman and others.2 The same strategy was used to call MS insertions or deletions: the original sequence files were reviewed to ensure that the insertion or deletion was not a result of PCR error.

Analysis of field isolates. Field isolates were collected as part of a larger study analyzing the predictive value of molecular markers of chloroquine resistance in Dakar, Senegal.20,21 This is an ongoing collaboration between Cheikh Anta Diop University in Dakar and the Harvard School of Public Health. The study has been reviewed and approved by the Human Subjects Committee at the Harvard School of Public Health and the Ethics Committee at Cheikh Anta Diop University.

Study site and population. The isolates used in this analysis were collected from August to December of 2001 from consenting adult patients presenting with uncomplicated malaria at an outpatient clinic in Pikine, a suburb of Dakar. Malaria transmission in Pikine is seasonal and hypoendemic, with the majority of transmission occurring from September to November. Study participants were ≥ 18 years old and positive for P. falciparum by thick blood smear. Patients were excluded from the study if there was evidence of another Plasmodium species in the blood smear, or if they tested positive for recent use of chloroquine as determined by the Saker-Solomon urine test. Patients were also excluded from the study if they had a second major illness such as acquired immunodeficiency syndrome, meningitis, chronic diarrhea, or tuberculosis. Study participants were treated with the Senegalese standard of care for uncomplicated malaria: 25 mg/kg of chloroquine for three days. Patients were monitored for 30 minutes after their dosing of chloroquine to ensure that it was well tolerated. If a patient agreed to participate in the study, 10 mL of venuous blood was removed prior to chloroquine treatment. Study members were subsequently followed-up from day 1 to day 7 and on days 14 and 28 for blood smears and finger prick blood samples that were deposited on Iso-code filter paper (Schleischer and Schuell, Keene, NH).

Determination of the 50% inhibitory concentration (IC50). The IC50s for chloroquine were determined using the double-site enzyme-linked lactate dehydrogenase (LDH) immunodetection (DELI) assay26,27 at Cheikh Anta Diop University in Dakar. The DELI assay is a nonisotopic, colorimetric-based enzymatic assay that measures the activity of the Plasmodium falciparum LDH gene. The IC50 was defined as 50% inhibition of LDH activity. An IC50 of 100 nM was used as the cutoff value for chloroquine sensitivity.

Extraction and analysis of DNA. Parasite DNA was extracted from blood samples using a standard phenol:chloroform extraction protocol.20 The DNA was used in PCR assays and restriction fragment length polymorphism (RFLP) analysis to determine the predictive value of molecular markers of chloroquine resistance in the coding regions of pfcrt and pfmdr1. All isolates were also genotyped using merozoite surface protein 1 (msp1) to test for the presence of multiple infections. To confirm the PCR and RFLP data, a subset of isolates were cloned and sequenced as described earlier in this report.

Analysis of pfmdr1 5' polymorphic region. The initial primers used in this study were -381F, 288F/R, and 505R. However, due to difficulty with amplification in this highly A-T rich region, four extra primer pairs were designed to amplify DNA from the field isolates. They were used either independently, or in a nested format. These primers were -436F 5'-CAATTCTTCTAATAAAAATAATAAACTAG-3',-176F/R 5'-CTAAATATCAAAAGATTCTGTTTTC-3', 4F/R 5'-CATAGATTATTTTATATATATG-3', and 270F/R 5'-GTTTGTTCGGAAGAATTAT-3'.

If the standard PCR program did not work, a program with a reduced annealing temperature was used, and if necessary, bands of the expected size were gel purified. The conditions for this program were an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of 95°C for 1 minute, 46°C for 45 seconds, and 62°C for 1 minute, and a final extension at 62°C for 10 minutes. Successful PCR products were cloned into the TA vector, and sequenced using the same methods described for the laboratory strains. However, 2–3 clones were sequenced for each PCR product derived from field isolate DNA. Thus, the total sequence coverage was 4–6 fold, rather than 18 fold.

Statistical analysis. TA skew was determined using the calculation of Lobry and others: (T–A)/(T+A).28 A negative value indicates the presence of more adenines than thymines in the sequence. A positive value indicates T skew, in which there are more thymines than adenines in the sequence. Nucleotide diversity ({pi}) and nucleotide polymorphism ({theta}) were calculated with the DnaSP software program.29 Nucleotide diversity was calculated using the Jukes and Cantor model, which calculates the average number of polymorphisms per site in a pairwise comparison.29,30 Nucleotide polymorphism was calculated using the Watterson and Nei equation29 {theta} = 4Nµ, where N is the population size and µ is the mutation rate per nucleotide site.30

All statistical tests for association between chloroquine resistance levels were conducted with GraphPad Prism 3.0 software (GraphPad Inc., San Diego, CA). An unpaired t-test was used to determine the association between SNP frequency and drug resistance in the laboratory strains. The Spearman rank correlation was used to test the correlation between number of SNPs and chloroquine resistance in the laboratory isolates. To test whether the localization of the SNPs to a 462-basepair region was significant, a random chance calculation was performed (Bachetti P, Division of Biostatistics, University of California at San Francisco). To assess the chance that four SNPs would all be within a span of ≤ 462 basepairs by chance, we randomly generated simulated sets of four SNPs with each having an equal chance of occurring anywhere along the length of the 5'-UTR. We then tabulated how often more than 10,000 such simulations the span between the first and last was ≤ 462 basepairs. Dividing this count by the total number of simulations then produced a P value for the observed span. The Wilcoxon rank sum and Fisher’s exact tests were used to test the association of the chloroquine IC50 and different allelic combinations in the field isolates.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
High degree of monomorphism in the 5' upstream region of pfmdr1. The pfmdr1 5' upstream region is highly monomorphic, with a total of four unique SNPs being identified among all of the laboratory strains. Table 1Go provides a breakdown of SNPs and MS polymorphisms for each of the laboratory strains compared with the 3D7 reference sequence. Muz12.4 had a total of three SNPs in the upstream region. Strains 7G8, W2, and HB3 had the same T->C nucleotide change at position 328, which was linked to a deletion of four thymine residues within the same MS repeat, as shown in Figure 1Go. Interestingly, the D6 sequence did not differ from that of the 3D7 reference strain. Microsatellite repeats were defined in the same manner as Volkman and others, ≥ eight tandem repeats of a sequence 1–8 basepairs in length.2 Due to the process of replication slippage, the frequency of polymorphisms in MS repeats is thought to be higher than that of higher complexity sequence.2 Three of the four SNPs were found in MS repeats, confirming the higher frequency of polymorphism in these regions. Three of the SNPs were also transitions. These are expected to occur at a higher rate than transversions, and are often found within MS repeats.31



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  		    FIGURE 1. Clustal W alignments of all strains of Plasmodium falciparum compared with strain 3D7 showing the sequence context of the 328 T->C single nucleotide polymorphism (SNP) identified in the diversity study. This SNP cosegregates with a deletion of four thymine residues within the same microsatellite repeat. The polymorphisms and adjacent deletions are underlined and in bold.

 
Identification of a polymorphic hotspot in the 5'-UTR of pfmdr1. The most striking aspect of these SNPs was that they were localized to a 462-basepair region proximal to the transcription start site of pfmdr1, which is located 1.94 kb upstream of the start methionine.24 The results of a random chance calculation indicate that the clustering of these mutations in this region was not random (P = 0.015). Neither the putative promoter nor the rest of the 5'-UTR contained any single nucleotide change. The one SNP identified upstream of the transcription start site in the Muz12.4 isolate is located beyond the limits of the pfmdr1 promoter as defined by transient transfection analysis and Northern blot data.24 Microsatellite repeat polymorphisms were distributed throughout this region, as shown in Table 1Go, but in seven discrete loci. Only two strains, HB3 and D6, contained MS polymorphism in the promoter region of pfmdr1.

The frequency of SNPs and MS repeat polymorphisms was calculated for each individual laboratory isolate, and then compared with the total frequency for all of the isolates (Table 2Go). The combined SNP frequency for all six laboratory strains is 1/1,670 basepairs. This value is much lower than the non-coding SNP frequency determined by Mu and others in an analysis of the genetic diversity of chromosome 3,32 and the 5' upstream region SNP frequency determined by Volkman and others (unpublished data). The frequency of SNPs of each laboratory isolate is significantly lower than other estimates for non-coding SNPs,2,6,8 which range from 1/250 to 1/1,000 basepairs. These data suggest that the 5'-UTR and promoter region of pfmdr1 have a low level of nucleotide diversity compared with other P. falciparum noncoding regions.


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TABLE 2
Frequency of SNPs and MS polymorphisms in the pfmdr1 5' upstream region*
 
Lack of a reduction of the frequency of polymorphism by TA skew. Noncoding regions of P. falciparum are highly A-T rich, and characterized by long stretches of low complexity repetitive sequence. The pfmdr1 5'-UTR and promoter is 90% A-T rich. In their study of the genetic diversity of introns of genes on chromosomes 2 and 3, Volkman and others found a low level of polymorphism.2 They attributed this low frequency to a recent origin of P. falciparum, resulting in less time for SNPs to arise. Jongwutiwes and others have argued that the low level of polymorphism found in introns may be due to the TA skew of these noncoding regions.8 This arose from a finding that higher nucleotide diversity in noncoding sequence was significantly correlated to negative TA skew, which indicates that there are more adenines than thymines in the sequence.8 The A-T content of the polymorphic region of the pfmdr1 5'-UTR was compared with that of the flanking DNA. There was no correlation between nucleotide diversity and TA skew. The A-T content of the hotspot is 91%, and the TA skew was 0.014. The 5' flanking sequence of the hotspot had an AT content of 92% and a TA skew of –0.014, and the 3' flanking sequence had an AT content of 89% and a TA skew of 0.06. Neither flanking sequence possessed any SNPs. Thus, in the context of the 5'-UTR of pfmdr1, TA skew does not appear to have an effect on the amount of polymorphism.

Higher frequency of SNPs in chloroquine-resistant strains than in chloroquine-sensitive strains. The low level of nucleotide diversity in the 5' upstream region of pfmdr1 complemented the results of Wootton and others, who identified pfmdr1 as a locus containing low levels of MS marker polymorphism in a genome-wide scan of genetic diversity.4 These investigators did not find a significant correlation between chloroquine-sensitive and chloroquine-resistant haplotypes and MS polymorphisms surrounding the pfmdr1 locus. We conducted an analysis of the SNPs identified in the pfmdr1 upstream region to determine if there was any correlation between SNPs and susceptibility to chloroquine. There was a negative correlation between number of SNPs and chloroquine sensitivity (Spearman rank correlation r = –0.80, P = 0.058); however, the significance of this result is limited by the small sample size.

To further examine the relationship between nucleotide diversity and drug resistance, a sample of 22 field isolates from Dakar, Senegal were selected for analysis. Isolates were collected from adult patients with uncomplicated P. falciparum malaria. The DELI assay was used to determine chloroquine IC50 levels. The cutoff value for in vitro chloroquine resistance was set at 100 nM, as had been used in previous studies.20,21 Parasite DNA was tested for the presence of multiple infections by genotyping with the msp1 gene. The DNA was also assayed for the pfcrt K76T and pfmdr1 N86Y resistance-associated polymorphisms. Discovery of the same SNPs in primary field isolates would also add credence to the SNP map generated from laboratory strains that may have developed mutations over generations of passage in the laboratory. We assumed that the 5'-UTR of field isolates would be similarly monomorphic to the laboratory strains, and limited this analysis to the 462-basepair polymorphic region. The isolate DNA was sequenced to a minimum of four times coverage from one PCR. Due to the low concentration of DNA in some of the samples, four new primer pairs were developed, and a nested strategy was used for isolates that could not be amplified with the original primer set used for the laboratory isolates.

Dominance of the T->C 328 SNP in a population of Senegalese field isolates. Our analysis determined that 82% of the isolates tested have the T->C change at position 328. Except for one, all of the isolates with the SNP also contain a deletion of four thymine residues at position 333. This the same thymine deletion detected in the laboratory strains (Figure 1Go). As shown in Table 3Go, the 328 SNP is prevalent throughout the population and is independent of in vitro chloroquine resistance levels. There was no correlation between this 328 SNP and the presence or absence of the resistance-associated pfmdr1 Y86 allele or the pfcrt T76 allele. Parasites with the 328 SNP and one or both of the mutant pfcrt and pfmdr1 alleles were not more or less likely to be chloroquine resistant (IC50 > 100 nM). Thus, this polymorphism appears to be dominant in this population of parasites, and does not correlate with either molecular markers of chloroquine resistance or chloroquine IC50 levels. This SNP was so prevalent in the population that a much larger sample size would be needed to determine any association with chloroquine resistance. Nevertheless, the appearance of the same SNP in a genetically diverse group of parasites suggests this mutation is of some importance to the parasite. One other nucleotide change was identified in one of these isolates: a G->A change at –88. This SNP is located in the putative promoter of pfmdr1, making it the only SNP found in the promoter region in this entire study.


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TABLE 3
Distribution of SNPs in field isolates by IC50*
 
Values for nucleotide diversity ({pi}) and nucleotide polymorphism ({theta}) within the 462-basepair SNP hotspot were calculated for all of the laboratory isolates, as well as a representative field isolate with the 328 SNP. The levels of {pi} and {theta} for the laboratory isolates were 0.0022 and 0.0026, respectively. The levels for a representative field isolate with the 328 SNP were both 0.0014. There was evidence of MS repeat polymorphism in the field isolates, which again was restricted to specific MS regions. The field isolates had a higher frequency of MS polymorphism localized within the promoter region of pfmdr1, compared with the laboratory isolates. There was no statistically significant association between the frequency of MS polymorphism in sensitive versus resistant isolates. With the exception of one isolate, the promoter regions of all of the field isolates analyzed do not have an SNP. This intriguing result suggests that the promoter region of pfmdr1 may be under negative selection.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this work was to analyze the genetic diversity of the 5' upstream region of pfmdr1 to determine if polymorphic regions might provide clues to biologic function. The results from this analysis confirm the finding of Wootton and others, which indicate that the pfmdr1 locus is in a chromosomal region of low genetic diversity.4 The SNP frequency and nucleotide diversity found in the upstream region of this gene was less than any of the current estimates of SNP frequency in intergenic regions, implying that this DNA is within a span of the genome that has been under selection.

A total of four unique SNPs were present among all the laboratory strains. All of the SNPs identified in this project were concentrated in a 462-basepair region proximal to the transcription start site of the gene. Statistical analysis suggests that the clustering of the four SNPs in this region was not random. There was no correlation between TA skew and the location of the polymorphisms. Indeed, there is no significant difference at the nucleotide level between this polymorphic hotspot and other sections of the 5' upstream region, suggesting that the concentration of SNPs in this area may be due to some functional requirement, such as a ribosomal binding site or secondary structure.

In the laboratory isolates, there was a slight correlation between the number of SNPs and chloroquine resistance; however, there was no association between any one SNP and resistance. When the study of the 462-basepair polymorphic zone was expanded to 22 field isolates from Senegal, it was found that the 328 T->C transition was prevalent throughout the population irrespective of in vitro chloroquine levels or the presence of pfcrt and pfmdr1 alleles associated with resistance. Thus, while this SNP merits interest due to its prevalence in both field and laboratory isolates, it does not appear to have a direct link to a resistant phenotype. The effect of this SNP on the transcriptional activity of the promoter should be tested in functional assays to determine if it is truly important, or if a diversity marker is present at a high prevalence in this population of Senegalese parasite isolates. Due to the downstream location of this SNP, it is also possible that it could have an effect on the translational activity of pfmdr1. It would be worthwhile to complete a SNP map for the entire 5' upstream region of a subset of the Senegalese isolates to determine if they possess any more nucleotide diversity.

The entire polymorphic map of the pfmdr1 upstream region is intriguing. Despite having an extremely long 5'-UTR of similar A-T content, all the SNPs cluster in a small area close to the transcription start site. The pfmdr1 promoter lacks polymorphism in a panel of geographically diverse isolates with different drug resistance profiles, suggesting that components of this DNA sequence are essential for the normal transcriptional activity of the gene.

Finally, except for one field isolate, no SNP was found in the promoter region of pfmdr in any of these isolates. This finding implies that the promoter region of this gene is highly conserved and may be under negative selection. One could argue that the lack of polymorphism is simply due to numbers, that given the low frequency of SNPs in the 5' upstream region, one would not expect another SNP past position 68 for at least another 1,000 basepairs. In fact, Muz12.4 was the only laboratory isolate that contained a SNP upstream of the transcription start site at position –899, which was beyond the start of the putative promoter. We propose that the lack of nucleotide diversity in the promoter region is not due to chance, but instead is an effect of negative selection. In other words, mutations in the promoter region have been selected against to preserve the essential function of the promoter. This hypothesis is being tested in functional assays via site-directed mutagenesis of the promoter region.

Received July 28, 2003. Accepted for publication July 7, 2004.

Acknowledgments: We thank Cathy Ndiaye and Abdoulaye Diallo for technical support. We also acknowledge the entire research team in Dakar, as well as the study participants, for their support. Dr. Peter Bachetti (Division of Biostatistics, University of California at San Francisco) made the random chance calculation.

Financial support: This work was supported by the Fogarty International Center, the National Institutes of Health (RO1 GM061351), and the Harvard Malaria Initiative.

Author’s addresses: Alissa Myrick, Department of Medicine, Building 10, Room 3402, University of California at San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110. Ousmane Sarr and Souleymane Mboup, Laboratory of Bacteriology and Virology, Le Dantec Hospital, Dakar, Senegal, Telephone: 221-821-6420, Fax: 221-822-5919. Therese Dieng and Omar Ndir, Laboratory of Parasitology, Faculty of Medicine and Pharmacy, Cheikh Anta Diop University, Dakar, Senegal, Telephone: 221-824-5588, Fax: 221-825-3668. Dyann F. Wirth, Department of Immunology and Infectious Diseases, Building 1, Room 704, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA, 02115, USA, Telephone: 617-432-1621, Fax: 617-432-4766, E-mail: dfwirth{at}hsph.harvard.edu.

Reprint requests: Department of Immunology and Infectious Diseases, Building 1, Room 704, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA, 02115.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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