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. 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 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 para- site. 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. How- ever, data from this research has revealed unique character- istics 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., lev- els 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 resis- tance 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. fal- ciparum chloroquine resistance transporter (pfcrt). 18,19 Woot- ton and others analyzed the level of polymorphism in micro- satellite (MS) markers flanking pfcrt in a global panel of iso- lates 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 re- duced diversity surrounding the pfmdr1 locus, but did not find a statistically significant association with a chloroquine resis- tance 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 identifi- cation 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 polymor- phisms 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 iso- lates were examined for any link between the presence of the identified polymorphism and drug resistance. MATERIALS AND METHODS Analysis of laboratory strains. To generate a comprehen- sive 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 1). Strain 3D7, which was used as the refer- ence 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 resis- tance 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 Primer design and PCR. The intergenic sequence flanked by pfmdr1 and PFE1145w was obtained from the P. falci- parum Genome Database. 23 A set of primers spanning 2.9 kb of sequence upstream of the pfmdr1 start ATG was gener- ated. Most primers were made in both the forward and re- verse direction so that sequences could easily be formed into Am. J. Trop. Med. Hyg., 72(2), 2005, pp. 182–188 Copyright © 2005 by The American Society of Tropical Medicine and Hygiene 182