Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum

Abstract

Mutations in the chloroquine resistance (CQR) transporter gene of Plasmodium falciparum (Pfcrt; chromosome 7) play a key role in CQR, while mutations in the multidrug resistance gene (Pfmdr1; chromosome 5) play a significant role in the parasite's resistance to a variety of antimalarials and also modulate CQR. To compare patterns of genetic variation at Pfcrt and Pfmdr1 loci, we investigated 460 blood samples from P. falciparum-infected patients from four Asian, three African, and three South American countries, analyzing microsatellite (MS) loci flanking Pfcrt (five loci [approximately 40 kb]) and Pfmdr1 (either two loci [approximately 5 kb] or four loci [approximately 10 kb]). CQR Pfmdr1 allele-associated MS haplotypes showed considerably higher genetic diversity and higher levels of subdivision than CQR Pfcrt allele-associated MS haplotypes in both Asian and African parasite populations. However, both Pfcrt and Pfmdr1 MS haplotypes showed similar levels of low diversity in South American parasite populations. Median-joining network analyses showed that the Pfcrt MS haplotypes correlated well with geography and CQR Pfcrt alleles, whereas there was no distinct Pfmdr1 MS haplotype that correlated with geography and/or CQR Pfmdr1 alleles. Furthermore, multiple independent origins of CQR Pfmdr1 alleles in Asia and Africa were inferred. These results suggest that variation at Pfcrt and Pfmdr1 loci in both Asian and African parasite populations is generated and/or maintained via substantially different mechanisms. Since Pfmdr1 mutations may be associated with resistance to artemisinin combination therapies that are replacing CQ, particularly in Africa, it is important to determine if, and how, the genetic characteristics of this locus change over time.

FIG. 1.

(A) Haplotype diversity and MPD of CQR Pfcrt allele-associated MS haplotypes. All Pfcrt MS haplotypes are ∼40 kb in length. The Pfcrt (72 to 76) allele(s) and total number of associated MS haplotypes (in parentheses) for each region are Asia, SVMNT1 (23); Africa, CVIET (14); and South America, SVMNT1 and/or SVMNT2 (4) and CVMET (2). The number of unique haplotypes/total number of haplotypes for each country is PNG, 12/20; Indonesia, 1/6; India, 1/4; Laos, 1/3; Kenya, 1/4; Uganda, 7/8; Ghana, 3/6; Brazil, 1/2; Colombia, 2/2; and Guyana, 1/2 (see Table S3a in the supplemental material). (B) Haplotype diversity and MPD of CQR Pfmdr1 allele-associated MS haplotypes. Pfmdr1 MS haplotypes are ∼5 kb in length in Asia and Africa and ∼10 kb in length in South America. The Pfmdr1 (86_184_1034_1042_1246) allele(s) and total number of associated MS haplotypes (in parentheses) for each region are Asia, YYSND (18); Africa, YYSND (19); and South America, NFCDY (3) and NFSDY (2). The number of unique haplotypes/total number of haplotypes for each country is PNG, 9/10; Indonesia, 1/2; India, 6/6; Uganda, 5/7; Ghana, 12/12; Brazil, 0/1; Colombia, 1/1; and Guyana, 0/1 (see Table S4 in the supplemental material). Laos was omitted from the analysis because all the Laotian samples carried the CQS Pfmdr1 86N allele.

FIG. 2.

Median-joining network of CQR Pfcrt allele-associated MS haplotypes. All Pfcrt MS haplotypes are ∼28 kb in length. H001 to H200, Asia; H201 to H300, Africa; and H301 to H400, South America (see Table S3b in the supplemental material); the associated Pfcrt allele and geographic distribution are indicated for each haplotype.

FIG. 3.

(A) Median-joining network of Pfmdr1 allele-associated MS haplotypes (∼5 kb in length). See Table S4 in the supplemental material for haplotype numbering information. Note that all samples, except those from Laos, carry CQR Pfmdr1 alleles. (B) Median-joining network of Pfmdr1 allele-associated MS haplotypes (∼10 kb in length). H001 is 9_7_8_2 (Brazil, n = 17, and Guyana, n = 12), H002 is 3_14_8_4 (Colombia, n = 11), H003 is 4_14_8_4 (Colombia, n = 1), H004 is 15_7_8_2 (Guyana, n = 1), H005 is 4_11_5_3 (Indonesia, n = 2), H006 is 4_11_8_1 (Indonesia, n = 1), H007 is 5_11_8_1 (Indonesia, n = 7), H008 is 5_11_8_2 (Indonesia, n = 1), H009 is 5_11_8_3 (Indonesia, n = 5), H010 is 7_11_5_3 (Indonesia, n = 1), H011 is 7_8_7_4 (Laos, n = 11), H012 is 7_8_6_4 (Laos, n = 1), and H013 is 7_11_7_4 (Laos, n = 1). While samples from Brazil, Guyana, Colombia, and Indonesia carry CQR Pfmdr1 alleles, Laotian samples carry the CQS Pfmdr1 86N allele.