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Large-scale survey for novel genotypes of Plasmodium falciparum chloroquine-resistance gene pfcrt.

Takahashi N, Tanabe K, Tsukahara T, Dzodzomenyo M, Dysoley L, Khamlome B, Sattabongkot J, Nakamura M, Sakurai M, Kobayashi J, Kaneko A, Endo H, Hombhanje F, Tsuboi T, Mita T - Malar. J. (2012)

Bottom Line: The number of CQ-mutant lineages observed in this study was identical to that found in previous studies.This supports the hypothesis that the emergence of novel CQ resistance is rare.The occurrence of new mutations flanking K76T may yield stronger resistance to CQ and/or a higher fitness than the original pfcrt mutant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of International Affairs and Tropical Medicine, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

ABSTRACT

Background: In Plasmodium falciparum, resistance to chloroquine (CQ) is conferred by a K to T mutation at amino acid position 76 (K76T) in the P. falciparum CQ transporter (PfCRT). To date, at least 15 pfcrt genotypes, which are represented by combinations of five amino acids at positions 72-76, have been described in field isolates from various endemic regions. To identify novel mutant pfcrt genotypes and to reveal the genetic relatedness of pfcrt genotypes, a large-scale survey over a wide geographic area was performed.

Methods: Sequences for exon 2 in pfcrt, including known polymorphic sites at amino acid positions 72, 74, 75 and 76, were obtained from 256 P. falciparum isolates collected from eight endemic countries in Asia (Bangladesh, Cambodia, Lao P.D.R., the Philippines and Thailand), Melanesia (Papua New Guinea and Vanuatu) and Africa (Ghana). A haplotype network was constructed based on six microsatellite markers located -29 kb to 24 kb from pfcrt in order to examine the genetic relatedness among mutant pfcrt genotypes.

Results: In addition to wild type (CVMNK at positions 72-76), four mutant pfcrt were identified; CVIET, CVIDT, SVMNT and CVMNT (mutated amino acids underlined). Haplotype network revealed that there were only three mutant pfcrt lineages, originating in Indochina, Philippines and Melanesia. Importantly, the Indochina lineage contained two mutant pfcrt genotypes, CVIET (n = 95) and CVIDT (n = 14), indicating that CVIDT shares a common origin with CVIET. Similarly, one major haplotype in the Melanesian lineage contained two pfcrt genotypes; SVMNT (n = 71) and CVMNT (n = 3). In Africa, all mutant pfcrt genotypes were the CVIET of the Indochina lineage, probably resulting from the intercontinental migration of CQ resistance from Southeast Asia.

Conclusions: The number of CQ-mutant lineages observed in this study was identical to that found in previous studies. This supports the hypothesis that the emergence of novel CQ resistance is rare. However, in the mutant pfcrt genotypes, amino acid changes at positions 72, 74 and 75 appear to have recently been generated at least several times, producing distinct pfcrt mutant genotypes. The occurrence of new mutations flanking K76T may yield stronger resistance to CQ and/or a higher fitness than the original pfcrt mutant.

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Haplotype network diagram in Plasmodium falciparum isolates harbouring pfcrt mutation. Network tree is shown according to pfcrt genotypes. The haplotype network was constructed for 196 P. falciparum isolates harbouring pfcrt mutation comprises 49 microsatellite haplotypes, based on allelic variations in six microsatellite loci flanking the pfcrt locus (see text for details). The size of each circle corresponds to the number of samples sharing the same haplotype, and the length of an edge is proportional to a variation in repeat number between two haplotypes. IC; Indochina lineage, M; Melanesian lineage, P; Philippine lineage.
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Figure 3: Haplotype network diagram in Plasmodium falciparum isolates harbouring pfcrt mutation. Network tree is shown according to pfcrt genotypes. The haplotype network was constructed for 196 P. falciparum isolates harbouring pfcrt mutation comprises 49 microsatellite haplotypes, based on allelic variations in six microsatellite loci flanking the pfcrt locus (see text for details). The size of each circle corresponds to the number of samples sharing the same haplotype, and the length of an edge is proportional to a variation in repeat number between two haplotypes. IC; Indochina lineage, M; Melanesian lineage, P; Philippine lineage.

Mentions: Among 227 isolates harbouring pfcrt mutant genotypes, 196 samples were successfully determined for MS haplotypes. In total, 49 MS haplotypes were identified, including the three previously known major haplotypes (IC1, M1 and P1) and those haplotypes similar to IC1, M1 or P1 (Figure 2). To assess the genetic relatedness of pfcrt mutants, a haplotype network based on size variations at all MS markers was constructed. The haplotype network clearly showed three distinct clusters of pfcrt mutants (Figure 3). The first lineage (Indochina lineage) consisted of the most prevalent haplotype IC1 (n = 57), having a combination of alleles of 152-180-182-150-203-191 at the MS loci of -29.268 kb, -10.833 kb, -2.814 kb, 0.59 kb, 10.389 kb and 23.576 kb, respectively, and those haplotypes closely related to IC1. Nearly all isolates in this lineage harboured the CVIET genotype. However, several isolates (IC1, IC5, IC6, IC9 and IC10; n = 14) contained the CVIDT genotype. This indicates that this mutant genotype shares a common origin with CVIET, and that MS haplotype IC1 contained two mutant pfcrt genotypes. The second lineage (Melanesian lineage) contained the most prevalent haplotype M1 (n = 36), having an allele combination of 152-172-182-152-206-187 and those haplotypes related to M1. All but M2 (CVMNT, n = 3) harboured the SVMNT genotype. This indicates that MS haplotype M1 also contained two mutant pfcrt genotypes. In the third lineage (Philippine lineage), all but one (P2) harboured an allele combination of 152-170-190-142-200-189 (P1). As previously observed in the Melanesian lineage [1], all isolates in the Philippine lineage harboured the SVMNT genotype.


Large-scale survey for novel genotypes of Plasmodium falciparum chloroquine-resistance gene pfcrt.

Takahashi N, Tanabe K, Tsukahara T, Dzodzomenyo M, Dysoley L, Khamlome B, Sattabongkot J, Nakamura M, Sakurai M, Kobayashi J, Kaneko A, Endo H, Hombhanje F, Tsuboi T, Mita T - Malar. J. (2012)

Haplotype network diagram in Plasmodium falciparum isolates harbouring pfcrt mutation. Network tree is shown according to pfcrt genotypes. The haplotype network was constructed for 196 P. falciparum isolates harbouring pfcrt mutation comprises 49 microsatellite haplotypes, based on allelic variations in six microsatellite loci flanking the pfcrt locus (see text for details). The size of each circle corresponds to the number of samples sharing the same haplotype, and the length of an edge is proportional to a variation in repeat number between two haplotypes. IC; Indochina lineage, M; Melanesian lineage, P; Philippine lineage.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369212&req=5

Figure 3: Haplotype network diagram in Plasmodium falciparum isolates harbouring pfcrt mutation. Network tree is shown according to pfcrt genotypes. The haplotype network was constructed for 196 P. falciparum isolates harbouring pfcrt mutation comprises 49 microsatellite haplotypes, based on allelic variations in six microsatellite loci flanking the pfcrt locus (see text for details). The size of each circle corresponds to the number of samples sharing the same haplotype, and the length of an edge is proportional to a variation in repeat number between two haplotypes. IC; Indochina lineage, M; Melanesian lineage, P; Philippine lineage.
Mentions: Among 227 isolates harbouring pfcrt mutant genotypes, 196 samples were successfully determined for MS haplotypes. In total, 49 MS haplotypes were identified, including the three previously known major haplotypes (IC1, M1 and P1) and those haplotypes similar to IC1, M1 or P1 (Figure 2). To assess the genetic relatedness of pfcrt mutants, a haplotype network based on size variations at all MS markers was constructed. The haplotype network clearly showed three distinct clusters of pfcrt mutants (Figure 3). The first lineage (Indochina lineage) consisted of the most prevalent haplotype IC1 (n = 57), having a combination of alleles of 152-180-182-150-203-191 at the MS loci of -29.268 kb, -10.833 kb, -2.814 kb, 0.59 kb, 10.389 kb and 23.576 kb, respectively, and those haplotypes closely related to IC1. Nearly all isolates in this lineage harboured the CVIET genotype. However, several isolates (IC1, IC5, IC6, IC9 and IC10; n = 14) contained the CVIDT genotype. This indicates that this mutant genotype shares a common origin with CVIET, and that MS haplotype IC1 contained two mutant pfcrt genotypes. The second lineage (Melanesian lineage) contained the most prevalent haplotype M1 (n = 36), having an allele combination of 152-172-182-152-206-187 and those haplotypes related to M1. All but M2 (CVMNT, n = 3) harboured the SVMNT genotype. This indicates that MS haplotype M1 also contained two mutant pfcrt genotypes. In the third lineage (Philippine lineage), all but one (P2) harboured an allele combination of 152-170-190-142-200-189 (P1). As previously observed in the Melanesian lineage [1], all isolates in the Philippine lineage harboured the SVMNT genotype.

Bottom Line: The number of CQ-mutant lineages observed in this study was identical to that found in previous studies.This supports the hypothesis that the emergence of novel CQ resistance is rare.The occurrence of new mutations flanking K76T may yield stronger resistance to CQ and/or a higher fitness than the original pfcrt mutant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of International Affairs and Tropical Medicine, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

ABSTRACT

Background: In Plasmodium falciparum, resistance to chloroquine (CQ) is conferred by a K to T mutation at amino acid position 76 (K76T) in the P. falciparum CQ transporter (PfCRT). To date, at least 15 pfcrt genotypes, which are represented by combinations of five amino acids at positions 72-76, have been described in field isolates from various endemic regions. To identify novel mutant pfcrt genotypes and to reveal the genetic relatedness of pfcrt genotypes, a large-scale survey over a wide geographic area was performed.

Methods: Sequences for exon 2 in pfcrt, including known polymorphic sites at amino acid positions 72, 74, 75 and 76, were obtained from 256 P. falciparum isolates collected from eight endemic countries in Asia (Bangladesh, Cambodia, Lao P.D.R., the Philippines and Thailand), Melanesia (Papua New Guinea and Vanuatu) and Africa (Ghana). A haplotype network was constructed based on six microsatellite markers located -29 kb to 24 kb from pfcrt in order to examine the genetic relatedness among mutant pfcrt genotypes.

Results: In addition to wild type (CVMNK at positions 72-76), four mutant pfcrt were identified; CVIET, CVIDT, SVMNT and CVMNT (mutated amino acids underlined). Haplotype network revealed that there were only three mutant pfcrt lineages, originating in Indochina, Philippines and Melanesia. Importantly, the Indochina lineage contained two mutant pfcrt genotypes, CVIET (n = 95) and CVIDT (n = 14), indicating that CVIDT shares a common origin with CVIET. Similarly, one major haplotype in the Melanesian lineage contained two pfcrt genotypes; SVMNT (n = 71) and CVMNT (n = 3). In Africa, all mutant pfcrt genotypes were the CVIET of the Indochina lineage, probably resulting from the intercontinental migration of CQ resistance from Southeast Asia.

Conclusions: The number of CQ-mutant lineages observed in this study was identical to that found in previous studies. This supports the hypothesis that the emergence of novel CQ resistance is rare. However, in the mutant pfcrt genotypes, amino acid changes at positions 72, 74 and 75 appear to have recently been generated at least several times, producing distinct pfcrt mutant genotypes. The occurrence of new mutations flanking K76T may yield stronger resistance to CQ and/or a higher fitness than the original pfcrt mutant.

Show MeSH