Plasmodium vivax dihydrofolate reductase point mutations from the Indian subcontinent

Acta Tropica 97 (2006) 174–180

Plasmodium vivax dihydrofolate reductase point mutations from the Indian subcontinent Suminder Kaur a , Surendra K. Prajapati b , Kavitha Kalyanaraman a , Asif Mohmmed a , Hema Joshi b , Virander S. Chauhan a,∗ a

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India b Genetics Division, Malaria Research Centre, 22, Sham Nath Marg, New Delhi 110054, India Received 18 March 2005; received in revised form 24 October 2005; accepted 24 October 2005 Available online 23 November 2005

Abstract Mutations in Dihydrofolate Reductase (dhfr) gene of Plasmodium vivax are known to be associated with resistance to antifolate drugs. To analyze the extent of these mutations in P. vivax population in India, dhfr gene was isolated and sequenced for 121 P. vivax isolates originating from different geographical regions of Indian subcontinent. These sequences were compared with the gene sequence that represent wild type sequence (accession no. X98123). P. vivax dhfr (Pvdhfr) sequences showed limited polymorphism and about 70% isolates showed wild type dhfr sequence. A total of 36 mutations were found at 11 positions in 121 isolates. A majority of mutant isolates showed double mutations at residues 58 (S → R) and 117 (S → N), known to be associated with pyrimethamine resistance, but only 19% showed double mutations at residues 57 (F → L) and 58 (S → R). Pvdhfr alleles showing quadruple mutation (F57L, S58R, T61M andS117T) were found in two isolates. Three other mutations reported earlier at residue 13, 33 and 173 were not found in any of the Isolates. Six novel mutations at residues 38 (R → G), 93 (S → C), 109 (S → H), 131 (R → G), 159 (V → A) and 188 (I → V) were observed in seven isolates. Whether these novel mutations are linked to pyrimethamine resistance remains to be established. © 2005 Elsevier B.V. All rights reserved. Keywords: Malaria; Plasmodium vivax; Dihydrofolate reductase; Antifolate; Pyrimethamine; Point mutations; Drug resistance

1. Introduction Development of resistance against antimalarial drugs is a major hurdle to combat malaria in most parts of the world. Due to the global spread of chloroquine resistance, the combination of sulfadoxine–pyrimethamine (S–P) becomes the first line therapy for complicated ∗

Corresponding author at: International Centre for Genetic Engineering and Biotechnology, PO Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India. Tel.: +91 11 2618 9358; fax: +91 11 2616 2316. E-mail address: [email protected] (V.S. Chauhan). 0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.10.003

Plasmodium falciparum malaria in areas of endemicity. Pyrimethamine, an effective drug, interacts with the folate synthesis pathway of the parasite where dihydrofolate reductase (DHFR) enzyme reduces dihydrofolate to tetrahydrofolate. Pyrimethamine competitively inhibits parasite DHFR thereby blocking thymidylate synthesis (Hyde, 2005). In P. falciparum, resistance to pyrimethamine is attributed to one or more point mutations in parasite’s dhfr gene (Anderson and Roper, 2005). Epidemiological studies, underpinned by biochemical analysis, strongly suggest that the drug pressure leads to the sequential appearance of point mutations and progressive selection of P. vivax parasites with increasing

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levels of resistance (Imwong et al., 2003; Hastings et al., 2004). In malaria endemic areas, prevalence of P. vivax often exceeds P. falciparum. Polymorphism in Pvdhfr gene is also known to be wide spread in different P. vivax endemic areas. Since P. vivax cannot be maintained in culture, therefore, the in vivo and in vitro evaluation of pyrimethamine resistance could not be established for this species of malaria. However, recent studies have shown that mutations at amino acid residues 57 (F → L), 58 (S → R) and 117 (S → N) confer resistance to pyrimethamine (Hastings et al., 2005). It has been suggested that these mutations emerge in the field populations of P. vivax in areas where S–P is used extensively for the treatment. Malaria in India contributes around 2–2.5 million cases every year. According to statistical data published by National Malaria Eradication Program (NMEP) in the year 1997, in India, the incidence of P. vivax malaria is 60–70%. For treatment of P. vivax malaria worldwide, the first choice of treatment still remains the combination of chloroquine–primaquine. In recent years chloroquine resistance P. vivax parasites have been reported in several locations with high-level resistance confirmed in parts of Indonesia, New Guinea and India (Bombay) (Imwong et al., 2003). However, there are very limited reports on extent of drug resistant genotypes among P. vivax population in Indian Subcontinent. In the present study, we have analyzed sequences of Pvdhfr genes from 121 P. vivax field isolates from different geographical regions of India. A small fraction of samples showed one to four mutations at different residues including mutations known to be responsible for pyrimethamine resistance. In addition, a number of unique and novel mutations were also identified in the present study. 2. Materials and methods 2.1. Geographical regions for sample collection Blood samples from patients having acute P. vivax infections were collected from various clinics/hospitals in different geographical locations in India (Fig. 1): New Delhi (39), Mohan Nagar (49), Mirzapur (8), Cuttack (3), Navi Mumbai (5), Goa (8), Chennai (8) and Car Nicobar (1). The diagnosis was made by microscopic examination of giemsa-stained thin and thick blood smears. All the patients were found to be only infected with P. vivax. These samples were collected during July–September 2004. All the blood samples were collected with the consent of each patient and approval of the protocol

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Fig. 1. Map of India showing P. vivax sampling sites.

was obtained from Human volunteer Research Ethical Committee of the International Center for Genetic Engineering and Biotechnology and Ethical Committee of Malaria Research Centre, New Delhi, prior to the study. 2.2. Parasite DNA extraction and amplification Template DNA was isolated from the blood spots of infected patients using QIAamp DNA extraction mini-kit (QIAGEN) following manufacturers’ protocol. The PvDHFR gene sequences were amplified from these DNA samples by primary and nested PCR following Imwong et al. (2001). Oligonucleotides were designed using a sequence of dhfr-ts gene of Plasmodium vivax (GenBank accession no. X98123). The primary amplification was performed using the external primers, VDT-OF (5 -ATGGAGGACCTTTCAGATGTATTTGACATT-3 ) and VDT-OR (5 -GGCGGCCATCTCCATGGTTATTTTATCGTG-3 ), wherein the entire P. vivax dwhfr-ts gene (1.8 kb) was amplified. This primary amplification product was then used for performing nested PCR, to amplify PvDHFR domain (711 bp). The oligonucleotide pair used were VDT-OF (5 -ATGGAGGACCTTTCAGATGTATTTGACATT-3 ) and VDFNR (5 -TCACACGGGTAGGCGCCGTTGATCCTCGTG-3 ). The PCR cycling parameters were as follows: initial denaturation at 95 ◦ C for 5 min, followed by 35 cycles

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of denaturation at 95 ◦ C for 30 s, annealing at 64 ◦ C for 30 s and extension at 72 ◦ C for 1 min. The amplified PCR products were then analyzed on 2% agarose gel. All the samples showed specific amplification in the present study. The amplified products were purified from agarose gel and cloned into pGEMT cloning vector (Promega, USA). For each sample, two to three clones were sequenced by automated DNA sequencer (ABI systems, Perkin-Elmer, France). Sequence alignments and analysis was carried out using DNASTAR software (Laser Gene, USA).

S31 and M5) showed mutation at residue 117 (S → N) only. In two isolates (CN1 and PK6), Ser (S) was mutated to Thr (T) instead of Asparagine (N) at residue 117. Earlier studies have also shown the prevalence of S → T rather than S → N mutation at residue 117 (Imwong et al., 2003). This mutation was found to be associated with mutations at residues 58 (S → R), 57 (F → L) and 61 (T → M). The combinations of double (residues 57 and 58) and quadruple mutations (residues 57, 58, 61 and 117) have also been reported earlier from Thai and Indonesian isolates, respectively (Eldin de Pecoulas et al., 2004; Hastings et al., 2004). Apart from these already known mutations, few novel mutations that lead to change in amino acid residues were observed in seven Indian isolates. In an isolate from eastern India (S3), an R → G mutation was found at position 131. Another novel mutation at residue 93 (S → C) was observed in five isolates from northern region of India (M1, M4 and M6). Three isolates (S10, S14 and 7848) were found to have single and novel mutation at position 109 (S → H). Novel mutations at residues 188 (I → V) and 159 (V → A), in isolates PK5 and SKP12, respectively, were found to be associated with mutation at 58 (S → R) and 117 (S → N) residues. Another isolate (S27) from North India showed a unique combination of mutations. This isolate showed presence of novel mutations at residue 109 (S → H) associated with two more novel mutations at positions 38 (R → G) and 159 (V → A). In addition this isolate also harbored the common mutation at position 58 (S → R) thereby showing a total of four mutations in all. Mutations at codons 13, 33 and 173, described earlier (Imwong et al., 2001, 2003) were not detected in any isolate in the present study. Two silent mutations at amino acids 8 and 9 were detected in 11 samples and one silent mutation at codon 9 was observed in one sample.

3. Results Pvdhfr gene sequences from a total of 121 P. vivax samples were amplified by primary and nested PCR. The amplified PCR fragments were cloned in pGEMT vector from three independent amplification reactions for each sample and three clones from each reaction were sequenced. The sequences of Pvdhfr obtained were compared with the gene sequence (GenBank accession no. X98123), representing wild type sequence. All the three sequences obtained of each isolate were found to be identical. A large fraction of samples i.e. 70% (85 of 121) represent gene sequence identical to wild type sequence whereas 36 samples showed mutations at 11 residues including mutations known to be responsible for pyrimethamine resistance (Table 1). The most prevalent combination among all the mutant isolates constitutes mutations at residues 58 (S → R) and 117 (S → N) in 19 of 121 samples. Nine isolates showed mutations only at residue 58 (S → R) and no change was observed at residue 117 as compared to the wild type sequence. In seven out of these nine isolates, the mutation at residue 58 was found in association with the mutation at residue 57 (F → L). Three isolates from North India (S28,

Table 1 Distribution of wild and mutant alleles for DHFR domain among Indian P. vivax isolates Sampling site

Goa Chennai Navi Mumbai Car Nicobar Cuttack Mirzapur New Delhi Mohan Nagar a b

Total no. of isolates

S/P drug used

Mutant type isolatesa

No. of isolates with sequence polymorphism for amino acid residueb 13

33

37

57

58

61

93

109

117

131

159

171

173

188

8 8 5 1 3 8 39 49

+ − + + + + − −

6 6 4 1 1 1 11 6

– – – – – – – –

– – – – – – – –

– – – – – 1 –

1 – – 1 – – 2 5

5 6 4 1 – 1 8 6

1 – – 1 – – – –

– – – – – – – 5

1 – – – – – 3 –

5 6 4 1 – 1 5 –

– – – – 1 – – –

– – 1 – – – 1 –

– – 1 – – – – –

– – – – – – – –

– 1 – – – – – –

PvDHFR sequence differed to wild type (GenBank accession no. X98123). Residues showing novel mutations are indicated in boldface.

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These mutations have not been reported in earlier studies. Sequence of all the other isolates showed no difference from the wild type sequence. 3.1. Tandem repeat region One of the unique features in the Pvdhfr gene is the presence of a tandem repeat region between amino acid residues 70–110. Size polymorphism in this repeat region of Pvdhfr gene resulting from a deletion or repetition of this six amino acid unit has been reported previously (Imwong et al., 2003). It is interesting to note that the various isolates also showed the presence of this repeat region in different combinations leading to the polymorphism in this region (Fig. 2). In addition, deletions of some of the residues of the repeat region were also observed among some isolates, in different combinations. Majority of the samples (86 out of 121 samples) showed similar pattern in the repeat region as that of the wild type. All the other 35 samples were distributed among seven different classes based on polymorphism in tandem repeat region (Fig. 2). A novel mutation at residue 93 (S → C) was observed in five of the isolates, where this novel mutation associated with mutations at 57 (F → L) and 58 (S → R) positions. This combination of triple mutation is unique for isolates but is not clear if this combination is responsible for drug resistance in P. vivax. According to the population profile of wild type versus mutants of pvdhfr gene across different geographical regions of India, Navi Mumbai represented the highest level of mutants (80%) followed by Goa and Chennai

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together, constituting 75% mutants (6 of 8 samples). Cuttack on the other hand showed 33% mutant types followed by New Delhi (28%) and Mirzapur (12.5%). The lowest level of mutants (12.2%) was found in Mohan Nagar where only 6 samples out of 49 showed mutant type Pvdhfr gene 4. Discussion Emergence of drug resistance phenotypes among P. vivax populations is a major hurdle in P. vivax endemic areas to combat malaria. The point mutations in Pvdhfr genes responsible for resistance against antifolate drugs are well characterized (Brega et al., 2004; Imwong et al., 2001). These mutations act as molecular markers to assess the extent of drug resistance in any P. vivax population. In the present study, we have analyzed Pvdhfr gene sequences from 121 P. vivax isolates from different geographical regions of India (Fig. 1). Analysis of Pvdhfr sequences showed limited polymorphism as compared to earlier studies, where 98 of 100 Thai samples, 30 of 32 Cambodian samples, and 11 of 12 Burmese samples were found to have double mutations (Eldin de Pecoulas et al., 1998b; Imwong et al., 2001, 2003). In the present study, 85 of 121 (approx. 70%) isolates yielded wild type dhfr sequence (Table 1) and 36 samples showed presence of mutation at one or more residues. Majority of the isolates carrying mutation showed presence of double mutations at residues 58 and 117 (52% of mutants). Mutations at codon 58 and 117 of PvDHFR are considered to be equivalent to mutation at residues 59 and 108

Fig. 2. Polymorphism observed in tandem repeat region of PvDHFR gene (70–110) of Indian isolates. Most of the samples showed wild type sequence whereas rest 35 samples showed eight different patterns. Two kinds of mutations were observed at one of the residues in the repeat for nine isolates. Each letter corresponds to standard amino acid residue, dashes represent deletions and non-synonymous mutations are in boldface.

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tions were present only once in the present isolates. These novel mutations were observed on residues 37 and 131 which are conserved between different species of plasmodium and also with human and E. coli dhfr genes (O’Neil et al., 2003; Yuvaniyama et al., 2003). These mutations (38, 93, 109, 131, 159, 188) are novel mutations proximity to the areas (105, 155, 172, 177) identified previously (Eldin de Pecoulas et al., 1998a). However superimposition of PvDHFR modeled structure with PfDHFR suggested that these residues may not be involved in interaction with antifolate directly, and also do not lie in predicted DHFR domain. Therefore involvement of these mutations in catalytic activity or antifolate resistance is not clear (Rastelli et al., 2003). Silent double mutations at codons 8 and 9 were observed among 11 samples whereas a single sample represented silent mutation at codon 9 only. Whether these mutations are linked to pyrimethamine resistance has yet to be established. Studies on point mutations in the Pvdhfr gene suggested that mutations at 58 and 117 appear together under drug pressure (Imwong et al., 2003), but it is not clear if the mutation at residue117 may precede that at 58, as in the case of homologous positions in PfDHFR. In

of PfDHFR, respectively that are known to be associated with pyrimethamine resistance. Double mutation at residue 57 and 58 were altered in 7 of 36 mutants (19.4%). The next mutant group of parasites consisted of those carrying 4 mutations in Pvdhfr. Besides carrying mutations at residue 58 and117; additional mutations at 61 and 57 were observed in two isolates (CN1 andPK6) (Table 2). Hastings et al. (2004) correlated this quadruple mutation allele with high risk of therapeutic failure. These authors also conferred that this allele has high levels of pyrimethamine resistance in vitro. Earlier reported mutations at residue 13, 33 and 173 were not detected in any of the isolates, whereas mutation at residue 171 was detected only in a parasite from Navi Mumbai (PK3). Six new mutations were also identified in the present study, which have not been reported previously. These mutations included R38G, S93C, S109H, R131G, V159A and I188V. These mutations were distributed among seven isolates, where mutation S93C was present among five isolates belonging to Mohan Nagar region of India. S109H on the other hand, was found among four isolates residing at different geographical regions of India. Two of the samples showed V159A mutation whereas the other three mutaTable 2 Polymorphism at different residues in PvDHFR among mutant isolates Collection sites Name of isolates

No. of isolates

Isolates showing mutations at specific residue in DHFR gene 37 (R) 57 F 58 S 61 T 93 (S) 109 (S) 117 S 131 (R) 159 (V) 171 F 188 (I)

Goa Goa Goa Chennai Chennai Navi Mumbai Navi Mumbai Navi Mumbai Car Nicobar Cuttack Mirzapur New Delhi New Delhi New Delhi New Delhi New Delhi New Delhi Mohan Nagar Mohan Nagar

7848 7844, P10M, PK12, SKP10 PK6 PK1, C62, P3M, P5M, P11M PK5 SKP12 P14M, SKP1 PK3 CN1 Ct1 Ct2 S10, S14 S21, S26, S30, D-5h S27 S28 S31 D12, D16 M1, M2, M3, M4, M6 M5

H

1 4

R

1 5 1 1 2 1 1 1 1 2 4 1 1 1 2 5 1

L

L

R R R R R R R

N M

T N V

N N N N T

M

A L G

R

N H

R R R

G

N H

A N

L L

R R

C

R

Region-wise depiction of 36 mutants showing mutations at 11 residues of PvDHFR. Residue at different positions in wild type sequence are indicated in the upper most panel and changed residue for these positions are depicted for each mutant. Novel mutations are shown in boldface. Earlier reported mutations at residues 13, 33 and 173 were not found in Indian isolates. Standard amino acid abbreviations are used.

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PvDHFR, mutations at residue 57 and 61 follow the mutation at 117 and 58, resulting in variants with quadruple mutations. Our results suggest that mutations at these residues can appear independently of each other, as mutations at 117 and 58 also appeared as single mutations in three different samples i.e. S28, S31 and M5, respectively. A single mutation in PvDHFR at residue 117 (S → N) has been shown to provide higher level of pyrimethamine and sulfadoxine resistance than the double mutations at 58 and117 residues, whereas single mutation at residue 58 provides moderate level of resistance (Leartsakulpanich et al., 2002). Studies by Hastings and Sibley (2002) showed that a single base pair mutation resulting in the substitution of serine to asparagine at codon 117 increased IC50 value for pyrimethamine more than 80 folds and together with the substitution of serine to arginine at codon 58, an enzyme that was more than 400-fold more resistant to pyrimethamine was produced. Similarly, mutation at residue 109 was observed alone in three of the isolates (7848, S10 and S14) whereas in a single isolate (S27), it has observed in association with mutation at residue 58. All of these isolates were collected from urban areas where antifolate drugs are being used for the treatment of malaria, suggesting that mutation at position 109 may appear before those at position 58 and 117 under drug pressure. Indian isolates also showed the presence of unique patterns within the repeat region (residues 70–110; Fig. 2) in different combinations. Though majority of the samples (86 of 121) showed wild type of combination, around 35 samples showed seven different combinations (Fig. 2). It is not clear if this polymorphism in the repeat region provides any resistance to antifolates. The population profile of wild type versus mutants of Pvdhfr gene showed that highest mutants (80%) were observed at Navi Mumbai, followed by Goa and Chennai (75% mutants). Cuttack, New Delhi, Mirzapur and Mohan Nagar represented 33, 28, 12.5 and 12.2% mutants, respectively. It is to be noted that the high proportion of alleles showing mutations responsible for drug resistance were found from coastal cities. In these areas, the use of S–P in combination with mefloquine is known for malaria treatment (National Vector Borne Disease Control Programme, http://www.namp.gov.in), and this drug pressure may be responsible for generation of these mutations. On the other hand, mutation at residue 58, which provides moderate level of S–P resistance (Hastings and Sibley, 2002), appeared in cities from plains where the drug pressure and self-medication might be relatively less.

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A validated molecular marker is essential to map current and changing pattern of drug resistance status of malaria parasite populations. Although our data shows limited polymorphism in Pvdhfr from Indian isolates, it provides basic information on the potential appearance of mutations responsible for S–P resistance as well as novel mutations under drug pressure in the geographical zones. Appearance of these S–P resistance genotypes in P. vivax demands a need for new antimalarial drugs that should preferably be effective against both P. vivax and P. falciparum malaria. The paucity of information on P. vivax underlines the need to extend molecular analyses to other areas where P. vivax is common, e.g. the Indian subcontinent, Central Asia, Middle East, America and Ethiopia (Hastings et al., 2005). The rationale behind assessment of dhfr alleles is to understand the molecular basis of point mutations that leads to resistance in pyrimethamine and finally addressing in better drug design. Acknowledgements This work was supported by INO-DEV project grant (contract no. ICA4-CT-2001-10077) from EU. Special thanks to Malaria Research Centre, New Delhi for help in collection of blood samples from patients. Technical assistance rendered by Ajay Kumar is gratefully acknowledged. References Anderson, T.J., Roper, C., 2005. The origins and spread of antimalarial drug resistance: lessons for policy markers. Acta Trop. 94, 269–280. Brega, S., de Monbrison, F., Severini, C., Udomsangpetch, R., Sutanto, I., Ruckert, P., Peyron, F., Picot, S., 2004. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob. Agents Chemother. 48 (7), 2581–2587. Eldin de Pecoulas, P., Tahar, R., Yi, P., Thai, K.H., Basco, L.K., 2004. Genetic variation of the dihydrofolate reductase gene in Plasmodium vivax in Snoul, northeastern Cambodia. Acta Trop. 92 (1), 1–6. Eldin de Pecoulas, P., Tahar, R., Ouatas, T., Mazabraud, A., Basco, L.K., 1998a. Sequence variations in the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene and their relationship with pyrimethamine resistance. Mol. Biochem. Parasitol. 92 (2), 265–273. Eldin de Pecoulas, P., Basco, L.K., Tahar, R., Ouatas, T., Mazabraud, A., 1998b. Analysis of the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene sequence. Gene 211 (1), 177–185. Hastings, M.D., Maguire, J.D., Bangs, M.J., Zimmerman, P.A., Reeder, J.C., Baird, J.K., Sibley, C.H., 2005. Novel Plasmodium vivax dhfr alleles from the Indonesian Archipelago and Papua New Guinea: association with pyrimethamine resistance determined

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