Research note

Allelic variation in the cg2 gene does not correlate with chloroquineresistance among Indian Plasmodium falciparum isolatesq

Indu Sharmaa, Manish K. Anejaa, Sukla Biswasb, Vas Devb, Musharraf A. Ansarib,S. Tazeen Pashac, Yagya D. Sharmaa,*

aDepartment of Biotechnology, All India Institute of Medical sciences, Ansari Nagar, New Delhi -110029, IndiabMalaria Research Centre, 22 Sham Nath Marg, Delhi -110054, India

cNational Institute of Communicable Diseases, 22 Sham Nath Marg, Delhi -110054, India

Received 23 May 2001; received in revised form 13 July 2001; accepted 20 July 2001

Abstract

The cg2 gene of Plasmodium falciparum has been proposed to be associated with chloroquine resistance. Here we describe PCR

amplification and sequencing of all the four repeat regions (kappa (k), gamma (g), psi (f) and omega (v)) of this gene, from Indian isolates.

There were variant forms for each of these repeat regions (two for k and g, and three for f and v) among the 123 Indian isolates of P.

falciparum. Among these isolates certain forms of f and v repeats were uniquely present while some of the reported forms of the k and v

repeats were absent. The pattern of combination of all four repeat regions of cg2 gene (genotype) was analysed from 52 isolates. A total of 11

different genotypes were observed among these cases, of which 10 were unique to Indian isolates. Certain genotypes were more common

than others. The nucleotide sequencing of all the four repeat regions revealed that Indian isolates have some unique repeating units within the

g and v domains. Altogether, the PCR and sequencing results showed that there was an unrelatedness between cg2 repeats and chloroquine

resistance. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Human malaria; Drug resistance; Genetic polymorphism; Plasmodium falciparum

Malaria affects millions of people each year and causes

large number of deaths in tropical countries. Most of these

deaths are due to Plasmodium falciparum, which is devel-

oping, resistance towards commonly used antimalarial

drugs. The drug resistant cases are increasing at an alarming

rate. Resistance towards the most widely used antimalarial

drug, chloroquine, is spreading rapidly in tropical countries

(Sharma et al., 1996). Molecular mechanisms as well as

monitoring of chloroquine resistance by molecular markers

remains elusive (Fidock et al., 2000b; Foote et al., 1989). Su

et al. (1997) reported an association between a gene (cg2)

encoding ~330 kDa protein and chloroquine resistance. This

cg2 encoded protein contains four peptide repeat regions

called k, g, f and v. Among these four domains, k and v

have been reported to be associated with chloroquine resis-

tance in Africa (Durand et al., 1999; McCutcheon et al.,

1999). We carried out a study on Indian isolates comprising

chloroquine resistant and sensitive parasite lines as well as

clinical isolates from different parts of the country to deter-

mine cg2 gene polymorphisms and their relationship with

chloroquine resistance. We used all four peptide repeat

regions of the cg2 gene for PCR amplification and

sequenced all variant forms of these domains observed

among Indian isolates. We report here the presence of

variant cg2 alleles in Indian isolates, but no correlation

with chloroquine resistance.

The chloroquine resistant and sensitive P. falciparum

lines from Indian isolates were generated and maintained,

as described earlier (Biswas et al., 1996). Clinical isolates

were obtained from different parts of the country. About 20

ml blood was collected in heparin from those individuals

who were microscopically positive for P. falciparum.

These individuals were informed about the study and

International Journal for Parasitology 31 (2001) 1669–1672

0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(01)00286-7

www.parasitology-online.com

q Note: Nucleotide sequences reported in this paper have been submitted

to the EMBL data base under the following accession numbers: k repeats

from M27(AJ311566), FSH (AJ311564), D9 (AJ311561), M28

(AJ311563), M30 (AJ311565), NE (AJ311562), A324 (AJ 311567) and

U409 (AJ311568); g repeats from M27(AJ311588), FSH (AJ311589), D9

(AJ311590), M28 (AJ311592), NE (AJ311591), A306 (AJ311593), A307

(AJ311594), A324 (AJ311595), A334 (AJ311596) and U409 (AJ311597);

f repeats from M27 (AJ308578), FSH (AJ308580), M25 (AJ308585), D9

(AJ308581), M14 (AJ308584), M28 (AJ308582), M30 (AJ308579) and

BSN8 (AJ308583); v repeats from M27 (AJ310615), FSH (AJ310616),

D9 (AJ310617), M28 (AJ310618), NE (AJ310619), A311 (AJ310620),

A339 (AJ310621), A340 (AJ310622) and M343 (AJ310623).

* Corresponding author. Fax: 191-11-6852286.

E-mail address: ydsharma@hotmail.com (Y.D. Sharma).

blood was collected with their full consent. The ethical

guidelines of the All India Institute of Medical Sciences

were followed for blood collection. The heparinised blood

was stored on ice and transported from the field to the

laboratory at Delhi. The samples were processed immedi-

ately upon their arrival for PCR amplification. DNA was

isolated from the cultured and field isolates by the method

of Foley et al. (1992) and PCR was carried out under the

conditions described earlier (Ranjit and Sharma, 1999).

PCR for all four repeat regions of the cg2 gene was carried

out using the following primers: k forward 5 0-GTA ATT

ATA TAA CCT CTC AGG AGG-3 0, k reverse 5 0-GGA

TGA TGG GAA TTA ATC GCT ATC A-3 0, g forward

5 0-ATC TGG AGA CAC AAC TAA AAA GGG-3 0, g

reverse 5 0-GGT ATT TTC TGG AAA GGT TCC TTT-3 0,

f forward 5 0-TGA GAG GAA CTA ATA TGG AAA AGG-

3 0, f reverse 5 0-TGA TAA TTT TGT TTA TCA AAG AAT

TAA-3 0, v forward 5 0-AAA TCT TAC TAT AAG AAG

GTA AAG G-3 0, v reverse 5 0-ATA TTC AAT GAG

CAA TTT ATT TCA CAG-3 0. The annealing temperature

for k and g primers was 688C whereas it was 668C and 628C

for f and v primers, respectively. The PCR products were

analysed on 12% polyacrylamide gel. PCR products were

also purified from the gel using a Qiaex II kit following

manufacturer’s instructions (Qiagen). The purified DNA

was then subjected to cycle sequencing using the above

mentioned primers and automated DNA sequencer (ABI

373A and 310 Genetic Analyser). The nucleotide sequence

was analysed using the DNAstar package.

We have analysed 123 P. falciparum samples for the

repeating domains of the cg2 gene. These samples included

91 field isolates and 32 cultured parasite lines (22 chloro-

quine resistant and ten chloroquine sensitive). Of these 123

samples, only 52 showed PCR amplification for all the four

repeats. The rest of the 71 samples were either positive for

one or more than one repeat region. The PCR positivity was

highest for the g repeat region where 117 of 123 samples

showed amplification. This positivity rate was 82/123 for f

repeats, 72/123 for v repeats and 68/123 for k repeats

(Table 1). The preferential amplification of certain domains

over the others could be attributed to the primer sequences.

This is because some primers bind more efficiently than

others on the same DNA preparation (Bhutani et al., 1998;

Ranjit and Sharma 1999). However, the mutation in the

I. Sharma et al. / International Journal for Parasitology 31 (2001) 1669–16721670

Table 1

Distribution of k, g, f and v alleles of P. falciparum cg2 gene among Indian isolates

Distribution of alleles

Parasite Lines/clinical isolates PCR positive lines/

clinical isolatesa

k alleles g alleles f alleles v alleles

460 bp 480 bp 275 bp 300 bp 220 bp 240 bp 260 bp 640 bp 675 bp 700 bp

1. Parasite lines

A.Chloroquine resistant 22 1 4 – 21 2 1 4 – 3 1

B. Chloroquine sensitive 10 1 4 – 10 3 2 3 – 5 1

2. Clinical isolates

A. Assam 59 3 36 6 48 2 26 17 3 33 8

B. Manipur 8 – 2 – 8 – – 4 – 2 –

C. Uttranchal 6 – 5 – 6 – – 5 – – 4

D. Delhi 18 – 12 – 18 – 1 12 – 8 4

Total 123 5 63 6 111 7 30 45 3 51 18

a Amplification achieved for one or more than one domain of the cg2 gene.

Table 2

Genotyping of the P. falciparum cg2 gene among Indian isolates

Parasite lines/isolates Size of PCR products (bp) Genotypea

k g f v

Chloroquine resistant and sensitive parasite linesb

M27 460 300 260 700 I

FSH, M28 480 300 220 675 II

M12, M19 480 300 260 675 III

NE 460 300 220 675 IV

Clinical isolates

Assam: A 324 460 300 260 700 I

Assam: A 267, A293,

A298, A303, A310,

A311,A312, A335

Manipur: M343, M354

Delhi:D386, D388,

D393,D396, D398, D399

480 300 260 675 III

Assam: A 265, A266,

A268, A269, A270,

A277, A292, A294,

A300, A325 Delhi: D402

480 300 240 675 V

Assam: A271 480 300 240 640 VI

Assam: A326, A328,

A332 Delhi: D397, D400,

D403, D405 Uttaranchal:

U408, U410, U411, U412

480 300 260 700 VII

Assam: A331 480 300 240 700 VIII

Assam: A334, A341 480 275 260 675 IX

Assam: A339 480 275 240 675 X

Assam: A340 480 275 220 640 XI

a Genotyping was derived from the combination pattern of different

domains.b M27 and FSH are chloroquine resistant, and M28, D9, M12, M19 and

NE are chloroquine sensitive parasite lines.

primer binding regions of the cg2 gene, in these isolates,

would also give rise to these results and can not be ruled out

at this stage.

Variant forms of these repeats were observed among

Indian isolates (Table 1). There were two forms for each

of the k (460 and 480 bp) and g (275 and 300 bp) repeats,

and three for each of the f (220, 240 and 260 bp) and v

(640, 675, and 700 bp) repeats. Distribution of all these

variant forms was not equal among isolates as some of

them were more common than others. Some of them (220

bp of f, and 640 and 675 bp of v repeats) were uniquely

present in these isolates of P. falciparum. There were 11

different combinations of these four domains (genotypes)

among 52 samples, of which ten were unique to Indian

isolates (Table 2). Only genotype VII was same as of Dd2

strain from Indochina (Su et al., 1997). Genotype III was

most common among these samples.

We sequenced these four domains of the cg2 gene from

various clinical isolates and parasite lines to cover all the

variant forms observed so far among Indian isolates (Fig. 1).

The sequenced regions also covered eight of 12 reported

point mutations of the cg2 gene (Su et al., 1997). The

sequencing results confirmed the presence of all the variant

forms, as detected by PCR, in all the four domains of the cg2

gene. The sequencing results revealed additional variation

in these domains as there was a polymorphism within the

DNA repeating units of these domains. There were certain

unique DNA repeating units within the g and v domains and

I. Sharma et al. / International Journal for Parasitology 31 (2001) 1669–1672 1671

Fig. 1. Amino acid sequence alignment of k (A), g (B), f (C) and v (D) domains of the P. falciparum cg2 gene from Dd2 (Indochina) with that of the Indian

isolates. The peptide repeat region is underlined. The same amino acid is marked by dots, gaps are indicated by dashes and different amino acids are defined.

The numbers at right hand side indicate the amino acid residue number. Site of point mutation is shaded and their position is indicated at the top of the sequence

(pt #). The following Indian isolates were used for sequencing: M25, M27, and FSH were chloroquine resistant parasite lines; M14, M28, M30, D9, NE and

BSN8 were chloroquine sensitive parasite lines. Clinical isolates A 306, A307, A311, A324, A334, A339 and A340 were from Assam, U409 from Uttaranchal,

and M343 from Manipur.

an alternate arrangements of peptide repeating units in the k

and v domains. The maximum sequence variation was

observed in the v domain and minimum in g (Fig.1).

Su et al. (1997) described the relationship between the P.

falciparum cg2 gene and chloroquine resistance. They have

observed that some of the repeats and point mutations could

be used as markers to monitor chloroquine resistant para-

sites. However, subsequent studies carried out on African P.

falciparum isolates using k and v repeats described variable

results (Durand et al., 1999; McCutcheon et al., 1999, 2000;

Fidock et al., 2000a). Here, we investigated the cg2 gene

polymorphism and its association with chloroquine resis-

tance from in-vitro established chloroquine resistant and

sensitive parasite lines and field isolates from India. Unlike

previous studies, we investigated all the four repeat domains

(k, g, f and v) of the cg2 gene as well as eight of 12 point

mutations. Comparison of the PCR product sizes of all the

four cg2 domains between chloroquine resistant and sensi-

tive parasite lines, did not yield any association with drug

susceptibility. All the variant forms were found to be present

in both chloroquine resistant and sensitive parasite lines.

The sequence comparison between chloroquine sensitive

and resistant parasite lines also did not yield any pattern

which could be associated with the drug susceptibility.

Similarly none of the point mutation (eight of 12 investi-

gated here) of the cg2 gene, previously reported to be asso-

ciated with chloroquine resistance, showed any unique

presence in resistant parasite lines of Indian isolates.

It is therefore concluded that the cg2 gene of P. falci-

parum shows a high degree of variation in the parasite

isolates. Maximum variation was noticed in the v repeat

domain of the gene. There were certain new alleles for f

and v repeats in the Indian isolates. Conversely, certain

alleles of k and v reported earlier in other isolates were

not found among these Indian isolates. The results of the

present study do not show any association of any of the

repeat domain of cg2 gene as well as eight point mutations

with that of chloroquine resistance.

While the present results were under compilation, yet

another gene named cg10 has been proposed as a marker

to monitor chloroquine resistance (Fidock et al., 2001;

Durand et al., 2001). However, these findings on cg10

should be confirmed by other laboratories, as the case has

been with cg2, before they are recommended for field use.

Acknowledgements

Partial financial support (to Y.D.S.) came from the Coun-

cil of Scientific and Industrial Research, and from the

Department of Biotechnology. We thank Mr D.S. Rawat,

Mr Manoj Kumar, Dr Ranjana Anand and Dr T.A. Singh

for their help.

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