Chloroquine resistance (CQR) in Plasmodium falciparum is associated with point mutations in Plasmodium falciparum chloroquine resistant transporter gene (pfcrt) in chromosome 7. The geographic origin of chloroquine resistance is associated with amino acid variations at position 72-76 and microsatellite haplotypes flanking this gene. Mutant SVMNT haplotype with PNG origin was reported to spread in many localities of India probably through SEA. In order to understand the gene flow of the SVMNT haplotype across India, seven microsatellite loci which are under chloroquine selection and three neutral polymorphic microsatellite loci were used. Further, the impact of transmission intensity on the genetic structure among 13 populations across India was also tested. The genetic admixture analysis and F-statistics detected genetically distinct groups in accordance to transmission intensity and the probable use of chloroquine. The distinct geographical groups identified ; Northeastern-Eastern-Island group (Assam, West Bengal, Orissa, Jharkhand, Chhattisgarh and Car Nicobar island), the Northwestern group (Uttar Pradesh, Rajasthan and Gujarat), the Central group (Madhya Pradesh and Maharashtra), the Southwestern group (Goa and Tamil Nadu). A large genetic break between the northeastern-eastern group and southwestern group was observed. A pattern of significant isolation by distance was observed in low transmission areas (P = 0.003, r = 0.49, N = 83, Mantel test), indicating restricted gene flow among the populations in accordance with geographical distances. The data also indicated gene flow within all the four groups. A significant genetic differentiation was observed between P. falciparum populations at high and low transmission areas and they have not differentiated substantially by genetic drift. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation in both neutral and adaptive loci at the geographic scale of India.
Introduction
Positive natural selection contributes to the fixation of beneficial alleles in a population. During this evolutionary process, it is not uncommon to observe a parallel increase in the frequency of specific neutral alleles, which are linked to these beneficial alleles by virtue of proximity. This carriage of neutral alleles during positive natural selection is called genetic hitchhiking (Smith, Haigh, 1974). Genetic hitchhiking typically results in a reduction of neutral variation in the population through a phenomenon called a selective sweep. The pattern of genetic hitchhiking largely depends on two factors: (i) the frequency of random mating within a population and (ii) the presence of geographic structure (genetically isolated by distance) in the population. The geographic structure of a population might simply delay the propagation of beneficial mutations by limited migration of parasites across different geographic space. Perhaps, the spread of beneficial mutations over a certain geographic distance results in weaker hitchhiking (Barton, 2000).
In Plasmodium falciparum, several genetic studies have investigated genetic hitchhiking around mutated genes thought to confer resistance to antimalarials like pyrimethamine (Nair et al., 2003; Roper et al., 2004), sulphadoxine (Alam et al., 2011; Vinayak et al., 2010) and chloroquine (Chen et al., 2005; Mehlotra et al., 2005; Wootton et al., 2002) . A few other studies have also established that geographic structure might impact the extent of a selective sweep associated with this drug resistance (McCollum et al., 2007; Mu et al., 2005; Pearce et al., 2005). For chloroquine resistance, these studies have found: (i) multiple foci for the origin of chloroquine resistance, one in Asia spreading to Africa, two in South America, one in Papua New Guinea (PNG), and one in the Philippines; (ii) Southeast Asia and Africa share the same mutant allele CVIET (amino acids from 72 to 76 with mutations underlined) in Plasmodium falciparum chloroquine resistant transporter gene (pfcrt), suggesting the role of human migration in conjunction with the spread of chloroquine resistance; and (iii) mutant SVMNT haplotype characterizes the South American type, which is prevalent throughout South America and the Pacific region. More recent studies from India have examined the genetic variation within microsatellite loci flanking pfcrt gene and reported the invasion of the PNG type (SVMNT) throughout India (Lumb et al., 2012; Mixson-Hayden et al., 2010). Expansion of the same mutant type into Pakistan has also been reported (Rawasia et al., 2012). However, studies connecting this genetic footprint with the existing population structure in India have not yet been made. In absence of any knowledge of population structure of P. falciparum in India, the gene flow of the SVMNT mutant across India has not yet been explored, nor has the spread of neutral alleles around the pfcrt gene across India been investigated. Understanding the patterns of a selective sweep in a population may reveal a hidden geographic structure within the population (Kim, Maruki, 2011).
Analyzing the genetic variation within neutral microsatellite loci provides an effective means to determine parasite population structure, can help to begin unraveling any potential links between the epidemiological pattern and genetic structure of P. falciparum (Abdel-Muhsin et al., 2004; Anderson et al., 2000). Studies also inferred that P. falciparum populations are poorly described as a single random mating population model and it is likely to observe distinct clusters of population (Anderson et al., 2000; Mu et al., 2005). Malaria exhibits a great epidemiological diversity in India, in terms of pathogen prevalence and distribution of Plasmodium species, transmission intensity, vector distribution, and ecology of vector breeding sites. The presence of local parasite population structure has been suggested to be a factor of exposure-related immunity, identifiable through differing seroprevalance frequencies to P.falciparum surface antigens, even among two closely spaced localities (Biswas et al., 2008), in contradictory identical genetic structure has been reported between two distant localities i.e, eastern and northeastern India (Joshi et al., 2007). Previous work has shown an association between the genetic diversity within the pfcrt gene and the various transmission intensity of India (Mallick et al., 2012). Moreover, there is limited information available on how the genetic diversity of P. falciparum is structured within the Indian population and it has yet to be established whether or not variability within malaria epidemiological factors across different geographic locations provides a basis for the evolutionary plasticity and local adaptation. However, these diverse epidemiological conditions and limited genetic studies involving surface antigens and drug resistance are impetus/stimulating to suspect a population structure in India.
This study aims to assess the genetic diversity and population structure of P. falciparum subpopulations, which have, over time, sustained different levels of chloroquine selection across India. Testing the correlation between transmission intensity and population structure, and analyzing the footprints of selection on a genomic level, may facilitate a deeper understanding of how drug utilization shapes the structure of P. falciparum in India. This study uses putative neutral microsatellite loci and pfcrt-flanking microsatellite loci assumed to be under chloroquine selection. Population structure analyses and hierarchical F-statistics revealed substantial geographical structure of P. falciparum populations within India and with strong parasite genetic differentiation between high transmission areas (HTA) and low transmission areas (LTA). Here, ultimately the spread of hitchhiking associated with chloroquine resistance across India is highlighted.
