Population genetics and genetic polymorphisms in Plasmodium falciparum parasites from Colombian Pacific region before the introduction of artemisinine compounds

Diego Fernando Echeverri Garcia, Purdue University

Abstract

Malaria is the most important tropical vector-borne disease of humans. Despite the implementation of different initiatives for malaria control and eradication, malaria is still a severe problem for global public health. Plasmodium falciparum have developed resistance to virtually all antimalarials, including artemisinine derivates which are the most potent antimalarials. The antimalarial drug resistance represents a threat for malaria control and eradication programs. As P. falciparum antimalarial resistance emerged and spread from areas of low malaria transmission (i.e., South America and Southeast of Asia), studies of population genetics and characterization of novel polymorphisms, single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) in these areas, may provide new insight to explain mechanisms of antimalarial resistance. However, population of parasites from South America has scarce information in this regard. The present research was conducted to understand the population structure and characterize genomic diversity in Colombian P. falciparum parasites. We conducted a population genetic study using P. falciparum samples collected between 1993 and 2007 from the four provinces of the Colombian Pacific region. A total of 384 SNPs were genotyped in 447 P. falciparum samples to evaluate clonality, relatedness population structure, clonal persistence and linkage disequilibrium (LD). Most infections (81%) contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs), with 32% of MLGs recovered from multiple (2 − 28) independent subjects. We observed extremely low genotypic richness (R=0.42) and long persistence of MLGs through time (median=537 days, range=1 − 2,997 days). There was a high probability (>5%) of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations ( FST=0.084 − 0.279) were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD) decayed more rapidly (r2=0.17 for markers <10 kb>apart) than observed previously in South American samples. In this research, we provided a comprehensive study of population genetics on P. falciparum Colombian parasites which suggests that Colombian parasite populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies. To characterize SNPs and CNVs in parasites from the Colombian Pacific region, a sub-sample of these 447 P. falciparum parasites was selected. Seventeen P. falciparum samples from this region were successfully adapted to culture (each sample represent a different MLG), and whole genome sequenced showing a 24X average coverage. SNPs and CNVs were called using automatic and manual predictions in the 17 Colombian samples and compared against 11 parasites previously sequenced by Welcome Trust Sanger Institute (WTSI). Analyses focused on 138 genes of interest (permeome genes and genes associated with drug resistance). Three datasets of informative SNPs were used in the comparative analyses: 18,649 SNPs called in Purdue University, 5,493 from WTSI and 501 SNPs called in Purdue University located in the coding region of the genes of interest only. Relatedness analyses and the distribution of mutant alleles in the samples suggest important differences between Colombian and non-Colombian parasites; in fact, 3,360 and 213 SNPs from the Colombian samples were unique, when compared with a global catalogue of SNPs from the old world (435,765 high quality and 2,189,263 exploratory SNPs, respectively). Analyses of CNVs using Control-Freec software also revealed 32 unique putative CNVs in the Colombian samples. From the total of 138 genes of interest, 30 shown identical sequences to that 3D7 reference genome. A total of 21/108 polymorphic genes (i.e., their sequences were different to that 3D7 reference genome) were identified to be under predicted directional selection and includes the well know genes associated with drug resistance such as Pfcrt, Pfmdr1, Pfmrp1 and Pfdhfr. These 17 whole genome sequences of P. falciparum from Colombian may be used as reference genomes before the introduction of artemisinine combinatory therapy (ACT) in the country. Parasites from Colombia have a unique and extensive repertoire of SNPs and CNVs when compared with parasites from other regions suggesting high divergence between them. These new polymorphisms are a starting point for genome-wide maps of genetic diversity in parasites from the Americas and will be useful to increase the catalogue of global P. falciparum diversity of MalariaGen. This new set of potential SNPs from low malaria endemic areas may help to improve the design of population genetics and genome-wide association studies (GWAs) studies for traits of interest, including antimalarial drug resistance.

Degree

Ph.D.

Advisors

Hill, Purdue University.

Subject Area

Genetics|Bioinformatics|Parasitology

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