Microsatellite genotyping and genome-wide single nucleotide polymorphism-based indices of Plasmodium falciparum diversity within clinical infections.

BACKGROUND: In regions where malaria is endemic, individuals are often infected with multiple distinct parasite genotypes, a situation that may impact on evolution of parasite virulence and drug resistance. Most approaches to studying genotypic diversity have involved analysis of a modest number of polymorphic loci, although whole genome sequencing enables a broader characterisation of samples. METHODS: PCR-based microsatellite typing of a panel of ten loci was performed on Plasmodium falciparum in 95 clinical isolates from a highly endemic area in the Republic of Guinea, to characterize within-isolate genetic diversity. Separately, single nucleotide polymorphism (SNP) data from genome-wide short-read sequences of the same samples were used to derive within-isolate fixation indices (F ws), an inverse measure of diversity within each isolate compared to overall local genetic diversity. The latter indices were compared with the microsatellite results, and also with indices derived by randomly sampling modest numbers of SNPs. RESULTS: As expected, the number of microsatellite loci with more than one allele in each isolate was highly significantly inversely correlated with the genome-wide F ws fixation index (r = -0.88, P < 0.001). However, the microsatellite analysis revealed that most isolates contained mixed genotypes, even those that had no detectable genome sequence heterogeneity. Random sampling of different numbers of SNPs showed that an F ws index derived from ten or more SNPs with minor allele frequencies of >10 % had high correlation (r > 0.90) with the index derived using all SNPs. CONCLUSIONS: Different types of data give highly correlated indices of within-infection diversity, although PCR-based analysis detects low-level minority genotypes not apparent in bulk sequence analysis. When whole-genome data are not obtainable, quantitative assay of ten or more SNPs can yield a reasonably accurate estimate of the within-infection fixation index (F ws).

Genome-wide analysis of selection on the malaria parasite Plasmodium falciparum in West African populations of differing infection endemicity.

Locally varying selection on pathogens may be due to differences in drug pressure, host immunity, transmission opportunities between hosts, or the intensity of between-genotype competition within hosts. Highly recombining populations of the human malaria parasite Plasmodium falciparum throughout West Africa are closely related, as gene flow is relatively unrestricted in this endemic region, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Genome-wide analysis of sequence variation was undertaken on a sample of 100 P. falciparum clinical isolates from a highly endemic region of the Republic of Guinea where transmission occurs for most of each year and compared with data from 52 clinical isolates from a previously sampled population from The Gambia, where there is relatively limited seasonal malaria transmission. Paired-end short-read sequences were mapped against the 3D7 P. falciparum reference genome sequence, and data on 136,144 single nucleotide polymorphisms (SNPs) were obtained. Within-population analyses identifying loci showing evidence of recent positive directional selection and balancing selection confirm that antimalarial drugs and host immunity have been major selective agents. Many of the signatures of recent directional selection reflected by standardized integrated haplotype scores were population specific, including differences at drug resistance loci due to historically different antimalarial use between the countries. In contrast, both populations showed a similar set of loci likely to be under balancing selection as indicated by very high Tajima's D values, including a significant overrepresentation of genes expressed at the merozoite stage that invades erythrocytes and several previously validated targets of acquired immunity. Between-population FST analysis identified exceptional differentiation of allele frequencies at a small number of loci, most markedly for five SNPs covering a 15-kb region within and flanking the gdv1 gene that regulates the early stages of gametocyte development, which is likely related to the extreme differences in mosquito vector abundance and seasonality that determine the transmission opportunities for the sexual stage of the parasite.

Population genetic structure of Plasmodium falciparum across a region of diverse endemicity in West Africa.

BACKGROUND: Malaria parasite population genetic structure varies among areas of differing endemicity, but this has not been systematically studied across Plasmodium falciparum populations in Africa where most infections occur. METHODS: Ten polymorphic P. falciparum microsatellite loci were genotyped in 268 infections from eight locations in four West African countries (Republic of Guinea, Guinea Bissau, The Gambia and Senegal), spanning a highly endemic forested region in the south to a low endemic Sahelian region in the north. Analysis was performed on proportions of mixed genotype infections, genotypic diversity among isolates, multilocus standardized index of association, and inter-population differentiation. RESULTS: Each location had similar levels of pairwise genotypic diversity among isolates, although there were many more mixed parasite genotype infections in the south. Apart from a few isolates that were virtually identical, the multilocus index of association was not significant in any population. Genetic differentiation between populations was low (most pairwise F(ST) values < 0.03), and an overall test for isolation by distance was not significant. CONCLUSIONS: Although proportions of mixed genotype infections varied with endemicity as expected, population genetic structure was similar across the diverse sites. Very substantial reduction in transmission would be needed to cause fragmented or epidemic sub-structure in this region.

High prevalence of hemoglobin disorders and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Republic of Guinea (West Africa).

Reliable and accurate epidemiological data is a prerequisite for a cost effective screening program for inherited disorders, which however, is lacking in a number of developing countries. Here we report the first detailed population study in the Republic of Guinea, a sub-Saharan West African country, designed to assess the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies, including screening for thalassemia. Peripheral blood samples from 187 Guinean adults were screened for hemoglobin (Hb) variants by standard hematological methods. One hundred and ten samples from males were screened for G6PD deficiency by the fluorescent spot test. Molecular analysis was performed for the most common α-thalassemia (α-thal) deletions, ß-globin gene mutations, G6PD variants B (376A), A (376G), A- (376G/202A) and Betica (376G/968C), using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) or sequencing. Of the 187 subjects screened, 36 were heterozygous for Hb S [ß6(A3)Glu→Val, GAG>GTG] (allele frequency 9.62%). Sixty-four subjects were heterozygous and seven were homozygous for the -α(3.7) kb deletion (allele frequency 20.85%). ß-Thalassemia alleles were detected in five subjects, four with the -29 (A>G) mutation (allele frequency 1.07%) and one with codon 15 (TGG>TAG) (allele frequency 0.96%). The G6PD A- and G6PD Betica deficient variants were highly prevalent with a frequency of 5.7 and 3.3%, respectively. While we did not test for ferritin levels or α(0)-thal, four females (5.2%) had red cell indices strongly suggestive of iron deficient anemia: Hb <9.7 g/dL; MCH <19.3 pg; MCV <68.2; MCHC <31.6 g/dl; RDW >19.8%. Our results are consistent with high frequency of alleles such as Hb S, α-thal and G6PD deficient alleles associated with malaria resistance. Finding a 9.6% Hb S allele frequency supports the notion for a proficient neonatal screening to identify the sickle cell patients, who might benefit from early prophylactic treatment for infections. The incidence of significant iron deficient anemia in women is lower than expected in an under developed country.

SLC40A1 Q248H allele frequencies and associated SLC40A1 haplotypes in three West African population samples.

BACKGROUND: Ferroportin is a transmembrane protein responsible for iron export from enterocytes and macrophages. Mutation c.744G → T (Q248H), located in exon 6 of the ferroportin gene SLC40A1, is found as a polymorphism in populations of African origin. This mutation has been extensively analysed in African-Americans, but poorly studied in native African populations. AIM: To increase information about Q248H mutation frequency in native sub-Saharan populations examining three West African populations. SUBJECTS AND METHODS: Samples from S. Tomé e Príncipe (n = 115), Angola (n = 156) and Republic of Guinea (n = 170) were analysed for Q248H mutation and for two polymorphisms, IVS1( - 24)G → C and microsatellite (CGG)(n), using standard molecular methodology. RESULTS: The estimated frequencies of Q248H allele were 2.2% in S. Tomé e Príncipe, 3.5% in Angola and 4.1% in Republic of Guinea. Analysis of polymorphisms IVS1( - 24)G → C and (CGG)(n) showed mutation allele c.744T to be strongly associated with haplotype IVS1( - 24)G/(CGG)(7). CONCLUSIONS: This study confirmed the presence of Q248H mutation at polymorphic frequencies in three native sub-Saharan populations. Analysis of two additional markers in the same gene support a single origin of the mutant allele c.744T in the haplotype background IVS1( - 24)G/(CGG)(7).

Breakdown in the process of incipient speciation in Anopheles gambiae.

Understanding genetic causes and effects of speciation in sympatric populations of sexually reproducing eukaryotes is challenging, controversial, and of practical importance for controlling rapidly evolving pests and pathogens. The major African malaria vector mosquito Anopheles gambiae sensu stricto (s.s.) is considered to contain two incipient species with strong reproductive isolation, hybrids between the M and S molecular forms being very rare. Following recent observations of higher proportions of hybrid forms at a few sites in West Africa, we conducted new surveys of 12 sites in four contiguous countries (The Gambia, Senegal, Guinea-Bissau, and Republic of Guinea). Identification and genotyping of 3499 A. gambiae s.s. revealed high frequencies of M/S hybrid forms at each site, ranging from 5 to 42%, and a large spectrum of inbreeding coefficient values from 0.11 to 0.76, spanning most of the range expected between the alternative extremes of panmixia and assortative mating. Year-round sampling over 2 years at one of the sites in The Gambia showed that M/S hybrid forms had similar relative frequencies throughout periods of marked seasonal variation in mosquito breeding and abundance. Genome-wide scans with an Affymetrix high-density single-nucleotide polymorphism (SNP) microarray enabled replicate comparisons of pools of different molecular forms, in three separate populations. These showed strong differentiation between M and S forms only in the pericentromeric region of the X chromosome that contains the molecular form-specific marker locus, with only a few other loci showing minor differences. In the X chromosome, the M/S hybrid forms were more differentiated from M than from S forms, supporting a hypothesis of asymmetric introgression and backcrossing.