Adaptive Copy Number Evolution in Malaria Parasites

Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2–11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation

Plotting haplotype-specific linkage disequilibrium patterns by extended haplotype homozygosity

Summary: Association studies may request more details of a specific haplotype. Haplotype-specific decay of linkage disequilibrium is such a crucial and versatile characteristic. It may be used, e.g. to search for signals of natural selection in a risk haplotype. Here, we present a web-based tool to explore the relationship between population frequency and extended linkage disequilibrium measured as haplotype homozygosity of observed haplotypes within a specified candidate region

Contraintes sélectives et adaptation chez l'homme : histoire évolutive des senseurs microbiens

Les Pattern-recognition receptors (PRRs) jouent un rôle clé dans la reconnaissance des microbes par l'hôte. La détection de sélection naturelle sur les gènes codant les PRRs permet de distinguer ceux qui sont essentiels de ceux qui montrent des fonctions plus redondantes. Nous nous intéressons ici à deux familles de PRRs cytosoliques humains: les NOD-like receptors (NLRs) qui reconnaissent a priori des bactéries et des signaux de danger cellulaire et les RIG-I-like receptors (RLRs) détectant les ARN provenant majoritairement de virus. Nous avons séquencé leurs 24 gènes dans un panel d'individus représentatif de la population mondiale. Nous avons d'une part mis en évidence que la majorité des NALPs, l'une des sous-familles de NLRs, était très contrainte, montrant un déficit en mutations non-synonymes. Cela suggère qu'ils ont joué un rôle essentiel et pourraient être impliqués dans des maladies graves ; ils devraient donc être étudiés en priorité dans une perspective médicale. Au contraire, la plupart des NOD/IPAF, autre sous-famille des NLRs, ainsi que les 3 RLRs semblent être impliqués dans des fonctions moins importantes ou plus redondantes, accumulant un grand nombre de changements dans la protéine. Ces données, ajoutées à celles des Toll-like-receptors (TLRs), nous ont permis de proposer un modèle hiérarchique, traduisant les contributions relatives des différentes familles de senseurs microbiens à notre survie. D'autre part, nous avons identifié certains gènes (NLRP1 en particulier) et variants comme étant sous sélection positive : ceux-ci pourraient expliquer les différences de résistance qui existent actuellement face à certaines maladies infectieuses.Pattern-recognition receptors (PRRs) constitute key actors in the recognition of microbes by the host. Detecting how natural selection has targeted their genes represents a useful tool to delineate those that are essential with respect to those that are more redundant in immune responses. Here, we studied the levels of naturally-occurring variation of two major families of human intracellular PRRs, the NOD-like Receptors (NLRs), mainly sensing bacteria and cellular danger signals and the RIG-I-like receptors (RLRs), essentially involved in the sensing of viral RNA. To this aim, we sequenced their 24 genes in a panel representative of worldwide diversity. First, we showed that most NALPs, a subfamily of NLRs, were strongly constrained, exhibiting a deficit in non-synonymous mutations. This suggests that they have played a major role in our survival and could be involved in severe diseases; their study should be therefore prioritized from a medical perspective. By contrast, most NOD/IPAF, another subfamily of NLRs, and the 3 RLRs seem to have less important or redundant functions, accumulating a high level of amino acid changes. This data, together with those of the Toll-like-receptors (TLRs), allowed to propose a hierarchical model, highlighting the relative contributions of the different families of microbial sensors to our survival. Furthermore, we identified some genes (NLRP1 in particular) and variants as under positive selection: they could explain some of our actual differences of resistance to infectious diseases

Genetic characterization of nucleoside analogue transporters ABCC4 and ABCC5 gene loci

Ph.DDOCTOR OF PHILOSOPH

The origin, genetics and dispersal of drug-resistant Plasmodium falciparum in Kenya

Three sets of molecular markes were used to investigate the population genetics of three populations of Plasmodium falciparum from Kenya; Mwea (low transmission), Tiwi (moderate transmission) and Bondo (high transmission). One set of markers codes for polymorphic antigens while the other two are microsatellite markers; one set located in non coding regions of the genome while the other set is located in the regions flanking two genes whose products are targets of the antimalarial drug sulphadoxine/pyrimethamine (SP). A comparison of the effectiveness of antigen-coding and the unlinked microsatellite loci in differentiating recrudescence from reinfection revealed that both sets of markers are equally effective. The microsatellite loci however, revealed more alleles per population than the antigen-coding loci possibly due to their different mutation rates. An analysis of the three populations using the neutral microsatellite loci revealed high levels of diversity, lack of linkage disequilibrium and virtually no population substructuring (FST<0.008) in the Kenyan P. falciparum populations even with the geographical areas being as much as 800 km apart. This indicates a lot of gene flow among these populations a factor that can only be explained by movement of people between the areas studied. An analysis of the same samples from the three areas at the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) gene loci that code for targets of the antimalarial drug SP revealed high prevalence of the multiply substituted alleles associated with SP resistance in the three regions. An analysis of ~17 kb regions flanking both sides dhfr reveal a strong selective sweep of the 108N/51I/59R triple mutation alleles associated with pyrimethamine resistance. The work presented also demonstrates that alleles of the dhfr gene, especially the triple mutant allele, isolated from the three different areas are closely related to one another and probably share a common and very recent ancestor. Most notable is the finding that dhfr triple mutants seem to be imported into the country through immigration from elsewhere. An equivalent region flanking the dhps gene also revealed a strong selective sweep of the 437G/540E double mutation allele associated with sulphadoxine resistance in two of the three sites. However, double mutation dhps alleles from Mwea revealed no selection at all. While the three populations reveal no geographic substructuring using the results of the unlinked microsatellite loci, they seem to be highly structured in their drug resistance patterns. While it would be expected that these populations would have the same prevalence of drug resistance mutations (due to the apparent panmixia), the Mwea population appears quite different in regard to selection for drug resistance-associted alleles. This is possibly due to the diet, other drug interactions or the hosts' genetics in this area. A simplistic model on the rate of spread of drug resistance in the three populations reveals that the selection for drug resistance alleles is faster in the lower transmission area of Mwea (selection coefficient, s = 0.26) and slowest in Bondo (s = 0.10) indicating selection for drug resistant alleles is favoured by low transmission. These observations have implications for malaria drug resistance surveillance programs due to the fact that if treatment failure spreads faster in low transmission areas where almost all the population has low immunity, malaria epidemics are bound to occur resulting in huge morbidity and mortality

Behavioural and evolutionary responses of Anopheles gambiae S.S. to bednets and pyrethoids

Vector control by insecticides is fundamental to the global strategy foOr malaria prevention. Insecticide treated bednets (ITNs) are central to this approach but with only pyrethroids available for ITN use, resistance to this class of insecticides is a major threat. Knock-down resistance to pyrethroids is strongly associated with the presence of a single amino acid substitution (kdr) in the voltage-gated sodium channel in many insects, including African malaria vectors. With the long-term aim of extending the effective lifespan of pyrethroids and ITNs in particular, this study investigated knock-down resistance and behaviour at the bednet interface in Anopheles gambiae s.s. Specifically, the molecular studies aimed to improve the reliability of kdr detection methods, investigate the origins and spread of kdr mutations and determine the strength of the selection pressures acting on them; the behavioural studies investigated non-contact repellency by pyrethroids and arrival patterns ofhost-seeking mosquitoes at a human-baited bednet. Detection of kdr alleles is important in order to monitor the spread of resistance in the field, and a novel kdr detection method (Hot Oligonucleotide Ligation Assay or HOLA) was developed to improve on existing techniques. This method differentiated homozygous and heterozygous individuals for both kdr alleles and was transferred to a resource poor laboratory. HOLA was also used to confirm the first occurrence ofa phenylalanine heterozygous specimen. population genetic based approach was usedserine/ to investigate the relative age ofthe kdr mutations and selection pressures acting upon them. Sequencing ofthe voltage-gated sodium channel gene allowed the identification of 29 novel single nucleotide polymorphisms that were used to screen populations of field collected An. gambiae s.s. from seven locations in Africa. Analysis of the extended haplotypes revealed the signature of the selective sweeps associated with the kdr alleles, and suggested that the serine kdr mutation found in Kenya pre-dated the kdr mutations in West Africa, possibly the result of selection by the historic use of DDT rather than the more recent use of pyrethroids. Data indicated that the spread of the serine mutation in Gabon was recent, possibly due to a selective advantage conferred by co-expression with the phenylalanine kdr mutation and, confirming published data, that it had probably arisen at least twice through novel mutation events. The phenylalanine mutation in West and Central Africa is likely to have been the result of at least two separate mutation events, both ofwhich have been subjected to a strong selective sweep

Analyses of Signatures of Selection in the Bovine Genome

This thesis presents a novel method called meta-selection-scores (MSS) to construct a meta-assembly of genome-wide signatures of selection within breeds (n = 24) and across geographical archetypes (European, African, Zebu and composite). The meta-assembly and review of published studies highlight the historical selection events, the role of underlying genes for various traits, limitations of the available bovine genomic resources and implications of using different methodologies. This study also developed another new method called composite selection signals (CSS) – to improve the power of selection signature scans – in which multiple pieces of evidence for selection derived from the rank distribution of individual tests are combined in a single score. CSS has shown improved power by localizing the genomic regions under selection for major traits in multi-breed panels of cattle and sheep. CSS was also used to detect regions associated with the complex trait of bovine stature and implicated 12 (nine were novel) regions harbouring multiple candidate genes in contrasting cohorts within the European and African cattle. Without phenotypic records on individual animals, the CSS method provides a firsthand scan of the genome to detect putative regions associated with complex traits. The genome-wide CSS scans for individual breeds (n = 60) of cattle using previously available and new genotypes from 50K SNP chip assay identified 177 genomic regions under selection. Finally, an ultra-high density dataset (1.6 million SNPs) was investigated with CSS for Angus and Holstein. A high (8 of 9) reproducibility of the 50K SNPs based CSS regions and up to 14 fold additional genomic regions under selection were detected using 1.6 million SNPs. Overall, this study provides a detailed investigation about the core traits influenced by the historical selection events in worldwide cattle breeds and presents novel insights about the hotspots of positive selection in the bovine genome