Background: National Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures. Methods: Samples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs. Results: GenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs. Conclusions: GenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community. Funding: The GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Host genetic factors can confer resistance against malaria, raising the question of whether this has led to evolutionary adaptation of parasite populations. In this study we investigated the correlation between host and parasite genetic variation in 4,171 Gambian and Kenya children ascertained with severe malaria due to Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and variation in three regions of the parasite genome, including nonsynonymous variants in the acyl-CoA synthetase family member PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The HbS-associated parasite alleles are in strong linkage disequilibrium and have frequencies which covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome, and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Host genetic factors can confer resistance against malaria1, raising the question of whether this has led to evolutionary adaptation of parasite populations. Here we searched for association between candidate host and parasite genetic variants in 3,346 Gambian and Kenyan children with severe malaria caused by Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and three regions of the parasite genome, which is not explained by population structure or other covariates, and which is replicated in additional samples. The HbS-associated alleles include nonsynonymous variants in the gene for the acyl-CoA synthetase family member2-4 PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The alleles are in strong linkage disequilibrium and have frequencies that covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii . We analysed whole genomes of 1,142 individual mosquitoes sampled from the wild in 13 African countries, and a further 234 individuals comprising parents and progeny of 11 lab crosses. The data resource includes high confidence single nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We used these data to analyse genetic population structure, and characterise genetic diversity within and between populations. We also illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance, and for accelerating research and development of new vector control tools.

We conducted a genome-wide association study of host resistance to severe Plasmodium falciparum malaria in over 17,000 individuals from 11 malaria-endemic countries, undertaking a wide ranging analysis which identifies five replicable associations with genome-wide levels of evidence. Our findings include a newly implicated variant on chromosome 6 associated with risk of cerebral malaria, and the discovery of an erythroid-specific transcription start site underlying the association in ATP2B4 . Previously reported HLA associations cannot be replicated in this dataset. We estimate substantial heritability of severe malaria ( h 2 ~ 23%), of which around 10% is explained by the currently identified associations. Our dataset will provide a major building block for future research on the genetic determinants of disease in these diverse human populations.

Background Antimalarial failure is rapidly spreading across parts of Southeast Asia where dihydroartemisinin-piperaquine (DHA-PPQ) is used as first line treatment. The first published reports came from western Cambodia in 2013. Here we analyse genetic changes in the Plasmodium falciparum population of western Cambodia in the six years prior to that. Methods We analysed genome sequence data on 1492 P. falciparum samples from Southeast Asia, including 464 collected in western Cambodia between 2007 and 2013. Different epidemiological origins of resistance were identified by haplotypic analysis of the kelch13 artemisinin resistance locus and the plasmepsin 2-3 piperaquine resistance locus. Findings We identified over 30 independent origins of artemisinin resistance, of which the KEL 1 lineage accounted for 91% of DHA-PPQ-resistant parasites. In 2008, KEL 1 combined with PLA 1, the major lineage associated with piperaquine resistance. By 2012, the KEL 1/ PLA 1 co-lineage had reached over 60% frequency in western Cambodia and had spread to northern Cambodia. Interpretation The KEL 1/ PLA 1 co-lineage emerged in the same year that DHA-PPQ became the first line antimalarial drug in western Cambodia and spread aggressively thereafter, displacing other artemisinin-resistant parasite lineages. These findings have significant implications for management of the global health risk associated with the current outbreak. Funding Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, UK Department for International Development, and Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health.

The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

Background As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond.Methods We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC(50)s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin piperaquine treatment.Findings Piperaquine IC(50)s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC(50)s: 20.0 nmol/L [IQR 13.7-29.0] vs 39.2 nmol/L [32.8-48.1] for Ratanakiri, 19.3 nmol/L [15.1-26.2] vs 66.2 nmol/L [49.9-83.0] for Preah Vihear, and 19.6 nmol/L [11.9-33.9] vs 81.1 nmol/L [61.3-113.1] for Pursat; all p <= 10(-3); Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC(50)s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdrl gene on chromosome 5 and a novel amplification of the plasmepsin 2 and plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC(50)s. After adjusting for covariates, both exo-E415G and plasmepsin 2-3 markers significantly associate (p=3.0 x10(-8) and p=1.7 x10(-7), respectively) with decreased treatment efficacy (survival rates 0.38 [95% CI 0.25-0.51] and 0.41 [0.28-0.53], respectively).Interpretation The exo-E415G SNP and plasmepsin 2-3 amplification are markers of piperaquine resistance and dihydroartemisinin piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since plasmepsins are involved in the parasite's haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, plasmepsin 2-3 amplification probably mediates piperaquine resistance. Copyright (C) The Author(s). Published by Elsevier Ltd.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6-kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003-2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms.

Artemisinin resistant Plasmodium falciparum is advancing across Southeast Asia in a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of standing variation that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

Background Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis.Aims and methods To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing.Results and conclusions We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype-phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes.

The molecular basis of cytochrome c oxidase (COX, complex IV) deficiency remains genetically undetermined in many cases. Homozygosity mapping and whole-exome sequencing were performed in a consanguineous pedigree with isolated COX deficiency linked to a Leigh syndrome neurological phenotype. Unexpectedly, affected individuals harbored homozygous splice donor site mutations in NDUFA4, a gene previously assigned to encode a mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase) subunit. Western blot analysis of denaturing gels and immunocytochemistry revealed undetectable steady-state NDUFA4 protein levels, indicating that the mutation causes a loss-of-function effect in the homozygous state. Analysis of one- and two-dimensional blue-native polyacrylamide gels confirmed an interaction between NDUFA4 and the COX enzyme complex in control muscle, whereas the COX enzyme complex without NDUFA4 was detectable with no abnormal subassemblies in patient muscle. These observations support recent work in cell lines suggesting that NDUFA4 is an additional COX subunit and demonstrate that NDUFA4 mutations cause human disease. Our findings support reassignment of the NDUFA4 protein to complex IV and suggest that patients with unexplained COX deficiency should be screened for NDUFA4 mutations.

IMPORTANCE Isolated cytochrome-c oxidase (COX) deficiency is one of the most frequent respiratory chain defects seen in human mitochondrial disease. Typically, patients present with severe neonatal multisystem disease and have an early fatal outcome. We describe an adult patient with isolated COX deficiency associated with a relatively mild clinical phenotype comprising myopathy; demyelinating neuropathy; premature ovarian failure; short stature; hearing loss; pigmentary maculopathy; and renal tubular dysfunction.OBSERVATIONS Whole-exome sequencing detected 1 known pathogenic and 1 novel COX10 mutation: c.1007A>T; p.Asp336Val, previously associated with fatal infantile COX deficiency, and c.1015C>T; p.Arg339Trp. Muscle COX holoenzyme and subassemblies were undetectable on immunoblots of blue-native gels, whereas denaturing gels and immunocytochemistry showed reduced core subunit MTCO1. Heme absorption spectra revealed low heme aa(3) compatible with heme A: farnesyltransferase deficiency due to COX10 dysfunction. Both mutations demonstrated respiratory deficiency in yeast, confirming pathogenicity. A COX10 protein model was used to predict the structural consequences of the novel Arg339Trp and all previously reported substitutions.CONCLUSIONS AND RELEVANCE These findings establish that COX10 mutations cause adult mitochondrial disease. Nuclear modifiers, epigenetic phenomenon, and/or environmental factors may influence the disease phenotype caused by reduced COX activity and contribute to the variable clinical severity related to COX10 dysfunction.