The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients

The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the “REGISTRY” cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Study of cellular and molecular mechanisms of Plasmodium falciparum involved in Artemisinin-based Combination Treatment resistance

Les combinaisons thérapeutiques à base d’artémisinine (ou CTAs) sont une des clés de voûte des stratégies actuelles de lutte contre le paludisme : ces thérapies ont en effet joué un rôle important dans la réduction de l’impact du paludisme au cours de la dernière décennie. Cependant, ces progrès sont aujourd’hui compromis par l’émergence de parasites Plasmodium falciparum résistants aux dérivés de l’artémisinine (ART). Les premiers parasites résistants ont été détectés pour la première fois en 2008 dans l’ouest du Cambodge, puis dans plusieurs pays avoisinants d’Asie du Sud-Est. Le problème de la multirésistance aux antipaludiques s’est récemment aggravé au Cambodge : plusieurs études ont rapporté l’émergence de parasites résistants à la pipéraquine (PPQ), la dernière génération de molécule partenaire utilisée en combinaison avec la dihydroartémisinine, entraînant des taux alarmants d’échecs cliniques. Pour préserver l’efficacité de ces combinaisons, la surveillance et la compréhension de ces deux types de résistance sont cruciales afin d’éviter la dissémination de parasites multirésistants en dehors d’Asie du Sud-Est, et particulièrement en Afrique où les conséquences sanitaires seraient désastreuses. À cette fin, le développement de nouveaux outils est nécessaire pour étudier les mécanismes biologiques et moléculaires impliqués dans la résistance aux CTAs et pour surveiller la distribution géographique des parasites multirésistants. Dans la première partie de cette thèse, axée sur la résistance à l’artémisinine, nous présentons tout d’abord la découverte de mutations au sein du gène K13, marqueur moléculaire pour la résistance à l'ART. Puis nous confirmons leur rôle comme déterminant majeur de cette résistance. Enfin, nous analysons l’étendue de cette résistance au travers d'une cartographie mondiale des polymorphismes de K13. Dans la seconde partie de cette thèse, nous présentons nos travaux portant sur l’émergence de la résistance à la pipéraquine au Cambodge, en confirmant dans un premier temps que des parasites multirésistants à l’ART et à la PPQ circulent désormais dans le pays. Puis en détaillant la mise en point d’un nouveau test phénotypique pour la détection des parasites PPQ-résistants, le Piperaquine Survival Assay ou PSA. Pour finir, nous exposons la découverte de l’amplification du gène PfPM2 comme marqueur moléculaire pour la résistance à la PPQ.Artemisinin-based combination therapies (ACTs) are one of the pillars of the current strategies implemented for fighting malaria. Over the last decade, ACTs have played a major role in decreasing malaria burden. However, this progress is being jeopardized by the emergence of artemisinin-resistant Plasmodium falciparum parasites. Artemisinin (ART) resistance was first detected in western Cambodia in 2008 and has since been observed in neighboring countries in Southeast Asia. The problem of antimalarial drug resistance has recently worsened in Cambodia, with reports of parasites resistant to piperaquine (PPQ), the latest generation of partner drug used in combination with dihydroartemisinin, leading to worrying rates of clinical treatment failure. The monitoring and the comprehension of both types of resistance are crucial to prevent the spread of multi-drug resistant parasites outside Southeast Asia, and particularly to Africa, where the public health consequences would be catastrophic. To this end, new tools are required for studies of the biological and molecular mechanisms underlying resistance to antimalarial drugs and for monitoring the geographic distribution of the resistant parasites.In the first section of this thesis, centered on artemisinin resistance, we first present the discovery of mutations within the K13 gene as a molecular marker for ART resistance. Then we confirm their role as a major determinant of such a resistance. Finally, we analyze the extent of ART resistance through a global mapping of K13 polymorphisms. In the second section, we present our work on the emergence of piperaquine resistance in Cambodia, by initially confirming that multiresistant parasites now circulate in the country. Then we detail the development of a new phenotypical test for the detection of PPQ-resistant parasites, the Piperaquine Survival Assay or PSA. Lastly, we report the discovery of the PfPM2 gene amplification as a candidate molecular marker for PPQ-resistance

Etudes des mécanismes cellulaires et moléculaires de Plasmodium falciparum impliqués dans les résistances aux combinaisons à bases de dérivés de l’artémisinine

Artemisinin-based combination therapies (ACTs) are one of the pillars of the current strategies implemented for fighting malaria. Over the last decade, ACTs have played a major role in decreasing malaria burden. However, this progress is being jeopardized by the emergence of artemisinin-resistant Plasmodium falciparum parasites. Artemisinin (ART) resistance was first detected in western Cambodia in 2008 and has since been observed in neighboring countries in Southeast Asia. The problem of antimalarial drug resistance has recently worsened in Cambodia, with reports of parasites resistant to piperaquine (PPQ), the latest generation of partner drug used in combination with dihydroartemisinin, leading to worrying rates of clinical treatment failure. The monitoring and the comprehension of both types of resistance are crucial to prevent the spread of multi-drug resistant parasites outside Southeast Asia, and particularly to Africa, where the public health consequences would be catastrophic. To this end, new tools are required for studies of the biological and molecular mechanisms underlying resistance to antimalarial drugs and for monitoring the geographic distribution of the resistant parasites.In the first section of this thesis, centered on artemisinin resistance, we first present the discovery of mutations within the K13 gene as a molecular marker for ART resistance. Then we confirm their role as a major determinant of such a resistance. Finally, we analyze the extent of ART resistance through a global mapping of K13 polymorphisms. In the second section, we present our work on the emergence of piperaquine resistance in Cambodia, by initially confirming that multiresistant parasites now circulate in the country. Then we detail the development of a new phenotypical test for the detection of PPQ-resistant parasites, the Piperaquine Survival Assay or PSA. Lastly, we report the discovery of the PfPM2 gene amplification as a candidate molecular marker for PPQ-resistance.Les combinaisons thérapeutiques à base d’artémisinine (ou CTAs) sont une des clés de voûte des stratégies actuelles de lutte contre le paludisme : ces thérapies ont en effet joué un rôle important dans la réduction de l’impact du paludisme au cours de la dernière décennie. Cependant, ces progrès sont aujourd’hui compromis par l’émergence de parasites Plasmodium falciparum résistants aux dérivés de l’artémisinine (ART). Les premiers parasites résistants ont été détectés pour la première fois en 2008 dans l’ouest du Cambodge, puis dans plusieurs pays avoisinants d’Asie du Sud-Est. Le problème de la multirésistance aux antipaludiques s’est récemment aggravé au Cambodge : plusieurs études ont rapporté l’émergence de parasites résistants à la pipéraquine (PPQ), la dernière génération de molécule partenaire utilisée en combinaison avec la dihydroartémisinine, entraînant des taux alarmants d’échecs cliniques. Pour préserver l’efficacité de ces combinaisons, la surveillance et la compréhension de ces deux types de résistance sont cruciales afin d’éviter la dissémination de parasites multirésistants en dehors d’Asie du Sud-Est, et particulièrement en Afrique où les conséquences sanitaires seraient désastreuses. À cette fin, le développement de nouveaux outils est nécessaire pour étudier les mécanismes biologiques et moléculaires impliqués dans la résistance aux CTAs et pour surveiller la distribution géographique des parasites multirésistants. Dans la première partie de cette thèse, axée sur la résistance à l’artémisinine, nous présentons tout d’abord la découverte de mutations au sein du gène K13, marqueur moléculaire pour la résistance à l'ART. Puis nous confirmons leur rôle comme déterminant majeur de cette résistance. Enfin, nous analysons l’étendue de cette résistance au travers d'une cartographie mondiale des polymorphismes de K13. Dans la seconde partie de cette thèse, nous présentons nos travaux portant sur l’émergence de la résistance à la pipéraquine au Cambodge, en confirmant dans un premier temps que des parasites multirésistants à l’ART et à la PPQ circulent désormais dans le pays. Puis en détaillant la mise en point d’un nouveau test phénotypique pour la détection des parasites PPQ-résistants, le Piperaquine Survival Assay ou PSA. Pour finir, nous exposons la découverte de l’amplification du gène PfPM2 comme marqueur moléculaire pour la résistance à la PPQ

Plasmodium copy number variation scan: gene copy numbers evaluation in haploid genomes.

International audienceIn eukaryotic genomes, deletion or amplification rates have been estimated to be a thousand more frequent than single nucleotide variation. In Plasmodium falciparum, relatively few transcription factors have been identified, and the regulation of transcription is seemingly largely influenced by gene amplification events. Thus copy number variation (CNV) is a major mechanism enabling parasite genomes to adapt to new environmental changes. Currently, the detection of CNVs is based on quantitative PCR (qPCR), which is significantly limited by the relatively small number of genes that can be analysed at any one time. Technological advances that facilitate whole-genome sequencing, such as next generation sequencing (NGS) enable deeper analyses of the genomic variation to be performed. Because the characteristics of Plasmodium CNVs need special consideration in algorithms and strategies for which classical CNV detection programs are not suited a dedicated algorithm to detect CNVs across the entire exome of P. falciparum was developed. This algorithm is based on a custom read depth strategy through NGS data and called PlasmoCNVScan. The analysis of CNV identification on three genes known to have different levels of amplification and which are located either in the nuclear, apicoplast or mitochondrial genomes is presented. The results are correlated with the qPCR experiments, usually used for identification of locus specific amplification/deletion. This tool will facilitate the study of P. falciparum genomic adaptation in response to ecological changes: drug pressure, decreased transmission, reduction of the parasite population size (transition to pre-elimination endemic area)

Evidence of plasmodium falciparum Malaria multidrug resistance to artemisinin and piperaquine in Western Cambodia : dihydroartemisinin-piperaquine open-label multicenter clinical assessment

Western Cambodia is recognized as the epicentre of Plasmodium falciparum multidrug resistance. Recent reports of dihydroartemisinin-piperaquine efficacy, the latest generation of ACTs recommended by the WHO, prompted further investigations. Clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 piperaquine plasma concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene and copy number of the mdr-1 gene (ACTRN12614000344695). 425 patients were recruited in 2011-2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) compared to eastern Cambodia (2.5%, p<10-3). Day 7 PP plasma concentrations and PP median IC50 were independent of treatment outcomes, contrarily to mefloquine median IC50 which was found lower in recrudescent patient isolates (18.7 vs. 39.7 nM, p=0.005). The most significant risk factor associated with DHA-PP treatment failure was patients infected by parasites carrying the K13 mutant allele (OR=17.5, 95% CI: 1-308, p=0.04). Our data support evidence of falciparum PP resistance in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies are needed urgently and must be tested such as the use of triple ACT