Etude génétique et fonctionnelle de l’ataxie spastique autosomique récessive de Charlevoix-Saguenay (ARSACS)

ARSACS is a recessive autosomal neurodegenerative condition characterized by cerebellar ataxia, spastic paraplegia and demyelinating sensitivo-motor polyneuropathy. ARSACS is caused by mutations in the SACS gene identified in 2000. Since then, cases have been reported worldwide. SACS encodes sacsin, a protein of still unknown function in spite of the description of numerous protein domains and of a recent focus on a potential implication in the regulation of mitochondrial physiology. Aims of this thesis were to identify new mutations in a large population of ataxic patients and then to functionally analyze their cellular effects in the mitochondrial compartment. We identified 2 variants in SACS in 10% of analyzed cases collected through the international SPATAX network. We also confirmed mutations in SACS in 5 patients identified using next-generation sequencing in the ATXAIC project, and studied a last case harbouring a large deletion encompassing the entire SACS gene identified by pangenomic CGH-array. For functional analyses, primary cultures of fibroblasts were obtained in 11 patients. A drastic and recurrent alteration of the mitochondrial network was observed in all patients except one. These anomalies seem very useful for the diagnosis of ARSACS when molecular results are difficult to interpret (missense variants). We therefore propose a grading diagnostic definition using clinical, genetic and cellular criteria for ARSACS. Finally, we started to study the mechanistic of the mitochondrial function of sacsin. Our preliminary data may suggest a potential role at the mitochondrial turn over level.ARSACS est une maladie neurodégénérative autosomique récessive caractérisée par une ataxie cérébelleuse, une paraplégie spastique et une polyneuropathie sensitivo-motrice démyélinisante. Le gène SACS, responsable de la maladie, a été identifié en 2000. Depuis, de nombreux cas ont été décrits dans le monde entier. Le gène SACS code pour la sacsine, dont la fonction reste inconnue malgré l’identification de nombreux domaines protéiques et surtout la description récente d’un rôle dans la physiologie mitochondriale. Les objectifs de ma thèse étaient d’identifier des mutations dans SACS au sein d’une large cohorte de patients atteints d’ataxie grâce à plusieurs collaborations puis d’étudier leurs effets au niveau du compartiment mitochondrial en utilisant essentiellement des cellules de malades. Nous avons pu identifier 2 variants de SACS dans 10% des cas analysés recrutés via le réseau international SPATAX. Nous avons parallèlement confirmé les mutations de SACS identifiées par séquençage haut-débit chez 5 autres patients via le PHRC ATAXIC et analysé un dernier cas porteur d’une délétion complète de SACS découverte par CGH-array pangénomique. Des cultures primaires de fibroblastes ont été obtenues chez 11 patients pour l’analyse fonctionnelle. Une altération de la morphologie des mitochondries a été observée chez tous les patients sauf un. Ces anomalies du réseau mitochondrial semblent très utiles pour cheminer vers le diagnostic d’ARSACS qui peut s’avérer complexe dans les situations non exceptionnelles où les résultats moléculaires sont délicats à interpréter (mutations faux-sens). Nous proposons une définition graduelle d’ARSACS reposant sur des critères cliniques, génétiques et cellulaires intégrant ces anomalies du réseau. Enfin, nous avons débuté l’exploration des mécanismes impliqués dans cette altération de la dynamique du réseau mitochondrial. Nos résultats préliminaires font évoquer une action potentielle de la sacsine au niveau du renouvellement des mitochondries

Genetic and functional studies of autosomique recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

ARSACS est une maladie neurodégénérative autosomique récessive caractérisée par une ataxie cérébelleuse, une paraplégie spastique et une polyneuropathie sensitivo-motrice démyélinisante. Le gène SACS, responsable de la maladie, a été identifié en 2000. Depuis, de nombreux cas ont été décrits dans le monde entier. Le gène SACS code pour la sacsine, dont la fonction reste inconnue malgré l’identification de nombreux domaines protéiques et surtout la description récente d’un rôle dans la physiologie mitochondriale. Les objectifs de ma thèse étaient d’identifier des mutations dans SACS au sein d’une large cohorte de patients atteints d’ataxie grâce à plusieurs collaborations puis d’étudier leurs effets au niveau du compartiment mitochondrial en utilisant essentiellement des cellules de malades. Nous avons pu identifier 2 variants de SACS dans 10% des cas analysés recrutés via le réseau international SPATAX. Nous avons parallèlement confirmé les mutations de SACS identifiées par séquençage haut-débit chez 5 autres patients via le PHRC ATAXIC et analysé un dernier cas porteur d’une délétion complète de SACS découverte par CGH-array pangénomique. Des cultures primaires de fibroblastes ont été obtenues chez 11 patients pour l’analyse fonctionnelle. Une altération de la morphologie des mitochondries a été observée chez tous les patients sauf un. Ces anomalies du réseau mitochondrial semblent très utiles pour cheminer vers le diagnostic d’ARSACS qui peut s’avérer complexe dans les situations non exceptionnelles où les résultats moléculaires sont délicats à interpréter (mutations faux-sens). Nous proposons une définition graduelle d’ARSACS reposant sur des critères cliniques, génétiques et cellulaires intégrant ces anomalies du réseau. Enfin, nous avons débuté l’exploration des mécanismes impliqués dans cette altération de la dynamique du réseau mitochondrial. Nos résultats préliminaires font évoquer une action potentielle de la sacsine au niveau du renouvellement des mitochondries.ARSACS is a recessive autosomal neurodegenerative condition characterized by cerebellar ataxia, spastic paraplegia and demyelinating sensitivo-motor polyneuropathy. ARSACS is caused by mutations in the SACS gene identified in 2000. Since then, cases have been reported worldwide. SACS encodes sacsin, a protein of still unknown function in spite of the description of numerous protein domains and of a recent focus on a potential implication in the regulation of mitochondrial physiology. Aims of this thesis were to identify new mutations in a large population of ataxic patients and then to functionally analyze their cellular effects in the mitochondrial compartment. We identified 2 variants in SACS in 10% of analyzed cases collected through the international SPATAX network. We also confirmed mutations in SACS in 5 patients identified using next-generation sequencing in the ATXAIC project, and studied a last case harbouring a large deletion encompassing the entire SACS gene identified by pangenomic CGH-array. For functional analyses, primary cultures of fibroblasts were obtained in 11 patients. A drastic and recurrent alteration of the mitochondrial network was observed in all patients except one. These anomalies seem very useful for the diagnosis of ARSACS when molecular results are difficult to interpret (missense variants). We therefore propose a grading diagnostic definition using clinical, genetic and cellular criteria for ARSACS. Finally, we started to study the mechanistic of the mitochondrial function of sacsin. Our preliminary data may suggest a potential role at the mitochondrial turn over level

Plasma oxysterols: biomarkers for diagnosis and treatment in spastic paraplegia type 5

IF 10.292International audienceThe hereditary spastic paraplegias are an expanding and heterogeneous group of disorders characterized by spasticity in the lower limbs. Plasma biomarkers are needed to guide the genetic testing of spastic paraplegia. Spastic paraplegia type 5 (SPG5) is an autosomal recessive spastic paraplegia due to mutations in CYP7B1, which encodes a cytochrome P450 7α-hydroxylase implicated in cholesterol and bile acids metabolism. We developed a method based on ultra-performance liquid chromatography electrospray tandem mass spectrometry to validate two plasma 25-hydroxycholesterol (25-OHC) and 27-hydroxycholesterol (27-OHC) as diagnostic biomarkers in a cohort of 21 patients with SPG5. For 14 patients, SPG5 was initially suspected on the basis of genetic analysis, and then confirmed by increased plasma 25-OHC, 27-OHC and their ratio to total cholesterol. For seven patients, the diagnosis was initially based on elevated plasma oxysterol levels and confirmed by the identification of two causal CYP7B1 mutations. The receiver operating characteristic curves analysis showed that 25-OHC, 27-OHC and their ratio to total cholesterol discriminated between SPG5 patients and healthy controls with 100% sensitivity and specificity. Taking advantage of the robustness of these plasma oxysterols, we then conducted a phase II therapeutic trial in 12 patients and tested whether candidate molecules (atorvastatin, chenodeoxycholic acid and resveratrol) can lower plasma oxysterols and improve bile acids profile. The trial consisted of a three-period, three-treatment crossover study and the six different sequences of three treatments were randomized. Using a linear mixed effect regression model with a random intercept, we observed that atorvastatin decreased moderately plasma 27-OHC (∼30%, P < 0.001) but did not change 27-OHC to total cholesterol ratio or 25-OHC levels. We also found an abnormal bile acids profile in SPG5 patients, with significantly decreased total serum bile acids associated with a relative decrease of ursodeoxycholic and lithocholic acids compared to deoxycholic acid. Treatment with chenodeoxycholic acid restored bile acids profile in SPG5 patients. Therefore, the combination of atorvastatin and chenodeoxycholic acid may be worth considering for the treatment of SPG5 patients but the neurological benefit of these metabolic interventions remains to be evaluated in phase III therapeutic trials using clinical, imaging and/or electrophysiological outcome measures with sufficient effect sizes. Overall, our study indicates that plasma 25-OHC and 27-OHC are robust diagnostic biomarkers of SPG5 and shall be used as first-line investigations in any patient with unexplained spastic paraplegia

New practical definitions for the diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay.

International audienceOBJECTIVE:Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the SACS gene. SACS encodes sacsin, a protein whose function remains unknown, despite the description of numerous protein domains and the recent focus on its potential role in the regulation of mitochondrial physiology. This study aimed to identify new mutations in a large population of ataxic patients and to functionally analyze their cellular effects in the mitochondrial compartment.METHODS:A total of 321 index patients with spastic ataxia selected from the SPATAX network were analyzed by direct sequencing of the SACS gene, and 156 patients from the ATAXIC project presenting with congenital ataxia were investigated either by targeted or whole exome sequencing. For functional analyses, primary cultures of fibroblasts were obtained from 11 patients carrying either mono- or biallelic variants, including 1 case harboring a large deletion encompassing the entire SACS gene.RESULTS:We identified biallelic SACS variants in 33 patients from SPATAX, and in 5 nonprogressive ataxia patients from ATAXIC. Moreover, a drastic and recurrent alteration of the mitochondrial network was observed in 10 of the 11 patients tested.INTERPRETATION:Our results permit extension of the clinical and mutational spectrum of ARSACS patients. Moreover, we suggest that the observed mitochondrial network anomalies could be used as a trait biomarker for the diagnosis of ARSACS when SACS molecular results are difficult to interpret (ie, missense variants and heterozygous truncating variant). Based on our findings, we propose new diagnostic definitions for ARSACS using clinical, genetic, and cellular criteria

Dataset for 2020 Illegal Killing of Non-Game Wildlife and Recreational Shooting in Conservation Areas

Illegal killing of non-game wildlife is a global yet poorly documented problem. The prevalence and ecological consequences of illegal killing are often underestimated or completely unknown. We review the practice of legal recreational shooting and present data gathered from telemetry, surveys, and observations on its association with illegal killing of wildlife (birds and snakes) within conservation areas in Idaho, USA. In total, 33% of telemetered long-billed curlews (Numenius americanus) and 59% of other bird carcasses found with known cause of death (or 32% of total) were illegally shot. Analysis of spatial distributions of illegal and legal shooting is consistent with birds being shot illegally in the course of otherwise legal recreational shooting, but snakes being intentionally sought out and targeted elsewhere, in locations where they congregate. Preliminary public surveys indicate that most recreational shooters find abhorrent the practice of illegal killing of wildlife; viewed through this lens, our data may imply only a small fraction of recreational shooters are responsible for this activity. This study highlights a poorly known conservation problem that could have broad implications for some species and populations of wildlife