Les maladies des petites artères cérébrales sont un groupe hétérogène de maladies qui affectent les petites artères, artérioles, veines et/ou capillaires du cerveau. La plupart des patients sont des cas sporadiques, mais plusieurs formes héréditaires ont été identifiées.Toutefois, 15 % seulement des patients atteints d’une cSVD familiale sont porteurs d’une mutation dans l’un de ces gènes, suggérant l’implication d’autres gènes. Dans cette thèse, nous avons montré que des mutations hétérozygotes du gène HTRA1 étaient responsables d’environ 5 % des cSVD familiales. L’analyse fonctionnelle de ces mutations a montré un effet perte de fonction. L’âge de début chez les sujets hétérozygotes était beaucoup plus tardif que chez les patients CARASIL, où les deux allèles d’HTRA1 sont mutés. Ensuite, nous avons identifié 2 familles (incluant la famille rapportée sous l’acronyme PADMAL / Pontine Autosomal Dominant Microangiopathy and Leukoencephalopathy) portant deux mutations distinctes dans un site d’accrochage du microRNA miR-29, dans la partie 3’UTR du gène COL4A1.Quatre autres patients index porteurs du même type de mutations ont été identifiés dans notre cohorte de cas cSVD. L’analyse fonctionnelle de ces mutations a mis en évidence une up-régulation de l’expression du gène COL4A1. Le phénotype observé était très stéréotypé, caractérisé par la survenue d’infarctus pontiques dans la 3ème décade. L’identification des bases moléculaires de ces deux nouvelles formes de cSVD héréditaire a des applications diagnostiques immédiates. Elle renforce par ailleurs l’hypothèse du rôle essentiel d’une altération du matrisome dans les mécanismes physiopathologiques des cSVD / Cerebral small vessel diseases (cSVD) are a heterogeneous group of disorders affecting small arteries, arterioles, veins, and/or capillaries of the brain. In most cases cSVD are sporadic, but several hereditary monogenic forms have been identified. Nevertheless, only 15% of familial cSVD patients sent for genetic screening are carriers of mutations in one of these genes, suggesting the implication of other genes. In this thesis work, we showed that heterozygous mutations in HTRA1 are found in 5% of familial cSVD cases. Functional analysis of these mutations showed that most of them behave as loss-of-function mutations. Disease onset was much later (>25 years) than in CARASIL patients, in which both2 HTRA1 alleles are mutated. Afterwards, we identified 2 informative families (including the original family reported to be affected by PADMAL / Pontine Autosomal Dominant Microangiopathy and Leukoencephalopathy) harboring two different mutations in the binding site of miR-29 microRNA within the 3’UTR of COL4A1 gene. Four other index patients carrying the same type of mutations were identified in our patient cohort. Functional analysis of these mutations showed an up-regulation of COL4A1 gene expression. The observed phenotype was highly stereotyped in all patients, characterized by pontine infarcts appearing in the 3rd decade. Identification of the molecular defects underlying these two novel hereditary cSVD forms provides tools to improve the molecular diagnosis of cSVD. Besides, it reinforces the hypothesis of an essential role of matrisome alteration in cSVD pathophysiological mechanisms

Age-related macular degeneration (AMD) is the leading cause of blindness worldwide, affecting an estimated 50 million individuals aged over 65 years. Environmental and genetic risk-factors contribute to the development of AMD. An AMD-risk locus on chromosome 10q26 spans two genes, ARMS2 and HTRA1, and controversy exists as to which variants are responsible for increased risk of disease. Recent work suggests that HTRA1 expression levels are significantly increased in carriers of the risk haplotype associated with AMD. However, relatively little is known about the interactions, substrate specificity and roles in disease played by this secreted serine protease. This thesis aims to elucidate the potential role played by HTRA1 in AMD pathogenesis. A combination of tandem affinity purification (TAP) and yeast two-hybrid techniques was used to identify interacting partners of HTRA1. A number of proteins, with diverse roles in the alternative complement pathway, cell signaling, cell-matrix interactions, inflammation, angiogenesis and fibrosis, were identified. These are attractive candidates for further study as such processes are disturbed in AMD, implicating HTRA1 and its binding partners in disease development. One interacting partner, Complement Factor D (CFD), is a key activator in the alternative complement pathway. CFD, a 24 kDa serine protease, is expressed as an inactive zymogen, from which a signal peptide and activation peptide are cleaved before release of the mature, active protein into the circulation. In vitro studies show that CFD interacts with, and can be a substrate for, HTRA1. The interacting domain between the two proteins is localised to a region of 30 amino acids at the N-terminal end of proCFD. The 5 amino acid pro-peptide of CFD appears to be both necessary and sufficient for proteolysis of CFD by HTRA1. Investigation of the functional relevance of the interaction between HTRA1 and CFD shows that proCFD is cleaved by HTRA1, whilst mature CFD is not subjected to proteolysis. HTRA1-mediated cleavage of CFD forms an active protease, leading to activation of factor B in the alternative complement pathway in in vitro assays. Furthermore, a normal complement response is restored to CFD-depleted serum by addition of proCFD activated by HTRA1. Thus, an HTRA1- mediated increase in alternative complement pathway activity may explain a proportion of the AMD-risk attributed to the chr10q26 locus. Genetic and protein-based approaches were used to study the potential role of CFD in AMD pathogenesis, independent of an interaction with HTRA1. An intronic SNP, rs3826945, was significantly associated with increased risk of AMD in two British case-control cohorts, and in a combined meta-analysis with 4 additional cohorts from North America and Europe (p-value = 0.032, Odds Ratio = 1.112 in 4765 cases and 2693 controls). Assessment of copy number variation and sequencing of CFD did not identify any functional variants which may explain the association with disease. However, plasma levels of CFD were measured by ELISA in 751 AMD cases and 474 controls, and were found to be significantly elevated in AMD cases compared to controls (p-value = 0.00025). This further implicates complement activation in AMD pathogenesis, and makes CFD an attractive candidate for therapeutic intervention. An alteration in the level of activated CFD, possibly mediated via an interaction with HTRA1, either at the systemic or local tissue level, may play a role in disease development and progression.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13291号 / 論医博第2189号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 小池 薫, 教授 妻木 範行 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Localisé entre l'Epithélium Pigmentaire Rétinien (EPR) et les segments externes des photorécepteurs, l'espace sous-rétinien est une zone immunosuppressive ; régulée par des signaux comme la thrombospondine-1 (TSP-1) ou Fas Ligand (FasL), qui empêchent l'accumulation des phagocytes mononucléés (PMs), en particulier des monocytes inflammatoires. La Dégénérescence Maculaire Liée à l'Age (DMLA) est associée à une rupture de l'immunosuppression de cet espace, et s'accompagne d'une accumulation de PMs ; causant la mort des photorécepteurs, la dédifférenciation de l'EPR et une néovascularisation pathologique. Des études d'associations génétiques ont établi un lien entre la DMLA et un haplotype qui affecte le locus 10q26, qui contient trois gènes : PLEKHA1, ARMS2 et HTRA1. L'haplotype est associé à une augmentation de la transcription de HTRA1 dans les lymphocytes ou les cellules de l'EPR. HTRA1 code pour une sérine protéase qui a une multitude de substrats ; mais le mécanisme par lequel elle pourrait être impliquée dans la pathogenèse de la DMLA reste inconnu. TSP-1 est une glycoprotéine exprimée par l'EPR, les macrophages résidents et inflammatoires. Le domaine C-terminal de TSP-1 contient deux séquences VVM qui peuvent chacune interagir avec un récepteur CD47. Dans cette étude, nous montrons que HTRA1 clive TSP-1 et inhibe l'élimination des PMs régulée par l'interaction entre TSP-1 et CD47 à l'état physiologique, in vitro et in vivo. L'activation pharmacologique de CD47 nous a permis d'annuler les effets pro-inflammatoires de HTRA1 et pourrait représenter un espoir thérapeutique pour le contrôle de la progression de la DMLA chez les patients porteurs de l'haplotype à risque. / Localized between the Retinal Pigment Epithelium (RPE) and the photoreceptors outer segments, the subretinal space is an immunosuppressive zone, mediated by signals such as Thrombospondin-1 (TSP-1), Fas Ligand (FasL) that prevent the accumulation of Mononuclear Phagocytes (MPs) and in particular pathogenic inflammatory monocytes. Age related Macular Degeneration (AMD) is associated with a breakdown of this immunosuppressivity and an accumulation of MPs, which causes photoreceptor degeneration, RPE dedifferentiation and pathological neovascularization. Genome association studies showed a strong link between AMD and a relatively common haplotype of 10q26 locus that contains the PLEKHA1, ARMS2 and HTRA1 genes. The disease haplotype is associated with increased HTRA1 transcription in cell types such as lymphocytes and RPE cells. HTRA1 is a serine protease with a number of substrates, but the mechanism by which it might be involved in AMD pathogenesis is unknown. TSP-1 is a glycoprotein expressed by RPE, resident macrophages and inflammatory macrophages. The C-terminal domain of TSP-1 contains two VVM sequences that can each interact with a CD47 receptor. We show that HTRA1 induced subretinal MP accumulation is dependent on TSP-1 deactivation in an RPE/Mo co-culture model and in a laser induced inflammation model in vivo. This pathogenic effect of HTRA1 was reversible by synthetic CD47 agonists. Our study reveals a comprehensive mechanism how the risk-allele 10q26 participates in the pathogenesis of AMD and opens new therapeutic avenues to restore subretinal immunosuppressivity and inhibit the inflammation-dependent neurodegeneration.

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A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Genetic association studies simplify genotype‐phenotype relationship investigation by considering only the presence of a given polymorphism and the presence or absence of a given downstream phenotype. Although such associations do not indicate causation, collections of phenotypes sharing association with a single genetic polymorphism may provide valuable mechanistic insights. In this thesis we explore such genetic pleiotropy with Deep Phenotype Association Studies (DeePAS) using data from the Age‐Related Eye Study 2 (AREDS2). We also employ a novel text mining approach to extract pleiotropic associations from the published literature as a hypothesis generation mechanism. Is it possible to identify pleiotropic genetic associations across multiple published abstracts and validate these in data from AREDS2? Data from the AREDS2 trial includes 123 phenotypes including AMD features, other ocular conditions, cognitive function and cardiovascular, neurological, gastrointestinal and endocrine disease. A previously validated relationship extraction algorithm was used to isolate descriptions of genetic associations with these phenotypes in MEDLINE abstracts. Results were filtered to exclude negated findings and normalize variant mentions. Genotype data was available for 1826 AREDS2 participants. A DeePAS was performed by evaluating the association between selected SNPs and all available phenotypes. Associations that remained significant after Bonferroni‐correction were replicated in AREDS. LitWAS analysis identified 9372 SNPs with literature support for at least two distinct phenotypes, with an average of 3.1 phenotypes/SNP. PheWAS analyses revealed that two variants of the ARMS2‐HTRA1 locus at 10q26, rs10490924 and rs3750846, were significantly associated with sub‐retinal hemorrhage in AMD (rs3750846 OR 1.79 (1.41‐2.27), p=1.17*10‐7). This associated remained significant even in populations of participants with neovascular AMD. Furthermore, odds ratios for the development of sub‐retinal hemorrhage in the presence of the rs3750846 SNP were similar between incident and prevalent AREDS2 sub‐populations (OR: 1.94 vs 1.75). This association was also replicated in data from the AREDS trial. No literature‐defined pleiotropic associations tested remained significant after multiple‐testing correction. The rs3750846 variant of the ARMS2‐HTRA1 locus is associated with sub‐retinal hemorrhage. Automatic literature mining, when paired with clinical data, is a promising method for exploring genotype‐phenotype relationships.