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Les déficits en facteurs de la coagulation sont des pathologies hémorragiques congénitales rares. Le développement récent des techniques de biologie moléculaire ont permis l'indentification de nombreuses anomalies génétiques dans les gènes codant les facteurs de coagulation. Cependant, la détermination de l'impact clinique réel de ces nouveaux variants est souvent un défi pour le biologiste moléculaire.La première partie de ce travail a consisté à l'étude par analyse chromosomique sur puce à ADN de grands réarrangements génomiques identifiés chez des patients hémophiles A ou B ayant parfois des phénotypes cliniques atypiques. Nous avons montré que certains gènes voisins des gènes F8 et F9 étaient associés, lorsqu'ils sont concernés par ces réarrangements de grande taille, à des déficits mentaux (SOX3) ou des pathologies cardiovasculaires (BRCC3).La seconde partie de cette étude a été centrée sur l'étude des variants de signification indéterminée localisés au niveau des sites d'épissage. Nous avons démontré par l'utilisation conjointe d'algorithmes informatiques et de tests in vitro de type minigène la pathogénicité de 21 variants identifiés chez des hémophiles A.La dernière partie de ce travail a consisté en la description de nouveaux mécanismes moléculaires responsables de pathologies hémorragiques. Nous avons identifié une délétion intronique chez 6,1% des hémophiles A mineurs de notre cohorte. Nous avons montré que cette anomalie était probablement responsable d'une dérégulation de l'hnRNP C ce qui entrainait l'insertion d'une séquence AluY dans les transcrits du gène F8. Enfin, nous avons décrit les mécanismes moléculaires responsables de la présence de concentrations très élevées de thrombomoduline soluble dans les plasmas de patients porteur de la mutation du gène THBD c.1611C>A. Ces travaux ont permis de détecter et de mieux appréhender certains mécanismes moléculaires responsables de pathologies rares hémorragiques / Inherited coagulation disorders are caused by a large number of genetic abnormalities. However, the determination of the clinical impact for some of these genetic variations is challenging for the molecular biologist.In a first part, we characterized large genomic rearrangements in haemophilia patients using cytogentic microarray analysis. In a first study, we delineated six F9 complete deletions in order to investigate genotype/phenotype correlations. We identified SOX3 as a candidate gene for intellectual disability (ID) found Haemophilia B patients. In a second study, we described five complex Xq28 rearrangements in Haemophilia A (HA) patients. We showed that several F8 neighbouring genes are involved in these rearrangements and some of these genes are involved in other pathologies such as ID, physical abnormalities and cardiovascular disease. Such investigations would be particularly useful for genetic counseling in female carriers to assess the risk of transmitting haemophilia associated with other diseases.In a second, we developed a minigene assay to characterise putative splice site mutations in F8 and we showed that 21 out of the 26 variations studied are associated with splicing defect.In the third part, we described two original molecular mechanisms leading to coagulation disorders. In a first case, we reported a recurrent deep intronic deletion in mild HA. We gave some evidences that this deletion promoted AluY exonization in F8 transcrits. Due to its high prevalence (6.1%), this deletion must be investigated in all patients with mild HA in whom no mutation has been detected by standard genetic analysis. In the second study we investigated the mechanism responsible for bleeding tendency in patient carrying the THBD c.1611C>A and having high levels of soluble thrombomoduline (TM). We showed that a higher sensitivity of the mutated TM to the proteolysis by metalloproteases and a defect of TM synthesis seemed responsible for TM shedding

Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) is an emerging food- and water-borne pathogen, causing approximately 73,000 annual infections in the United States and an estimated 1.5 million infections globally. E. coli O157:H7, the most frequently associated EHEC strain, is primarily transmitted through consumption of contaminated ground beef and produce and leads to hemorrhagic colitis in humans. In 5% to 15% of infected patients, circulating Shiga toxins (Stx1, Stx2) cause hemolytic uremic syndrome (HUS), characterized by the presence of thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy, contributing to acute kidney injury (AKI). Current treatment is supportive and antibiotic therapy is contraindicative as it increases toxin production. Therapeutics for EHEC-induced HUS need to be identified to minimize kidney injury and uncontrolled coagulopathy. Well-characterized animal models of HUS and EHEC infection are available and provide avenues for potential therapeutic discovery. Baboons (Papio) challenged with endotoxin-free Shiga toxins develop full spectrum HUS, and mice infected with Stx2-producing Citrobacter rodentium (Cr Stx2+), a genetically modified enteric mouse pathogen, develop severe Stx2-mediated kidney injury. Initial studies have shown that soluble thrombomodulin (sTM), an anti-coagulant, is a promising therapeutic in preventing severe kidney injury in pediatric patients. In these studies, we determined whether complement was activated in baboons challenged with Shiga toxins, and evaluated whether intraperitoneal injection of sTM would reduce disease severity from mice infected with Cr Stx2+. D-dimer and cell injury markers (HMGB1, histones) confirmed the presence of coagulopathy and cell injury in Stx challenged baboons. Studies revealed that complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these pre-clinical models. Soluble thrombomodulin treatment in Cr Stx2+ infected mice significantly decreased colonization but did not alter mortality. However, gene expression of kidney injury markers (NGAL, KIM-1) decreased significantly compared to no treatment indicating sTM-associated cytoprotectivity. The C. rodentium mouse model does not develop the coagulopathy seen in HUS patients and sTM treatment may be more effective in the baboon toxemia model. Soluble thrombomodulin is a promising therapeutic for EHEC-induced HUS and should be further evaluated in Stx challenged baboons.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成5年9月14日 水谷大裕氏の博士論文として提出された

Stuttgart, Univ., Diss., 2001.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23071号 / 医博第4698号 / 新制||医||1049(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 伊達 洋至, 教授 YOUSSEFIAN Shohab, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20965号 / 医博第4311号 / 新制||医||1026(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 伊達 洋至, 教授 平井 豊博, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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