Détermination des mécanismes physiopathologiques d'anomalies rares de la coagulation à l'aide de modèles in vitro et d'approches génétiques innovantes

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 sheddingLes 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 hémorragique

Physiopathological mechanism determination of rare coagulation abnormalities using in vitro experiments and innovative genetic approaches

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 hémorragiquesInherited 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 sheddin

Whole F9 gene sequencing identified deep intronic variations in genetically unresolved hemophilia B patients

International audienceBackground: The disease-causative variant remains unidentified in approximately 0.5% to 2% of hemophilia B patients using conventional genetic investigations, and F9 deep intronic variations could be responsible for these phenotypes.Objectives: This study aimed to characterize deep intronic variants in hemophilia B patients for whom genetic investigations failed.Methods: We performed whole F9 sequencing in 17 genetically unsolved hemophilia B patients. The pathogenic impact of the candidate variants identified was studied using both in silico analysis (MaxEntScan and spliceAI) and minigene assay.Results: In total, 9 candidate variants were identified in 15 patients; 7 were deep intronic substitutions and 2 corresponded to insertions of mobile elements. The most frequent variants found were c.278-1806A>C and the association of c.278-1244A>G and c.392-864T>G, identified in 4 and 6 unrelated individuals, respectively. In silico analysis predicted splicing impact for 4 substitutions (c.278-1806A>C, c.392-864T>G, c.724-2385G>T, c.723+4297T>A). Minigene assay showed a deleterious splicing impact for these 4 substitutions and also for the c.278-1786_278-1785insLINE. In the end, 5 variants were classified as likely pathogenic using the American College of Medical Genetics and Genomics guidelines, and 4 as of unknown significance. Thus, the hemophilia B-causing variant was identified in 13/17 (76%) families.Conclusion: We elucidated the causing defect in three-quarters of the families included in this study, and we reported new F9 deep intronic variants that can cause hemophilia B

Why patients with THBD c.1611C>A (p.Cys537X) nonsense mutation have high levels of soluble thrombomodulin?

Recently our group has described a new autosomal dominant bleeding disorder characterized by very high plasma levels of soluble thrombomodulin (TM). The THBD c.1611C>A (p.Cys537X) mutation in heterozygous state was found in the propositus. This mutation leads to the synthesis of a truncated TM which has lost the last three amino-acids of the transmembrane domain and the cytoplasmic tail.We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c.1611C>A mutation.Complementary DNA of TM wild type (TM-WT) was incorporated into a pcDNA3.1 vector for transient transfection in COS-1 cells. Mutagenesis was performed to create the c.1611C<A (TM1-536) mutant and 4 other TM mutants (TM1-515, TM1-525, TM1-533 and TM1-537) with a transmembrane domain having different lengths. The effect of shear stress, metalloprotease inhibitor, certain proteases and reducing agents were tested on TM shedding.Western blot and immunofluorescent analysis showed that TM1-536 was produced and a certain amount of TM1-536 was anchored on the cell membrane. A significantly higher levels of soluble TM was observed in the TM1-536 cell medium in comparison with TM-WT (56.3 +/- 5.2 vs 8.8 +/- 1.6 ng/mL, respectively, p = 0.001). The shedding of TM1-536 was 75% decreased in cells cultured in the presence of a metalloprotease inhibitor. No difference was observed between TM1-536 and TM-WT shedding after cell exposure to cathepsin G, elastase, several reducing agents and high shear stress (5000 s-1). Significantly higher levels of soluble TM were observed in the cell media of TM1-533, TM1-525, TM1-515 in comparison with TM-WT (p < 0.05).The mechanism responsible for TM shedding is complex and is not completely understood: higher sensitivity of the TM1-536 to the proteolysis by metalloproteases and a defect of synthesis due to the decreased size of the transmembrane domain might explain the high levels of soluble TM in plasma of the carriers

Thrombomodulin shedding under different conditions.

<p>Levels of released of thrombomodulin into cell culture medium were measure by ELISA. (A to D) Effect of proteases on TM-WT and TM_1-536 release. Cells were treated with elastase and cathepsin G at final concentration of 1, 2.5 and 5 μg/mL for 90 minutes. (E and F) Effect of reducing agents on TM-WT and TM_1-536 release. Cells were treated with 2mM H₂O₂; 10mM N-acetyl-L-cystein (NAC) and 5mM reduced glutathione (GSH) for 90 minutes. (G) Effect of GM6001, a broad-spectrum metalloproteinase inhibitor, on TM-WT and TM_1-536 release. COS-1 cells were cultured during 48h after transfection in medium containing GM6001 (20μM). Results are presented as means +/- standard error (n>3). * p<0.05 versus control without protease inhibitor. (H) Thrombomodulin shedding after shear stress exposition. Forty-eight hours after transfection by TM-WT and TM_1-536 expression vectors, COS-1 cells were exposed to shear stress (5000s^-1) using “cone and plate” viscometer during 0, 2, 5 and 10 minutes. Thrombomodulin concentrations in the cell supernatants were measured after exposure to shear stress in order to evaluate the thrombomodulin shedding.</p

cell surface thrombomodulin immunofluorescent labelling and confocal microscopy analysis of transfected COS-1 cells.

<p>COS-1 cells were transiently transfected by TM-WT and TM_1-536 expression vectors and were incubated in Labtek chambers for 48h. The immunofluorescent labeling was performed as described in the materials and methods section. Cell nuclei were labeled using DAPI (blue). Cell membranes were labelled using Alexa Fluor 594® conjugated Wheat Germ Agglutinin (red) and thrombomodulin were labelled using Alexa Fluor Fluor 588® antibodies (green). Superimposed images were obtained and revealed that both TM-WT and TM_1-536 were expressed at the cell surface of COS-1 cells.</p

Characterization of the secreted and intracellular thrombomodulin from COS-1 cells.

<p>(A) Cell lysates western blotting of the expressed thrombomodulin constructs. SDS-PAGE under reducing conditions and immunoblotting of cell lysates expressing each of the mutant p.Cys536X (TM_1-536), the wild-type thrombomodulin (TM-WT) or the non-transfected cells (NT). Immunodetection was performed with mouse monoclonal anti-TM antibody followed by HRP-conjugated anti-mouse antibody and enhanced chimoluminescence detection. (B) Thrombomodulin ELISA dosages in cell lysates and supernatants. (C) Cell supernatants western blotting (reducing conditions) of the expressed thrombomodulin constructs. (D) Thrombomodulin activities in cell supernatant. Thrombomodulin activities were evaluated measuring the decrease of thrombin generation of normal platelet poor plasma.</p