Deep intronic variations in hemophilia : identification and description of pathophysiological mechanisms

Les hémophilies A et B sont des pathologies hémorragiques à transmission récessive liée à l’X causées respectivement par des variants des gènes F8 ou F9. Avec les stratégies de diagnostic standard, 1-5% d’hémophiles A et 1-2% d’hémophiles B demeurent sans anomalie causale identifiée. Des variants introniques profonds du gène F8 ont été décrits comme responsables d’hémophilie A par anomalie d’épissage. La première partie de ce travail a consisté en l’étude des ARNm ectopiques leucocytaires du gène F8 chez des patients hémophiles A en échec de diagnostic génétique. Nous avons identifié 3 variants introniques profonds dont l’impact délétère sur l’épissage a été confirmé par analyse in vitro en minigène. Du fait des limites de l’étude des ARNm, nous avons dans un second temps développé le séquençage total des gènes F8 et F9 par NGS, couplé à des analyses fonctionnelles in vitro (étude des ARNm ou minigène) des variants candidats identifiés. Cette seconde approche nous a permis d’identifier 33 variants introniques profonds du gène F8 et 9 du gène F9, responsables de l’hémophilie et associés à des anomalies d’épissage. Des mécanismes plus complexes qu’une création de novo ou une augmentation de force d’un site d’épissage semblent être impliqués, notamment l’implication de séquences régulatrices. Pour les variants n’altérant pas l’épissage, d’autres mécanismes, comme une modification de la transcription, sont suspectés. Ces mécanismes sont en cours d’étude dans notre équipe afin de pouvoir mieux les comprendre et les appréhender. Ces travaux ont donc permis de mettre en avant de nouveaux mécanismes physiopathologiques potentiellement associés aux variants introniques profonds.Haemophilias A and B are hereditary X-linked bleeding disorders caused by F8 or F9 genes variations, respectively. Conventional genetic investigation fails to identify the causal variant in about 1-5% of haemophilia A and 1-2% of haemophilia B patients. Deep intronic variants in the F8 have been reported as disease causing through splice alteration. In a first part, we performed F8 ectopic mRNA analysis in genetically unresolved haemophilia A patients. We identified 3 deep intronic variants for which in vitro minigene analysis confirmed the deleterious impact on splicing. Given the limits of the mRNA study, we then developed F8 and F9 whole gene sequencing by NGS, combined with functional analysis of the candidate variants identified (mRNA study or minigene). We identified 33 haemophilia causing deep intronic variants in the F8 and 9 in the F9, leading to splice alteration. Aside from de novo creation or strengthening of a splice site, more complex mechanisms, such as regulation sequences modification, seem to be involved. Other pathophysiological mechanisms, especially transcription alteration, are suspected for variants that do not affect splicing. Our laboratory is currently studying these mechanisms in order to provide with a better understanding of them. We highlighted that new pathophysiological mechanisms could be associated with deep intronic variants in haemophilia

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