Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non-severe haemophilia A.

INTRODUCTION: Conventional genetic investigation fails to identify the F8 causal variant in 2.5%-10% of haemophilia A (HA) patients with non-severe phenotypes. In these cases, F8 deep intronic variants could be causal. AIM: To identify pathogenic F8 deep intronic variants in genetically unresolved families with non-severe HA analysed in the haematology laboratory of the Hospices Civils de Lyon. METHODS: The whole F8 was analysed by next generation sequencing. The pathogenic impact of candidate variants identified was assessed using both in silico analysis (MaxEntScan and spliceAI) and functional analysis (RNA or minigene assay). RESULTS: Sequencing was performed in 49/55 families included for which a DNA sample from a male propositus was available. In total, 33 candidate variants from 43 propositi were identified. These variants corresponded to 31 single nucleotide substitutions, one 173-bp deletion, and an 869-bp tandem triplication. No candidate variant was found in six propositi. The most frequent variants found were the association of [c.2113+1154G>C and c.5374-304C>T], identified in five propositi, and the c.2114-6529C>G identified in nine propositi. Four variants had been previously described as HA-causing. Splicing functional assay found a deleterious impact for 11 substitutions (c.671-94G>A, c.788-312A>G, c.2113+1154G>C, c.2114-6529C>G, c.5999-820A>T, c.5999-786C>A, c.5999-669G>T, c.5999-669G>A, c.5999-669G>C, c.6900+4104A>C, and c.6901-2992A>G). The HA-causing variant was identified in 33/49 (67%) cases. In total, F8 deep intronic variants caused 8.8% of the non-severe HA among the 1643 families analysed in our laboratory. CONCLUSION: The results emphasise the value of whole F8 gene sequencing combined with splicing functional analyses to improve the diagnosis yield for non-severe HA.

Identification of new F8 deep intronic variations in patients with haemophilia A.

INTRODUCTION: With current molecular diagnosis, about 1 to 5% of haemophilia A (HA) patients remain genetically unresolved. In these cases, deep intronic variation or structural variation disrupting the F8 gene could be causal. AIM: To identify the causal variation in four genetically unresolved mild-to-severe HA patients using an F8 mRNA analysis approach. METHODS: Ectopic F8 mRNA analysis was performed in four unrelated HA patients. An in vitro minigene assay was performed in order to confirm the deleterious splicing impact of each variation identified. RESULTS: In all probands, mRNA analysis revealed an aberrant splicing pattern, and sequencing of the corresponding intronic region found a deep intronic substitution. Two of these were new variations: c.2113+601G>A and c.1443+602A>G, while the c.143+1567A>G, found in two patients, has previously been reported. The c.1443+602A>G and the c.143+1567A>G variants both led to the creation of a de novo acceptor or donor splice site, respectively. Moreover, the c.143+1567A>G was found in 3/6 patients with genetically unresolved moderate HA registered in our laboratory. Haplotype analysis performed in all patients carrying the c.143+1567A>G variation suggests that this variation could be a recurrent variation. The c.2113+601G>A led to the exonization of a 122-bp antisense AluY element by increasing the strength of a pre-existing cryptic 5' splice site. For each point variation, in vitro splicing analysis confirmed its deleterious impact on splicing of the F8 transcript. CONCLUSION: We identified three deep intronic variations, leading to an aberrant mRNA splicing process as HA causing variation.

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

BACKGROUND: 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.

[Long-term automated Bayesian management of IQC results: The experience of the Lyon Hospitals Board hemostasis laboratory]. / Déploiement d'une gestion automatisée au long cours des résultats de CIQ en approche bayésienne : retours d'expérience du Laboratoire d'hémostase aux Hospices Civils de Lyon.

The Lyon Hospitals Board (HCL) hemostasis laboratory has shifted from a frequentist to a long-term Bayesian approach to IQC results management, using the Hemohub® software of the Werfen corporation, which hosts the requisite Bayesian tools. IQC plans based on supplier specifications proved effective in managing analytic risk in line with the ISO 15189 standard. Long-term Hemohub® control and monitoring has been validated by acceptable feedback from the EQA organization used by the hemostasis community.

Comprehensive analysis of F8 large deletions: Characterization of full breakpoint junctions and description of a possible DNA breakage hotspot in intron 6.

BACKGROUND: Large F8 deletions represent 3-5% of the variations found in severe hemophilia A patients, but only a few deletion breakpoints have been characterized precisely. OBJECTIVES: Resolving at the nucleotide level 24 F8 large deletions to provide new data on the mechanisms involved in these rearrangements. METHODS: Breakpoint junctions of 24 F8 large deletions were characterized using a combination of long-range polymerase chain reaction, whole F8 NGS sequencing, and Sanger sequencing. Repeat elements, non-B DNA, and secondary structures were analyzed around the breakpoints. RESULTS: Deletions ranged from 1.667 kb to 0.5 Mb in size. Nine involved F8 neighboring genes. Simple blunt ends and 2-4 bp microhomologies were identified at the breakpoint junctions of 10 (42%) and 8 (33%) deletions, respectively. Five (21%) deletions resulted from homeologous recombination between two Alu elements. The remaining case corresponded to a more complex rearrangement with an insertion of a 19 bp-inverted sequence at the junction. Four different breakpoints were located in a 562-bp region in F8 intron 6. This finding suggested that this region, composed of two Alu elements, is a DNA breakage hotspot. Non-B DNA and secondary structures were identified in the junction regions and may contribute to DNA breakage. CONCLUSION: Molecular characterization of deletion breakpoints revealed that non-homologous non-replicative DNA repair mechanisms and replication-based mechanisms seemed to be the main causative mechanisms of F8 large deletions. Moreover, we identified a possible F8 DNA breakage hotspot involved in non-recurrent rearrangements.