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.
Introduction
Data on failure to identify the molecular mechanism underlying FXI deficiency by Sanger analysis and the contribution of gene segment deletions are almost inexistent.
Aims and methods
...Prospective and retrospective analysis was conducted on FXI‐deficient patients’ DNA via Next Generation Sequencing (NGS), or Sanger sequencing and Multiplex Probe Ligation‐dependent Assay (MLPA) to detect cryptic causative gene variants or gene segment deletions.
Results
Sanger analysis or NGS enabled us to identify six severe and one partial (median activity 41 IU/dl) FXI deficient index cases with deletions encompassing exons 11–15, the whole gene, or both. After Sanger sequencing, retrospective evaluation using MLPA detected seven additional deletion cases in apparently homozygous cases in non‐consanguineous families, or in previously unsolved FXI‐deficiency cases. Among the 504 index cases with a complete genetic investigation (Sanger/MLPA, or NGS), 23 remained unsolved (no abnormality found n = 14 or rare intronic variants currently under investigation, n = 9). In the 481 solved cases (95% efficiency), we identified F11 gene‐deleted patients (14 cases; 2.9%). Among these, whole gene deletion accounted for four heterozygous cases, exons 11–15 deletion for five heterozygous and three homozygous ones, while compound heterozygous deletion and isolated exon 12 deletion accounted for one case each.
Conclusion
Given the high incidence of deletions in our population (2.9%), MLPA (or NGS with a reliable bioinformatic pipeline) should be systematically performed for unsolved FXI deficiencies or apparently homozygous cases in non‐consanguineous families.
Essentials
No F8 genetic abnormality is detected in about 2% of severe hemophilia A patients.
Detection of F8 structural variants remains a challenge.
We identified a new F8 rearrangement in a severe ...hemophilia A patient using nanopore sequencing.
We highlight the value of single‐molecule long‐read sequencing technologies in a genomics laboratory.
Background
No F8 genetic abnormality is detected in about 2% of severe hemophilia A patients using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal.
Objective
To characterize, in a genetically unresolved severe hemophilia A patient, a new Xq28 rearrangement disrupting F8 using comprehensive molecular techniques including nanopore sequencing.
Results
Long‐range polymerase chain reaction (PCR) performed throughout F8 identified a nonamplifiable region in intron 25 indicating the presence of a genomic rearrangement. F8 messanger ribonucleic acid (mRNA) analysis including 3′rapid amplification of complementary deoxyribonucleic acid (cDNA) ends and nanopore sequencing found the presence of a F8 fusion transcript in which F8 exon 26 was replaced by a 742‐bp pseudoexon corresponding to a noncoding region located at the beginning of the long arm of chromosome X (Xq12; chrX: 66 310 352‐66 311 093, GRCh37/hg19). Cytogenetic microarray analysis found the presence of a Xq11.1q12 gain of 3.8 Mb. The PCR amplification of junction fragments and fluorescent in situ hybridization (FISH) analysis found that the Xq11q12 duplicated region was inserted in the F8 intron 25 genomic region.
Conclusion
We characterized a novel genomic rearrangement in which a 3.8‐Mb Xq11.1q12 gain inserted in the F8 intron 25 led to an aberrant fusion transcript in a patient with severe hemophilia A (HA), using comprehensive molecular techniques. This study highlights the value of single‐molecule long‐read sequencing technologies for molecular diagnosis of HA especially when conventional genetic approaches have failed.
Background
Despite a high prevalence of angiodysplasia, no specific guidelines are available for the modalities of endoscopic exploration of gastrointestinal (GI) bleeding in von Willebrand disease ...(VWD). Whether VWD patients could benefit from video capsule endoscopy (VCE) looking for angiodysplasia eligible to endoscopic treatment or at high risk of bleeding is unknown.
Objectives
To assess the diagnostic efficacy for angiodysplasia and the prognostic value of VCE on top of conventional endoscopy in VWD patients with GI bleeding.
Patients/Methods
A survey was sent to the 30 centers of the French‐network on inherited bleeding disorders to identify VWD patients referred for endoscopic exploration of GI bleeding from January 2015 to December 2017. Data obtained included patient characteristics, VWD phenotype/genotype, GI bleeding pattern, results of endoscopic investigations, and medical management applied including endoscopic therapy. We assessed by Kaplan‐Meier analysis the recurrence‐free survival after the first GI bleeding event according to endoscopic categorization and, in patients with angiodysplasia, to the presence of small‐bowel localizations on VCE exploration.
Results
GI bleeding source localization was significantly improved when including VCE exploration (P < .01), even in patients without history of angiodysplasia (P < .05). Patients with angiodysplasia had more GI bleeding recurrences (P < .01). A lower recurrence‐free survival was observed in patients with angiodysplasia (log‐rank test, P = .02), and especially when lesions were located in the small bowel (log‐rank test, P < .01), even after endoscopic treatment with argon plasma coagulation (log‐rank test, P < .01).
Conclusion
VCE should be more systematically used in VWD patients with unexplained or recurrent GI bleeding looking for angiodysplasia eligible to endoscopic treatment or at high risk of relapse.
Adeno-associated virus (AAV)-mediated gene therapy may provide durable protection from bleeding events and reduce treatment burden for people with hemophilia A (HA). However, pre-existing immunity ...against AAV may limit transduction efficiency and hence treatment success. Global data on the prevalence of AAV serotypes are limited. In this global, prospective, noninterventional study, we determined the prevalence of pre-existing immunity against AAV2, AAV5, AAV6, AAV8, and AAVrh10 among people ≥12 years of age with HA and residual FVIII levels ≤2 IU/dL. Antibodies against each serotype were detected using validated, electrochemiluminescent-based enzyme-linked immunosorbent assays. To evaluate changes in antibody titers over time, 20% of participants were retested at 3 and 6 months. In total, 546 participants with HA were enrolled at 19 sites in 9 countries. Mean (standard deviation) age at enrollment was 36.0 (14.87) years, including 12.5% younger than 18 years, and 20.0% 50 years of age and older. On day 1, global seroprevalence was 58.5% for AAV2, 34.8% for AAV5, 48.7% for AAV6, 45.6% for AAV8, and 46.0% for AAVrh10. Considerable geographic variability was observed in the prevalence of pre-existing antibodies against each serotype, but AAV5 consistently had the lowest seroprevalence across the countries studied. AAV5 seropositivity rates were 51.8% in South Africa (
= 56), 46.2% in Russia (
= 91), 40% in Italy (
= 20), 37.2% in France (
= 86), 26.8% in the United States (
= 71), 26.9% in Brazil (
= 26), 28.1% in Germany (
= 89), 29.8% in Japan (
= 84), and 5.9% in the United Kingdom (
= 17). For all serotypes, seropositivity tended to increase with age. Serostatus and antibody titer were generally stable over the 6-month sampling period. As clinical trials of AAV-mediated gene therapies progress, data on the natural prevalence of antibodies against various AAV serotypes may become increasingly important.
The objectives were to describe the peri-operative management of people with inherited bleeding disorders in oral surgery and to investigate the association between type of surgery and risk of ...developing bleeding complications.
This retrospective observational study included patients with haemophilia A or B, von Willebrand disease, Glanzmann thrombasthenia or isolated coagulation factor deficiency such as afibrinogenemia who underwent osseous (third molar extraction, ortho-surgical traction, dental implant placement) or nonosseous oral surgery between 2014 and 2021 at Bordeaux University Hospital (France). Patients and oral surgery characteristics were retrieved from medical records. Odds ratio (OR) and 95% confidence interval (CI) were estimated using logistic regression.
Of the 83 patients included, general anaesthesia was performed in 16%. Twelve had a bleeding complication (14.5%) including six after osseous surgery. The most serious complication was the appearance of anti-FVIII inhibitor in a patient with moderate haemophilia A. All bleeding complications were managed by a local treatment and factor injections where indicated. No association was observed between type of surgery (osseous vs. nonosseous) and risk of bleeding complications after controlling for sex, age, disease type and severity, multiple extractions, type of anaesthesia and use of fibrin glue (OR: 3.21, 95% CI: .69-14.88).
In this study, we have observed that bleeding complications after oral surgery in people with inherited bleeding disorders were moderately frequent and easily managed. However, in this study, we observed a serious complication highlighting the necessity of a thorough benefit-risk balance evaluation during the preoperative planning of the surgical and medical protocol.
Abstract
Cardiovascular diseases (CVDs) are a major issue in aging patients with hemophilia (PWHs). Antithrombotic agents are widely used in the general population for CVD treatment, but this ...recommendation is not fully applicable to PWHs. To improve treatment strategies, a prospective case–control study (COCHE) that analyzed CVD management and follow-up (2 years/patient) in PWHs was performed in France from 2011 to 2018. In total, 68 PWHs (median age: 65 years 39–89; 48 mild, 10 moderate, and 10 severe hemophilia) were included (
n
= 50 with acute coronary syndrome,
n
= 17 with atrial fibrillation,
n
= 1 with both). They were matched with 68 control PWHs without antithrombotic treatment. In our series, bleeding was significantly influenced by (1) hemophilia severity, with a mean annualized bleeding ratio significantly higher in COCHE patients than in controls with basal clotting factor level up to 20%, (2) antihemorrhagic regimen (on-demand vs. prophylaxis) in severe (hazard ratio HR = 16.69 95% confidence interval, CI: 8.2–47.26;
p <
0.0001) and moderate hemophilia (HR = 42.43 95% CI: 1.86–966.1;
p
= 0
.
0028), (3) type of antithrombotic treatment in mild hemophilia, with a significantly higher risk of bleeding in COCHE patients than in controls for dual-pathway therapy (HR = 15.64 95% CI: 1.57–115.8;
p
= 0
.
019), anticoagulant drugs alone (HR = 9.91 95% CI: 1.34–73.47;
p
= 0
.
0248), dual antiplatelet therapy (HR = 5.31 95% CI: 1.23–22.92;
p
= 0
.
0252), and single antiplatelet therapy (HR = 3.76 95% CI: 1.13–12.55;
p
= 0
.
0313); and (4) HAS-BLED score ≥3 (odds ratio OR = 33 95% CI: 1.43–761.2;
p
= 0
.
0065). Gastrointestinal bleeding was also significantly higher in COCHE patients than in controls (OR = 15 95% CI: 1.84–268;
p
= 0
.
0141). The COCHE study confirmed that antithrombotic treatments in PWHs are associated with increased bleeding rates in function of hemophilia-specific factors and also of known factors in the general population.
Introduction
Factor X deficiency is a rare inherited bleeding disorder. To date, 181 variants are reported in the recently updated F10‐gene variant database.
Aim
This study aimed to describe new F10 ...variants.
Method
The F10 gene was analysed in 16 consecutive families with FX deficiency by a targeted high‐throughput sequencing approach, including F10, F9, F8 genes, and 78 genes dedicated to haematological malignancies.
Results
We identified 19 variants (17 missense, one nonsense and one frameshift) and two copy number variations. Two patients presenting a combined FVII‐FX deficiency showed a loss of one F10 gene copy (del13q34) associated with a missense variant on the remaining allele, leading to a FX:C significantly lower than the FVII:C level and explaining their unusual bleeding history. We reported five novel variants. Three missense variants (p.Glu22Val affecting the signal peptide cleavage site, p.Cys342Tyr removing the disulphide bond between the FX heavy and light chains, and p.Val385Met located in FX peptidase S1 domain) were detected at compound heterozygosis status in three patients with severe bleeding symptoms and FX:C level below 10 IU/dL. Two truncating variants p.Tyr279* and p.Thr434Aspfs*13 leading to an altered FX protein were found at heterozygous state in two patients with mild bleeding history.
Conclusion
This study showed the feasibility and the interest of high‐throughput sequencing approach for rare bleeding disorders, enabling the report of F10 gene screening in a 3‐weeks delay, suitable for clinical use. The description of five new variants may contribute to a better understanding of the phenotype‐genotype correlation in FX deficiency.
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