Familial transmission of chromoanagenesis leads to unpredictable unbalanced rearrangements through meiotic recombination.

Chromoanagenesis is a cellular mechanism that leads to complex chromosomal rearrangements (CCR) during a single catastrophic event. It may result in loss and/or gain of genetic material and may be responsible for various phenotypes. These rearrangements are usually sporadic. However, some familial cases have been reported. Here, we studied six families in whom an asymptomatic or paucisymptomatic parent transmitted a CCR to its offspring in an unbalanced manner. The rearrangements were characterized by karyotyping, fluorescent in situ hybridization, chromosomal microarray (CMA) and/or whole genome sequencing (WGS) in the carrier parents and offspring. We then hypothesized meiosis-pairing figures between normal and abnormal parental chromosomes that may have led to the formation of new unbalanced rearrangements through meiotic recombination. Our work indicates that chromoanagenesis might be associated with a normal phenotype and normal fertility, even in males, and that WGS may be the only way to identify these events when there is no imbalance. Subsequently, the CCR can be transmitted to the next generation in an unbalanced and unpredictable manner following meiotic recombination. Thereby, prenatal diagnosis using CMA should be proposed to these families to detect any pathogenic imbalances in the offspring.

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.

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A.

INTRODUCTION: Depending on the location of insertion of the gained region, F8 duplications can have variable clinical impacts from benign impact to severe haemophilia A phenotype. AIM: To characterize two large Xq28 duplications involving F8 incidentally detected by chromosome microarray analysis (CMA) in two patients presenting severe intellectual disability but no history of bleeding disorder. METHODS: Whole genome sequencing (WGS) was performed in order to characterize the two large Xq28 duplications at nucleotide level. RESULTS: In patient 1, a 60-73 kb gained region encompassing the exons 23-26 of F8 and SMIM9 was inserted at the int22h-2 locus following a non-homologous recombination between int22h-1 and int22h-2. We hypothesized that two independent events, micro-homology-mediated break-induced replication (MMBIR) and break-induced replication (BIR), could be involved in this rearrangement. In patient 2, the CMA found duplication from 101 to 116-kb long encompassing the exons 16-26 of F8 and SMIM9. The WGS analysis identified a more complex rearrangement with the presence of three genomic junctions. Due to the multiple micro-homologies observed at breakpoints, a replication-based mechanism such as fork stalling and template switching (FoSTeS) was greatly suspected. In both cases, these complex rearrangements preserved an intact copy of the F8. CONCLUSION: This study highlights the value of WGS to characterize the genomic junction at the nucleotide level and ultimately better describe the molecular mechanisms involved in Xq28 structural variations. It also emphasizes the importance of specifying the structure of the genomic gain in order to improve genotype-phenotype correlation and genetic counselling.

Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization.

Incorporation of distant intronic sequences in mature mRNA is an underappreciated cause of genetic disease. Several disease-causing pseudoexons have been found to contain repetitive elements such as Alu elements. This study describes an original pathological mechanism by which a small intronic deletion leads to Alu exonization. We identified an intronic deletion, c.2113+461_2113+473del, in the F8 intron 13, in two individuals with mild hemophilia A. In vivo and in vitro transcript analysis found an aberrant transcript, with an insertion of a 122-bp intronic fragment (c.2113_2114ins2113+477_2113+598) at the exon 13-14 junction. This out-of-frame insertion is predicted to lead to truncated protein (p.Gly705Aspfs∗37). DNA sequencing analysis found that the pseudoexon corresponds to antisense AluY element and the deletion removed a part of the poly(T)-tail from the right arm of these AluY. The heterogenous nuclear riboprotein C1/C2 (hnRNP C) is an important antisense Alu-derived cryptic exon silencer and binds to poly(T)-tracts. Disruption of the hnRNP C binding site in AluY T-tract by mutagenesis or hnRNP C knockdown using siRNA in HeLa cells reproduced the effect of c.2113+461_2113+473del. The screening of 114 unrelated families with mild hemophilia A in whom no genetic event was previously identified found a deletion in the poly(T)-tail of AluY in intron 13 in 54% of case subjects (n = 61/114). In conclusion, this study describes a deletion leading to Alu exonization found in 6.1% of families with mild hemophila A in France.

Compliance with Early Long-Term Prophylaxis Guidelines for Severe Hemophilia A.

OBJECTIVES: To evaluate the applicability and compliance with guidelines for early initiation of long-term prophylaxis in infants with severe hemophilia A and to identify factors associated with guideline compliance. STUDY DESIGN: This real-world, prospective, multicenter, population-based FranceCoag study included almost all French boys with severe hemophilia A, born between 2000 and 2009 (ie, after guideline implementation). RESULTS: We included 333 boys in the study cohort. The cumulative incidence of long-term prophylaxis use was 61.2% at 3 years of age vs 9.5% in a historical cohort of 39 boys born in 1996 (ie, before guideline implementation). The guidelines were not applicable in 23.1% of patients due to an early intracranial bleeding or inhibitor development. Long-term prophylaxis was delayed in 10.8% of patients. In the multivariate analysis, 2 variables were significantly associated with "timely long-term prophylaxis" as compared with "delayed long-term prophylaxis": hemophilia treating center location in the southern regions of France (OR 23.6, 95% CI 1.9-286.7, P = .013 vs Paris area) and older age at long-term prophylaxis indication (OR 7.2 for each additional year, 95% CI 1.2-43.2, P = .031). Long-term prophylaxis anticipation was observed in 39.0% of patients. Earlier birth year (OR 0.5, 95% CI 0.3-0.8, P = .010 for birth years 2005-2009 vs 2000-2004) and age at first factor replacement (OR 1.9 for each additional year, 95% CI 1.2-3.0, P = .005) were significantly associated with "long-term prophylaxis guideline compliance" vs "long-term prophylaxis anticipation." CONCLUSIONS: This study suggests that long-term prophylaxis guidelines are associated with increased long-term prophylaxis use. However, early initiation of long-term prophylaxis remains a challenge.

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.

Splicing analysis of 26 F8 nucleotide variations using a minigene assay.

BACKGROUND: Classically, the study of splicing impact of variation located near the splice site is performed by both in silico and mRNA analysis. However, RNA sample was rarely available. OBJECTIVE: To characterize a panel of putative haemophilia A splicing variations. MATERIALS AND METHODS: Twenty-six F8 variations identified from a cohort of 2075 haemophilia A families were studied using both bioinformatic tools and in vitro minigene assays in HeLa and Huh7 cells. RESULTS: An aberrant splicing was demonstrated for 21/26 tested sequence variations. A good correlation between in silico and in vitro analysis was obtained for variations affecting donor splice site (12/14) and for the synonymous variations located inside an exon (6/6). Conversely, no concordant results were observed for the six variations affecting acceptor splice sites. The variations resulted more frequently in exon skipping (n = 13) than in activation of nearby cryptic splice sites (n = 5), in use of a de novo splice site (n = 2) or in insertion of large intronic sequences (n = 1). This study allowed to reclassify 5 synonymous substitutions c.1167A>G (p.Gln389Gln), c.1569G>T (p.Leu523Leu), c.1752G>A (p.Gln584Gln), c.5586G>A (p.Leu1862Leu) and c.6066C>T (p.Gly2022Gly) as splicing variations. The pathological significance of five variations remained unclear (c.222G>A [p.Thr74Thr], c.237C>T [p.Asn79Asn], c.240C>T [p.Ile80Ile], c.2113+5_2113+8del and c.2113+5G>A). DISCUSSION: The minigene assay herein gave additional evidences for the clinical significance of 21/26 F8 putative splice site mutations. Such investigation should be performed for each F8 putative splice site variation for which no mRNA sample is available, notably to greatly improve the genetic counselling given to female carriers.

Analyses of the FranceCoag cohort support differences in immunogenicity among one plasma-derived and two recombinant factor VIII brands in boys with severe hemophilia A.

Around one third of boys with severe hemophilia A develop inhibitors (neutralizing antibodies) against their therapeutic factor VIII product. This adverse effect may result in more life-threatening bleeding, disability, impaired quality of life, and costly care. We compared the incidence of inhibitors in boys treated with the three factor VIII products most used in France: one plasma-derived (Factane) and two recombinant products (Advate and Kogenate Bayer). A previously untreated cohort of patients was created in 1994 to investigate risk factors for inhibitor development. We selected boys with severe hemophilia A (factor VIII <1 IU/dL) first treated with one of the three factor VIII products studied. Details of product infusions, inhibitor assays and main fixed and time-varying inhibitor risk factors were recorded for the first 75 exposure days. Three outcomes (all inhibitors, high-titer inhibitors and subsequently treated inhibitors) were analyzed by univariate and multivariate Cox models. We studied 395 boys first treated between 2001 and 2016 (131, 137, and 127 with Factane, Advate, and Kogenate Bayer, respectively). Clinically significant inhibitors were diagnosed in 121 patients (70 high-titer). The incidence of high-titer inhibitors was significantly associated with the factor VIII product received (P=0.005): the cumulative incidence at 75 exposure days was 12.7% (95% CI: 7.7-20.6) with Factane, 20.4% (95% CI: 14.0-29.1) with Advate, and 31.6% (95% CI: 23.5-41.7) with Kogenate Bayer. The low inhibitor incidence observed with Factane is concordant with recent findings from the SIPPET randomized trial. These consistent results from observational and experimental studies should lead to improved care for previously untreated patients and cost savings for healthcare systems worldwide.

Differential diagnosis of neonatal alloimmune thrombocytopenia: Type 2B von Willebrand disease.

At birth, severe thrombocytopenia without context of infection should mainly suggest neonatal alloimmune thrombocytopenia (NAIT), especially in case of a platelet count below 20 GL-1. We report two cases of severe neonatal thrombocytopenia, first suspected as being NAIT. Both had a platelet count below 20 GL-1 with platelet clumps. The absence of alloantibodies and failure of platelet transfusion and intravenous immunoglobulins to improve the platelet count led to question the diagnosis and to evoke inherited bleeding disorders. Measurements of Von Willebrand factor (VWF) levels showed a marked reduction of VWF:RCo and a normal VWF:Ag, suggesting a type 2B Von Willebrand disease (VWD2B). Ristocetin-induced platelet aggregation could not be performed because of the very low platelet count. In the first case, after sequencing VWF exon 28, a heterozygous p.Leu1460Pro mutation was found consistent with VWD2B. In the second case, the genetic analysis of VWF exon 28 identified a homozygous mutation: p.Pro1337Leu confirming type VWD2B and also the p.Arg854Gln homozygous mutation in exon 20 confirming type 2N (ratio FVIII/VWF:Ag <0.5). The two cases underline that, even if NAIT remains the most common diagnosis in severe neonatal thrombocytopenia, it should be challenged in the absence of documented incompatibility, chronic evolution, or treatment failure. Diagnosis of VWD2B should be considered in early thrombocytopenia, even without familial history. In the cases presented, genotyping confirmed the subtype of VWD and helped to guide the therapeutic management of bleeding episodes.

Recombinant factor VIII products and inhibitor development in previously untreated boys with severe hemophilia A.

Six recombinant factor VIII (rFVIII) products have been marketed worldwide. In 2013, the Research of Determinants of Inhibitor Development (RODIN) study group reported an unexpectedly high risk of inhibitor development with a second-generation full-length rFVIII (Product D) in previously untreated patients (PUPs) with severe hemophilia A (HA). In 1994, French public health authorities established a prospective cohort to monitor hemophilia treatment safety. A PUP subgroup was designed to investigate inhibitor risk factors. We analyzed this subcohort in view of the RODIN findings. After excluding 50 patients who participated in the RODIN study, the primary analysis focused on 303 boys with severe HA first treated with a rFVIII product. A clinically significant inhibitor was detected in 114 boys (37.6%). The inhibitor incidence was higher with Product D vs the most widely used rFVIII product (adjusted hazard ratio [aHR], 1.55; 95% confidence interval [CI], 0.97-2.49). Similar results were found for high-titer inhibitors and in 10 sensitivity analyses. No heterogeneity was observed between RODIN and our results. Combined aHRs were 1.58 (95% CI, 1.17-2.14) for all inhibitors and 1.70 (95% CI, 1.15-2.52) for high-titer inhibitors. Our results confirm the higher immunogenicity of Product D vs other rFVIII products in PUPs with severe HA.

Expanding the Mutation Spectrum Affecting αIIbß3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbß3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real-time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbß3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbß3 expression; included was a heterozygous c.1440-13_c.1440-1del in intron 14 of ITGA2B causing exon skipping in seven unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and ß3 domain structure across both subunits, thereby interfering with integrin maturation and/or function. Our study extends knowledge of GT and the pathophysiology of an integrin.

Whole F8 gene sequencing identified pathogenic structural variants in the remaining unsolved patients with severe hemophilia A.

BACKGROUND: No F8 genetic abnormality is detected in approximately 1% to 2% of patients with severe hemophilia A (HA) using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. OBJECTIVES: The study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed. METHODS: We performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction. RESULTS: A structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5. CONCLUSION: All the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.

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.

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.

The highly prevalent deletions in F8 intron 13 found in French mild hemophilia A patients result from both founder effect and recurrent de novo events.

BACKGROUND: Recently, our group has reported a 13-bp deletion in a poly(T)-track in the F8 intron 13 as the causative variant in approximately 6% of all cases of mild haemophilia A (HA) in France. The systematic screening of mild HA patients for this deletion identified individuals carrying deletions from 9 to 14-bp in the same region. AIMS: To demonstrate that these highly prevalent deletions could result from a recurrent molecular mechanism and to determine the clinical significance of deletions other than 13-bp in size. METHODS: Haplotype analysis using five polymorphic markers was performed in 71 unrelated French mild hemophilia A patients. Minigene analysis was performed to study the splicing impact of deletions from 1 to 14-bp. RESULTS: A peculiar haplotype (H1) was identified in 22.5% of patients carrying the 13-bp deletion. Haplotypes differing from H1 only for the two most distal markers were found in more than the half of patients. These results confirmed the founder effect origin for the 13-bp deletion. However, the 9 patients carrying other sizes of deletion had a different haplotype suggesting that these deletions arose independently. Supporting the recurrent mechanism hypothesis, similar deletions were also found in 3/19 genetically unresolved mild Canadian patients. In vitro splicing analysis confirmed that deletions larger than 9-bp had a deleterious impact on splicing of F8 transcript. CONCLUSION: We demonstrated that the poly(T)-track in F8 intron 13 is a deletion hotspot. We recommend that deletions in this region should be specifically investigated in all genetically unresolved mild HA patients.

Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing.

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.

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

BACKGROUND: 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. OBJECTIVE: We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c.1611C>A mutation. METHODS: 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

Characterisation of 96 mutations in 128 unrelated severe haemophilia A patients from France. Description of 62 novel mutations.

Direct sequencing of the coding region of factor VIII (F8) gene was used to determine the mutations responsible for severe haemophilia A (FVIII<1%) in 128 unrelated haemophiliacs A, negative for intron 22 and intron 1 inversions. A mutation was found in 122/128 patients (95%). Ninety-six distinct mutations were identified in this cohort, 62 of these are novel. They consisted of deletions (7 large and 24 small deletions), insertions (n = 9), associations of insertion/deletion (n = 2), association of deletion/substitution (n = 1), and single nucleotide substitutions (53 point mutations consisting of 31 missense, 20 nonsense, and 2 splicing mutations). Twenty-two patients had developed inhibitors, and among this subgroup 3 large deletions, 6 frameshift, 9 nonsense and 4 missense mutations were detected. For 6 patients, among which one developed an anti-FVIII inhibitor, no mutations were detected in the coding and splicing regions of factor VIII gene. Different approaches of molecular modelling were performed in addition to familial linkage analysis to determine the pathophysiological responsibility of these novel missense mutations.

A case of Glanzmann's thrombasthenia successfully treated with recombinant factor viia during a surgical procedure: observations on the monitoring and the mechanism of action of this drug.

Recombinant factor VIIa (rFVIIa) has been shown to be efficient for the treatment of haemorrhages in patients with Glanzmann's thrombasthenia presenting anti-glycoprotein IIb-IIIa antibodies, but the mechanism of action is not well established and there is no routine laboratory test for the monitoring of rFVIIa. In this study, thrombin generation (TG) test was used to assess the efficacy of rFVIIa ex vivo in a Glanzmann patient with inhibitor, who had a surgery for cholesteatoma. The day before surgery, TG capacity in platelet rich plasma was significantly diminished (Endogenous thrombin potential = 637nM x min) in comparison with the normal control group (1338+/-353 nM x min). Thirty minutes after the first infusion of 90 microg/kg of rFVIIa, TG was increased by 59% (1010 nM x min). rFVIIa was administered as intravenous bolus injection of 90 microg/kg q x 2h during the first 24h, than 66microg/kg q x 2h during 24h and 53 microg/kg q x 2h on the post-operative day 3. Residual TG capacity measured before rFVIIa administration mostly remained above 1000nM x min and the coagulation capacity was not significantly modified after a new injection of rFVIIa. The fibrin network was studied with 3D confocal microscopy using clots obtained with TG test. After rFVIIa infusion, the fibrin network was tighter in comparison with the sample before rFVIIa injection. These results provide further ex vivo evidence on haemostatic efficacy of rFVIIa in Glanzmann's patients.