Search

Chromoanagenesis are complex chromosomal rearrangements that are supposed to occur during a single catastrophic event. They may result in loss 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 which a healthy parent carrying a chromoanagenesis transmitted its rearrangement in an unbalanced manner to its descent. The rearrangements were characterized by karyotype, fluorescent in situ hybridization, chromosomal microarray and whole genome sequencing (WGS) in the parents and their offspring. We then hypothesized meiosis-pairing figures of parental chromosomes that may have led to meiotic recombination between normal and abnormal homologous chromosomes, resulting in the formation of new unbalanced rearrangements. This work underlines that chromoanagenesis can be associated with a normal phenotype and a normal fertility, even in males, and that WGS may be the unique way to identify these rearrangements in some cases. Chromoanagenesis can be transmitted in an unbalanced and unpredictable way because of meiotic recombination. Thus, genetic counseling and technical choice for prenatal diagnosis are complex. Finally this work questions the meiotic pairing mechanisms of complex rearrangements, still poorly understood.

The lack of quality control for patient point-of-care (POC) INR devices is an issue that has led the French health authorities to make recommendations: a laboratory INR (lab INR) has to be performed at the same time as the POC INR every 6 months. However, the differences observed between the two INRs, POC and lab INRs, are not necessarily due to a failure of the POC INR device. We present here a review of the different causes of discrepancies between INR results, which are the basis of the proposals of the Groupe français d'études sur l'hémostase et la thrombose (GFHT) on the management of lab and POC INR discrepancies. Pre-analytical conditions may account for discrepancies (sampling, transport and storage conditions), as well as analytical factors (mainly the nature of the thromboplastin used) and the clinical context (inflammatory or autoimmune diseases, polycythaemia...). The interpretation of INR discrepancies is not always easy and these proposals aim at standardizing the procedure to be followed in order to make the most appropriate decision for the patient.

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.Resolving at the nucleotide level 24 F8 large deletions to provide new data on the mechanisms involved in these rearrangements.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.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.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.

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.

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.

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.

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.

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.

International audience; 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.

Contexte : Mise en place en France en septembre 2010, la Premiere Annee Commune des Etudes de Sante (PACES) est caracterisee par un enseignement de masse, un concours tres selectif et une grande quantite d’information a memoriser. Dans ce contexte tres particulier, nous avons, pour la premiere fois, determine les approches d’apprentissage specifiquement adoptees par les etudiants et etudie leur impact sur la reussite au concours. Materiel et methodes : Une version francaise du questionnaire R-SPQ-2F de Biggs a ete administree aux etudiants de PACES 2011-2012 avant les partiels en decembre. Les approches d’apprentissage specifiques ont ete determinees apres analyse factorielle exploratoire du R-SPQ-2F et selection des items pertinents dans ce contexte. L’association entre les approches et la bonne reussite aux partiels a ete etudiee par un modele de regression binomiale. Resultats : Au total, 1977 etudiants sont inclus dans l’analyse. Les etudiants qui ont le plus de chance de reussir sont i) ceux qui s’interessent a leurs etudes (risque relatif (RR) de reussite de 1,5) et ii) ceux qui se limitent au contenu du cours (approche de surface, RR = 1,4). Les etudiants qui apprennent par cœur (approche de surface) ont 1,6 plus de risque d’echouer que les autres. Conclusion : Ce concours de PACES tres selectif et tres stressant a pour effet d’encourager une certaine approche de surface tres forte en vue de la reussite au concours au detriment d’une approche plus en profondeur indispensable a une formation solide.

INTRODUCTION Haemophilia A (HA) and haemophilia B (HB) are X-linked recessive diseases, caused by a large number of pathogenic variants in the F8 and F9 genes. With the exception of introns 22 and 1 inversions which are frequent in severe HA cases, about 2000 unique variants in F8 and 1000 in F9 have been described in databases and their recurrence remains limited. AIM AND METHODS During routine analysis, we identified two recurrent missense variants, the F8 gene c.1244C>T, p.Ala415Val variant in 27 HA patients and the F9 gene c.835G>A, p.Ala279Thr variant in 34 HB patients, in two groups of haemophiliac patients from two different regions of France. We aimed to identify whether these variants result from a founder effect. We performed haplotype reconstruction after analysis of extragenic and intragenic polymorphic markers. The ESTIAGE programme was used to estimate the age of the variant. RESULTS We identified a common ancestral haplotype HA1, in all the HA patients sharing the p.Ala415Val variant, and HB1 for 22 of 34 HB patients sharing the p.Ala279Thr variant. The estimated time of occurrence of the founder variant was between the 13th and 17th century (95% CI: 16 to 29 generations) for the F8 variant and between the 3rd and the 11th century for the F9 variant (95% CI: 44 to 72 generations). CONCLUSION This study supports a founder effect for these two variants in the two largest reported cohorts of haemophilia patients with an identical variant. These pathogenic variants are among the three most early reported variants in haemophilia.

cited By 1; Article in Press; International audience; 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+461₂113+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.2113₂114ins2113+477₂113+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+461₂113+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. © 2017 American Society of Human Genetics.

International audience; A round 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 lifethreatening 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.

Haemophilia A (HA) is an X-linked recessive bleeding disorder, caused by a wide variety of mutations in the factor VIII (F8) gene, leading to deficiency in the activity of coagulation FVIII. These mutations can affect all the F8 exons from the initiation codon to the termination codon, however, only few molecular changes in the promoter region of the F8 gene were reported so far. Here, we describe six nucleotide variations (c.-51G>A, c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A and c.-664G>A) detected in the F8 promoter and their correlation with clinical phenotype of the patients. Potential role of these mutations in HA was also assessed. Causality was demonstrated with transient transfection experiments using luciferase reporter gene plasmids and computational analysis. Two molecular changes (c.-51G>A and c.-664G>A) did not seem to affect the promoter function of the F8 gene whereas c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A mutations had an impact on the F8 promoter function and were responsible for HA. Furthermore, these mutations were associated with resistance to 1-deamino-8-D-argininevasopressin (desmopressin) therapy when they were causative. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to desmopressin and this clinical implication should be considered by clinicians.

Background Genomic inversions are usually balanced, but unusual patterns have been described in haemophilia A (HA) patients for intron 22 (Inv22) and intron 1 (Inv1) inversions leading to the hypothesis of more complex rearrangements involving deletions or duplications. Aim To characterize five abnormal patterns either in Southern blot and long-range PCR for Inv22 or in PCR for Inv1. Materials and methods All patients were studied using cytogenetic microarray analysis (CMA). Results In all cases, CMA analysis found that each inversion was associated with complex Xq28 rearrangement. In three patients, CMA analysis showed large duplication ranging from 230 to 1302 kb and encompassing a various number of contiguous genes among which RAB39B. RAB39B duplication is a strong candidate gene for X-linked intellectual disability (XLID). Surprisingly, none of the severe HA patients with RAB39B duplication reported in this study or in the literature exhibited XLID. We hypothesise that F8 complex rearrangement down regulated RAB39B expression. In the two remaining patients, CMA analysis found Xq28 large deletion (from 285 to 522 kb). Moyamoya syndrome was strongly suspected in one of them who carried BRCC3 deletion. Conclusion Because several F8 neighbouring genes are associated with other pathologies such as XLID and cardiovascular disease, all HA patients where complex Xq28 rearrangement was suspected should be referred to a geneticist for possible utility of a pangenomic study. Such investigation should be carefully considered in genetic counselling in female carriers to assess the risk of transmitting severe HA with a “contiguous gene syndrome”.

Background: Genetic variations that are found at a relatively high frequency can be the consequence of a founder effect or repeated de novo events. Recently, our group has identified an intronic deletion, c.2113+461_2113+473del [DEL13T], in the F8 intron 13, in two mild haemophilia A patients. This deletion removed a part of the poly(T)-tail from the right arm of antisens AluY element and led to AluY exonisation. Then, deletions in the poly(T)-tail of AluY in F8 intron 13, from 10 to 14 pb in size, were found in approximately 6% of all cases of mild haemophilia in France. Aim: In this study, we determined whether these highly prevalent deletions are the result of recurrent molecular mechanism or of a founder effect. Methods: Haplotype reconstruction was performed after analysis of F8 extragenic and intragenic polymorphic markers in 71 unrelated French mild haemophilia A patients carrying a deletion in the poly(T)-tail of AluY in F8 intron 13 (c.2113+460_2113+473del [DEL14T] n=1; DEL13T, n=62; c.2113+462_2113+473del [DEL12T], n=2; c.2113+463_2113+473del [DEL11T], n=5 and c.2113+464_2113+473del, n=1 [DEL10T]) and in 50 non-haemophilia A subjects. The ESTIAGE tool was used to estimate the age of the DEL13T. Nineteen genetically unresolved mild Haemophilia A patients from Queen's University, Canada, were also included in the study. All patients and controls gave informed consent for genetic studies. Results: Two intragenic (STR13 and STR22) and 3 extragenic (DXS8061, ST14 and DF2) microsatellites were investigated in the DNA of the 71 mild haemophilia A patients. This set of polymorphisms markers covered a genomic region of 2,779,113 nucleotides (4.17 cM). Among the 62 patients carrying the DEL13T, 60 patients had the same allele for the marker directly flanking the deletion on the centromeric side (STR13, genetic distance 0.0062 cM). Two of them differed from the others at the next centromeric maker STR22 (genetic distance 0.2047 cM) and 20 patients differed from the others at ST14 (genetic distance 3.4237 cM). On the telomeric side, only 8 patients differed from the others at DF2 (genetic distance 0.0292 cM). None of control individuals shared such haplotypes with these 60 patients. These results provided evidence that the founder effect hypothesis was very plausible for the variant DEL13T. The ESTIAGE tool estimated that the DEL13T occurred about 61 generations ago (95% CI : 51-74 generations). Assuming that a generation spanned 25 years, the French common ancestor carrying the c.2113+461_2113+473del was estimated to have lived between the 2th and the 8th century. However, two patients carrying the DEL13T and 9 patients carrying the other deletions (DEL10T, DEL11T, DEL12T and DEL14T) had a different haplotype suggesting that these deletions arose independently. In order to support the hypothesis of a recurrent molecular mechanism, we have investigated the presence of these deletions in other geographies. F8 intron 13 deletions were found in 3/19 Canadian patients included in this study (DEL13T, n=2 and DEL10T, n=1). Haplotype analysis performed in these three patients suggested a de novo mechanism for two of them. Conclusion: This study supports both a founder effect for the c.2113+461_2113+473del in the French mild haemophilia A patients and a recurrent molecular mechanism leading to deletion in the poly(T)-tail of AluY in F8 intron 13. We recommended that these deletions be specifically investigated in all mild haemophilia A patients in whom no genetic abnormality has been detected by standard genetic analysis. Finally, these results suggest that large poly(T)-tail of inverted Alu elements may be a mutational hot spot and such deletions leading to alu-exonization, could occur in other genes. Disclosures Negrier: Sobi/Bioverativ: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; LFB: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta/Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Resume Le deficit combine en facteurs V et VIII (F5F8D) de la coagulation est decrit depuis 1954 comme une entite se distinguant des deficits classiques de la coagulation concernant un seul facteur. La description de familles atteintes de ce deficit combine a permis dans un premier temps de connaitre la symptomatologie de cette pathologie, puis de caracteriser deux genes responsables de la diminution simultanee des facteurs V et VIII . La connaissance de ces deux genes a permis de mettre en evidence une voie de transport intracellulaire des facteurs V et VIII entre le reticulum endoplasmique et l'appareil de Golgi, et donc de mieux apprehender le metabolisme intracellulaire aboutissant a la secretion de ces deux facteurs de la coagulation dans la circulation sanguine. Ces connaissances permettent d'envisager de nouvelles cibles therapeutiques pour reguler la coagulation sanguine en agissant sur la production des facteurs V et VIII .

Introduction Haemophilia A (HA) is a bleeding disorder due to an absence or a reduced activity of coagulation factor VIII (FVIII) caused by mutations in F8 gene. Missense mutations represent approximately 45% of the reported molecular defects in HA. However, only few missense mutations in FVIII B domain have been described. Aim The aim of this study was to characterize five genetic variations (three novels and two previously reported) localized in the FVIII B domain. In all cases, an additional missense variation located outside the FVIII B domain was found. We investigated each of these variations separately and in combination too for their contribution to HA phenotype. Methods F8 variants were transiently expressed in COS-1 cells. Media and cell lysates were collected after 72 h. Then, FVIII activity, secretion and thermostability were analysed and compared to FVIII wild-type. Results The 5 FVIII B domain variants showed normal FVIII:C (98.5–128.5%) and FVIII:Ag (97.7–154%). No synergistic effect was observed between the B domain variant and their associated mutations. In contrast, the variants located outside the B domain, p.V682L, p.S714L, p.V592D and p.C573F revealed significantly decrease of FVIII:C with values in the range 3.5–44.5% (p < 0.05). However, the p.G224R variant showed FVIII:C and FVIII:Ag values no significantly different from FVIII-WT. Conclusion The FVIII B domain variants, p.D963N, p.S806T, p.G873D, p.H998Q and p.Q1225R may be considered as polymorphism or non-pathologic mutations. In five patients, clinical phenotype could be explained by the additional causative missense mutation. For the p.G224T variant further splicing studies are necessary to determine its pathogenicity.

Resume La thromboelastographie est une technique qui existe depuis 1948 et qui permet une evaluation globale et rapide de la coagulation depuis l'initiation, la formation jusqu'a la stabilite du caillot. Son utilisation dans le diagnostic des pathologies de la coagulation a ete progressivement abandonnee au profit des tests plasmatiques standardises. Plus recemment, des ameliorations techniques ont permis sa reintroduction pour le diagnostic de la coagulopathie et le suivi therapeutique en transplantation hepatique et en chirurgie cardiaque. A la fin des annees 1990 est apparue la thromboelastometrie rotative (ROTEM®), qui, par une miniaturisation de l'appareil et une standardisation des reactifs contenant des activateurs, permet une utilisation en biologie delocalisee. Cette nouvelle version a egalement montre son interet en chirurgie cardiaque et hepatique mais son utilisation dans d'autres domaines, en particuliers pour le suivi therapeutique des polytraumatises ou en pathologie obstetricale doivent faire l'objet d'autres evaluations. Une etude realisee au laboratoire a partir de 270 echantillons provenant de 89 polytraumatises montre qu'a partir de certains parametres du ROTEM® (amplitudes du caillot) obtenus en 15 minutes, il est possible de definir le degre d'hypocoagulation du polytraumatise ainsi que l'aspect qualitatif du deficit (CIVD plus ou moins associee a une hyperfibrinolyse).

Resume Le sepsis et les infections severes sont des causes encore frequentes de morbidite et mortalite dans les services de reanimation. Les marqueurs biologiques de sepsis utilisables 24 heures sur 24 sont rares et onereux. Une anomalie sur le TCA realise sur l'automate MDAII (bioMerieux) et appelee FlagA2 a une valeur diagnostique et pronostique dans la prise en charge des patients avec sepsis severe.

SummaryDirect sequencing of the coding region of factor VIII (F8) gene was used to determine the mutations responsible for severe haemophilia A (FVIII

BACKGROUND Binder syndrome is a maxillonasal dysostosis characterized by midface and nasal hypoplasia, sometimes associated with short terminal phalanges of fingers and toes and transient radiological features of chondrodysplasia punctata. Warfarin- or phenytoin-induced vitamin K deficiency during early pregnancy is a well-established etiology for this syndrome, which occurs nevertheless sporadically in most cases. CASE(S) We describe here the first case, to our knowledge, of Binder syndrome in a child whose mother presented with biliary lithiasis in early pregnancy. The mother proved to have a decrease in clotting factors II, VII, and X, and in prothrombin time, at 11 weeks of gestation, which was highly suggestive of vitamin K deficiency. CONCLUSIONS The biliary lithiasis–induced vitamin K deficiency in early pregnancy is likely to have resulted in Binder syndrome. This observation should prompt physicians to carefully check for vitamin K deficiency in pregnant women presenting with biliary lithiasis, in order to prevent Binder syndrome in the fetus by providing intravenous vitamin K supplementation as soon as possible. Finally, reassuring genetic counseling regarding the genetic risk for future pregnancies is to be provided to the parents. Birth Defects Research (Part A), 2005. © 2005 Wiley-Liss, Inc.

Summary. Activated protein C (APC) resistance phenotype/genotype discrepancy is a very rare event. The objective of this study was to characterize the molecular mechanisms in two cases of APC phenotype/genotype discrepancy. An approach using direct sequencing of each exon and splicing junctions of the factor V gene showed that two novel factor V null mutations combined with heterozygous factor V Leiden mutation were responsible for this discrepancy. Our results suggest the necessity to use both phenotypic and genotypic analyses in some cases to determine an accurate diagnosis.

UNLABELLED This study aims to determine the way to predict the haemophilia A (HA) carrier status and the potential severity in six females with low FVIII C levels (

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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 expression; included was a heterozygous c.1440-13_c.1440-1del in intron 14 of ITGA2B causing exon skipping in 7 unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits thereby interfering with integrin maturation and/or function. Our study extends knowledge of Glanzmann thrombasthenia and the pathophysiology of an integrin. This article is protected by copyright. All rights reserved.

Glanzmann thrombasthenia is the most common inherited disorder of platelets that may induce severe bleeding complications. Molecular biology techniques have offered the possibility to assess the basis of this chronic haemorrhagic disease at the molecular level. However, the accessibility of mRNA in platelets is limited by the availability of the patient's blood samples and the relatively weak amount of this material in these cells. Taking advantage of the genetic phenomenon of illegitimate transcription, we have demonstrated that glycoprotein IIb and glycoprotein IIIa mRNA could be detected in lymphoblastoid cell lines issued from normal EBV-transformed lymphoblasts. We further analysed the sequences of the two glycoprotein transcripts in lymphoblastoid cell lines from two previously characterized patients presenting with Glanzmann thrombasthenia. The results showed that illegitimate transcripts presented similar molecular abnormalities to those found in platelets. These data demonstrated that the nucleotide sequences of illegitimate transcripts were identical to tissue-specific mRNA found in platelets. We applied this methodology to screen for the genetic defect in a new thrombasthenic patient, and found a homozygous nonsense mutation GCA--TGA converting Arg8 to stop in the glycoprotein IIIa gene. This immortalized source of genetic material is therefore particularly useful for molecular genetic studies in inherited platelet disorders, avoiding repetitive and large blood samplings in frequently anaemic patients.

Introduction Hemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder. Aim In this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum. Methods We screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure. Results We identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles. Conclusion The mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715

Introduction The identification of molecular defects in haemophilia is essential for the optimization of patient treatment and the formal characterization of female carriers. The Sanger method is the gold standard for sequencing F8 and F9 genes but is time-consuming and expensive. We aimed to develop a high-throughput method to genotype haemophilia A (HA) and B (HB) patients using the Next-Generation Sequencing (NGS) technology for an exhaustive and less expensive analysis of F8 and F9 genes. Material & Methods We developed a small panel containing F8 and F9 for exons and introns/exons junctions sequencing. We used two different methods for library preparation (AmpliSeq™, Life Technologies™ and HaloPlex™, Agilent™), performed in the same PCR emulsion system (Ion One Touch 2™, Life Technologies™) and sequenced with a Ion 316™ chip in a PGM™ Ion Torrent sequencer, or a Ion PI™ chip in a Proton™ sequencer (Life Technologies™) respectively. The promoter and 3' regions of F8 and F9 were always studied by Sanger. NGS analysis was first performed in 62 samples (HA: n= 42; HB: n=13; carriers: n=7) previously characterized for F8 or F9 mutations by Sanger method or Multiplex Ligation-Probe Amplification (MLPA). All types of mutations were studied (nonsense, missense, splice, small insertion/deletion and exons deletion/duplication) and were distributed in all exons of F8 and F9. NGS analysis was further performed in 42 haemophilia patients (HA: n=31, HB: n=11; of which 36 presented a mild phenotype), with unknown mutation status. All patients were included by the local Comprehensive Care Haemophilia Center of Lille University Hospital after written informed consent. Data were analyzed with SeqNext™ software (JCI Medical System™). A Normalized Reads Depth (NRD) ratio was used to detect exons deletion/duplication. Results All exons were well covered by AmpliSeq™ (average number of reads, ANR = 300) and overall by HaloPlex™ (ANR = 15000, except small parts of exons 14 and 19 in F8). The average quality value for mutation detection was 60 (risk of false result In previously-genotyped patients, 92% (57/62) of F8 and F9 mutations were detected by AmpliSeq™ and 85% (53/62) by HaloPlex™. The detection rate of small insertion/deletion in homopolymers of exon 14 in F8 was only 20% (1/5) with both methods and 71% (5/7) in other exons of F8. Four deletions and one duplication of exons accounting for a severe haemophilia phenotype were identified (3 HA, 1 HB and 1 HA-carrier). In 5 uncharacterized patients by Sanger method, neither AmpliSeq™ nor HaloPlex™ were able to find a mutation suggesting that the molecular defect is located in introns of F8 or F9. In never-genotyped patients, a mutation was detected in 90% (38/42) of cases (including 34 missense, 2 nonsense and 2 splice mutations in both F8 and F9). A duplication of exons 10 to 14 was also detected in a severe HA patient and was confirmed by MLPA. In NGS negative patients, no mutation was found in promoter or 3' regions. Of the ten candidate mutations identified in our cohort, seven were predicted to be deleterious by in silico analysis and/or co-segregation studies. No mutation was found in 10% (4/42) of never-genotyped patients with mild haemophilia A, in consistence with the available data for the mild phenotype. The technical development and laboratory protocol was easier and less expensive ($530 vs $602 including reagents and technical/medical staff) with AmpliSeq™ than HaloPlex™. Conclusion We confirmed that NGS is able to detect the main types of mutations in F8 and F9 genes, albeit with a lower mutation detection rate with HaloPlex™ compared to AmpliSeq™. These detections were associated with an important depth of reads and high Quality Values, except for exons 14 and 19 in F8 with HaloPlex™. AmpliSeq™ seems also an interesting screening method for the detection of exons deletion/duplication using the NRD ratio. However, both strategies fail to detect small insertion/deletion located in homopolymers of exon 14 in F8, whom identification will still rely on Sanger sequencing. AmpliSeq™ protocol performed in the PGM™ sequencer appears as a new interesting tool in genotyping of HA and HB patients of the Lille University Hospital. Disclosures Zawadzki: Pfizer Pharmaceutical Company: Research Funding.

Glycoprotein IIb and IIIa contain antigenic determinants involved in the potential production of allo- or autoantibodies directed against platelets, that may result in severe thrombocytopenia. Most of these epitopes appear to be supported by single nucleotide substitutions. We have used denaturing gradient gel electrophoresis (DGGE) to identify sequence variations within the promoter and the coding regions of the glycoprotein IIb and glycoprotein IIIa genes. Using genomic DNA from 60 unrelated normal individuals, we have amplified short domains that encompass the coding sequences and the exon-intron boundaries of both genes that were further separated according to their melting behaviour during the denaturant electrophoretic migration. Only the fragments with an abnormal migration pattern were sequenced. We confirmed the sensitivity of this method by recognizing both previously described Human Platelet Antigen polymorphisms and mutations affecting either the glycoprotein IIb or the glycoprotein IIIa genes in thrombasthenic patients. We also identified four other polymorphisms. Two were located in the glycoprotein IIb gene, involving intron 21 (C--G at nucleotide 10480) and first codon of exon 30 (codon GTC--GTT coding for residue Val 990), and two in the glycoprotein IIIa gene (exon 6 CCC--CCT coding for residue Pro 268; intron 14 C--T at position 37126). The screening of the GPIIIa promoter also revealed three different polymorphisms located at position-468 (A/T polymorphism), -425 (A/C polymorphism) and-400 (A/C polymorphism), which could influence the expression of the complex at the cell surface. Denaturing gradient gel electrophoresis appears to be a sensitive and specific technique for identifying polymorphisms and mutations in the GPIIb and GPIIIa genes.

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.

We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.

Activated protein C (APC) resistance phenotype/genotype discrepancy is a very rare event. The objective of this study was to characterize the molecular mechanisms in two cases of APC phenotype/genotype discrepancy. An approach using direct sequencing of each exon and splicing junctions of the factor V gene showed that two novel factor V null mutations combined with heterozygous factor V Leiden mutation were responsible for this discrepancy. Our results suggest the necessity to use both phenotypic and genotypic analyses in some cases to determine an accurate diagnosis.

This study describes the genetic mechanisms responsible for the de novo occurrence of severe and mild hemophilia A in monozygotic twin females. Both twins were found to carry a previously known factor VIII mutation (Tyr16Cys) in the heterozygous state which most probably arose in the paternal germ line. Both twins showed concordant skewing of X inactivation toward the maternally derived normal X chromosome, the most severely affected twin exhibiting a higher percentage of inactivation of the normal X chromosome. The degree of skewing of X inactivation closely correlated with both the coagulation parameters and the clinical phenotype of the twins. Since these twins were monochorionic, such results suggest that the twinning event in this case has occurred after the onset of the X-inactivation period.

In this study we have used denaturing gradient gel electrophoresis (DGGE) for identifying sequence alterations in glycoprotein (GP) IIb and IIIa genes from 20 patients affected by Glanzmann's thrombasthenia. These patients were from 16 different families. Using computer modelling, we divided the promoters, coding sequences and flanking splicing regions, in 31 segments for the GPIIb gene and 19 domains for the GPIIIa gene. We were able to find a mutation potentially affecting GPIIb-IIIa expression or function in 16 patients out of 20. In six patients from three families, the gypsy mutation modifying the splice donor site of intron 15 of the GPIIb gene was detected. In the other patients, 10 novel mutations were characterised, which were located either in the GPIIb gene (nine cases) or in the GPIIIa gene (one case). The type of mutation was nonsense mutation (one case), missense mutation (five cases), small insertion of 1 bp (one case) and splicing modifications (three cases). Among these genetic events, three were directly responsible for Glanzmann's thrombasthenia, four were localised in regions known to be involved in GPIIb-IIIa complex expression and three mutations were potentially responsible for Glanzmann's thrombasthenia.

Rapid diagnosis of the French gypsy mutation in Glanzmann thrombasthenia using high-resolution melting analysis

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder, caused by a quantitative or qualitative defect of the GPIIb-IIIa integrin (alpha IIb beta 3), which functions as the platelet fibrinogen receptor. We report a case of type I GT due to a homozygous mutation resulting in Ser 870 to stop codon substitution. This residue is located near the proteolytic cleavage site of proGPIIb. The mutation results in a GPIIb truncated of 138 amino acids, including transmembrane and intracytoplasmic domains. Cotransfection of an expression vector containing the mutant GPIIb and wild-type GPIIIa showed that the mutant Ser 870-->stop GPIIb was able to associate to GPIIIa. However, this heterodimer failed to mature as shown by endoglycosidase-H digestion and was therefore not expressed at the COS-7 cell surface. This report is the first description of a homozygous nonsense mutation in the GPIIb gene and highlights the role of the GPIIb light chain.

SummaryMultiple coagulation disorders are unusual. We report here a combination of haemophilia B Leyden with type 1 von Willebrand disease (vWD) affecting different members of the same family. Haemophilia B Leyden was due to a -6 G → A mutation within the promoter of the factor IX gene and was responsible for a mild haemophilia in the father of the proband. The proband and her sister (age 4 and 6) exhibited a twofold lower level of factor IX activity (0.4 IU/ml) than the paternal grandmother (0.95 IU/ml). The differences in FIX levels in the three carriers of the same -6 G → A mutation suggest the implication of an age-related mechanism responsible for the increase in factor IX plasma level. Haemophilia B Leyden patient and carriers suffered also from a mild von Willebrand disease. The diagnosis of this associated type 1 vWD was performed by assaying plasma von Willebrand factor together with multimer electrophoretic studies and DDAVP test. The inheritance of this vWD was investigated by haplotype analysis of the vWF gene. Individuals affected by such an association are actually asymptomatic, but per- and post-operative bleeding risk remains to be evaluated.

We have recently described an unusual situation which involved a combination of a factor IX and a protein C deficiency in a young child who presented, according to the bleeding tendency, as a hemophilia B patient. In this particular hemophiliac, baseline prothrombin fragment F1 + 2 levels were unexpectedly elevated and increased after an injection of a very high purity factor IX concentrate. This observation raised a question regarding the substitution schedule in the case of repeated injections of factor IX, since the thrombotic tendency has been a major concern with some factor IX concentrates. We monitored factor IX, prothrombin fragment F1 + 2, and D-dimer plasma levels before and during the 6 hr following the injection of an immunopurified factor IX concentrate. The results showed an increase in the F1 + 2 levels after the factor IX injection, but an increase lower than previously observed with an ion-exchange chromatography-purified concentrate. Furthermore, the F1 + 2 level returned to baseline value 6 hr after administration. This factor IX concentrate seems to be best for use in the patient where repeated injections are involved (as employed during surgery). Moreover, the data point out the advantage of a monoclonal antibody-purified factor IX concentrate over less purified concentrates in a specific situation, with regard to the thrombogenic risk.

Hemophilia A is a common inherited bleeding disorder, caused by factor VIII (FVIII) deficiency as a result of mutations in the factor VIII gene (F8 gene). Intron 1 and 22 inversions, small insertions/deletions and point mutations are the most common genetic defects responsible for severe hemophilia A. However, a F8 gene mutation is not found in 2–5% of patients with severe hemophilia A (FVIII:C