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Background: One of the major problems for patients with severe hemophilia A (HA) is the development of neutralizing antibodies against factor VIII. This study aimed to analyze the molecular and clinical profiles of patients with severe HA and to determine if certain genetic variants predispose to inhibitor development in these patients., Methods: A single-center study was conducted among patients with severe HA between March 20, 2000, and June 31, 2023. Demographic data and laboratory results of patients were collected. The inverse-shifting PCR (IS-PCR) technique was initially used to screen patients for intron 22 and 1 inversions (Inv-22 and Inv-1)., Results: A total of 480 patients with severe HA (408 without inhibitors and 72 with inhibitors) were enrolled in this study. The median age of the patients at the time of diagnosis was 6 months (IQR: 3 months to 18 months). Inv-22 was observed in 199 (41.5 %) of the cases. Among those patients who developed inhibitors, 53 (73.6 %) were classified as high-titer and 19 (26.4 %) as low-titer. Inv-22, positive family history of inhibitor formation, and history of intense injections revealed a statistically significant association with the risk of inhibitor development., Conclusion: The results of this study confirm the important role of different genetic variants, family history of inhibitor formation, and history of intense injections for the formation of inhibitors in patients with severe HA. This would allow us to stratify the patients which can have important clinical implications, especially in terms of their management and outcome., Competing Interests: Conflicts of interest The authors declare no potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Abstract: A2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 μg/kg) was equal to that of the WT (2 μg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Aim: To characterise non-severe haemophilia A (HA) patients enrolled on the Australian Bleeding Disorders Registry (ABDR) treated through a state-wide Haemophilia Treatment Centre (HTC) with respect to their mutational profile, inhibitor risk and health-care burden., Method: We conducted a single-centre observational study of all non-severe HA patients treated at the Alfred Health HTC registered on the ABDR as of the 26th July 2023. Data were extracted from the ABDR and electronic medical record (EMR) regarding demographics, severity, genetic testing, treatment, inhibitors, bleeding events and procedures. Inhibitor risk was calculated as a function of exposure days (EDs) of FVIII replacement., Results: There were 289 non-severe HA patients treated at the Alfred HTC registered on the ABDR as of July 2023, all of whom were adult patients aged > 18 years old. Genotyping had been performed in 228/289 (78.9%). Of the inhibitor analysis population, 14/193 (7.3%) had an inhibitor. The cumulative incidence of inhibitor development at 75 EDs was 31% (95% CI 13%-46%). The median cost of bypassing agents per inhibitor patient was $57,087.50/year., Conclusion: These results demonstrate a relatively high inhibitor prevalence and incidence risk in non-severe HA compared to previously published work, although this may partly reflect a smaller population size. High rates of genotyping have allowed representative mutational characterisation. The burden of care imposed by non-severe HA in terms of bleeding events, procedures and bypassing agent cost is larger than expected, particularly within the inhibitor population., (© 2024 The Authors. Haemophilia published by John Wiley & Sons Ltd.)

Introduction: The diagnosis of hemophilia A (HA) is based on the measurement of factor VIII activity (VIII:C). About one-third of non-severe HA patients show a discrepancy of VIII:C measured by one-stage (VIII:C 1st) and chromogenic (VIII:C chr) assays. Different mutations in the F8 gene may cause the discrepancy in results of the FVIII activity assay. The aim of this study was to investigate F8 gene mutations in patients with assay discrepancies and to evaluate their impact on the results of VIII:C assays., Methods: Mutation analysis was performed on 41 individuals with a discrepancy in VIII:C 1st and FVIII: C chr assays by direct sequencing. In addition, the effect of the variants on FVIII macromolecule structure was investigated by in silico and bioinformatics tools., Results: Genetic analysis disclosed 22 different variants, of which 19 were identified for the first time to be involved in the phenotype of VIII:C discrepancy. Most of the variants related to the higher VIII:C 1st were found in A1, A2, A3 domains. The variant related to VIII:C chr > VIII:C 1st was located in the thrombin cleavage site. In silico analysis showed the effect of variants on FVIII macromolecule stability, which may be the possible mechanism causing the discrepancy., Conclusion: Our data shed light on the impact of genetic defects on VIII:C assay and provided evidence that the consideration of these mutations may open a new window to the proper diagnosis and treatment monitoring of non-severe HA patients., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by "redirecting" polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants' causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches., Competing Interests: Declaration of interests M.P. is the inventor of a patent (PCT/IB2011/054573) on modified U1snRNAs. All other authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Hemophilia A is an X-linked inherited blood coagulation disorder caused by the production and circulation of defective coagulation factor VIII protein. People living with this condition receive either prophylaxis or on-demand treatment, and approximately 30% of patients develop inhibitor antibodies, a serious complication that limits treatment options. Although previous studies performed targeted mutations to identify important residues of FVIII, a detailed understanding of the role of each amino acid and their neighboring residues is still lacking. Here, we addressed this issue by creating a residue interaction network (RIN) where the nodes are the FVIII residues, and two nodes are connected if their corresponding residues are in close proximity in the FVIII protein structure. We studied the characteristics of all residues in this network and found important properties related to disease severity, interaction to other proteins and structural stability. Importantly, we found that the RIN-derived properties were in close agreement with in vitro and clinical reports, corroborating the observation that the patterns derived from this detailed map of the FVIII protein architecture accurately capture the biological properties of FVIII.

Some genetic and treatment-related factors are risk factors for inhibitor development in patients with hemophilia A (PwHA). However, the genotype distribution of the factor VIII gene ( F8 ) and genetic impact on inhibitor development in Japanese PwHA remain unknown. In 2007, the Japan Hemophilia Inhibitor Study 2 (J-HIS2) was organized to establish a nationwide registry system for hemophiliacs and to elucidate risk factors for inhibitor development, designed for prospective investigation following a retrospective study (J-HIS1) which had already finished. Patients, newly diagnosed after January 2007, were enrolled in J-HIS2 and followed up for inhibitor development and clinical environments since 2008 onward. In the present study, F8 genotypes of PwHA were investigated in the patients recruited from the J-HIS2 cohort as well as those with inhibitor from the J-HIS1 cohort. F8 variants identified in 59 PwHA with inhibitor in J-HIS1 were: 20 intron-22 inversions, 5 intron-1 inversions, 9 large deletions, 4 nonsense, 8 missense, 11 small in/del, and 2 splice-site variants. F8 variants identified in 267 (67 with inhibitor) PwHA in J-HIS2 were: 76(28) intron-22 inversions, 3(2) intron-1 inversion, 1(0) duplication, 8(5) large deletions, 21(7) nonsense, 109(7) missense, 40(11) small in/del, and 9(7) splice-site variants. Forty variants were novel. The cumulative inhibitor incidence rate in the severe group with null changes was 42.4% (95% confidence interval [CI]: 33.7-50.8), higher than that with nonnull changes (15.6% [95%CI: 6.8-27.8]), in J-HIS2. Relative risk for inhibitor development of null changes was 2.89. The spectrum of F8 genotype and genetic impact on inhibitor development in Japanese PwHA were consistent with the previous reports., Competing Interests: K.S. was an endowed assistant professor funded by CSL Behring. K.Y. teaches a course endowed by Takeda Co. Ltd. T.K. received research funding from Chugai Pharmaceutical Co., Ltd. and Bayer AG, and a lecture reward from Chugai Pharmaceutical Co., Ltd., Takeda Co. Ltd., Novo Nordisk A/S, Sanofi S.A., Bayer A/G, and Sysmex Corporation. K.N. has received grants from Takeda Co. Ltd. and Sanofi S.A., and research funding from Chugai Pharmaceutical Co., Ltd., F. Hoffmann-La Roche Ltd., Bayer A/G, Novo Nordisk A/S, KM Biologics, and also honoraria from Takeda Co. Ltd., Bayer A/G, Novo Nordisk A/S, Sanofi S.A., CSL Behring, Chugai Pharmaceutical Co., Ltd., F. Hoffmann-La Roche Ltd. outside the submitted work. M.T. has received research funding from Chugai Pharmaceutical Co., Ltd., F. Hoffmann-La Roche Ltd., Sanofi S.A., CSL Behring, and Novo Nordisk A/S and also honoraria from Chugai Pharmaceutical Co., Ltd., CSL Behring, Bayer AG, Takeda Co. Ltd., Sanofi S.A., and Novo Nordisk A/S outside the submitted work and listed as an entity's board of directors or advisory committee member for Chugai Pharmaceutical Co., Ltd., Novo Nordisk A/S, Sanofi S.A., and Bayer AG. K.F. has received grants and personal fees from Takeda Co. Ltd. (Baxalta/Shire), Bayer, Pfizer, CSL Behring, Novo Nordisk, Sanofi S.A. (Biogen/Bioverativ), KM Biologics (Kaketsuken) and Chugai Pharmaceutical Co. LTD, and grants from Japan Blood Products Organization, personal fees from SRL Inc., LSI Medience, Roche Diagnostics, Siemens, Sekisui Medical, Fujirebio Inc., Torii Pharmaceuticals, Octapharma, Sysmex, MSD, and other from CIMIC, outside the submitted work. A.Y. has received payment for a manuscript and consulting fee from Japan Blood Products Organization, and honoraria for lectures from Takeda Co., Ltd., Bayer A/G, and Chugai Pharmaceutical Co., Ltd. A.S. has no conflict of interest. M.S. received research funding from Chugai Pharmaceutical Co., Ltd., F. Hoffmann-La Roche Ltd., Sanofi S.A., Takeda Co. Ltd., CSL Behring, KM Biologics Co., Ltd., and Novo Nordisk A/S; and consulting fee from Chugai Pharmaceutical Co., Ltd.; and payment for lectures on speaker's bureau from Chugai Pharmaceutical Co., Ltd., Sanofi S.A., Bayer AG, and Sysmex corporation; and is listed as an entity's board of directors or advisory committee member for Chugai Pharmaceutical Co., Ltd., F. Hoffmann-La Roche Ltd., BioMarin Pharmaceutical Inc., Bayer AG, and Sanofi S.A.; and is an inventor of patents related to anti-FIXa/FX bispecific antibodies., (Thieme. All rights reserved.)

Background:  Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them., Methods:  The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential., Results:  Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays., Conclusion:  There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8 , and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA., Competing Interests: J.P.A. has received research grants from Shire, honoraria from Stago, Siemens, Sysmex, Roche, Baxter, Sobi, and acts on advisory boards for Sobi and Novo Nordisk. M.B. acts on the advisory board for CSL Behring, has had consultant assignments for Novo Nordisk, and received lecturer honoraria from Sobi., (Thieme. All rights reserved.)

Haemophilia is an X-linked genetic disorder in which A and B types are the most common that occur due to absence or lack of protein factors VIII and IX, respectively. Severity of the disease depends on mutation. Available Machine Learning (ML) methods that predict the mutational severity by using traditional encoding approaches, generally have high time complexity and compromised accuracy. In this study, Haemophilia 'A' patient mutation dataset containing 7784 mutations was processed by the proposed Position-Specific Mutation (PSM) and One-Hot Encoding (OHE) technique to predict the disease severity. The dataset processed by PSM and OHE methods was analyzed and trained for classification of mutation severity level using various ML algorithms. Surprisingly, PSM outperformed OHE, both in terms of time efficiency and accuracy, with training and prediction time improvement in the range of approximately 91 to 98% and 80 to 99% respectively. The severity prediction accuracy also improved by using PSM with different ML algorithms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Hemophilia A (HA) is an X-linked recessive bleeding disorder characterized by qualitative and quantitative deficiency of factor VIII (FVIII). The development of inhibitor antibodies against FVIII is the most challenging complication of treatment. Mutations in the FVIII gene is one of the genetic factors that leads to development of FVIII inhibitors especially intron 22 inversion (Inv22)., Objectives: This study was carried out to assess the frequency of Inv22 of FVIII gene in Egyptian patients with hemophilia A and its role as a risk factor for developing inhibitors., Patients and Methods: Seventy-two patients with severe HA and 48 patients with moderate HA were enrolled in the current study. All patients were treated on demand with either plasma-derived factor VIII or recombinant factor VIII concentrates. Genotyping of FVIII Inv22 was performed by LD-PCR while the presence and magnitude of inhibitor activity in blood was determined by the Bethesda assay., Results: Around 23% of all hemophilia cases had positive Inv22. Intron 22 inversion mutation was detected in 6 and 33% of patients with moderate and severe HA respectively. Twenty-one cases (18%) of all hemophilic patients developed inhibitors. Thirty-7% of patients with Inv22 had inhibitor in their blood, almost all, but one, had severe HA. The risk of an inhibitor development during replacement therapy was four folds higher among Inv22 positive cases as compared with mutation negative peers (OR 4.3, 95% CI 1.6-11.9, P = 0.003)., Conclusions: The prevalence of Inv22 of F VIII in Egyptian hemophiliacs is nearly like that of other population. This mutation was more frequently detected among severe hemophilic patients as compared with moderately affected peers. The presence of Inv22 mutation significantly predispose to FVIII inhibitor development.

Objective: Hemophilia A (HA) is the most severe X-linked inherited bleeding disorder caused by hemizygous mutations in the factor 8 (F8) gene. The aim of this study is to determine the mutation spectrum of the F8 gene in a large HA cohort from Turkey, and then to establish a phenotype-genotype correlation., Materials and Methods: All HA cases (270 patients) analyzed molecularly in the Ege University Pediatric Genetics Molecular Laboratory between March 2017 and March 2018 were included in this study. To identify intron 22 inversion (Inv22), intron 1 inversion (Inv1), small deletion/insertions, and point mutations, molecular analyses of F8 were performed using a sequential application of molecular techniques., Results: The mutation detection success rate was 95.2%. Positive Inv22 was found in 106 patients (39.3%), Inv1 was found in 4 patients (1.5%), and 106 different disease-causing sequence variants were identified in 137 patients (50.6%). In 10 patients (3.7%), amplification failures involving one or more exonic regions, considered to be large intragenic deletions, were identified. Of 106 different F8 mutations, 36 were novel. The relationship between F8 genotype and inhibitor development was considered significant., Conclusion: A high mutation detection rate was achieved via the broad molecular techniques applied in this study, including 36 novel mutations. With regard to mutation types, mutation distribution and their impact on clinical severity and inhibitor development were found to be similar to those previously reported in other hemophilia population studies.

Introduction: Molecular analysis in haemophilia is currently used in the diagnosis, treatment and prognosis of this disease. Hispanic populations in Latin America have been of interest to researchers due to the reportedly high prevalence of inhibitors in these patients., Aim: To perform next-generation sequencing (NGS) in a cohort of Mexican patients with HA and HB and correlate with clinical phenotypes., Methods: Patients with Haemophilia A (HA) or haemophilia B (HB), were evaluated using NGS with an Ion AmpliSeq Custom Panel. Odds ratios (ORs) for associations between F8 variants and inhibitors were obtained., Results: A total of 85 patients (60 with HA and 25 with HB) were included. Pathogenic variants in F8 were found in 93.3% of HA patients and in F9 in 96% of HB patients. Twelve novel potentially pathogenic variants were found. Inhibitors were observed in 20% of patients with severe HA. Four patients clinically diagnosed with HA were negative for F8 variants., Conclusion: Overall detection rate of pathogenic variants in F8 and F9 genes was 94.6%. We identified 12 non previously reported variants and pathogenic variants in other coagulation related genes. Molecular diagnosis of HA and HB permits better options for management, assessment and genetic counseling., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Hemophilia A (HA) is associated with mutations in the F8 gene that expresses factor VIII (FVIII). Unexpectedly, HA also manifests in a small subset of individuals with no mutations (exonic or intronic) in their F8 gene. MicroRNAs (miRNAs) cause translational interference, affecting protein quality and stoichiometry. Here, by analyzing miRNAs of two patients from this subset, we evaluated miRNA-based FVIII suppression as a testable hypothesis to explain FVIII deficiency in patients with HA with no F8 gene mutations., Study Design and Methods: To test the hypothesis, miRNA sequencing from two patients with mild and moderate HA with no mutations in their F8 gene, followed by experimental verification, was used to identify a group of upregulated miRNAs in patients with HA compared to normal controls; with binding sites in the 3' untranslated region (UTR) of F8 messenger RNA (mRNA), a prerequisite for miRNA-based gene regulation. From this pool, miR-374b-5p and miR-30c-5p, known to be expressed in human liver, where FVIII is expressed, were subjected to extensive characterization., Results: In two cell lines that constitutively express FVIII, we demonstrated that overexpression of miR-374b or miR-30c decreased FVIII expression, while an miR-30c inhibitor partially restored FVIII expression., Conclusion: These data support a role for microRNAs in fine-tuning F8 gene regulation. Based on our findings, our current model suggests that in HA cases where the F8 gene is normal and is predicted to express normal levels of FVIII, F8 mRNA 3' UTR targeting miRNAs may be responsible for a FVIII-deficiency phenotype clinically manifesting as HA., (© 2019 AABB.)

Compensated pathogenic deviations (CPDs) are sequence variants that are pathogenic in humans but neutral in other species. In recent years, our molecular understanding of CPDs has advanced substantially. For example, it is known that their impact on human proteins is generally milder than that of average pathogenic mutations and that their impact is suppressed in non-human carriers by compensatory mutations. However, prior studies have ignored the evolutionarily relevant relationship between molecular impact and organismal phenotype. Here, we explore this topic using CPDs from FVIII and FIX and data concerning carriers' hemophilia severity. We find that, regardless of their molecular impact, these mutations can be associated with either mild or severe disease phenotypes. Only a weak relationship is found between protein stability changes and severity. We also characterize the population variability of hemostasis proteins, which constitute the genetic background of FVIII and FIX, using data from the 1000 Genome project. We observe that genetic background can vary substantially between individuals in terms of both the amount and nature of genetic variants. Finally, we discuss how these results highlight the need to include new terms in present models of protein evolution to explain the origin of CPDs.

Background: In 10%-18% of mild-type hemophilia A (HA) patients, mutations cannot be found by routine DNA analysis., Objective: We aimed to identify the genetic defects by mRNA analysis of F8 gene in mild HA patients without mutation in exonic DNA., Patients and Methods: From 2006 to 2016, we identified F8 exon mutations in 39 of 49 mild HA patients using routine genetic testing. We then evaluated the 10 remaining patients from six unrelated families without exonic DNA mutation by performing cDNA sequence analysis., Results: Nine of the 10 (90%) patients were confirmed to have F8 gene mutation. Eight patients from four unrelated families were notably found to have presence of an aberrant 675-bp fragment. Sequencing of this fragment showed that there were two separate new alternative splicing exons of 35 bp and 55 bp within intron 18, which formed a 90-bp insertion between exon 18 and exon 19 (E18ins90bpE19) in the mRNA. Based on direct sequencing, this alternative splicing transcript appears to have resulted from deep intronic variant c.5999-277G>A of intron 18., Conclusions: Our study suggests that deep intronic variant of c.5999-277G>A may be a hot spot mutation for mild hemophilia patients without mutation in exonic DNA., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII) gene. Genome-editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty in preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Haemophilia A (HA), inherited via an X-linked recessive pattern, is the most common severe lifelong bleeding disorder caused by mutations in the coagulation factor VIII gene (F8). It has significant socio-economic effects due to its long course of disease and high cost of care. These impacts argue for a more accurate genetic diagnosis in an increasingly complex clinical environment., Methods: A three-generation Han-Chinese family with mild HA was recruited in the study. Exome sequencing was performed in the index case to detect potential disease-causing mutations, and Sanger sequencing was applied to verify the mutation in the family., Results: A hemizygous c.602G > A variant in the F8 gene, leading to a single amino acid substitution at codon 201 from glycine to glutamic acid (p.G201E) within the factor VIII (FVIII) A1 domain, was identified in the HA family. This mutation detected in the proband was found in his affected sibling, while it was absent in the unaffected family member and the two hundred ethnically-matched controls. The mutation affects an evolutionary conserved residue, which may impact the tertiary structure of FVIII., Conclusion: The study findings should provide for more dependable and precise genetic counseling which may assist in perfecting family management.

: Hemophilia A is a bleeding disorder caused by coagulation factor VIII protein deficiency or dysfunction, which is classified into severe, moderate, and mild according to factor clotting activity. An overwhelming majority of missense and nonsense mutations occur in exons of F8 gene, whereas mutations in introns can also be pathogenic. This study aimed to investigate the effect of an intronic mutation, c.6430-3C>G (IVS22-3C>G), on pre-mRNA splicing of the F8 gene. We applied DNA and cDNA sequencing in a Chinese boy with hemophilia A to search if any pathogenic mutation in the F8 gene. Functional analysis was performed to investigate the effect of an intronic mutation at the transcriptional level. Human Splicing Finder and PyMol were also used to predict its effect. We found the mutation c.6430-3C>G (IVS22-3C>G) in the F8 gene in the affected boy, with his mother being a carrier. cDNA from the mother and pSPL3 splicing assay showed that the mutation IVS22-3C>G results in a two-nucleotide AG inclusion at the 3' end of intron 22 and leads to a truncated coagulation factor VIII protein, with partial loss of the C1 domain and complete loss of the C2 domain. The in-silico tool predicted that the mutation induces altered pre-mRNA splicing by using a cryptic acceptor site in intron 22. The IVS22-3C>G mutation was confirmed to affect pre-mRNA splicing and produce a truncated protein, which reduces the stability of binding between the F8 protein and von Willebrand factor carrier protein due to the loss of an interaction domain.

Essentials A residual factor VIII synthesis is likely to be protective towards inhibitor (INH) development. Mutation type-inhibitor risk association was explored in 231 patients with severe hemophilia A. A 2-fold increase in INH development for in silico null vs. non-null mutations was found. A 3.5-fold increase in INH risk for antigen negative vs. antigen positive mutations was found., Summary: Background The type of F8 mutation is the main predictor of inhibitor development in patients with severe hemophilia A. Mutations expected to allow residual synthesis of factor VIII are likely to play a protective role against alloantibody development by inducing immune tolerance. According to the expected full or partial impairment of FVIII synthesis, F8 variants are commonly classified as null and non-null. Objectives To explore the mutation type-inhibitor risk association in a cohort of 231 patients with severe hemophilia A enrolled in the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) randomized trial. Methods The genetic defects in these patients, consisting of inversions of intron 22 (n = 110) and intron 1 (n = 6), large deletions (n = 16), and nonsense (n = 38), frameshift (n = 28), missense (n = 19) and splicing (n = 14) variants, of which 34 have been previously unreported, were reclassified according to two additional criteria: the functional effects of missense and splicing alterations as predicted by multiple in silico analyses, and the levels of FVIII antigen in patient plasma. Results A two-fold increase in inhibitor development for in silico null mutations as compared with in silico non-null mutations (hazard ratio [HR] 2.08, 95% confidence interval [CI] 0.84-5.17) and a 3.5-fold increase in inhibitor development for antigen-negative mutations as compared with antigen-positive mutations (HR 3.61, 95% CI 0.89-14.74] were found. Conclusions Our findings confirm an association between the synthesis of minute amounts of FVIII and inhibitor protection, and underline the importance of investigating the residual FVIII antigen levels associated with causative variants in order to understand their clinical relevance., (© 2018 International Society on Thrombosis and Haemostasis.)

Hemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the factor VIII gene ( F8). Our aim is to identify the causative mutations in a large HA cohort from China. We studied 216 unrelated HA families. Molecular analyses of F8 were performed using a combination of molecular techniques, including polymerase chain reaction, direct sequencing, and multiplex ligation-dependent probe amplification. The deleterious consequences of the unreported missense mutations were evaluated using various bioinformatics approaches. Causative mutations in F8 were identified in 209 families, intron 22 inversion (Inv22) was identified in 89 severe families, and intron 1 inversion (Inv1) was positive in 5 severe families; 95 mutations were detected among 115 noninversion families, of which 42 were novel, including 29 null variations and 13 missense mutations for which causality was demonstrated via bioinformatics. Among the 53 previously reported mutations, more nonsense (5 of 9) and missense (10 of 26) mutation sites were found to occur at Arginine (Arg) sites and multiple small deletions/insertions (5 of 10) located within the poly-A runs of the B domain. The majority of these sequence variants frequently recurred in the database. The odds ratios for the likelihood of developing inhibitors significantly increased in the presence of nonsense mutation. Our F8 defect spectrum was heterogeneous. Small deletions/insertions in the poly-A runs of the B domain and nonsense and missense mutations at Arg sites were identified as mutation hot spots. Nonsense mutation increased the risk of developing inhibitors.

Hemophilia A (HA) is an X‑linked recessive hereditary disorder caused by defects in the coagulation factor VIII (FVIII) gene. In order to diagnose patients with presymptomatic HA and carriers, the present study conducted direct gene diagnosis for the common abnormalities in FVIII and subsequently performed indirect gene diagnosis for the other abnormalities in FVIII for Chinese HA pedigrees. Direct gene diagnosis was performed in 10 HA pedigrees using inverse shifting‑polymerase chain reaction to detect intron 22 inversion (inv22), intron 22 deletion, intron 22 duplication and inv1 of FVIII. Pedigrees with no detected mutations were further analyzed using indirect genetic diagnosis (haplotype linkage analysis), where the genetic markers of FVIII included one variable number of tandem repeat, seven short tandem repeats and three restriction fragment length polymorphisms. The results of three pedigrees were taken as examples. Pedigree 1 underwent direct gene diagnosis, which demonstrated that the proband was inv22 distal pattern hemophiliac and the mother was an inv22 distal pattern carrier. The other two pedigrees were subjected to indirect gene diagnosis. In pedigree 2, the detection of DXS52, 13(CA) n, DXS9901(GT) n, intron (int)18, int19 and int22 confirmed the proband's baby brother was normal, the proband's maternal aunt was a carrier and her baby son was normal. Detection of DXS9901(GT)n, int18, int19 and int22 in pedigree 3 demonstrated that the proband's maternal grandmother was not a carrier. As the maternal grandfather was not affected by the disease, it was deduced that a mutation of FVIII occurred in the proband's mother. The combination of direct and indirect gene diagnoses provides reliable evidence for the use of genetic counseling in HA pedigrees, particularly for screening presymptomatic males and female carriers with normal offspring.

: Defects in the coagulation factor VIII gene cause haemophilia A, which is the most common X-linked recessive bleeding disorder. In total, 45 affected families were investigated to elucidate the factor VIII gene mutation spectrum. The families were subjected to clinical, biochemical, and molecular analyses. Inverse-shifting PCR was first applied to severe haemophilia A patients to identify inversions in introns 22 and 1. Then, next-generation sequencing was performed to detect mutations in inversion-negative patients with severe haemophilia A and moderate-mild haemophilia A patients. Finally, multiplex ligation-dependent probe amplification was utilized to identify rare cases with large fragment duplications or deletions in the factor VIII gene. In total, 41 mutations were identified, 19 of which (c.24C>A, c.49T>C, c.170_171delTT, c.533T>C, c.1126delG, c.1495delA, c.1660A>C, c.1736A>G, c.2711-2712insAATCT, c.3077C>G, c.3846delA, c.4238C>G, c.4349delG, c.4828G>C, c.5821A>C, c.6190C>T, c.6656T>C, c.6902T>G, and c.1904-2A>T) were novel and 80% (44/55) of the pathogenic mutations fell into the categories of missense (43.6%), nonsense (16.36%), frameshift (14.55%), and splice (5.45%) mutations. Additionally, 10 (18.18%) patients displayed inversions in intron 22 or 1 and one case (1.82%) exhibited a 3059-bp large fragment deletion in factor VIII. This study aimed to provide insight contributing to the genetic diagnosis of haemophilia A and to fill gaps in the factor VIII mutation spectrum in northern China.

Objective: To conduct genetic diagnosis for a family affected with hamophilia A., Methods: Potential mutations of the F8 gene were analyzed with PCR and Sanger sequencing. Carriers of the mutation were identified through linkage analysis using short tandem repeat (STR) markers. Suspected mutations were verified among 100 healthy controls to rule out genetic polymorphism. Prenatal diagnosis was provided based on the above results., Results: Sequencing analysis has identified two mutations, c.1 A>T and c.4 C>T, which have replaced the start codon (ATG) with leucine (TTG) and glutamine (GAA) with the stop codon (TAA), respectively. The same mutations were not found among the 100 healthy controls. The patient's mother and sister were heterozygous for the same mutations. Upon prenatal diagnosis, the fetus was determined as a male and did not harbor the above mutations. Linkage analysis also confirmed that the fetus has inherited the non-risk X chromosome from his maternal grandfather., Conclusion: Detection of pathogenic mutations can enable prenatal diagnosis for the disease.

Aims: The frequency of X chromosome pericentric inversion is much less than that of autosome chromosome. We hereby characterise a pericentric inversion of X chromosome associated with severe factor VIII (FVIII) deficiency in a sporadic haemophilia A (HA) pedigree., Methods: PCR primer walking and genome walking strategies were adopted to identify the exact breakpoints of the inversion. Copy number variations (CNVs) of the F8 and the whole chromosomes were detected by AccuCopy and Affymetrix CytoScan High Definition (HD) assays, respectively. A karyotype analysis was performed by cytogenetic G banding technique., Results: We identified a previously undescribed type of pericentric inversion of the X chromosome [inv(X)(p11.21q28)] in the proband with FVIII:C <1%. One breakpoint was located in the intron 7 of the F 8, which disrupted the transcription of the F8, and the other located in the upstream of the PFKFB1 of the X chromosome. The inversion segment was approximately 64.4% of the total chromosomal length. The karyotype analysis of the X chromosome confirmed the pericentric inversion of the X chromosome in the proband and his mother. A haplotype analysis traced the inversion to his maternal grandfather, who was not a somatic mosaic of the inversion. This finding indicated that the causative mutation may originate from his germ cells or a rare possibility of germ-cell mosaicism., Conclusions: The characterisation of pericentric inversion involving F8 extended the molecular mechanisms causing HA. The pericentric inversion rearrangement involves F8 by non-homologous end joining is responsible for pathogensis of severe HA., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The epidemiology of X-linked recessive diseases, a class of genetic disorders, is modeled with a discrete-time, structured, non linear mathematical system. The model accounts for both de novo mutations (i.e., affected sibling born to unaffected parents) and selection (i.e., distinct fitness rates depending on individual's health conditions). Assuming that the population is constant over generations and relying on Lyapunov theory we found the domain of attraction of model's equilibrium point and studied the convergence properties of the degenerate equilibrium where only affected individuals survive. Examples of applications of the proposed model to two among the most common X-linked recessive diseases (namely the red and green color blindness and the Hemophilia A) are described.

Introduction: As there is currently no complete cure for hemophilia A (HA), the identification of pathogenic mutations in factor VIII (FVIII) gene from HA patients and carriers, which can contribute to genetic counseling prenatal diagnosis, and preimplantation genetic diagnosis (PGD), is an important step to prevent HA., Methods: A total of 14 unrelated Chinese HA subjects (FVIII activity <40%), 20 carrier subjects, three fetuses, and one PGD were included in this study. We first screened for the presence of FVIII intron 22 and intron 1 inversions. Second, the coding region of the FVIII gene was sequenced. For the novel mutations, FVIII mRNA expression was detected by real-time PCR and the protein structures were analyzed by bioinformatic tools., Results: Five novel mutations (c.1A>C, c.304&#95;305insA, c.1594T>A, c.6045G>A, and c.2645&#95;2646insG) were found. The real-time PCR showed that the expression of FVIII mRNAs was lower in HA patients than in normal subjects. Prenatal diagnosis and PGD were successfully performed: Two of three fetuses and four of eight blastomeres were confirmed to be normal., Conclusion: In conclusion, genetic diagnosis of 14 unrelated HA subjects, 20 carrier subjects, three fetuses, and one PGD was successfully performed in our study., (© 2016 John Wiley & Sons Ltd.)

Essentials Factor VIII levels vary in mild and moderate hemophilia A (MHA) patients with the same mutation. We aimed to estimate the variation and determinants of factor VIII levels among MHA patients. Age and genotype explain 59% of the observed inter-individual variation in factor VIII levels. Intra-individual variation accounted for 45% of the variation in the three largest mutation groups., Summary: Background The bleeding phenotype in patients with mild/moderate hemophilia A (MHA) is inversely associated with the residual plasma concentration of factor VIII (FVIII:C). Within a group of patients with the same F8 missense mutation, baseline FVIII:C may vary, because, in healthy individuals, von Willebrand factor (VWF) levels, ABO blood group and age are also known to influence baseline FVIII:C. Our understanding of the pathophysiologic process of the causative genetic event leading to reduced baseline FVIII:C in MHA patients is still limited. Objectives To estimate the variation and determinants of baseline FVIII:C among MHA patients with the same F8 missense mutation. Methods Three hundred and forty-six patients carrying mutations that were present in at least 10 patients in the cohort were selected from the INSIGHT and the RISE studies, which are cohort studies including data of 3534 MHA patients from Europe, Canada, and Australia. Baseline FVIII:C was measured with a one-stage clotting assay. We used Levene's test, univariate and multivariate linear regression, and mixed-model analyses. Results For 59% of patients, the observed variation in baseline FVIII:C was explained by age and genotype. Compared to FVIII:C in patients with Arg612Cys, FVIII:C was significantly different in patients with eight other F8 missense mutations. Intra-individual variation explained 45% of the observed variance in baseline FVIII:C among patients with the same mutation. Conclusion Our results indicate that baseline FVIII:C levels are not exclusively determined by F8 genotype in MHA patients. Insights into other factors may provide potential novel targets for the treatment of MHA., (© 2016 International Society on Thrombosis and Haemostasis.)

Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

The molecular diagnosis of haemophilia A (HA) patients has many benefits including diagnosis confirmation and inhibitor risk development prediction. In female carries of a mutation, the molecular diagnosis allows for genetic counseling and prenatal diagnosis, which have become part of the comprehensive care for HA in many countries. Therefore, the aim of this study was to determine the F8 mutations in severe HA (sHA) patients and female carriers. In 12 patients with sHA, the presence of the intron 22 and intron 1 inversions was investigated using an inverse and a conventional PCR method, respectively. In patients negative for the inversions, the F8 gene was screened through conformation sensitive gel electrophoresis (CSGE) and further sequencing. The causative mutation was successfully identified in 6/12 patients, including the novel mutation p.G190C. The mothers of these six patients and those of seven other sHA patients molecularly diagnosed in a previous work were investigated for the presence of the genetic alterations found in their sons. All mothers were found to be carriers. This is the first study conducted in Venezuela which directly analyzes the F8 gene in potential carrier mothers to specifically identify the presence of the mutation that was detected in their sons, and complements a previous study on sHA patients. Our findings will facilitate the implementation of regular screening of HA carriers in our country and will allow a better care of bleeding symptoms and genetic counseling.

Unlabelled: Many newly diagnosed Swedish severe haemophilia A (HA) patients are sporadic cases. Some genotypically non-carrier mothers have gone on to have two descendants with the same mutation, presumably because of mosaicism., Aims: To define the origin of mutation in sporadic cases of HA, reveal possible sex-specific differences in mutagenesis and identify potential mosaics among non-carrier mothers., Method: Sanger sequencing characterized the mutations and microsatellite haplotyping determined the origin of the X chromosome carrying the mutation in 3 generations of 45 families with sporadic severe HA. Droplet digital polymerase chain reaction (ddPCR) was used in five cases to reveal that mosaicism mutations are not found on conventional DNA sequencing., Results: In 23 out of 45 families, the mother carried the mutation and in 5 out of 28 families, the grandmother was also a carrier. The X chromosome was of grandpaternal origin in 17 out of 23 cases. One of five tested mothers was a mosaic with a mutation frequency of 7%., Conclusion: In 40 out of 45 families, the sporadic case resulted from a mutation in the last two generations. In 82% (23/28), the carrier mothers had a de novo mutation where the X chromosome was of paternal origin in 74% (17/23). ddPCR is a potentially powerful and promising analysis for mosaicism in HA., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most problematic and costly complication of FVIII replacement therapy that affects up to 30% of previously untreated patients with severe hemophilia A. The development of inhibitors is a multifactorial complication involving environmental and genetic factors. Among the latter, F8 gene mutations, ethnicity, family history of inhibitors, and polymorphisms affecting genes involved in the immune response have been previously investigated. To identify novel genetic elements underling the risk of inhibitor development in patients with severe hemophilia A, we applied whole-exome sequencing (WES) and data analysis in a selected group of 26 Italian patients with (n = 17) and without (n = 9) inhibitors. WES revealed several rare, damaging variants in immunoregulatory genes as novel candidate mutations. A case-control association analysis using Cochran-Armitage and Fisher's exact statistical tests identified 1364 statistically significant variants. Hierarchical clustering of these genetic variants showed 2 distinct patterns of homozygous variants with a protective or harmful role in inhibitor development. When looking solely at coding variants, a total of 28 nonsynonymous variants were identified and replicated in 53 inhibitor-positive and 174 inhibitor-negative Italian severe hemophilia A patients using a TaqMan genotyping assay. The genotyping results revealed 10 variants showing estimated odds ratios in the same direction as in the discovery phase and confirmed the association of the rs3754689 missense variant (OR 0.58; 95% CI 0.36-0.94; P = .028) in a highly conserved haplotype region surrounding the LCT locus on chromosome 2q21 with inhibitor development., (© 2016 by The American Society of Hematology.)

Current screening methods for factor VIII gene (F8) mutations can reveal the causative alteration in the vast majority of haemophilia A patients. Yet, standard diagnostic methods fail in about 2% of cases. This study aimed at analysing the entire intronic sequences of the F8 gene in 15 haemophilia A patients by next generation sequencing. All patients had a mild to moderate phenotype and no mutation in the coding sequence and splice sites of the F8 gene could be diagnosed so far. Next generation sequencing data revealed 23 deep intronic candidate variants in several F8 introns, including six recurrent variants and three variants that have been described before. One patient additionally showed a deletion of 9.2 kb in intron 1, mediated by Alu-type repeats. Several bioinformatic tools were used to score the variants in comparison to known pathogenic F8 mutations in order to predict their deleteriousness. Pedigree analyses showed a correct segregation pattern for three of the presumptive mutations. In each of the 15 patients analysed, at least one deep intronic variant in the F8 gene was identified and predicted to alter F8 mRNA splicing. Reduced F8 mRNA levels and/or stability would be well compatible with the patients' mild to moderate haemophilia A phenotypes. The next generation sequencing approach used proved an efficient method to screen the complete F8 gene and could be applied as a one-stop sequencing method for molecular diagnostics of haemophilia A.

Background: Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in factor VIII (FVIII). von Willebrand disease (VWD) is characterized by a quantitative or qualitative defect in von Willebrand factor (VWF). Patients with VWD with severely low VWF or VWD Type 2N (VWD2N), a VWD subtype distinguished by defective VWF binding to FVIII, may have reduced FVIII levels secondary to their VWD. These patients superficially resemble patients with HA and pose a potential for misdiagnosis., Objectives: To investigate the unexplained cause of bleeding in HA patients without known FVIII mutations by assessing plasma VWF antigen (VWF:Ag), FVIII binding capacities and VWF genotypes., Patients/methods: Thirty-seven of 1027 patients with HA studied as part of the Hemophilia Inhibitor Research Study lacked identifiable F8 mutations. These patients (cases) and 73 patients with identified F8 mutations (controls) were evaluated for VWF:Ag, a patient's VWF capacity to bind FVIII (VWF:FVIIIB) and VWF sequence., Results: Four cases had VWF:Ag < 3 IU dL(-1) and VWF mutations consistent with Type 3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type 1 VWD (VWD1) (n = five cases and one control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n = 1 case). One control had VWF:Ag < 30 IU dL(-1) and seven patients (four cases and three controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB., Conclusions: These data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy., (© 2015 International Society on Thrombosis and Haemostasis.)

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 (<0.50 IU mL(-1) ), F8 gene variations and without family history of HA. Except p.Ser577Tyr, F8 gene variations that we reported have never been described (p.Leu107His, p.Pro521Leu, p.Val682Leu, p.Leu2032Pro, p.Ala315dup). Prediction of their potential causal impact was studied by two strategies: bioinformatics approaches and site-directed mutagenesis followed by FVIII cellular expression into COS-1 cell. FVIII clotting assay (, Fviii: C) and antigen (, Fviii: Ag) were assayed in vitro. In silico analysis showed the probably damaging effect of all substitutions and the full conservation of the residues across mammalian species, except for p.Leu2032Pro. The in vitro variant expression model showed abnormal intra and/or extracellular, Fviii: C and, Fviii: Ag levels for five mutations, which suggest their causality in HA and provide informations about the involved mechanism. We suspect a defect in synthesis and secretion for p.Leu107His, p.Ala315dup and p.Pro521Leu. The mutation p.Val682Leu only affects the FVIII function while p.Ser577Tyr alters function and synthesis. The variant p.Leu2032Pro is probably a polymorphism because no alteration of the FVIII protein expression was observed in vitro. In vitro results suggest that mutations p.Ser577Tyr and p.Ala315dup could led to a severe HA in men. This study demonstrates the ability of this in vitro cellular expression model to contribute to the diagnosis strategy for female suspected of being HA carrier, without HA family history and with a novel F8 gene variation and to provide new criteria for the genetic counselling., (© 2015 John Wiley & Sons Ltd.)

Background: The recessive X-linked disorder hemophilia A (HA) is rarely expressed in female carriers, most of whom express about half of normal factor VIII activity (, Fviii: C)., Objective: To propose an integrative assessment model for the binary role of the phase between the mutated F8 and the active X-chromosome (Xa) in, Fviii: C in HA carriers., Methods: We studied 67 females at risk of severe HA, comprising five symptomatic females (, Fviii: C < 1.5 IU dL(-1) ) and 14 controls. A correlation study between, Fviii: C (observed vs. expected) and X-chromosome inactivation (XCI) patterns (XIPs; androgen receptor gene [AR] system) in blood leukocyte DNA was performed in carriers, by comparison of a model correlating, Fviii: C and XIP with arbitrary models devoid of biological significance, and with, Fviii: C levels in non-carriers (mean model) as a proxy from background data dispersion not influenced by XIP., Results: We provide proof-of-concept example from a family presenting with extremely skewed XIPs in which the severe HA phenotype appeared in a heterozygous carrier of a crossover between AR and F8 loci that phased the mutated F8 with the maternally inherited Xa. Furthermore, four cases of severe HA affected women who had a combination of a heterozygous F8 mutation and extremely skewed XIPs in leukocytes or oral mucosa are presented. Correlation analyses between, Fviii: C levels and XIPs in carriers (n = 38) but not in non-carriers (n = 20) showed highly significant differences between the proposed correlation model and models without biological significance. The data support a binary influence of XCI, either increasing or decreasing the, Fviii: C, subject to the underlying phase set between the F8 mutation and XCI., Conclusions: Our evidence suggests that the phase between XCI and mutated F8 acts as a molecular switch conditioning, Fviii: C levels and HA expression in carriers., (© 2015 International Society on Thrombosis and Haemostasis.)

Mutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5&#95;1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1&#95;601+5del5 and c.1444-8&#95;1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %, 14.2 %, 5.2 %, 19.2 %, 1.8 % and 2.5 % of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.

Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making., (© 2015 John Wiley & Sons Ltd.)

About 10% of mutations in haemophilia A cases generate a premature termination codon in the factor VIII gene (F8). Upon therapeutic FVIII substitution, it was noted that the risk of developing inhibitors is higher when the nonsense mutation is located in the light chain (LC) of the factor VIII (FVIII) protein than in the heavy chain (HC). We analysed the impact of six different nonsense mutations distributed over the six FVIII domains on recombinant FVIII expression to elucidate the process of inhibitor formation in haemophilic patients. Full-length F8 mRNA was transcribed from all constructs despite the presence of nonsense mutations. Polyclonal antigen assays revealed high antigen levels in transfection experiments with constructs truncated in LC whereas low antigen was detected from constructs truncated in HC. Those results were supported by FVIII localization experiments. These findings suggest that F8 transcription occurs in a usual way despite nonsense mutations, whereas translation appears to be interrupted by the premature stop codon. We hypothesize that the inclusion of the B domain enables proteins truncated in LC to accumulate in the ER. Proteins truncated in HC are mainly degraded or may pass through the ER and be secreted into the blood circulation, thus presumably preventing inhibitor formation after therapeutic FVIII substitution. The LC is known to have higher immunogenicity than the HC. Moreover, translation of the F8B gene comprising F8 exons 23-26 may be dependent on the position of the premature stop codon and thus contributes to the immune response of truncated FVIII proteins., (© 2014 John Wiley & Sons Ltd.)

Objective: The present cohort study was performed to investigate the impact of the factor 5 rs6025 [F5] and the factor 2 rs1799963 [F2] mutations on high-titer inhibitor development [HRI] in patients with severe/moderate-severe hemophilia A [HA]., Patients and Methods: 216 patients with F8<2% born between 1980 and 2011 were followed after initial HA diagnosis over the first 200 exposure days. The first HA patient per family who presented for diagnosis was included in the present study., Results: 32 of 216 children [14.8%] tested for F5/F2 carried either the F5 or the F2 variant. HRI occurred in 14 out of 32F5/F2-carriers compared with 40 of 184 without F5/F2. Multivariate analysis adjusted for F8 genotype, treatment intensity, first-line use of plasma derived FVIII versus recombinant FVIII concentrates revealed that the presence of F5/F2 independently increases the risk of HRI development to odds [OR] of 3.4. Large deletions in the F8 gene [OR: 5.10], patients from Israel [OR: 4.0], the increase of FVIII per one IU/kgbw [OR: 1.05] and birth year [OR: 1.12] were significantly associated with the risk to develop HRI., Conclusion: Data presented here suggest that HRI development is of multifactorial origin and that F5 and F2 mutations may contribute to this risk., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)