Novel Severe Hemophilia a Mouse Model with Factor VIII Intron 22 Inversion

Hemophilia A (HA) is an X-linked recessive blood coagulation disorder, and approximately 50% of severe HA patients are caused by F8 intron 22 inversion (F8I22I). However, the F8I22I mouse model has not been developed despite being a necessary model to challenge pre-clinical study. A mouse model similar to human F8I22I was developed through consequent inversion by CRISPR/Cas9-based dual double-stranded breakage (DSB) formation, and clinical symptoms of severe hemophilia were confirmed. The F8I22I mouse showed inversion of a 391 kb segment and truncation of mRNA transcription at the F8 gene. Furthermore, the F8I22I mouse showed a deficiency of FVIII activity (10.9 vs. 0 ng/mL in WT and F8I22I, p < 0.0001) and severe coagulation disorder phenotype in the activated partial thromboplastin time (38 vs. 480 s, p < 0.0001), in vivo bleeding test (blood loss/body weight; 0.4 vs. 2.1%, p < 0.0001), and calibrated automated thrombogram assays (Thrombin generation peak, 183 vs. 21.5 nM, p = 0.0012). Moreover, histological changes related to spontaneous bleeding were observed in the liver, spleen, and lungs. We present a novel HA mouse model mimicking human F8I22I. With a structural similarity with human F8I22I, the F8I22I mouse model will be applicable to the evaluation of general hemophilia drugs and the development of gene-editing-based therapy research.

HLA Variants and Inhibitor Development in Hemophilia A: A Retrospective Case-Controlled Study Using the ATHNdataset

In hemophilia A (HA) patients, F8 gene-defects as genetic risk-factors for developing inhibitors to Factor VIII have been extensively studied. Here we provide estimates of inhibitor-risk associated with the patient's Human Leukocyte Antigen (HLA). We used next generation sequencing for high-resolution HLA Class II typing of 997 HA patients. Using inhibitor prevalence reports from the My Life Our Future (MLOF) research repository, we calculated Odds Ratios (OR) for inhibitor development in a multivariate model considering HLA-DRB1/3/4/5, HLA-DPB1, HLA-DQB1, race, F8 pathogenic variant type, and age. Participants with 1 HLA variant (DPB1*02:02) had developed inhibitors at a higher rate while participants with 2 HLA variants (DRB1*04:07; DRB1*11:04) had developed inhibitors at a lower rate. Additionally, patients with missense variants had developed inhibitors at a lower rate and participants with large structural changes (&gt;50 bp) had developed inhibitors at a higher rate (both compared to Intron 22 inversion). Using a cohort of participants with a distribution of HLA-DRB1 alleles comparable to that in the North American population we show that the HLA repertoire of a HA patient can be a risk-factor for inhibitor development.

Real Time PCR; A new, fast and accurate technique in diagnosing the severe type of hemophilia A patients

Introduction: Hemophilia A (HA) is one of the most deleterious X-linked bleeding disorders in male patients. Early and rapid detection could provide management strategies for this disease. This study aims to improve the classic method for detection of important intron 22 inversion mutations (INV22). Materials and Methods: Whole blood samples were taken from 21 male children with a history of severe HA, referred to the Iranian comprehensive hemophilia care center. Two groups were involved for detection of INV22 and two methods were examined in a double-blind manner. The first method used a classical method, inverse PCR, and the second method was Real-time inverse PCR. Results: Results showed that both methods could separately detect the INV22 in 11 out of 21 patients with severe HA (52%) in the same accuracy, but with this difference that only one day was needed for detecting of INV22 by Real-time inverse PCR and on other hand 3 days were needed for the classical methods. Concusion: Because of the medical importance of early detection or prenatal/postnatal diagnosis of this disease, this study recommends the Real-time inverse PCR technique for the rapid detection of this mutation in well-equipped genetic laboratories everywhere.

Factor VIII Intron 22 Inversion in Severe Hemophilia A Patients in Palestine

Background. Hemophilia A is an X-linked recessive bleeding disorder caused by mutations in FVIII gene with an incidence of 1 in 5,000 to 10,000 live born males. The Inv22 mutation is a major cause of the disease worldwide, accounting for up to 40%–50% of severe FVIII mutations. The aim of the present study was to screen Inv22 of the FVIII gene in Palestinian patients with severe HA and reveal its role as a predisposing factor for the development of inhibitors. Materials and Methods. A cohort of 77 HA individuals including 5 carrier females from 52 unrelated families registered at governmental hemophilia centers in the West Bank area of Palestine was investigated. The demographic data and the clinical history were retrieved from medical files. Molecular analysis of Inv22 mutation in severe HA (30 cases) from Palestine was performed using the subcycling polymerase reaction (S-PCR). FVIII coagulant activities were carried out on an aPTT-based 1-stage clotting assay. FVIII inhibitors were quantified using the Nijmegen modification of the Bethesda assay. Result. Overall, 41.7% (30/72) of the studied cases were classified as having severe HA, 22.2% (16/72) had moderate HA, and 36.1% (26/72) had mild HA. Five randomly selected carrier mothers were screened for the Inv22 mutation to confirm its transmission to their sons. The Inv22 mutation was detected in 11 severe HA patients (36.6%). Among the severe HA patients with positive Inv22, 45.5% (5/11) had developed inhibitors. The current study showed that there was no association (p=0.53) between inhibitor development and the Inv22 mutation. Conclusion. Findings on Inv22 are in agreement with worldwide reports, being a major genetic mutation in severe HA. The S-PCR is a simple, rapid, and cost-effective method for the diagnosis of Inv22 in severe HA patients. Although the Inv22 mutation was associated with 36.6% of severe HA phenotype cases, it was not a major predisposing factor for inhibitor formation.

Factor VIII inhibitor development in Egyptian hemophilia patients: does intron 22 inversion mutation play a role?

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.

FVIII Protein Is Not Detectable in Human PBMCs or Livers from Dogs with an Intron-22 Inversion Mutation: Implications for FVIII Immunogenicity and Tolerance

A clinically relevant question is whether partial FVIII proteins are expressed in tissues from patients with severe hemophilia A (HA) due to an intron-22 inversion mutation. If so, this could in principle confer central immune tolerance to the expressed FVIII regions, thereby lowering the risk of anti-FVIII immune responses. In 2013, Pandey et al. reported detection of FVIII proteins in peripheral blood mononuclear cells (PBMCs), B cells and liver tissues from an intron-22 inversion subject and non-HA controls. They concluded that partial FVIII proteins were translated from inverted mRNA encoding F8 exons 1-22, and also from the F8B transcript, which contains F8 exons 23-26 (Nature Medicine 19(10), 1318-24). In 2014, the Montgomery and Ginsburg labs reported that FVIII protein is expressed principally, and possibly exclusively, in endothelial cells (ECs). These studies utilized mouse models with EC-directed deletion of F8 exons 17-18, or of the FVIII transporter protein LMAN1, respectively. The F8-EC-knockout mice had a severe hemophilia A (HA) phenotype. Earlier studies from several labs, including Pandey et al., relying primarily on antibody staining, had reported FVIII expression in additional cell types, including PBMCs and hepatocytes. The present study tests the hypothesis that partial FVIII proteins are expressed from inverted mRNA encoding F8 exons 1-22 and/or from F8B mRNA. A panel of FVIII-specific polyclonal and monoclonal antibodies (mAbs) was tested for FVIII specificity by staining permeabilized PBMCs, monocytes, T cells, blood outgrowth ECs, monocyte-derived macrophages and dendritic cells, HUVECs and B-cell lines. Positive and negative controls included FVIII-expressing BHK cells and non-engineered BHK cells. Specificity was further tested by immunoprecipitation (IP) of cell lysates followed by SDS-PAGE, Western blots and mass spectrometry to define proteins pulled down by the anti-FVIII antibodies. In addition, immunohistochemistry (IHC) staining was carried out for liver sections from HA dogs with an intron-22 inversion mutation and from otherwise normal (non-HA) control dogs. IP followed by SDS-PAGE, Westerns and/or mass spec revealed that a significant fraction of the anti-FVIII antibodies bound to other proteins besides FVIII in the tissues and cells examined. A cocktail of 4 anti-FVIII mAbs that were validated using (+) and (-) control samples was used to stain isolated and cultured cells and tissues. Cross-recognition of canine FVIII was confirmed by IP + Western blots. IHC using the mAb cocktail followed by HRP-conjugated secondary antibodies produced variable staining of liver tissues from HA and non-HA dogs, regardless of tissue preparation methods. However, use of fluorescent-labeled secondary mAbs produced signals well above background in ECs of control dog livers but not in the HA livers. Multiple confocal images were selected randomly, and average MFI values of 10 non-HA liver sections were well above those of 10 HA sections (p = 0.006). A second staining method, the Duolink Proximity Ligation Assay, was employed as an independent test to detect interactions between FVIII and VWF. Again, only normal control liver sections showed fluorescence above background (p =0.0004). F8B mRNA was not detected in canine liver and was not expected based on the canine intron-22 DNA sequence. We conclude that if protein is expressed from inverted mRNA encoding F8 exons 1-22, it is below the detection limit of these assays. Permeabilized human cells were tested for FVIII expression by staining using the mAb cocktail, or validated anti-FVIII-C2 mAbs to detect the putative protein encoded by F8B, followed by fluorescent detection. Cells were also stimulated with histamine followed by a chromogenic FVIII activity assay of supernatants. Results indicated that if FVIII or F8B protein are expressed in non-ECs, they are below the detection limit of these assays. We also note that if the putative F8B-encoded protein confers tolerance, immune responses of HA patients to the FVIII C2 domain would be quite rare, which is not the case. Although neonatal thymic expression of partial FVIII proteins may occur, immune tolerance to self-antigens requires repeated exposures of the immune system to the antigens. We conclude it is unlikely that HA patients with an intron-22 inversion mutation have universally acquired central tolerance to partial proteins encoded by inverted F8 mRNA or F8B. Disclosures Ragni: Shire/Takeda: Consultancy, Other: Study drug; Sangamo: Research Funding; OPKO: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; ICER: Consultancy; Biomarin: Consultancy, Research Funding; Alnylam/Sanofi: Consultancy, Research Funding; Spark Therapeutics: Consultancy, Research Funding. Pratt:Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding.

Severe haemophilia A in a neonate presenting as haemopneumothorax after tracheo-oesophageal fistula-oesophageal atresia repair

A male infant with oesophageal atresia and distal tracheo-oesophageal fistula (TEF type C) underwent right thoracotomy and transpleural repair of TEF on day 4 of life. He did not have a family history of coagulation disorders. A preoperative finding of prolonged partial thromboplastin time (PTT)>200 s was overlooked, and he went to surgery. There were no concerns with haemostasis prior to and even during the operation. The prolonged PTT was treated with one 10 mL/kg dose of fresh frozen plasma in the immediate postoperative period. On the fourth postoperative day, the infant developed a right haemopneumothorax, requiring fresh frozen plasma and packed cell transfusions. He was subsequently diagnosed with severe haemophilia A due to intron 22 inversion in the factor VIII gene, with factor VIII level <0.01 IU/mL.

MUTATIONAL PROFILES OF F8 AND F9 IN A COHORT OF HAEMOPHILIA A AND HAEMOPHILIA B PATIENTS IN THE MULTI-ETHNIC MALAYSIAN POPULATION

Background: Haemophilia A (HA) and Haemophilia B (HB) are X-linked blood disorders that are caused by various mutations in the factor VIII (F8) and factor IX (F9) genes respectively. Identification of mutations is essential as some of the mutations are associated with the development of inhibitors. This study is the first comprehensive study of the F8 mutational profile in Malaysia.Materials and methods: We analysed 100 unrelated HA and 15 unrelated HB patients for genetic alterations in the F8 and F9 genes by using the long-range PCR, DNA sequencing, and the multiplex-ligation-dependent probe amplification assays. The prediction software was used to confirm the effects of these mutations on factor VIII and IX proteins.Results: 44 (53%) of the severe HA patients were positive for F8 intron 22 inversion, and three (3.6%) were positive for intron 1 inversion. There were 22 novel mutations in F8, including missense (8), frameshift (9), splice site (3), large deletion (1) and nonsense (1) mutations. In HB patients, four novel mutations were identified including the splice site (1), small deletion (1), large deletion (1) and missense (1) mutation.Discussion: The mutational spectrum of F8 in Malaysian patients is heterogeneous, with a slightly higher frequency of intron 22 inversion in these severe HA patients when compared to other Asian populations. Identification of these mutational profiles in F8 and F9 genes among Malaysian patients will provide a useful reference for the early detection and diagnosis of HA and HB in the Malaysian population.