Factor V Research Articles

Association evaluations of oral anticoagulants with dementia risk based on genomic and real-world data

BackgroundSeveral observational studies have suggested that oral anticoagulants (OACs) might reduce the risk of dementia in the elderly, but the evidence is inconclusive. And the consistency of this relationship across different OAC classes and dementia subtypes is still uncertain. MethodsTo comprehensively evaluate this association, we applied Mendelian randomization (MR) combined with pharmacovigilance analysis. MR was used to assess the associations between genetic proxies for three target genes of OACs (VKORC1, F2, and F10) and dementia, including Alzheimer's disease (AD) and vascular dementia (VaD). This genetic analysis was supplemented with real-world pharmacovigilance data, employing disproportionality analysis for more reliable causal inference. ResultsIncreased expression of the VKORC1 gene was strongly associated with increased risk of dementia, especially for AD (OR = 1.28, 95% CI = 1.14–1.43; p value < 0.001). Based on pharmacovigilance data, vitamin K antagonists (VKAs, inhibitors targeting VKORC1) exhibited a protective effect against dementia risk (ROR = 0.43, 95% CI = 0.28–0.67). Additional sensitivity analyses, including different MR models and cohorts, supported these results. Conversely, no strong causal associations of genetically proxied F2 and F10 target genes with dementia and its subtypes were found. ConclusionsThis study reveals that the inhibition of genetically proxied VKORC1 expression or VKAs exposure is associated with a reduced risk of Alzheimer's dementia. However, there is little evidence to support similar associations with direct oral anticoagulants (F2 inhibitors and F10 inhibitors). Further research is warranted to clinically validate our findings.

Leiden mutation (rs6025) in a severe COVID-19 pneumonia patient with Down syndrome: a clinical case

COVID-19 was first reported in December 2019 in Wuhan (Hubei Province, China). Later, the pandemic of this disease took the world by storm, challenging the medical community. Its clinical manifestations vary from asymptomatic to a severe course that requires hospitalization and intensive therapy with oxygen support. The mortality rate in patients with a severe course of COVID-19 can exceed 50% The majority of fatal cases of COVID-19 were associated with thrombotic events, despite the prophylactic use of anticoagulant therapy. Numerous theoretical overviews and research articles indicate the need for genetic testing in patients with COVID-19 to determine the genetic profile of proteins involved in thrombophilia. According to the researchers, the Leiden mutation (G1691A, rs6025) of the FV gene is one of the promising candidates for testing. The aim of the work was to demonstrate the clinical features of the severe course of COVID-19 in the presence of the Leiden mutation. 58 patients with COVID-19 of the intensive care unit were genotyped. The Leiden mutation in the heterozygous state was found only in one patient, who had Down syndrome. The Leiden mutation was detected with a frequency of 1.72% in the investigated group. The described clinical case clearly showed that individuals with Down syndrome, associated with hereditary thrombophilia are at risk of undesirable clinical consequences in the treatment of COVID-19. The condition of the patient with the Leiden mutation was severe when admitted to the hospital. The score according to the sequential organ failure assessment scale was 2 points. Bilateral multisegmental pneumonia was detected on the X-ray. On the second day after admission, due to the development of acute respiratory distress syndrome and multiple organ failure, the patient was transferred to the intensive care unit, where he received oxygen therapy through a facial mask. Medical treatment was carried out according to the protocol: non-steroidal anti-inflammatory drugs, antibacterial therapy, anticoagulants, sympatho¬mimetics, and glucocorticosteroids. Despite the medical measures taken, progression of respiratory failure, renal failure, and portal hypertension was noted. On the 11th day, the patient developed asystole. Resuscitation measures were unsuccessful. Thus, the described case of a severe course of COVID-19 in a carrier of a heterozygous variant of the Leiden mutation with Down syndrome confirms the recommendations regarding the need for genetic testing for thrombophilia in high-risk groups and the appointment of personalized measures to prevent complications.

Variant spectrum of F8 and F9 in hemophilia patients from southern China and 26 novel variants.

Hemophilia, an X-linked recessive disorder, is characterized by spontaneous or trauma-induced prolonged bleeding. It is classified as hemophilia A when caused by variants in the F8 gene, and hemophilia B when caused by F9 variants. Few studies have described hemophilia variants in the Chinese population. This study aimed to investigate the clinical and genetic profiles of 193 hemophilia patients from southern China. Utilizing Sanger sequencing, multiplex ligation-dependent probe amplification, gap detection, long-range PCR, and multiplex PCR, we identified both F8 and F9 gene variants. Pregnant women with a history of hemophilia A offspring underwent amniocentesis or villus sampling for the variant detection. Variants in F8 and F9 were pinpointed in 183 patients, with 26 being novel discoveries. Notably, genetic testing was absent in the initial evaluation of 133 out of 161 patients, leading to a protracted average definitive diagnosis timeline of 2years. Remarkably, two hemophilia A cases with anticipated severe phenotypes due to protein-truncating variants presented with only moderate or mild clinical manifestations. Among the 40 fetuses tested, 34 were males, with 17 exhibiting hemizygous variants in the F8 gene. Our results contribute to the broader understanding of F8 and F9 variant spectrum and highlight the underuse of genetic analyses in southern China.

Genotype-Phenotype Relationship among 785 Unrelated White Women with Inherited Congenital Factor VII Deficiency: A Three-Center Database Study.

Congenital factor VII (FVII) deficiency, a rare bleeding disorder resulting from mutations in the F7 gene with autosomal recessive inheritance, exhibits clinical heterogeneity that lacks a strong correlation with FVII:C levels. The objective of this study was to discern genetic defects and assess their associations with the clinical phenotype in a substantial cohort comprising 785 white women exhibiting FVII:C levels below the age-dependent cut-off percentage. Individuals with verified inherited factor VII deficiency underwent i) genotyping using the Sanger method and multiplex ligation-dependent probe amplification (MLPA) to identify F7 mutations, including common polymorphic variants. Additionally, they were ii) categorized based on clinical bleeding scores (BS). Thrombophilic variants and blood groups were also determined in the study participants. The probands in this study encompassed both asymptomatic individuals (referred for a laboratory investigation due to recurrent prolonged prothrombin time; n = 221) and patients who manifested mild, moderate, or severe bleeding episodes (n = 564). The spectrum of bleeding symptoms included epistaxis, gum bleeding, gastrointestinal bleeding, hematuria, postoperative bleeding, and gynecologic hemorrhage. The median ISTH bleeding score (BS) recorded within a two-year period prior to the work-up was 2 (0-17). Notably, this score was significantly higher in symptomatic women compared to their asymptomatic counterparts (3 versus 0; p < 0.001). The corresponding PBAC score before hormonal treatment stood at 225 (5-1200), exhibiting a positive correlation with the ISTH BS (rho = 0.38; p = 0.001). Blood group O was more prevalent in symptomatic women compared to asymptomatic individuals (58 versus 42%; p = 0.01). Among the 329 women (42%), known and novel mutations in the F7 gene, encompassing coding regions, exon/intron boundaries, and the promoter region, were identified, while common polymorphisms were detected in 647 subjects (95%). Logistic regression analysis, adjusted for clinical and laboratory data (including blood group, FVII activity, the presence of F7 gene mutations and/or polymorphisms, thrombophilia status, and additional factor deficiencies) revealed that older age at referral (increase per year) (odds/95% CI: 1.02/1.007-1.03), the presence of blood group O (odds/95% CI: 1.9/1.2-3.3), and the coexistence of further bleeding defects (odds/95% CI: 1.8/1.03-3.1) partially account for the differences in the clinical bleeding phenotype associated with FVII deficiency. The clinical phenotype in individuals with FVII deficiency is impacted by factors such as age, blood group, and the concurrent presence of other bleeding defects.

Analysis of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ deficiency due to compound heterozygous variants of F12 gene

To analyze the clinical phenotypes and genetic variants of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. A pedigree presented at the First Affiliated Hospital of Air Force Medical University on December 24,2021 was selected as the study subject. Activated partial thromboplastin time (APTT) and coagulation factor Ⅻ activity (FⅫ:C) were determine by a clotting method, and FⅫ antigen was detected with an ELISA assay. Following the extraction of genomic DNA, all exons and flanking regions of the F12 gene were subjected to Sanger sequencing. Clustalx-2.1-win, PROVEAN and Swiss-PDB Viewer software was used to analyze the conservation of amino acids at the variant sites, impact of of the variants on the amino acid substitutions and the protein structure. The APTT of the proband has prolonged to 70.2 s. Her FⅫ:C and FⅫ:Ag have decreased to 12% and 13%, respectively. DNA sequencing revealed that the proband has harbored c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) heterozygous compound missense variants in exons 5 and 13 of the F12 gene, respectively. Her father and sister were heterozygous carriers for the c.346G>A (p.Gly97Ser) variant, whilst her mother and brother were heterozygous for the c.1583C>A (p.Ser509Tyr) variant. The c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) compound heterozygous variants of the F12 gene probably underlay the pathogenesis of hereditary coagulation FⅫ deficiency in this pedigree.

Homozygous missense variant F12 (Gly506Asp) associated with severe factor XII deficiency: a case report

BackgroundFactor XII deficiency can be related to either homozygous or compound heterozygous pathogenic variants in the F12 gene. The disease is commonly known as Hageman trait and is inherited in both autosomal recessive or dominant patterns. Clinically, factor XII deficiency is not associated with bleeding but conversely has been linked to thrombotic events, recurrent pregnancy loss, and hereditary angioedema. Molecular data of F12 deficiency are scarce and have revealed varying results between cases. However, most of the reported variants are missense mutations, gross deletions, or small insertion. Factor XII deficiency has been reported in the Saudi population in several studies, either as isolated case reports or included within the studies of rare bleeding factors deficiency. However, molecular data are lacking as no case report of genetic studies related to factor XII deficiency has been published in our local population, to the best of our knowledge.Case reportHerein we describe a homozygous missense variant involving exon 12 within F12 gene (5:176,830,269 G>A; p.Gly506Asp) in a 36-year-old Saudi multiparous female referred from the surgical clinic with significantly high activated partial thromboplastin time during preoperative assessment for sleeve gastrectomy. The patient had no history of bleeding episodes during the previous deliveries nor any tooth extractions. She had single event of spontaneous abortion during the 15th week of gestation without any bleeding complication. There was no history of thrombosis or skin manifestations, and she was not taking any medicines. There was no family history of bleeding or thrombosis. Family history revealed consanguinity as the parents are first-degree cousins. Physical examination was unremarkable. Upon investigation, the prolonged activated partial thromboplastin time was fully corrected by a 1:1 mixing study with normal pool plasma while lupus anticoagulant tests were negative. Factor assays and von Willebrand factor tests are all within normal ranges except for factor XII, which was severely deficient. A homozygous missense variant involving exon 12 within F12 gene (5:176,830,269 G>A; p.Gly506Asp) was identified.ConclusionF12 (5:176,830,269 G>A; p.Gly506Asp) variant is likely to be a pathogenic variant among homozygous factor XII-deficient patients. Genetic counseling and management of the patients and families should be based on clinical evaluation.

To Test or Not to Test: Routine Thrombophilia Diagnostic Screening of Women with Reproductive Failures.

Recurrent reproductive failure is a global health issue affecting a significant number of women. Thrombophilias have been implicated as a possible cause. Inherited thrombophilias include a single nucleotide variant on factor V Leiden and prothrombin. The aim of this study was to evaluate the association between the following single nucleotide variants: factor V Leiden (c.1601G>A), the prothrombin gene (c.*97G>A) and the reproductive failure in the Polish population. The study was conducted in a group of 545 patients with recurrent pregnancy loss, RPL (≥2 miscarriages), and in a group of 641 patients with infertility. The distribution of genotypes for the selected variants were determined by RFLP-PCR and by the real-time PCR method. A variant of the F5 gene was found in 5.14% of patients with RPL and in 6.08% of infertile women. A variant of the F2 gene was identified in 0.73% of patients with RPL and in 2.03% of women with infertility. The frequency in the study groups did not differ from that in the general population. No association between the studied variants of the F5 gene or the F2 gene and the predisposition to reproductive wastage was found. Recommendations for routine thrombophilia testing in women with recurrent miscarriages should be revisited. The decision regarding testing should be made individually depending on additional factors indicating an increased risk of venous thromboembolism.

RAAV8-PC Gene Therapy for Severe Protein C Deficiency Using a Novel Murine Model

Introduction Protein C (PC) is a vitamin K-dependent protein, synthesized in the liver. The PROC gene is located on chromosome 2 at position 2q13-14 and encodes 9 exons. Severe protein C deficiency (SPCD) is a rare devastating autosomal recessive disease that presents at birth with purpura fulminans (PF) and is associated with high rates of morbidity and mortality. As current management options are limited and burdensome, there is a large unmet need for new treatments including curative therapies. Because small increases in PC may significantly ameliorate the clinical presentation of SPCD and potentially prevent PF, we hypothesize that SPCD could be treated by PC gene therapy (GT). Aim To establish a murine model enabling the survival of SPCD mice and to evaluate PC expression after recombinant adeno-associated viral vector- PC (rAAV8-PC) gene therapy. Methods The study was conducted with C57Bl/6 PC heterozygous mice (a kind gift of Prof. Weiler, [Wisconsin]), severe hemophilia A (HA) mice and wild type C57Bl/6 mice. Genotyping was performed by PCR using appropriate primers for murine PROC and F8 genes. rAAV8-PC was produced by transfecting HEK293 cells with PX-680 as rAAV helper, AAV8 and PC plasmid with an alpha-1 antitrypsin promotor. Extraction and purification were performed using a commercial kit. Viral concentration was determined by a designated commercial qPCR assay. rAAV8 - PC (10 12 /kg Vg of AAV8-PC) was injected via the tail vein to 5-8 weeks old mice. Blood samples were collected from the facial vein into 1.5 ml tubes containing 0.109M sodium citrate at blood: anticoagulant ratio of 1:1 (vol/vol). PC plasma levels were measured prior and following rAAV8-PC infusion using a commercial ELISA assay for PC antigen. Thrombin generation was measured in plasma samples using a modification of the calibrated automated thrombinoscope assay. Results As expected, breeding of heterozygous PC mice yielded only 3/200 homozygous SPCD mice (PC levels= 0), that survived for less than 24 hours. Thus, GT for heterozygous PC mice was considered; however, PC plasma levels in heterozygous PC mice were not substantially different from those in WT mice (median levels in heterozygous PC and WT mice: 216 ng/ml vs. 190 ng/ml, respectively), which precluded this option. Hence, with the aim of increasing the survival rate and duration of homozygous SPCD mice, we decided to rebalance the severe thrombotic disorder of SPCD, by induction of a genetically engineered combination with the severe coagulopathy of Hemophilia. We established an alternative SPCD murine model by breeding severe HA mice with heterozygous PC mice. This approach yielded homozygous SPCD mice with severe HA (PROC-/F8-), exhibiting a significantly prolonged survival compared to homozygous SPCD mice with normal F8 levels (over 90% survived over 6 weeks). Genotyping and ELISA of PC levels confirmed PC homozygosity (null mutation) as well as severe HA. Nineteen PROC-/F8- mice were injected with rAAV8-PC into the tail vein and followed for at least 12 weeks. Nine mice died during blood sampling at different time points and were excluded from further analysis. Individual responses to PC- GT were noted among the remaining 10 mice, reflected by non-uniform PC expression measured by ELISA (initial PC increase, time to peak and PC peak plasma levels). An increase in PC level was noted at week 2-5 post vector infusion, reaching PC peak level between week 2-10 post GT. Table 1 summarizes the individual patterns of 10 PROC-/F8-mice that underwent GT. The functional activity of PC in the murine plasma following GT was supported by thrombin generation assays. Analyses performed in PROC-/F8-mice before and after GT demonstrated endogenous thrombin potential (ETP) and peak hight decrease following GT, as compared to untreated PROC-/F8-mice. Conclusion We present here a novel murine model of combined PC and factor VIII deficiency that supports the survival of SPCD mice and enables PC gene therapy studies. Our results provide the first proof of concept that infusion of rAAV8-PC results in increased PC plasma levels in mice, and may serve as a basis for future studies investigating GT for SPCD. The concept of modulating a severe genetic prothrombotic disorder, which is nearly incompatible with life, by genetic combination with severe coagulopathy may offer a valuable tool enabling investigation and potential development of treatment modalities for the prothrombotic disorder.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro

BackgroundHemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. MethodsInitially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression. ResultsWe identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro. ConclusionsKnock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.

Four-Year Follow-up of the Alta Study, a Phase 1/2 Study of Giroctocogene Fitelparvovec (PF-07055480/SB-525) Gene Therapy in Adults with Severe Hemophilia Α

Introduction: Hemophilia A is an X-linked ( F8 gene) recessive disorder of hemostasis that results in insufficient factor VIII (FVIII) activity. Adeno-associated virus (AAV)-based gene therapy enables delivery of a modified F8 cDNA, allowing synthesis of functional endogenous FVIII, which prevents bleeding events. We present updated results with nearly 4 years of follow-up on an ongoing gene therapy study in participants with severe hemophilia A (FVIII activity &amp;lt;1%). Methods:The phase 1/2 Alta study (NCT03061201) is a dose-ranging study of giroctocogene fitelparvovec (PF-07055480, previously called SB-525), a recombinant AAV serotype 6 vector encoding a modified B-domain-deleted F8 coding sequence. Four ascending doses of giroctocogene fitelparvovec (9e11, 2e12, 1e13, and 3e13 vg/kg) were infused into adults aged ≥18 years with severe hemophilia A across 4 cohorts (n=2 each). The high-dose (3e13 vg/kg) cohort was expanded to 5 participants. Key endpoints included safety, circulating FVIII activity, use of FVIII replacement therapy, and frequency of bleeding events. Results: Eleven male participants were enrolled in the study (mean [SD] age, 30.3 [7.8] years; White, 81.8%). As of the cutoff date (May 19, 2023), participants had been followed for 153 to 290 weeks. Two participants left the study after Week 156. Of the remaining, 1 participant had not yet completed 4 years (208 weeks). The most common treatment-related adverse events (AEs) reported in the high-dose cohort (n=5) were elevated liver enzymes and infusion-related reactions: increased alanine aminotransferase (ALT; n=3 [60.0%]), increased aspartate aminotransferase (AST; n=2 [40.0%]), pyrexia (n=3 [60.0%]), and tachycardia (n=2 [40.0%]). Treatment-related serious AEs were reported in 1 participant in the high-dose cohort who experienced hypotension and fever, with onset ≈6 h after infusion; the events fully resolved with treatment. AEs (all causality) of ALT increases requiring ≥7 days of corticosteroids were observed in 4 of 5 participants in the high-dose cohort. ALT elevations were managed with tapering courses of corticosteroids (median duration: 56 days; range: 7-135 days), with maintenance of efficacious levels of FVIII activity. Participants in the high-dose cohort have not required steroids since Week 65, have had ALT values in the normal range (follow-up: 156-208 weeks) and normal findings via liver MRI (follow-up: 104-208 weeks). No participant developed a confirmed inhibitor to FVIII. No thrombotic events or liver masses have been detected. Of the 5 participants in the high-dose cohort, 2 had data available through Week 208 and FVIII activity was maintained in the mild to normal range ( Table), consistent with Week 156 results. Of those without Week 208 data, 2 had data through Week 182. One participant maintained FVIII activity in the mild range (14.1% and 24.1% of normal, measured with a chromogenic and 1-stage assay, respectively); the other had FVIII activity of 3.1% and 7.2%. The remaining participant left the study after Week 156, with FVIII activity maintained in the mild range (11.8% and 22.9%). In the high-dose cohort, the mean annualized total bleeding rate [(number of all bleeding episodes starting 3 weeks after study drug infusion) / (observation period in years)] was 0 for the first year post infusion and 1.2 (SD 2.58) throughout the total duration of follow-up. In this cohort, the participant with the lowest FVIII activity level experienced a total of 22 bleeds, with 21 necessitating treatment (8 traumatic; 7 spontaneous; 6 unknown). The other 4 participants had no or very minimal bleeds, including 1 who experienced a bleed in a target joint. No participants in the high-dose cohort have resumed prophylaxis. Conclusion:A single infusion of giroctocogene fitelparvovec gene therapy in participants with severe hemophilia A remains generally well tolerated over a period of nearly 4 years post infusion, with associated increases in FVIII levels in the moderate to normal range, without sustained AEs and with no AEs associated with increased liver function tests since Week 59. The ongoing phase 3 study (NCT04370054) in a larger cohort will provide more long-term data on the safety and durability of giroctocogene fitelparvovec in participants with moderately severe to severe hemophilia A.

Factor XI Deficiency in Chinese Patients: Results from a Nationwide Multicenter Retrospective Study

Background: Factor XI(FXI) deficiency is a rare and relatively mild bleeding disorder. To date, data for China is limited. Our study aimed to investigate the clinical phenotype, F11 genotype and management of individuals diagnosed with FXI deficiency from throughout China. Methods: The study retrospectively analyzed patients with FXI activity (FXI:C) &amp;lt;50% registered from the National Hemophilia Registration System. The study collected patient characteristics, F11 gene mutations, clinical history, bleeding events, and treatment strategy by telephone follow-up. RESULTS: The study included 234 patients throughout 27 provincial districts registered in 38 Chinese hemophilia treatment centers. Among these patients, 192 (82.1%) had severe FXI deficiency (FXI:C &amp;lt;15%), and 42 (17.9%) had non-severe FXI deficiency (15%≤FXI:C&amp;lt;50%). The median age was 30 (1-87) years at diagnosis. There were 19 patients (8.1%) who reported a family history of bleeding. Five patients (2.1%) reported having consanguineous marriages in their families. There were 57 patients (24.4%) had their F11 gene tested. We detected 44 kinds of variants on 99 alleles, including nine that have never been reported to the F11 database. The most frequent variants were missense (44, 44.4%) and nonsense mutations (35, 35.4%). Among the genotyped subjects, F11 mutations were homozygous in 12 (21.0%), compound heterozygous in 29 (50.9%), and heterozygous in 16 (28.1%) patients. The FXI:C among patients with homozygous/compound heterozygous mutations was significantly lower than among patients with heterozygous mutations (2.1±2.3% vs. 14.8±12.9%, P&amp;lt;0.001). In total, 91 (38.9%) reported bleeding symptoms to varying degrees. Among these patients, 79 (33.8%) experienced spontaneous bleeding, with menorrhagia, epistaxis, and dermatorrhagia being the most prevalent manifestations. Twenty-one (26.6%) patients were administered on-demand replacement therapy. There were 129 patients underwent 220 invasive procedures with a total of 22 bleeding episode. The risk of bleeding was found to be significantly higher when sites with high fibrinolysis were involved, as compared to sites without fibrinolysis (Chi2=3.976, P=0.046). The prophylactic replacement treatment was administered to 50 cases (22.8%), and three bleeding events occurred, compared to 19 bleeding events in the 170 cases without prophylaxis. It was observed that prophylaxis did not reduce bleeding events (Chi2=1.150, P=0.283). A total of 83 deliveries were recorded in 58 female patients, and four bleeding events occurred. The blood group-O is a risk factor for bleeding [odds ratio (OR)=2.429, 95% confidence interval (CI) 1.139-5.182]. Conclusion: Most patients had no or mild bleeding symptoms. Most F11 variants were missense. The FXI:C among patients with homozygous/compound heterozygous mutations was significantly lower than among patients with heterozygous mutations. The risk of bleeding was higher when procedure sites with high fibrinolysis were involved. Prophylactic repalcement treatment could not reduce the risk of hemorrhages after invasive procedure. There is increased risk of bleeding in patients with blood group-O. Table 1 Table 2

Development of an Ex Vivo Precision Gene Engineered B Cell Medicine That Produces Active and Sustained Levels of FIX for the Treatment of Hemophilia B

Hemophilia B is an X-linked recessive bleeding disorder that affects approximately 1:20,000 males. It is caused by mutations in the F9 gene that encodes for the factor IX (FIX) protein, an essential enzyme in the coagulation cascade. The current recommended therapy for hemophilia B patients is prophylactic administration of exogenous FIX derived from recombinant protein. The short biological half-life of FIX requires frequent infusions to maintain therapeutic level. More recently, an adeno-associated virus (AAV) vector-based gene therapy, etranacogene dezaparvovec, has been approved for some adults as a potential new option. Despite these advances, there remains a significant unmet medical need in hemophilia B. Intravenous infusions of weekly (or more frequently, 3 times per week) factor replacements are an enormous burden to patients and their caregivers. Gene therapy, while a promising option for some, carries potential for significant risk. Importantly, the limitations associated with increasing the dose of FIX needed to treat pediatric patients as they age make immunogenic AAV-based gene therapy inappropriate for this patient population. Terminally differentiated human plasma cells derived from genetically engineered B cells (termed B Cell Medicines, BeCMs), potentially offer natural longevity (persisting for decades), capacity for high levels of protein secretion (thousands of Ig molecules/cell/sec), the ability to engraft without host preconditioning, and the ability to re-dose, making them an attractive platform for the sustained supply of biologics where continuous dosing is required to achieve therapeutic benefit. We have developed an ex vivo precision engineered BeCM platform with modularity and broad therapeutic utility. In this study, primary human B cells were isolated, activated, and engineered by CRISPR/Cas9 genome editing followed by AAV-mediated homology directed repair (HDR) insertion of human F9 gene into the C-C chemokine receptor type 5 (CCR5) safe harbor locus. The cells were then further expanded and differentiated towards the plasma cell lineage, resulting in FIX-producing BeCMs. We achieved approximately 40% targeted integration as measured by droplet digital PCR (ddPCR). Engineered BeCMs secreted up to 60 ng/1e6 cells/hour of FIX protein, approaching 40% of IgG secretion rate as measured by ELISA. BeCM-produced FIX was analyzed by LC-MS which demonstrated gamma carobxylation of FIX protein Gal-domain. Vitamin K-dependent activated partial thromboplastin time (aPTT), using the one stage clotting assay, was employed to verify biological activity of BeCM-produced FIX. Similarly, FIX-expressing BeCMs exhibited vitamin K-dependent activity in vitro in the chromogenic assay. FIX-expressing BeCMs were transferred into immunodeficient NOG-hIL6 mice, with FIX production demonstrated at least 20 weeks in vivo. The safety of FIX-expressing BeCMs has been characterized based on 28-day and 5-month in vivo studies in NOG-hIL6 mice. Neither abnormal clinical observations nor mortality were observed in those studies. Biodistribution of the FIX expressing BeCM product was assessed using a qPCR assay. The qPCR data confirms the expected biodistribution of FIX-expressing BeCMs, which engraft and persist in bone marrow tissue over time. In summary, we have developed an ex vivo precision gene engineered B cell medicine that produces active and sustained levels of FIX for the treatment of hemophilia B. The potential therapeutic application of this unique biologic delivery system could afford a novel treatment modality for hemophilia B.

Reduced Vitamin C Levels in Patients with Bleeding of Unknown Cause and Generalized Joint Hypermobility

Introduction Vitamin C is essential for the synthesis of collagen. Ehler-Danlos syndrome (EDS) is a group of collagen disorders associated with generalized joint hypermobility (HM), bleeding and bruising. The genetic cause of the HM EDS subtype (hEDS) is unknown. Referral to haematology clinics may result in the diagnosis: bleeding of unknown cause (BUC), where no specific haemostatic treatment is available. However, it is a clinical experience, published in many textbooks, that bleeding, and bruising associated with EDS improve by oral intake of vitamin C. No previous studies examined vitamin C levels in patients with BUC. The aim of the study is to investigate the association of vitamin C levels with HM and vascular fragility in patients with BUC. Methods Patients with bleeding tendency are referred to our Haemophilia Center. Following ISTH-BAT score, normal coagulation test (INR, APTT, fibrinogen, F8, F9, F11, F13, VWFAg, VWFRist) and inconclusive results of flow cytometry analysis of platelet function, patients are diagnosed with BUC. Patients with a high ISTH-BAT score &amp;gt; 10 were included in this study. We performed clinical examinations by a tourniquet test for capillary fragility and a Beighton score for generalized HM. A skin extensibility test was performed by pulling on the skin off the forearm and the lateral side of the throat until resistance was met. The test was considered positive (abnormal) if the skin extended more than 2 cm in both sites when pulled. Germline DNA was analyzed by whole genome sequencing (WGS) analysis and an in silico panel of 170 genes associated with bleeding and generalized HM was examined. Additionally, homozygous polymorphism in vitamin C genes ( GULOP, SLC23A1, SLC23A2, SVCT1, HP, GST and SOD2) known to cause reduced vitamin C levels, were assessed Vitamin C analysis: The participants received oral and written guidelines regarding intake of vitamin C in relation to the blood sample. The guidelines included no vitamin C supplements for 3 days before the blood sampling and no intake of vitamin C rich food on the day of the appointment. Plasma samples were immediately acidified by addition of 10% meta-phosphoric acid to ensure the stability of ascorbic acid, and subsequently analyzed by high-performance liquid chromatography. Severe vitamin C deficiency was defined as &amp;lt;11µM, deficiency as &amp;lt; 23 µM, suboptimal status as 23-49 µM and adequate status as &amp;gt;49 µM. Results Sixty probands and 20 healthy age- and gender-matched controls (HC) were included. Most patients were women (56/60, 93%), median age 48 (range 21- 75) and median BAT score 13 (range 10-21). By WGS a 67-year woman with Beighton score 7 was diagnosed with Noonan syndrome due to a pathogenic variant in PTPN11 (c.922A&amp;gt;G). Also, a 62-year woman without HM was diagnosed with mild FV deficiency due to a heterozygous variant in F5 (c.449A&amp;gt;G). No other causal variants were identified. In a patient with a homozygous variant in SLC23A1, vitamin C level was suboptimal 42µM.Suboptimal vitamin C levels were found in 19/60 patients and 2/20 HC. One patient was severely vitamin C deficient (7µM). Vitamin C levels were significantly lower in patients compared to HC (median 56 µM vs 76 µM; p = 0.012). Beighton score was positive in 29/60 patients and 1/20 HC (p&amp;lt;0.001). In 18/60 patients versus 0/20 HC the skin extensibility test was abnormal (p = 0.01). In 12/29 patients with a positive Beighton score the test for skin extensibility was also positive indicating a diagnosis of hEDS. Tourniquet test was positive (&amp;gt; 10 petechiae) in 18/60 patients and 4/20 HC (p&amp;gt;0.5). Patients with positive Beighton scores had significantly higher BAT scores than patients without HM (P= 0.014). No significant associations were found between vitamin C levels, a positive Beighton score, a positive Tourniquet test or a high BAT score respectively. Conclusions We found that a high proportion of patients with BUC have generalized HM, abnormal skin extensibility and positive tourniquet test. Indeed, patients with HM had increased ISTH-BAT scores compared to patients with negative Beighton scores. Our data support that hEDS may be an underlying cause of BUC. Further studies are needed to determine the genetic background of hEDS. Levels of vitamin C were reduced in patients with BUC compared to age and sex matched control. Our results could pave the way for a randomized study of vitamin C supplements versus placebo for bleeding and bruising in patients with generalized HM and BUC.

Thrombosis Caused By a Novel Coagulation Factor IX Mutation (FIX Shanghai II)

Most thrombophilia patients are caused by insufficient suppression of coagulation reactions, which could be either mediated by deficiencies of protein C, protein S,antithrombin, or inhibition-resistant coagulation factor mutants, such as Factor V Leiden and prothrombin Yukuhashi. The thrombosis caused by coagulation factor mutants with increased procoagulant activity is very rare and so far only two Factor IX (FIX) variants involving residue Arg384, FIX Padua (p.Arg384Leu) and Shanghai (p.Arg384Gln), which increased FIX activity by up to 8 folds, had been reported to be associated with the incidence of thrombosis. In a young male venous thrombosis patient we had identified a novel F9 gene missense mutation c.1018G&amp;gt;A, resulting in p. Glu340Lys substitution (designated as FIX Shanghai II). The patient's APTT time was 28.9 seconds (normal reference 22.3~38.7s), and his PT was also within the normal reference range (11.5 seconds, normal reference 10.0~16.0s). Although the patient had the FIX: Ag comparable to the normal reference, 128.20±6.07%, the his FIX:C was around 201.69±10.38% of normal control (normal reference 50~150%). Thrombin generation assay (TGA) showed that compared with normal control, the patient had a shorter Lag time (2.67 vs 3.33 min), elevated ETP (1649.43 vs 1311.62 nM*min), and higher Peak value (387.34 vs 200.86 nM), indicating increased global coagulation activity of the patient. The activity of protein C, protein S, and antithrombin were all within the normal reference ranges and the patient was negative for antiphospholipid antibodies. Consistent with the patient's coagulation laboratory manifestations, the purified recombinant FIX Glu340Lys mutant showed 1.82 folds higher procoagulant activity than wild type FIX as determined by FIX:C/FIX:Ag ratio. The recombinant FIX Shanghai II mutant can be converted into FIXa by activated factor XI (FXIa) in a similar fashion as FIX WT. The kinetic study of FIXa-catalyzed FX activation showed that p. Glu340Lys mutation led to accelerated FX activation by FIXa. The p.Glu340Lys mutation also enhanced the procoagulant activity of previously reported thrombosis-causing FIX mutants, FIX Shanghai and Padua, in a similar fashion as it did on FIX WT. The FIX:C/FIX:Ag ratio of double mutants containing Glu340Lys and Arg384Gln (FIX Shanghai) or Arg384Leu (FIX Padua) was about twice of FIX Shanghai or Padua alone, 11.48- and 22.53-folds vs 6.16- and 8.13- folds higher than FIX WT respectively.

Commercial human frozen plasmas for local, cross-site and long-term comparability of coagulation analysers.

Laboratory compliance implies to correlate instruments for coagulation parameter with a wide range of measure using patient samples or commercialized sets of frozen plasmas. The aim of this study was to evaluate the intra, inter-reproducibility and long-term stability of ExpertCor Routine (ECR) plasma sets (Stago) on different parameters. The study was realized in two laboratories on four different instruments. Inter-site and intra-site correlation of ECR sets for PT, aPTT, Fibrinogen, INR, factor V (FV) UFH and LMWH anti-Xa and intra-reproducibility of DDimer (DDI), factor VIII (FVIII:C) and antithrombin (AT) assays were tested. To evaluate ECR long-term stability, samples were tested until 180 after delivery in one laboratory. Intra-site evaluation correlation coefficients is around 1. All predefined criteria to fulfil good comparability between inter-site instruments are met with Passing slopes between 0.9 and 1.1 and intercepts ranging from -0.62 to 2.83%. Long-term stability evaluation does not show any deviation over 180 days for aPTT, fibrinogen, DDI, UFH, LMWH but a drift for FV with STA-NeoPTimal reagent. On contrary, AT and FVIII:C are not stable. PT in second has an excellent stability unlike PT in percentage. Our study validates the use of ECR sets for correlation between instruments and inter-sites agreement, as for parameters claimed on the products than for factor V and FVIII:C. The evaluation of stability confirming the possible extension of use for 180 days after delivery except for FVIII:C and AT. These plasmas sets are an excellent alternative to local plasma patient use to perform instrument comparison.

The Anticoagulant Systems Cooperate Synergistically on Phospholipid Vesicle and Endothelial Cell Membranes, but Not on Platelets

Introduction: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). TFPIα, APC, and PS function synergistically on the surface of phospholipid vesicles (PLs). We have also recently reported a shear-dependent interaction between PS and von Willebrand factor (VWF), which partially blocks its anticoagulant function. Here, we investigated the function of the TFPIα/APC/PS anticoagulant system on physiologic membrane surfaces (platelets and endothelial cells, or ECs), and the effect of VWF on prothrombinase inhibition in these different membrane environments. Methods: Thrombin generation was measured in plasma and platelet-rich plasma (PRP) by calibrated automated thrombography, purified prothrombinase activity was assayed using a chromogenic substrate, and FVa cleavage was monitored by immunoblotting. Experiments were performed with synthetic PLs, washed platelets, or cultured EA.hy926 cells, an EC line. Experiments involving VWF were performed in the presence or absence of shear. Results: In plasma TG, as we have previously published, PS anticoagulant activity was greatly enhanced by addition of thrombomodulin (TM), to promote protein C (PC) activation. TM decreased thrombin generation in a PS-dependent manner. The effect of TM and PS was enhanced by the addition of TFPIα, particularly at low PS concentrations. Conversely, inhibitory antibodies against TFPI and PC had an additive effect on plasma TG which was mimicked by inhibiting PS alone. Similar results were obtained in plasma supplemented with ECs (which provide TM) or in PRP. However, in PRP, the antibodies reversed TG beyond what was seen with tissue factor-initiation alone. In purified protein systems, performed using either PLs or ECs, physiologic TFPIα concentrations had no detectable effect on thrombin activation by prothrombinase, unless APC was present in the system. The greatest inhibitory activity was observed when TFPIα, APC, and PS were all present, and was similar on ECs and PLs. Similarly, inhibition of FXa by TFPIα was found to render FVa susceptible to proteolysis by APC on either PLs or ECs. However, prothrombinase assembled on activated platelets was protected from inhibition, even in the presence of TFPIα, APC, and PS, as measured either by thrombin activation or FVa cleavage. Finally, we assessed the functional consequences of the PS/VWF interaction. In purified systems with PLs, sheared VWF dose-dependently reversed FXa inhibition by PS/TFPIα, measured using a chromogenic FXa substrate. Conversely, APC cofactor function, as measured by FVa cleavage, was unaffected on either PLs or activated platelets. Consistent with this, thrombin activation by prothrombinase was unaffected by sheared VWF on these surfaces. Interestingly, sheared VWF promoted prothrombinase function on ECs, increasing the rate of thrombin activation beyond that observed in the absence of the anticoagulants. Conclusions: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. On PL or EC membranes in the plasma environment, APC, TFPIα, and PS cooperate synergistically to inhibit prothrombinase activity, preventing excessive clot propagation. Conversely, platelets protect prothrombinase from inhibition, supporting a procoagulant environment within the clot. The anticoagulant system appears to function similarly on PLs and on ECs, except that VWF has greater effect on ECs, suggesting that the protection mediated by platelets is due to a platelet component not present in ECs. We hypothesize that these regulatory mechanisms serve to localize procoagulant activity within the platelet plug and suppress it on ECs downstream of a clot.

Plain language summary of the GENEr8-1 clinical trial of valoctocogene roxaparvovec gene therapy for hemophilia A

What is this summary about? In healthy people, a protein called factor VIII (FVIII) helps blood to clot and prevents excessive bleeding. People with hemophilia A lack FVIII because a faulty F8 gene is giving the wrong instructions to the liver cells that make it. Valoctocogene roxaparvovec (ROCTAVIAN™) is a gene therapy designed to transfer working copies of the F8 gene into liver cells. This summary describes the GENEr8-1 study, which looked at how well valoctocogene roxaparvovec works for treating people with severe hemophilia A compared with their usual FVIII replacement therapy, and its safety. 134 men received valoctocogene roxaparvovec; results from the first 2 years are reported. What were the results? Valoctocogene roxaparvovec significantly improved bleed control in men with severe hemophilia A. A single infusion reduced average treated bleeding episodes per year from almost 5 while on FVIII prophylaxis to less than one after gene therapy. Eight out of every 10 participants had no bleeds needing treatment and 9 out of every 10 had no joint bleeds needing treatment; FVIII levels improved to normal or mild hemophilia ranges. All except 6 participants remained off regular FVIII prophylaxis for at least 2 years. All participants had at least one side effect and 22 (16%) reported serious adverse events. Nine out of every 10 participants (89%) showed increased blood levels of liver enzymes, indicating an expected immune response to the product's viral shell; this was manageable with steroids. Other common side effects included headache (41%), joint pain (40%), feeling sick (38%), and side effects to steroids (60%). What do the results mean? After a one-time dose of valoctocogene roxaparvovec, people with severe hemophilia A start producing their own FVIII and require fewer or no FVIII injections to protect them from bleeds. Results showing bleed control for at least 2 years have led to approvals of valoctocogene roxaparvovec in Europe and the USA for use in adults with severe hemophilia A who do not have antibodies against FVIII or AAV type 5. How well valoctocogene roxaparvovec works and the side effects over a longer period are still being studied.

Transcriptomic Profiling to Understand Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a

Background: When starting treatment with factor VIII (FVIII) concentrate in individuals with severe hemophilia A, minimizing the risk of FVIII inhibitor development is an important concern for clinicians, patients and their families. Despite increased understanding of some of the genetic and environmental risk factors for inhibitor formation from epidemiological studies, identification of previously untreated patients (PUPs) at risk of developing inhibitors remains a challenge. Prediction of inhibitor development may help optimize care in PUPs with hemophilia A. RNA sequencing (RNA-Seq) with next-generation sequencing (NGS) could be used to investigate the underlying mechanisms of inhibitor development. Aims: Toevaluate peripheral blood transcriptomic profiles prior to and during early FVIII treatment in severe hemophilia A patients who did or did not develop inhibitors to simoctocog alfain the prospective NuProtect study. Methods: The NuProtect study (NCT01712438; EudraCT 2012-002554-23) was a multicenter, multinational, open-label, non-controlled, phase III study that enrolled male PUPs with severe hemophilia A (FVIII:C &amp;lt;1%) of any age and ethnicity. Patients received simoctocog alfa for prophylaxis or on-demand treatment, as well as for surgical prophylaxis. RNA-Seq was performed on blood samples collected from baseline and every 3-4 exposure days (ED) up to 20 EDs or until inhibitor development to assess messenger RNA (mRNA) expression profiles. Bioinformatics analyses were performed for the following timepoints: baseline, early treatment and a later timepoint on treatment. Principal component analysis (PCA) was performed to identify differences in global mRNA expression profiles between inhibitor and non-inhibitor patients. Gene ontology (GO) analyses were performed to detect differences in GO clusters between inhibitor and non-inhibitor patients. Results: Within this first analysis of the full dataset, transcriptomic profiles from 78 of 108 severe hemophilia A PUPs were analyzed (inhibitors n=22, non-inhibitors n=56). Median (range) age at ED1 was 13 (0.5-146) months; 44.9% of patients had the intron 22 inversion F8 gene mutation. Although the PCA indicated no strong global signal for differential expression profiles between non-inhibitor and inhibitor patients, GO analyses demonstrated differences between these patient groups. Inhibitor patients demonstrated increased gene expression of metabolic processes (GO:0008152) and cellular processes (GO:0009987) at baseline. Upregulation of immune system processes (GO:0002376) was seen in inhibitor patients at the early timepoint compared with non-inhibitor patients. Expression of ribosomal proteins, including RPL17 and RPS8 were significantly upregulated at baseline in inhibitor patients (p&amp;lt;0.05). Inhibitor patients further demonstrated upregulation of proteins involved in adaptive immune responses: IGHV1-69-2 at all timepoints (p&amp;lt;0.01) and IGHV2-70D at baseline and the early timepoint (p&amp;lt;0.01) compared with non-inhibitor patients. Upregulation of the B-cell related factors DPP4 at baseline and the early timepoint (p&amp;lt;0.05) and CD20 at the late timepoint (p&amp;lt;0.05) was observed in inhibitor patients compared with non-inhibitor patients. Conclusion: Differential transcriptomic profiles were seen in severe hemophilia A patients who developed inhibitors compared with those who do not, even before commencing FVIII treatment. Inhibitor patients demonstrated alterations in ribosomal protein expression that could disrupt protein synthesis efficiency, protein folding and quality control processes, and trigger cellular stress responses. Inhibitor patients also showed an upregulation of B-cell mediated factors involved in adaptive immune mechanisms. Additional analyses are ongoing to evaluate dynamic changes in gene expression during commencement of FVIII replacement to further identify patients at risk of inhibitor development.

The Role of Coagulation Gene Polymorphisms in Sars-CoV2 Infection in the Bergamo Area

Introduction: Given the great variability of the individual response to SARS-CoV2 infection, it is important to understand whether and how biological and genetic factors might predispose to the infection and to the degree of severity, including the development of systemic coagulopathy and thrombosis. In this study, we evaluated the frequency of prothrombotic allelic variants (AVs) in coagulation genes in a population of COVID-19 patients, to verify whether these polymorphisms, alone or in combination, are associated to an increased susceptibility to develop COVID-19 and/or a greater severity. Methods: A cohort of 358 patients (257M/101F; median age: 55 years) from the Bergamo area with two different degrees of COVID-19 severity, i.e., severe disease (hospitalized, n=212) or mild disease (non-hospitalized, n=146), was enrolled from April 2020 to June 2021 and followed-up for disease outcome and thrombosis. A cohort of 377 healthy subjects (177M/200F; median age: 49 years) from the same area without COVID-19 acted as a control group. DNA was analyzed for a panel of prothrombotic/inflammatory single nucleotide polymorphisms (SNPs) including FII rs1799963, FV rs6025, FV rs118203907, FXIIIA1 rs5985, FGB rs1800790, MTHFR rs1801131 and MTHFR rs1801133 as well as two SNPs located in the coding region of angiotensin-converting enzyme (ACE2) gene, namely ACE2 rs140312271 and ACE2 rs41303171. Statistical analysis was performed using SPSS statistical software. Results: Among the AVs analyzed, the rs6025 polymorphism of FV gene (FV Leiden) was more frequent in COVID-19 patients than in the control group. By a logistic regression corrected for age and gender, FV Leiden was associated with a 3-fold risk of developing COVID-19 (OR: 3.2 [95% CI 1.02-10]). In contrast, the rs41303171 polymorphism of the ACE2 gene was more frequent in the control group and was a protective factor for COVID-19 (OR: 0.12 [95% CI 0.02-0.99]) even after correction for age and gender. No significant differences appeared in the frequency of the other SNPs alone or in combination. Within the COVID-19 patient cohort, the rs1801131 SNP of MTHFR gene was associated with less severe COVID-19 disease. In the hospitalized severe cohort, 17 (8%) patients developed an overt venous thromboembolic event, and 7 COVID-19-related deaths (4%) were recorded. No significant associations were found between any of the AVs analyzed and the outcomes of thrombosis and death. However, the analyses of circulating biomarkers of clotting (i.e. D-dimer, FVIII, fibrinogen, prothrombin fragment 1+2 [F1+2], and FXIII), fibrinolysis (i.e. tissue plasminogen activator [tPA], and plasminogen activator inhibitor-1 [PAI-1]) and endothelial cell activation (i.e. thrombomodulin [TM], and von Willebrand Factor [vWF]), and the neutrophil-to-lymphocyte ratio (NLR) as an inflammatory biomarker revealed thathaving fibrinogen (OR: 4.9 [95% CI 1.04-23.1]), vWF:Ag (OR: 11.8 [95% CI 2.1-66.9]) and NLR (OR: 26.1 [95% CI 2.9-237] levels above the 75th percentile was significantly associated with death. Of interest, in the same hospitalized patient group, but not in the control group, the presence of FXIII SNPs was significantly associated with high levels of F1+2. Conclusions: Our study shows that the FV Leiden polymorphism predisposes to COVID-19 infection, while the rs41303171 ACE2 polymorphism has a protective role. In addition, the rs1801131 SNP of gene MTHFR correlates with a milder COVID-19 disease. In patients with severe COVID-19, the inflammatory marker NLR, and the circulating hemostatic proteins vWF and fibrinogen are associated with mortality.