Factor V Research Articles

Comprehensive Analysis of Hemophilia A (CAHEA): Towards Full Characterization of the F8 Gene Variants by Long-Read Sequencing.

Hemophilia A (HA) is the most frequently occurring X-linked bleeding disorder caused by heterogeneous variants in the F8 gene, one of the largest genes known. Conventional molecular analysis of F8 requires a combination of assays, usually including long-range polymerase chain reaction (LR-PCR) or inverse-PCR for inversions, Sanger sequencing or next-generation sequencing for single-nucleotide variants (SNVs) and indels, and multiplex ligation-dependent probe amplification for large deletions or duplications. This study aimed to develop a LR-PCR and long-read sequencing-based assay termed comprehensive analysis of hemophilia A (CAHEA) for full characterization of F8 variants. The performance of CAHEA was evaluated in 272 samples from 131 HA pedigrees with a wide spectrum of F8 variants by comparing to conventional molecular assays. CAHEA identified F8 variants in all the 131 pedigrees, including 35 intron 22-related gene rearrangements, 3 intron 1 inversion (Inv1), 85 SNVs and indels, 1 large insertion, and 7 large deletions. The accuracy of CAHEA was also confirmed in another set of 14 HA pedigrees. Compared with the conventional methods combined altogether, CAHEA assay demonstrated 100% sensitivity and specificity for identifying various types of F8 variants and had the advantages of directly determining the break regions/points of large inversions, insertions, and deletions, which enabled analyzing the mechanisms of recombination at the junction sites and pathogenicity of the variants. CAHEA represents a comprehensive assay toward full characterization of F8 variants including intron 22 and intron 1 inversions, SNVs/indels, and large insertions and deletions, greatly improving the genetic screening and diagnosis for HA.

Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non-severe haemophilia A.

Conventional genetic investigation fails to identify the F8 causal variant in 2.5%-10% of haemophilia A (HA) patients with non-severe phenotypes. In these cases, F8 deep intronic variants could be causal. To identify pathogenic F8 deep intronic variants in genetically unresolved families with non-severe HA analysed in the haematology laboratory of the Hospices Civils de Lyon. The whole F8 was analysed by next generation sequencing. The pathogenic impact of candidate variants identified was assessed using both in silico analysis (MaxEntScan and spliceAI) and functional analysis (RNA or minigene assay). Sequencing was performed in 49/55 families included for which a DNA sample from a male propositus was available. In total, 33 candidate variants from 43 propositi were identified. These variants corresponded to 31 single nucleotide substitutions, one 173-bp deletion, and an 869-bp tandem triplication. No candidate variant was found in six propositi. The most frequent variants found were the association of [c.2113+1154G>C and c.5374-304C>T], identified in five propositi, and the c.2114-6529C>G identified in nine propositi. Four variants had been previously described as HA-causing. Splicing functional assay found a deleterious impact for 11 substitutions (c.671-94G>A, c.788-312A>G, c.2113+1154G>C, c.2114-6529C>G, c.5999-820A>T, c.5999-786C>A, c.5999-669G>T, c.5999-669G>A, c.5999-669G>C, c.6900+4104A>C, and c.6901-2992A>G). The HA-causing variant was identified in 33/49 (67%) cases. In total, F8 deep intronic variants caused 8.8% of the non-severe HA among the 1643 families analysed in our laboratory. The results emphasise the value of whole F8 gene sequencing combined with splicing functional analyses to improve the diagnosis yield for non-severe HA.

Polymorphism of coagulation factor genes in patients with type 2 diabetes mellitus and chronic heart failure with retained ejection fraction

BACKGROUND. Patients with type 2 diabetes mellitus (DM2) have disorders of cellular and plasma hemostasis independent of the level of glycemia, increased platelet activation, combined with microvascular angiopathy. The study of the role of genetic markers of hemostasis disorders in the formation and progression of chronic heart failure (CHF) in patients with type 2 diabetes will allow for prevention, possibly optimize treatment and improve prognosis.AIM. To reveal polymorphisms of genes of the hemostasis system in patients with type 2 diabetes mellitus and chronic heart failure with preserved ejection fraction.MATERIALS AND METHODS. The frequency of coagulation factor genetic polymorphisms was studied in patients with CHF-pEF and DM2 (52 people), CHF with reduced ejection fraction (CHF-rEF) and DM2 (49) and healthy volunteers (66), mean age 69.9±10.1 years old. DNA was isolated from venous blood according to the method of the manufacturer. Genetic polymorphisms were determined by real-time polymerase chain reaction.RESULTS. The frequencies of polymorphisms rs1799963 and rs6025 of the genes of blood coagulation factors F2 (prothrombin) and F5 (factor V of blood coagulation) in all three groups were insignificant and comparable in magnitude. In patients with CHF and DM2, the frequencies of the rs6046 polymorphism of the factor F7 gene in the heterozygous form were slightly higher (by 2.6 and 1.7 times, respectively) than in the control group, but the result was not statistically significant. The CHF-pEF and CHF-rEF groups differ in the frequencies of F13 (rs5985) and fibrinogen (rs1800790) genetic polymorphisms, but are more common in patients with CHF-rEF and DM2.CONCLUSION. Based on the results of the study, it follows that the groups of CHF-pEF and CHF-rEF differ significantly in the frequencies of polymorphisms of the studied genes, both among themselves and with the control group. The highest frequency of polymorphisms of genes, the products of which are involved in the coagulation and cellular components of hemostasis, is observed in the group of patients with DM2 and CHF-rEF.

Whole-exome sequencing reveals a novel frameshift mutation in a consanguineous family with a hereditary coagulation factor XII deficiency

Background and aimsWe aimed to elucidate a hereditary mutation of coagulation factor XII (FXII) in a consanguineous Chinese family. MethodsMutations were investigated using Sanger and whole-exome sequencing. FXII (FXII:C) activity and FXII antigen (FXII:Ag) were assessed using clotting assays and ELISA, respectively. Gene variants were annotated and the likelihood that amino acid mutations would affect protein function was predicted using bioinformatics. ResultsActivated partial thromboplastin time was prolonged to > 170 s (reference range, 22.3–32.5 s), and FXII:C and FXII:Ag were decreased to 0.3% and 1%, respectively, (normal range for both, 72%–150%) in the proband. Sequencing revealed a homozygous frameshift mutation c.150delC (p.Phe51Serfs*44) site in the F12 gene exon 3. This mutation results in premature termination of the encoded protein translation and the protein is truncated. Bioinformatic findings indicated a novel pathogenic frameshift mutation. ConclusionThe c.150delC frameshift mutation p.Phe51Serfs*44 in the F12 gene likely explains the low FXII level and the molecular pathogenesis of an inherited FXII deficiency in a consanguineous family.

Gene Therapy Approaches for the Treatment of Hemophilia B

In contrast to the standard enzyme-replacement therapy, administered from once per 7–14 days to 2–3 times a week in patients with severe hemophilia B, as a result of a single injection, gene therapy can restore F9 gene expression and maintain it for a prolonged time. In clinical research, the approach of delivering a functional copy of a gene using adeno-associated viral (AAV) vectors is widely used. The scientific community is actively researching possible modifications to improve delivery efficiency and expression. In preclinical studies, the possibility of genome editing using CRISPR/Cas9 technology for the treatment of hemophilia B is also being actively studied.

JAK2 V617F Mutation in Patients with Thrombosis

Background: Thrombosis is a major disorder with serious complications. The JAK2V617F mutation results in constitutive phosphorylation of JAK2 and activation of the cellular proliferation cascade.
 The present study aimed to clarify the prevalence of JAK2V617F mutation in patients with thrombosis.
 Subjects and Methods: This case-control study was conducted on 60 subjects subdivided into 2 groups: 30 healthy subjects as a control group (Group I) and 30 patients diagnosed with thrombosis (Group II). All subjects were subjected to full history taking, and clinical examination and investigated for Molecular detection of JAK2V617F mutation, FV (Factor V Leiden) – PTH (Prothrombin), and MTHFR (Methylene tetrahydro-folate reductase).
 Results: There was a statistically significant difference among the studied groups as regards the JAK2V617F mutation (P value < 0.05, odds ratio = 41.216, 95% C.I. 95% = 2.302 - 738.034, relative risk = 25, 95% C.I. 95% = 1.547– 404.012). This mutation was more significantly associated with venous thrombosis than arterial thrombosis (P = < 0.05).
 Conclusion: This study showed a significant association between JAK2V617F mutation and thrombosis with more significant evidence in the venous than the arterial thrombosis.

Impaired factor V-related anticoagulant mechanisms and deep vein thrombosis associated with A2086D and W1920R mutations.

Factor V (FV) plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, seem to be associated with deep vein thrombosis. However, the detailed mechanism(s) through which these mutations are linked to thrombophilia remains to be fully explored. The aim of this study was to clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) and the piggyBac transposon system. Susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with limited cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and protein S (P) with similar affinities to that of FV-WT. However, weakened interaction between FVa-A2086D and phospholipid membranes was evident through the prothrombinase assay. Moreover, addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and reduce prothrombin times. This inhibitory effect was independent of PC, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.

Genetic Analysis of Hereditary Coagulation Factor V Deficiency in Two Chinese Families Caused by Compound Heterozygous Mutations.

This study aims to provide a preliminary discussion of the molecular basis of FV deficiency caused by compound heterozygous mutations in two Chinese families. Relative coagulation index was measured by the one-stage clotting method and the FV:Ag was measured by ELISA. All exons and flanking regions of the F5 gene were amplified by PCR and directly sequenced. ClustalX-2.1-win was used to analyze the conservation of mutations. The online software was used to predict the pathogenicity of mutations. PyMOL was used to analyze the variation in the spatial structure of the FV protein before and after mutations. Calibrated automated thrombogram was used to analyze the function of the mutant protein. Phenotyping suggested that both probands had a simultaneous decrease in FV:C and FV:Ag. Their genetic tests showed that proband A had a missense mutation p.Ser111Ile in exon 3 and a polymorphism p.Arg2222Gly in exon 25. At the same time, the proband B had a missense mutation p.Asp96His in exon 3 and a frame-shift mutation p.Pro798Leufs*13 in exon 13. Meanwhile, the p.Ser111Ile is conserved among homologous species. The bioinformatics and protein model analysis revealed that p.Ser111Ile and p.Pro798Leufs*13 were pathogenic and could affect the structure of the FV protein. The thrombin generation test revealed that the clotting function of proband A and B had been affected. These four mutations may be responsible for the reduction of FV levels in two Chinese families. Moreover, the p.Ser111Ile mutation is a novel pathogenic variant that has not been reported.

#5958 VALUE OF EXOME SEQUENCING “FIRST” FOR AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common Mendelian kidney diseases, that can progress to end-stage kidney failure. Pathological variants in PKD1 or PKD2 genes are found in about 78% and 15% respectively. Additional variants in genes such as GANAB, DNAJB11, and ALG8/5 have been identified in ADPKD. The sequencing of PKD1 by short read sequencing technics with exome capture such as Exome sequencing (ES) has been describe as technically challenging given 6 pseudogenes with more than 98% homology in PKD1 exonic regions 1 to 33. Moreover, the presence of repeated motifs and GC-rich in PKD1/2 add difficulties. We study the relevance of exome sequencing (ES) “first-hand” during autosomal dominant polycystic kidney disease. Method ES was performed in 684 unrelated adult patients with kidney disease from the department of Nephrology of Sorbonne University, Paris, France. Genomic DNA was extracted and exonic coding regions (37 megabases) were enriched with the Twist Human Core Exome kit, and paired-end sequenced on NextSeq500 (Illumina) machine. Sequences were analyzed with in-house and the SeqOne v1.3,2019 pipelines according to GATK4 best practices. If necessary, co-segregation of pathological variants was studied by Sanger sequencing in relatives. Results Of the 684 patients, 143 had renal cysts. Pathogenic or probably pathogenic variations in the PKD1, PKD2, and DNAJB11 genes have been identified in 26 patients, all with cystic disease; with respectively 17 variants in PKD1, 6 variants in PKD2 and 3 variants in DNAJB11. In this cohort, 18.2% of the 17 pathogenic variants are reported in PKD1, and 14 (82%) are included in exons 1 to 33. All variants were eventually confirmed by Sanger sequencing without false positive. Moreover, in 5 of the 26 patients diagnosed (19%), meaningful additional genetic data have been found, falling either in CFTR, DHCR7, HFE, F8, or ACTA2 gene. Conclusion ES highlights PKD1 and PKD2 pathogenic variants detection without false positive. This strategy allowed us to provide appropriate genetic counseling (CFTR, DHCR7), as management of yet unexpected additional genetic diseases that can affect ADPKD (F8, HFE, and ACTA2) phenotype. Given these preliminary data, ES appears effective for PKD1/PKD2 variant detection, providing additional information for ADPKD management in 20% of cases.

Alu RNA fold links splicing with signal recognition particle proteins.

Transcriptomic diversity in primates was considerably expanded by exonizations of intronic Alu elements. To better understand their cellular mechanisms we have used structure-based mutagenesis coupled with functional and proteomic assays to study the impact of successive primate mutations and their combinations on inclusion of a sense-oriented AluJ exon in the human F8 gene. We show that the splicing outcome was better predicted by consecutive RNA conformation changes than by computationally derived splicing regulatory motifs. We also demonstrate an involvement of SRP9/14 (signal recognition particle) heterodimer in splicing regulation of Alu-derived exons. Nucleotide substitutions that accumulated during primate evolution relaxed the conserved left-arm AluJ structure including helix H1 and reduced the capacity of SRP9/14 to stabilize the closed Alu conformation. RNA secondary structure-constrained mutations that promoted open Y-shaped conformations of the Alu made the Alu exon inclusion reliant on DHX9. Finally, we identified additional SRP9/14 sensitive Alu exons and predicted their functional roles in the cell. Together, these results provide unique insights into architectural elements required for sense Alu exonization, identify conserved pre-mRNA structures involved in exon selection and point to a possible chaperone activity of SRP9/14 outside the mammalian signal recognition particle.

Full-scale network analysis reveals properties of the FV protein structure organization

Blood coagulation is a vital process for humans and other species. Following an injury to a blood vessel, a cascade of molecular signals is transmitted, inhibiting and activating more than a dozen coagulation factors and resulting in the formation of a fibrin clot that ceases the bleeding. In this process, the Coagulation factor V (FV) is a master regulator, coordinating critical steps of this process. Mutations to this factor result in spontaneous bleeding episodes and prolonged hemorrhage after trauma or surgery. Although the role of FV is well characterized, it is unclear how single-point mutations affect its structure. In this study, to understand the effect of mutations, we created a detailed network map of this protein, where each node is a residue, and two residues are connected if they are in close proximity in the three-dimensional structure. Overall, we analyzed 63 point-mutations from patients and identified common patterns underlying FV deficient phenotypes. We used structural and evolutionary patterns as input to machine learning algorithms to anticipate the effects of mutations and anticipated FV-deficiency with fair accuracy. Together, our results demonstrate how clinical features, genetic data and in silico analysis are converging to enhance treatment and diagnosis of coagulation disorders.

Acute Myocardial Infarction in Patients with Hereditary Thrombophilia-A Focus on Factor V Leiden and Prothrombin G20210A.

Factor V (FV) Leiden and prothrombin G20210A are the most common hereditary thrombophilias. While their role in venous thromboembolism is well known, there are still uncertainties regarding their relationship with arterial thrombotic events, especially coronary ones. Our research, based on an in-depth analysis of the available literature, provides up-to-date information on the relationship between FV Leiden and prothrombin G20210A and acute myocardial infarction. FV Leiden and prothrombin G20210A screening should be implemented only in select cases, such as acute coronary syndrome in young individuals and/or in the absence of traditional cardiovascular risk factors and/or in the absence of significant coronary artery stenosis at angiography. Their identification should be followed by the implementation of optimal control of modifiable traditional cardiovascular risk factors to reduce the risk of recurrent events and genotyping and genetic counseling of all family members of affected cases for proper prophylaxis. An extended dual antiplatelet therapy (DAPT) may be considered, given the lower risk of bleeding under DAPT conferred by FV Leiden.

WITHDRAWN: Mutational analysis of the F8 gene and phenotypic characterization of Hemophilia A.

This article has been withdrawn as the request of the author(s) and/or Editors. The Publisher apologizes for any inconvenience this may cause.

A high-throughput test enables specific detection of hepatocellular carcinoma

High-throughput tests for early cancer detection can revolutionize public health and reduce cancer morbidity and mortality. Here we show a DNA methylation signature for hepatocellular carcinoma (HCC) detection in liquid biopsies, distinct from normal tissues and blood profiles. We developed a classifier using four CpG sites, validated in TCGA HCC data. A single F12 gene CpG site effectively differentiates HCC samples from other blood samples, normal tissues, and non-HCC tumors in TCGA and GEO data repositories. The markers were validated in a separate plasma sample dataset from HCC patients and controls. We designed a high-throughput assay using next-generation sequencing and multiplexing techniques, analyzing plasma samples from 554 clinical study participants, including HCC patients, non-HCC cancers, chronic hepatitis B, and healthy controls. HCC detection sensitivity was 84.5% at 95% specificity and 0.94 AUC. Implementing this assay for high-risk individuals could significantly decrease HCC morbidity and mortality.

Impact of timing of prophylaxis commencement, F8 genotype and age on factor consumption and health-related quality of life in patients with severe haemophilia A.

The timing of prophylaxis and F8 genotype can impact treatment outcomes in adults with severe haemophilia A (HA). To investigate how F8 genotype, timing, and type of prophylaxis influence arthropathy, bleeding rates, factor consumption and health-related quality of life (HRQoL). Thirty-eight patients with severe HA were enrolled. Bleeding events were recorded retrospectively during median 12.5 months. F8 gene variants were classified as null or non-null. Joint health and HRQoL were assessed with HJHS and EQ-5D-5L, respectively. The median age at prophylaxis start was 1.25 years in the primary prophylaxis group (N=15, median age 26 years) and 31.5 years in the secondary group (N=22, 45 years), respectively. There were significant differences in the medians of HJHS (4 vs. 20, p<.001), EQ-5D-5L index (0.9647 vs. 0.904, p=.022), EQ VAS (87 vs. 75, p=.01) and FVIII consumption (3883 vs. 2737 IU/kg/year, p=.02), between the primary and secondary groups, respectively. Median annualized bleeding rate (ABR) was 0 for both groups. Twenty-five null and thirteen non-null F8 gene variants were identified. In the secondary prophylaxis group, lower median FVIII consumption (1926 vs. 3370 IU/kg/year) was shown for non-null compared to null variants, respectively,with similar ABR and HJHS. Delayed prophylaxis start with intermediate dose intensity prevents bleeds but at a cost of more arthropathy and reduced HRQoL, compared to higher intensity primary prophylaxis. Non-null F8 genotype may allow lower factor consumption with similar HJHS and bleeding rates, compared to null genotype.

Understanding etiopathology of high altitude induced venous thrombo-embolism (HA-VTE) by variant analysis using whole exome sequencing (WES) data

BackgroundVenous thrombo-embolsim(VTE) is a complex disease and a significant cause of morbidity and mortality worldwide. Its manifestation attributes to various acquired and genetic risk factors. Environmental condition at high altitude (HA) such as hypoxia (oxygen deprivation) has been regarded as independent risk factor for VTE. Individual variations in coagulation genes play a major role in predisposing a person towards VTE. MethodologyPresent study evaluates genetic variants in VTE patients from HA (n = 6) and from sea level(SL) (n = 15) in comparison to healthy controls (n = 19). To unfold a unique thrombophilia panel in Indian population, whole exome sequencing (WES) was performed on IlluminaHiSeq platform. Presence or absence of known variants was studied across the three study groups. Statistically significantly variants were enlisted for each study group. ResultsOur findings suggest that VTE pathology is associated with total 38 variants in pro-coagulant and anti-coagulant genes. These variants were distinct for HA-VTE and SL-VTE patients. Eighteen significant variants in HA-VTE group primarily included coagulation factors, rs6035 and rs6030 in F5 gene; rs3136434, rs216311, rs216902 and rs2192205 in F2 gene and anti-coagulants, rs2759328 in SERPINC1 and rs758232970 in PROS1 gene. Amongst 7 significant variants in SL-VTE, rs6035 was common with HA-VTE. Besides, 13 variants significantly appeared when the two patient groups, HA-VTE and SL-VTE were compared with each other. ConclusionWES study delineated variants in VTE patients, from HA and SL. Our findings have generated a unique thrombophilia panel which could be helpful in predicting individual's susceptibility towards VTE at HA and even at SL.

Survival analysis of coagulation disorders: A retrospective study with a 5-year follow-up

ObjectiveRare bleeding disorders (RBDs) are the diseases in which patients experience a deficiency of coagulation factors. In the management of these disorders, surveillance is a significant challenge. This study aimed to assess the survival of patients with RBDs in a five-year follow-up. Materials and methodsThis descriptive cross-sectional study was conducted on 146 patients with RBDs who had referred to Be'sat Hospital of Hamadan, Iran from July 2017 to August 2022. A computerized record search was performed to identify the patients. The surveillance time for a five-year follow-up was assessed with the Kaplan–Meier curve. A log-rank test also served to compare the survival rates according to the type of factor. ResultsOut of 146 patients, 117 (80.2%) were males and 29 (19.8%) were females. They were in the range of 2–59 years of age with a mean of 23.11 ± 14.6. The most common disorder was FVIII deficiency (65.8%), and the rarest one was FXIII deficiency (4.8%). The rate of survival for any reason was 54.42 ± 1.3 months. The survival in combined FV and FVIII deficiencies was found to be longer than in the other deficiencies (55.9 ± 5.7), but there was no significant difference (P ≥ 0.05). In contrast, the survival in FXIII deficiency was observed to be lower than the other cases (44 ± 9.6); however, no significant difference was found in this regard (P ≥ 0.05). ConclusionThe results of this study show that patients with RBDs have different rates of survival, which suggests that identifying high-risk patients may be helpful for the improvement of their survival time through timely therapeutic interventions.

A machine learning approach for identifying variables associated with risk of developing neutralizing antidrug antibodies to factor VIII

A key unmet need in the management of hemophilia A (HA) is the lack of clinically validated markers that are associated with the development of neutralizing antibodies to Factor VIII (FVIII) (commonly referred to as inhibitors). This study aimed to identify relevant biomarkers for FVIII inhibition using Machine Learning (ML) and Explainable AI (XAI) using the My Life Our Future (MLOF) research repository. The dataset includes biologically relevant variables such as age, race, sex, ethnicity, and the variants in the F8 gene. In addition, we previously carried out Human Leukocyte Antigen Class II (HLA-II) typing on samples obtained from the MLOF repository. Using this information, we derived other patient-specific biologically and genetically important variables. These included identifying the number of foreign FVIII derived peptides, based on the alignment of the endogenous FVIII and infused drug sequences, and the foreign-peptide HLA-II molecule binding affinity calculated using NetMHCIIpan. The data were processed and trained with multiple ML classification models to identify the top performing models. The top performing model was then chosen to apply XAI via SHAP, (SHapley Additive exPlanations) to identify the variables critical for the prediction of FVIII inhibitor development in a hemophilia A patient. Using XAI we provide a robust and ranked identification of variables that could be predictive for developing inhibitors to FVIII drugs in hemophilia A patients. These variables could be validated as biomarkers and used in making clinical decisions and during drug development. The top five variables for predicting inhibitor development based on SHAP values are: (i) the baseline activity of the FVIII protein, (ii) mean affinity of all foreign peptides for HLA DRB 3, 4, & 5 alleles, (iii) mean affinity of all foreign peptides for HLA DRB1 alleles), (iv) the minimum affinity among all foreign peptides for HLA DRB1 alleles, and (v) F8 mutation type.

Rare inherited coagulation and fibrinolytic defects that challenge diagnostic laboratories.

Coagulation factors, anticoagulants, and fibrinolytic proteins are important for hemostasis, and mutations affecting these proteins causes some rare inherited bleeding disorders that are particularly challenging to diagnose. This review provides current information on rare inherited bleeding disorders that are difficult to diagnose. A review of the literature was conducted for up to date information on rare and difficult to diagnose bleeding disorders. Some rare bleeding disorders cause an inherited deficiency of multiple coagulation factors (F), such as combined FV and FVIII deficiency and familial vitamin K-dependent clotting factor deficiency. Additionally, congenital disorders of glycosylation can affect a variety of procoagulant and anticoagulant proteins and also platelets. Some bleeding disorders reflect mutations with unique impairments in the procoagulant/anticoagulant balance, including those caused by F5 mutations that secondarily increase the plasma levels of tissue factor pathway inhibitor as well as THBD mutations that increase functional thrombomodulin in plasma or cause a consumptive coagulopathy due to thrombomodulin deficiency. Some bleeding disorders accelerate fibrinolysis due to loss-of-function mutations in SERPINE1 and SERPINF2 or in the case of Quebec platelet disorder, a duplication mutation that rewires PLAU and selectively increases expression in megakaryocytes, resulting in a unique platelet-dependent gain-of-function defect in fibrinolysis. Current information on rare and difficult to diagnose bleeding disorders indicates they have unique clinical and laboratory features, and pathogenic characteristics to consider for diagnostic evaluation. Laboratories and clinicians should consider rare inherited disorders, and difficult to diagnose conditions, in their strategy for diagnosing bleeding disorders.

CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B.

Hemophilia B (HB) is an X-linked recessive disease caused by F9 gene mutation and functional coagulation factor IX (FIX) deficiency. Patients suffer from chronic arthritis and death threats owing to excessive bleeding. Compared with traditional treatments, gene therapy for HB has obvious advantages, especially when the hyperactive FIX mutant (FIX-Padua) is used. However, the mechanism by which FIX-Padua works remains ambiguous due to a lack of research models. Here, in situ introduction of F9-Padua mutation was performed in human induced pluripotent stem cells (hiPSCs) via CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs). The hyperactivity of FIX-Padua was confirmed to be 364% of the normal level in edited hiPSCs-derived hepatocytes, providing a reliable model for exploring the mechanism of the hyperactivity of FIX-Padua. Moreover, the F9 cDNA containing F9-Padua was integrated before the F9 initiation codon by CRISPR/Cas9 in iPSCs from an HB patient (HB-hiPSCs). Integrated HB-hiPSCs after off-target screening were differentiated into hepatocytes. The FIX activity in the supernatant of integrated hepatocytes showed a 4.2-fold increase and reached 63.64% of the normal level, suggesting a universal treatment for HB patients with various mutations in F9 exons. Overall, our study provides new approaches for the exploration and development of cell-based gene therapy for HB.