Genetic Landscape of Factor VII Deficiency: Insights from a Comprehensive Analysis of Pathogenic Variants and Their Impact on Coagulation Activity.

Congenital factor VII (FVII) deficiency is a rare genetic bleeding disorder characterized by deficient or reduced activity of coagulation FVII. It is caused by genetic variants in the F7 gene. We aimed to evaluate the rate of detection of pathogenic variants in the F7 gene in a large group of patients with FVII deficiency and investigate the correlations between the F7 genotype and FVII activity (FVII:C). Moreover, the influence of the common genetic variant rs6046: c.1238G>A; p.(Arg413Gln), designated as the M2 allele, on FVII:C was investigated. Genetic analysis of the F7 gene was performed on 704 index patients (IPs) using either direct Sanger- or next-generation sequencing. Genetic variants were detected in 390 IPs, yielding a variant detection rate (VDR) of 55%. Notably, the VDR exhibited a linear decline with increasing FVII:C levels. We identified 124 genetic variants, of which 48 were not previously reported. Overall, the frequency of the M2 allele was considerably higher in patients with mild deficiency (FVII:C > 20 IU/dl). Furthermore, IPs lacking an identified pathogenic variant exhibited a significantly higher prevalence of the M2 allele (69%) compared to IPs with a disease-causing variant (47%). These results strongly support the association of the M2 allele with decreased FVII:C levels. This study shows the utility of FVII:C as a predictive marker for identifying pathogenic variants in patients with FVII deficiency. The M2 allele contributes to the reduction of FVII:C levels, particularly in cases of mild deficiency.

Heterozygosity in factor XIII genes and the manifestation of mild inherited factor XIII deficiency.

BACKGROUND: The characterization of inherited mild factor XIII deficiency is more imprecise than its rare, inherited severe forms. It is known that heterozygosity at FXIII genetic loci results in mild FXIII deficiency, characterized by circulating FXIII activity levels ranging from 20% to 60%. There exists a gap in information on 1) how genetic heterozygosity renders clinical bleeding manifestations among these individuals and 2) the reversal of unexplained bleeding upon FXIII administration in mild FXIII-deficient individuals. OBJECTIVES: To assess the prevalence and burden of mild FXIII deficiency among the apparently healthy German-Caucasian population and correlate it with genetic heterozygosity at FXIII and fibrinogen gene loci. METHODS: Peripheral blood was collected from 752 donors selected from the general population with essentially no bleeding complications to ensure asymptomatic predisposition. These were assessed for FXIII and fibrinogen activity, and FXIII and fibrinogen genes were resequenced using next-generation sequencing. For comparison, a retrospective analysis was performed on a cohort of mild inherited FXIII deficiency patients referred to us. RESULTS: The prevalence of mild FXIII deficiency was high (∼0.8%) among the screened German-Caucasian population compared with its rare-severe forms. Although no new heterozygous missense variants were found, certain combinations were relatively dominant/prevalent among the mild FXIII-deficient individuals. CONCLUSION: This extensive, population-based quasi-experimental approach revealed that the burden of heterozygosity in FXIII and fibrinogen gene loci causes the clinical manifestation of inherited mild FXIII deficiency, resulting in ''unexplained bleeding'' upon provocation.

Disruption of Structural Disulfides of Coagulation FXIII-B Subunit; Functional Implications for a Rare Bleeding Disorder.

Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine's, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.

Exploring the structural similarity yet functional distinction between coagulation factor XIII-B and complement factor H sushi domains.

Coagulation factor XIII (FXIII) covalently crosslinks pre-formed fibrin clots preventing their premature fibrinolysis. In plasma, FXIII circulates as a zymogenic heterotetramer composed of catalytic FXIII-A subunits, and carrier/regulatory FXIII-B subunits. FXIII-A is a well characterized component of this complex, and has been associated with several pleiotropic roles outside coagulation as well. In comparison only protective/regulatory roles towards the FXIII-A subunit have been identified for FXIII-B. Strong homology between FXIII-B and complement regulator Complement factor H suggests a putative role of FXIII-B in complement activation. In the current study we have analyzed the similarities and yet functional divergence of these two proteins using in silico sequence alignment and structural analysis. We have evaluated complement activation post reconstitution of FXIII components into FXIII deficient and CFH deficient plasma. We have also transiently expressed FXIII-B in SH-SY5Y cell lines and evaluated its effect on the endogenous complement activation. Our investigations show no effect of FXIII-B subunit on the rate of complement activation. Therefore we conclude that at a physiological level, FXIII-B subunit plays no role in the complement system, although a vestigial function in altered pathological states might still exist.

A rare case of unprovoked venous thromboembolism manifestation in a young patient with antithrombin Type IIB deficiency combined with inferior vena cava anomaly from Lithuania.

Hereditary antithrombin (AT) deficiency is an autosomal-dominant disorder predisposing to venous and arterial thrombosis. Homozygosity resulting in severe AT deficiency is not compatible with life, apart from homozygous mutations affecting the heparin-binding site representing the most severe thrombophilia. PATIENTS AND METHODS: A 12-year-old previously healthy boy of Romani origin presented with a swollen, painful left leg and fever. Imaging revealed signs of inferior vena cava (IVC) thrombosis, the presence of interrupted intrahepatic IVC with azygos continuation and bilateral iliofemoral thrombosis with enlargement of the azygous and hemiazygos venous system. In addition, right pleural effusion and signs of bilateral renal pericortical cysts, possibly caused by retroperitoneal lymphangiectasia, were disclosed. Thrombophilia screening involving protein C, Protein S, Antithrombin (chromogenic assays based on the inhibition of FIIa and FXa, antigen concentration), APC resistance, prothrombin mutation and Lupus anticoagulants was performed using standard laboratory methods. Genetic analysis of the SERPINC1 gene was done by direct sequencing. RESULTS: Thrombophilia screening showed isolated decreased AT activity at 21% (RR 80-120%). AT levels were retested and remained decreased (33-36%) on two consecutive occasions. SERPINC1 gene analysis revealed a previously described homozygous mutation (Budapest III) causing type IIB deficiency (c.391C>T; p.Leu131Phe). A family study confirmed the same mutation in both parents and three siblings. The patient improved significantly following warfarin therapy and over the past 2.5 years did not experience new thromboembolism. CONCLUSIONS: This case represents a rare combination of two risk factors provoking manifestation of spontaneous venous thromboembolism at a young age requiring permanent anticoagulant therapy.

Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5' splice site.

Disease-associated silent mutations are considered to affect the accurate pre-messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 5' splice site (5'ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 5'ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5'ss. This hairpin sequesters the 5'ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA.

Revisiting the mechanism of coagulation factor XIII activation and regulation from a structure/functional perspective.

The activation and regulation of coagulation Factor XIII (FXIII) protein has been the subject of active research for the past three decades. Although discrete evidence exists on various aspects of FXIII activation and regulation a combinatorial structure/functional view in this regard is lacking. In this study, we present results of a structure/function study of the functional chain of events for FXIII. Our study shows how subtle chronological submolecular changes within calcium binding sites can bring about the detailed transformation of the zymogenic FXIII to its activated form especially in the context of FXIIIA and FXIIIB subunit interactions. We demonstrate what aspects of FXIII are important for the stabilization (first calcium binding site) of its zymogenic form and the possible modes of deactivation (thrombin mediated secondary cleavage) of the activated form. Our study for the first time provides a structural outlook of the FXIIIA2B2 heterotetramer assembly, its association and dissociation. The FXIIIB subunits regulatory role in the overall process has also been elaborated upon. In summary, this study provides detailed structural insight into the mechanisms of FXIII activation and regulation that can be used as a template for the development of future highly specific therapeutic inhibitors targeting FXIII in pathological conditions like thrombosis.

Coagulation Factor XIIIA Subunit Missense Mutations Affect Structure and Function at the Various Steps of Factor XIII Action.

Inherited defects of coagulation Factor XIII (FXIII) can be categorized into severe and mild forms based on their genotype and phenotype. Heterozygous mutations occurring in F13A1 and F13B genes causing mild FXIII deficiency have been reported only in the last few years primarily because the mild FXIII deficiency patients are often asymptomatic unless exposed to some kind of a physical trauma. However, unlike mutations causing severe FXIII deficiency, many of these mutations have not been comprehensively characterized based on expression studies. In our current article, we have transiently expressed 16 previously reported missense mutations detected in the F13A1 gene of patients with mild FXIII deficiency and analyzed their respective expression phenotype. Complimentary to expression analysis, we have used in silico analysis to understand and explain some of the in vitro findings. The expression phenotype has been evaluated with a number of expression phenotype determining assays. We observe that the mutations influence different aspects of FXIII function and can be functionally categorized on the basis of their expression phenotype. We identified mutations which even in heterozygous form would have strong impact on the functional status of the protein (namely mutations p.Arg716Gly, p.Arg704Gln, p.Gln602Lys, p.Leu530Pro, p.His343Tyr, p.Pro290Arg, and p.Arg172Gln).

Characterization of a novel large deletion caused by double-stranded breaks in 6-bp microhomologous sequences of intron 11 and 12 of the F13A1 gene.

Coagulation Factor XIII is a heterotetrameric protransglutaminase which stabilizes preformed fibrin clots by covalent crosslinking them. Inherited homozygous or compound heterozygous deficiency of coagulation Factor XIII (FXIII) is a rare severe bleeding disorder affecting 1 in 2 million individuals. Most of the patients with inherited FXIII deficiency described in the literature carry F13A1 gene point mutations (missense, nonsense and splice site defects), whereas large deletions (>0.5 kb in size) are underrepresented. In this article we report for the first time the complete characterization of a novel homozygous F13A1 large deletion covering the entire exon 12 in a young patient with a severe FXIII-deficient phenotype from France. Using primer walking on genomic DNA we have identified the deletion breakpoints in the region between g.6.143,016-g.6.148,901 caused by small 6-bp microhomologies at the 5´ and 3´ breakpoints. Parents of the patient were heterozygous carriers. Identification of this large deletion offers the possibility of prenatal diagnosis for the mother in this family who is heterozygous for this deletion.

Structural and functional influences of coagulation factor XIII subunit B heterozygous missense mutants.

The coagulation factor XIII(FXIII) is a plasma circulating heterotetrameric protransglutaminase that acts at the end of the coagulation cascade by covalently cross-linking preformed fibrin clots (to themselves and to fibrinolytic inhibitors) in order to stabilize them against fibrinolysis. It circulates in the plasma as a heterotetramer composed of two homomeric catalytic Factor XIIIA2 (FXIIIA2) and two homomeric protective/carrier Factor XIIIB2 subunit (FXIIIB2). Congenital deficiency of FXIII is of two types: severe homozygous/compound heterozygous FXIII deficiency which results in severe bleeding symptoms and mild heterozygous FXIII deficiency which is associated with mild bleeding (only upon trauma) or an asymptomatic phenotype. Defects in the F13B gene (Factor XIIIB subunit) occur more frequently in mild FXIII deficiency patients than in severe FXIII deficiency. We had recently reported secretion-related defects for seven previously reported F13B missense mutations. In the present study we further analyze the underlying molecular pathological mechanisms as well as the heterozygous expression phenotype for these mutations using a combination of in vitro heterologous expression (in HEK293T cells) and confocal microscopy. In combination with the in vitro work we have also performed an in silico solvated molecular dynamic simulation study on previously reported FXIIIB subunit sushi domain homology models in order to predict the putative structure-functional impact of these mutations. We were able to categorize the mutations into the following functional groups that: (1) affect antigenic stability as well as binding to FXIIIA subunit, that is, Cys5Arg, Cys316Phe, and Pro428Ser (2) affect binding to FXIIIA subunit with little or no influence on antigenic stability, that is, Ile81Asn and Val401Gln c) influence neither aspects and are most likely causality linked polymorphisms or functional polymorphisms, that is, Leu116Phe and Val217Ile. The Cys5Arg mutation was the only mutation to show a direct secretion-based defect since the mutated protein was observed to accumulate in the endoplasmic reticulum.

Eight novel F13A1 gene missense mutations in patients with mild FXIII deficiency: in silico analysis suggests changes in FXIII-A subunit structure/function.

Mild FXIII deficiency is an under-diagnosed disorder because the carriers of this deficiency are often asymptomatic and reveal a phenotype only under special circumstances like surgery or induced trauma. Mutational reports from this type of deficiency have been rare. In this study, we present the phenotypic and genotypic data of nine patients showing mild FXIII-A deficiency caused by eight novel heterozygous missense mutations (Pro166Leu, Arg171Gln, His342Tyr, Gln415Arg, Leu529Pro, Gln601Lys, Arg703Gln and Arg715Gly) in the F13A1 gene. None of these variants were seen in 200 healthy controls. In silico structural analysis of the local wild-type protein structures (activated and non-activated) from X-ray crystallographic models downloaded from the protein databank identified potential structural/functional effects for the identified mutations. The missense mutations in the core domain are suggested to be directly influencing the catalytic triad. Mutations on other domains might influence other critical factors such as activation peptide cleavage or the barrel domain integrity. In vitro expression and subsequent biochemical studies in the future will be able to confirm the pathophysiological mechanisms proposed for the mutations in this article.

In vitro secretion deficits are common among human coagulation factor XIII subunit B missense mutants: correlations with patient phenotypes and molecular models.

Coagulation factor XIII (FXIII) proenzyme circulates in plasma as a heterotetramer composed of two each of A and B subunits. Upon activation, the B subunits dissociate from the A subunit dimer, which gains transglutaminase activity to cross-link preformed fibrin clots increasing mechanical strength and resistance to degradation. The B subunits are thought to possess a carrier/protective function before FXIII activation. Mutations in either A or B subunits are associated with pathological patient phenotypes characterized by mild to severe bleeding. In vitro expression of FXIII B subunit (FXIIIB) missense variants in HEK293T cells revealed impaired secretion for all seven variants studied. To investigate the likely molecular environments of the missense residues, we created molecular models of individual FXIIIB Sushi domains using phylogenetically similar complement factor H Sushi domain structural templates. Assessment of the local molecular environments for the models suggested surface or buried positions for each mutant residue and possible pathological mechanisms. The in vitro expression system and in silico analytical methods and models we developed can be used to further investigate the molecular basis of FXIIIB mutation pathologies.

A common F13A1 intron 1 variant IVS1+12(A) is associated with mild FXIII deficiency in Caucasian population.

Mild factor XIII deficiency is an underdiagnosed coagulation disorder. Considering the large number of coding and non-coding polymorphisms identified in the F13A1 gene, there is a possibility that some of these might result in alterations of plasma FXIII levels and cause mild FXIII deficiency. Recently, a homozygous F13A1 gene intron 1 variant (IVS1+12C>A) was found in a patient with FXIII deficiency. In vitro expression studies for this variant demonstrated its lowering effect on FXIII levels. In order to determine the impact of this variant on a population level, we analysed the prevalence of this variant in three clinically and genetically defined population cohorts: an apparently healthy control cohort C1 (n = 102), a mild FXIII deficiency cohort C2 with no detectable F13A1 or F13B gene mutations (n = 183) and a mild FXIII-A deficiency cohort C3 exhibiting heterozygous F13A1 mutations (n = 37). FXIII activity was determined using photometric assay on plasma samples. The F13A1 gene intron 1 variant was analysed by direct sequencing. The C1 cohort showed a normal distribution of FXIII activity (mean 114.1 ± 20.86%). Mean FXIII activity levels for the C2 and C3 cohorts were 54.45 ± 11.12% and 44.21 ± 10.16%, respectively. The frequencies of minor allele (A) were 0.07 in C1 cohort, 0.19 in C2 cohort and 0.11 in C3 cohort. The difference in minor allele frequencies for the C1 and C2 cohorts were highly significant (p < 0.001). The greater frequency of the IVS1+12(A) variant among C2 cohort patients suggests that this polymorphism is associated with mild FXIII deficiency.

Insights into pathological mechanisms of missense mutations in C-terminal domains of von Willebrand factor causing qualitative or quantitative von Willebrand disease.

The carboxyl-terminal domains of von Willebrand factor, D4-CK, are cysteine-rich implying that they are structurally important. In this study we characterized the impact of five cysteine missense mutations residing in D4-CK domains on the conformation and biosynthesis of von Willebrand factor. These variants were identified as heterozygous in type 1 (p.Cys2619Tyr and p.Cys2676Phe), type 2A (p.Cys2085Tyr and p.Cys2327Trp) and as compound heterozygous in type 3 (p.Cys2283Arg) von Willebrand disease. Transient expression of human cell lines with wild-type or mutant von Willebrand factor constructs was performed. The mutated and wild-type recombinant von Willebrand factors were quantitatively and qualitatively assessed and compared. Storage of von Willebrand factor in pseudo-Weibel-Palade bodies was studied with confocal microscopy. The structural impact of the mutations was analyzed by homology modeling. Homozygous expressions showed that these mutations caused defects in multimerization, elongation of pseudo-Weibel-Palade bodies and secretion of von Willebrand factor. Co-expressions of wild-type von Willebrand factor and p.Cys2085Tyr, p.Cys2327Trp and p.Cys2283Arg demonstrated defective multimer assembly, suggesting a new pathological mechanism for dominant type 2A von Willebrand disease due to mutations in D4 and B domains. Structural analysis revealed that mutations p.Cys2283Arg, p.Cys2619Tyr and p.Cys2676Phe disrupted intra-domain disulfide bonds, whereas p.Cys2327Trp might affect an inter-domain disulfide bond. The p.Cys2327Trp variant is distinguished from the other mutants by an electrophoretic mobility shift of the multimer bands. The results highlight the importance of cysteine residues within the carboxyl-terminal of von Willebrand factor on structural conformation of the protein and consequently multimerization, storage, and secretion of von Willebrand factor.

An update of the mutation profile of Factor 13 A and B genes.

Mutational reports over the past two decades have accumulated an immense amount of literature for inherited Factor XIII deficiency. However, the genotype and phenotype correlations for inherited Factor XIII deficiency are complicated. While many studies clearly prove a cause and effect relationship for the reported mutations, others are lacking in this regard. The F13B gene remains an elusive component as far as inherited Factor XIII deficiencies are concerned. Also, an in-depth analysis into the heterozygous state of this deficiency is also lacking. In this review we have tried to analyze and present an exhaustive amount of mutational data from the past three decades. The source of our mutational data is our website dedicated to Factor XIII deficiencies (www.F13-database.de) as well as literature search done on the Pubmed (www.ncbi.nlm.nih.gov/pubmed).

Identification of eight novel coagulation factor XIII subunit A mutations: implied consequences for structure and function.

BACKGROUND: Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer. DESIGN AND METHODS: We analyzed ten index patients and three relatives for factor XIII activity using a photometric assay and sequenced their F13A and F13B genes. Additionally, structural analysis of the wild-type protein structure from a previously reported X-ray crystallographic model identified potential structural and functional effects of the missense mutations. RESULTS: All individuals except one were heterozygous for factor XIIIA mutations (average factor XIII activity 51%), while the remaining homozygous individual was found to have severe factor XIII deficiency (<5% of normal factor XIII activity). Eight of the 12 heterozygous patients exhibited a bleeding tendency upon provocation. CONCLUSIONS: The identified missense (Pro289Arg, Arg611His, Asp668Gly) and nonsense (Gly390X, Trp664X) mutations are causative for factor XIII deficiency. A Gly592Ser variant identified in three unrelated index patients, as well as in 200 healthy controls (minor allele frequency 0.005), and two further Tyr167Cys and Arg540Gln variants, represent possible candidates for rare F13A gene polymorphisms since they apparently do not have a significant influence on the structure of the factor XIIIA protein. Future in vitro expression studies of the factor XIII mutations are required to confirm their pathological mechanisms.

The first case of combined coagulation factor V and coagulation factor VIII deficiency in Poland due to a novel p.Tyr135Asn missense mutation in the MCFD2 gene.

Congenital combined coagulation factor V and coagulation factor VIII deficiency (F5F8D) is a rare bleeding disorder due to mutations in the LMAN1 or MCFD2 genes. Here we report the first Polish family with F5F8 deficiency due to a mutation in the MCFD2 gene. The proposita suffered from mild bleeding including epistaxis, menorrhagia, bleeding after dental extraction, and bruising after minor traumas. The F5F8 deficiency was diagnosed due to an excessive postpartum bleeding at the age of 31. Analysis of further family members revealed a second affected individual. Sequencing of the MCFD2 gene and its flanking regions in both patients demonstrated a novel homozygous missense mutation within the second elongation factor hand domain resulting in a substitution of tyrosine by asparagine at amino acid position 135 (p.Tyr135Asn). This variant represents the third missense mutation found in the MCFD2 gene and most likely disrupts the MCFD2-LMAN1 interaction, thus leading to the disease phenotype.

International registry on factor XIII deficiency: a basis formed mostly on European data.

FXIII deficiency is known as one of the rarest blood coagulation disorders. In this study, the phenotypic and in part genotypic data of 104 FXIII-deficient patients recorded from 1993 - 2005 are presented. The most common bleeding symptoms were subcutaneous bleeding (57%) followed by delayed umbilical cord bleeding (56%), muscle hematoma (49%), hemorrhage after surgery (40%), hemarthrosis (36%), and intracerebral bleeding (34%). Prophylactic treatment was initiated in about 70% of all patients. FXIII-B subunit-deficient patients had a milder phenotype than patients with FXIII-A subunit deficiency. The most frequent mutation affecting the F13A gene was a splice site mutation in intron 5 (IVS5-1G>A). This mutation was found in eight (17%) of 46 analyzed families. The haplotype analysis of patients carrying the IVS5-1A allele was consistent with a founder effect. The international registry (http://www.f13-database.de) will provide clinicians and scientists working on FXIII deficiency with a helpful tool to improve patient care and direct future studies towards better understanding and treatment of the disease.