Fibrinogen Bonn (p. Arg510Cys) in the Aα-Chain Is Associated with High Risk of Venous Thrombosis.

INTRODUCTION: Inherited dysfibrinogenemia is a qualitative defect of fibrinogen caused by various mutations among three fibrinogen genes. Dysfibrinogenemia can be associated with an increased risk of thrombosis, bleeding, or both. Here, we report a 36-year-old female with dysfibrinogenemia who experienced two successful pregnancies under thromboprophylaxis after cerebral venous sinus thrombosis (CVST). PATIENTS AND METHODS: In addition to plasmatic coagulation tests, fibrinogen genes FGA, FGB, and FGG were screened using direct genomic DNA sequencing. The structural-functional implications of the detected mutation were analyzed in silico. RESULTS: Inherited dysfibrinogenemia was diagnosed in an index patient after CVST in a risk situation. Anticoagulation with warfarin was stopped after 12 months when the first pregnancy was planned. Pregnancy and spontaneous delivery (2020) was uncomplicated. A second pregnancy was interrupted because of acute cytomegalovirus infection and the third pregnancy was successful in 2022. Pregnancies were accompanied by thromboprophylaxis with enoxaparin 40 mg once daily until 6 weeks postpartum. Substitution of fibrinogen has not become necessary in the index patient so far. Genetic analysis revealed a novel missense mutation (p. Arg510Cys) in the FGA gene ("fibrinogen Bonn") in the index patient, as well as an asymptomatic sister, and their father who experienced recurrent pulmonary embolism. Surface exposure of wild-type Arg510 suggested the mutated Cys510 to form nonnative disulfide bonds with surface-exposed reactive cysteines from other plasma proteins like albumin leading to formation of aggregates and impaired fibrinolysis. CONCLUSIONS: Fibrinogen Bonn might be associated with an increased risk of thrombosis, possibly due to impaired polymerization.

Experiences in Routine Genetic Analysis of Hereditary Hemorrhagic, Thrombotic, and Platelet Disorders.

Hemostasis is a complex and tightly regulated system that attempts to maintain a homeostatic balance to permit normal blood flow, without bleeding or thrombosis. Hemostasis reflects the subtle balance between procoagulant and anticoagulant factors in the pathways of primary hemostasis, secondary hemostasis, and fibrinolysis. The major components in this interplay include the vascular endothelium, platelets, coagulation factors, and fibrinolytic factors. After vessel wall injury, the subendothelium is exposed to the blood stream, followed by rapid activation of platelets via collagen binding and von Willebrand factor-mediated platelet adhesion to the damaged vessel wall through platelet glycoprotein receptor Ib/IX/V. Activated platelets change their shape, release bioactive molecules from their granules, and expose negatively charged phospholipids on their surface. For a proper function of this process, an adequate number of functional platelets are required. Subsequently, a rapid generation of sufficient amounts of thrombin begins; followed by activation of the coagulation system and its coagulation factors (secondary hemostasis), generating fibrin that consolidates the platelet plug. To maintain equilibrium between coagulation and anticoagulation, the naturally occurring anticoagulants such as protein C, protein S, and antithrombin keep this process in balance. Deficiencies (inherited or acquired) at any level of this fine-tuned system result in pathologic bleedings or increased hypercoagulability states leading to thrombosis. This review will focus on genetic diagnosis of inherited bleeding, thrombotic, and platelet disorders, discussing strengths and limitations of existing diagnostic settings and genetic tools and highlight some important considerations necessary for clinical application.

Combined coagulation factor VIII and factor IX deficiency (CDF8F9) in a patient from Lithuania.

Haemophilia A (FVIII deficiency) and haemophilia B (FIX deficiency) are X-linked inherited bleeding disorders. It is a very rare event to identify both haemophilias in the same patient. So far, only two families with such combination are reported in the literature worldwide supported by genetic background. PATIENTS AND METHODS: Evaluation of clinical data, determination of FVIII and FIX levels and genetic analysis of F8 and F9 genes by direct sequencing. RESULTS: We report on a patient having severe haemophilia B (FIX:C <1 IU dl-1) and mild haemophilia A (FVIII:C 18 IU dl-1 ). FIX deficiency was known since childhood, whereas mild haemophilia A was confirmed at the age of 42 due to unexpected bleeding complications after dental extraction despite adequate substitution with plasma derived FIX concentrate. F9 gene analysis showed a point mutation in exon 2 (c.223C>T, p.R75X), whereas F8 gene analysis revealed a point mutation in exon 4 (c.545A>C, p.D182A). The mother of the patient was heterozygous for F8 mutation, but not for F9 mutation suggesting a de novo F9 mutation. Accidentally, further family from Germany with mild Haemophilia A was identified to have the same F8 mutation. F8 Haplotype analysis revealed that the p.D182A mutation most likely represents a founder mutation with common ancestors of the German and the Lithuanian family. CONCLUSIONS: Our results confirm the rare event of Haemophilia A and haemophilia B in the same patient originating from two distinct genetic defects in F8 and F9 genes.

Evidence of pathogenicity of a mutation in 3' untranslated region causing mild haemophilia A.

INTRODUCTION: Despite the high mutation detection rate, in a small group of haemophilia A patients, using current screening methods, no causal mutation in F8 can be detected. In such cases, the causal mutation might be in the non-coding sequences of F8. AIM: Rarely, mutations in non-coding sequences reveal a pivotal role. Here, we analysed a mild haemophilia A patient harbouring a mutation in the 3' untranslated region (UTR) of F8 and elucidated the molecular mechanism leading to haemophilia phenotype. METHODS: To find the causal mutation, the complete F8 genomic region was analysed by next generation sequencing. The effect of the identified alteration on F8 expression was evaluated in silico and analysed for the splicing effect at mRNA level. Moreover, in vitro studies using a luciferase reporter system were performed to functionally analyse the mutation. RESULTS: We identified an alteration in the 3' UTR (c.*56G>T) as the only change in F8 gene. Pedigree analysis showed a segregation pattern for three affected members for the presumptive mutation. Moreover, the variant was predicted in silico to create a new donor splice site, which was also detected at mRNA level, resulting in a 159 bp deletion in 3' UTR of F8. Finally, the variant showed reduced expression of the gene reporter firefly luciferase in cell line expression analysis. CONCLUSION: Our results advocate the patient specific c.*56G>T base change in the 3' UTR to be a disease-associated mutation leading to alternative splicing explaining the mild haemophilia A phenotype.

Identification and functional characterization of a novel F5 mutation (Ala512Val, FVB onn ) associated with activated protein C resistance.

UNLABELLED: Essentials Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. A novel missense mutation (Ala512Val - FVBonn ) was characterized in vitro and in silico. FVBonn is a new cause of APC resistance and venous thrombosis. FVBonn expresses additionally enhanced procoagulant activity in the absence of APC. SUMMARY: Background Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. This phenotype is most commonly associated with the factor V Arg506Gln mutation (FV Leiden), which impairs the APC-mediated inactivation of both activated FV (FVa) and activated FVIII (FVIIIa). Objectives Here, we report the identification and characterization of a novel FV mutation (Ala512Val, FVBonn ) in six patients with APC resistance and venous thrombosis or recurrent abortions. Methods FVBonn was expressed in a recombinant system and compared with recombinant wild-type (WT) FV and FV Leiden in several functional assays. Results FVBonn conferred APC resistance to FV-depleted plasma, both in the activated partial thromboplastin time (APTT)-based test (APC sensitivity ratio [APCsr] of 1.98 for FVBonn versus 4.31 for WT FV and 1.59 for FV Leiden) and in the thrombin generation-based test (normalized APCsr of 5.41 for FVBonn versus 1.00 for WT FV and 8.99 for FV Leiden). The APC-mediated inactivation of FVaBonn was slower than that of WT FVa (mainly because of delayed cleavage at Arg506), but was greatly stimulated by protein S. The APC cofactor activity of FVBonn in FVIIIa inactivation was ~ 24% lower than that of WT FV. In line with these findings, an in silico analysis showed that the Ala512Val mutation is located in the same loop as the Arg506 APC cleavage site and might hamper its interaction with APC. Moreover, FVBonn was more procoagulant than WT FV and FV Leiden in the absence of APC, because of an increased activation rate and, possibly, an enhanced interaction with activated FX. Conclusions FVBonn induces hypercoagulability via a combination of increased activation/procoagulant activity, decreased susceptibility to APC-mediated inactivation, and slightly reduced APC cofactor activity.

Novel characterization of a breakpoint in F8: an individualized approach to gene analysis when PCR and MLPA results contradict.

Haemophilia A is an X-linked bleeding disorder caused by heterogeneous mutations in the F8 gene. Two inversion hotspots in intron 22 and intron 1, as well as point mutations, small insertions and deletions in the F8 gene account for causal mutations leading to severe haemophilia A. Rarely, novel molecular mechanisms lead to a haemophilia A phenotype which cannot be completely characterized by routine molecular diagnostic methods. Here, we characterized the molecular abnormality in a boy with a severe haemophilia A phenotype. On investigation by PCR and DNA sequencing, exon 18 of F8 repeatedly failed to amplify. However, analysis by multiplex ligation-dependent probe amplification demonstrated the presence of exon 18 sequence, suggesting a more complex rearrangement than a single exon deletion. The analysis of exon 18 and its flanking regions by inverse PCR revealed a complex mutation comprising insertions of extragenic sequences from Xq28 along with a partial duplication of exon 18. Based on the successful analysis and characterization of the familial breakpoint, we developed a PCR-based diagnostic approach to detect this defect in family members in whom no diagnostic test could be offered until this time.

Genetic testing in bleeding disorders.

The aim of molecular genetic analysis in families with haemophilia is to identify the causative mutation in an affected male as this provides valuable information for the patient and his relatives. For the patient, mutation identification may highlight inhibitor development risk or discrepancy between different factor VIII assays. For female relatives, knowledge of the familial mutation can facilitate carrier status determination and prenatal diagnosis. Recent advances in understanding mutations responsible for haemophilia and methods for their detection are presented. For reporting of such mutations, participation in external quality assessment ensures that essential patient and mutation details are routinely included and that pertinent information is incorporated in the interpretation.

F8 genetic analysis strategies when standard approaches fail.

Haemophilia A is a common X-linked recessive disorder caused by mutations in F8 leading to deficiency or dysfunction of coagulant factor VIII (FVIII). Despite tremendous improvements in mutation screening methods, in a small group of patients with FVIII deficiency suffering from haemophilia A, no DNA change can be found. In these patients, analysis reveals no causal mutations even after sequencing the whole coding region of F8 including the flanking splice sites, as well as the promoter and the 3' untranslated region (UTR). After excluding the mutations mimicking the haemophilia A phenotype in interacting partners of the FVIII protein affecting the half life and transport of the protein, mutations or rearrangements in non-coding regions of F8 have to be considered responsible for the haemophilia A phenotype. In this review, we present the experiences with molecular diagnosis of such cases and approaches to be applied for mutation negative patients.

Deep intronic 'mutations' cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA.

BACKGROUND: In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and non-genetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. METHODS: We determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LR-PCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. RESULTS: All VWF tests were normal. The LR-PCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. CONCLUSION: By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

Identification of a third rearrangement at Xq28 that causes severe hemophilia A as a result of homologous recombination between inverted repeats.

BACKGROUND: Intrachromosomal homologous recombination between inverted repeats on the X chromosome account for about half of severe hemophilia A cases. Repeats in F8 intron 1 and intron 22 can recombine with homologous inverted repeats located about 200 kb upstream and 500 kb downstream of F8, respectively, resulting in partial sequence inversion of the F8 open reading frame and, subsequently, no functional protein production. OBJECTIVES: In the present study, we characterize a third novel homologous recombination at Xq28 consistent with absence of F8 transcription that we previously reported for the affected chromosome of the index patient as well as his mother and sister. RESULTS: The rearrangement occurs between a repeat in F8 intron 1 (Int1R-1) and an inverted identical repeat (Int1R-2d) in intron 2 of a duplicated copy of IKBKG located about 386 kb upstream of F8. The rearrangement was confirmed by Southern blot and inverse PCR and results in failure of PCR amplification across Int1R-1. CONCLUSION: We developed a PCR-based diagnostic method that can be used to screen for this genetic rearrangement in cases of severe hemophilia A for which mutations cannot be identified.