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
gene. We aimed ...to evaluate the rate of detection of pathogenic variants in the
gene in a large group of patients with FVII deficiency and investigate the correlations between the
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
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.
Molecular genetic analysis is widely applied in inherited bleeding disorders. The outcome of genetic analysis allows genetic counselling in affected families and helps to find a link between the ...genotype and phenotype. Genetic analysis in hemophilia A (HA) has tremendously improved in the past decades. Many new techniques and modifications as well as analysis software have become available, which has enabled genetic analysis and interpretation of data to become faster and more accurate. The advances in mutation detection strategies facilitate the identification of the causal mutation in up to 97% of patients with HA. This review discusses the milestones in genetic analysis of HA and highlights the importance of identification of the causative mutations for genetic counseling and particularly for the interpretation of the clinical presentation of HA patients.
Inactivation of thrombin by the endogenous inhibitor antithrombin (AT) is a central mechanism in the regulation of hemostasis. This makes hereditary AT deficiency, which is caused by SERPINC1 gene ...mutations, a major thrombophilic risk factor. Aim of this study was to assess to what extent AT mutations impair thrombin inhibition kinetics. The study population included 36 thrombophilic patients with 19 different mutations and mean AT levels of 65% in a thrombin-based functional assay, and 26 healthy controls. To assess thrombin inhibition kinetics, thrombin (3.94 mU/mL final concentration) was added to citrated plasma. Subsequently, endogenous thrombin inhibition was stopped by addition of the reversible thrombin inhibitor argatroban and the amount of argatroban-complexed thrombin quantified using an oligonucleotide-based enzyme capture assay. The plasma half-life of human thrombin was significantly longer in patients with AT mutations than in the controls (119.9 versus 55.9 s). Moreover, it was disproportionately prolonged when compared with preparations of wild type AT in plasma, in whom a comparable thrombin half-life of 120.8 s was reached at a distinctly lower AT level of 20%. These findings may help to better understand the increased thrombotic risk of SERPINC1 mutations with near normal AT plasma levels in functional assays.
Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. For genomic inversions in particular, the disease causing mechanism may not be clear, ...especially if the inversion border does not cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, in a mainly epigenetic context, may provide additional information regarding sequence-specific regulation of gene expression in human diseases. Herein, we study one such inversion hotspot at Xq28, which leads to the disruption of F8 gene and results in hemophilia A phenotype. To determine the epigenetic consequence of this rearrangement, we evaluated DNA methylation levels of 12 CpG rich regions with the coverage of 550 kb by using bisulfite-pyrosequencing and next-generation sequencing (NGS)-based bisulfite re-sequencing enrichment assay. Our results show that this inversion prone area harbors widespread methylation changes at the studied regions. However, only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (two regions), intron 22 inversion (two regions) and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, two CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. While we found age to be an influencing factor on methylation levels at some regions, covariate analysis still confirms the differential methylation induced by inversion, regardless of age. The hemophilia A methylation inversion "HAMI" assay provides an advantage over conventional PCR-based methods, which may not detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in the F8 (Xq28) region are associated with detectable changes in methylation levels and can be used as an epigenetic diagnostic marker.
Coagulation Factor VIII is activated by an ordered limited thrombin proteolysis with different catalytic efficiency at three P1 Arginine residues: Arg759> Arg1708>Arg391, indicating the flanking ...residues of the latter to be less optimal. This study aimed to investigate, in silico and in vitro, the impact of possessing hypothetically optimized residues at these three catalytic cleavage sites. The structural impact of the residues flanking Arginine cleavage sites was studied by in silico analysis through comparing the cleavage cleft of the native site with a hypothetically optimized sequence at each site. Moreover, recombinant FVIII proteins were prepared by replacing the sequences flanking native thrombin cleavage sites with the proposed cleavage-optimized sequence. FVIII specific activity was determined by assessing the FVIII activity levels in relation to FVIII antigen levels. We further investigated whether thrombin generation could reflect the haemostatic potential of the variants. Our in silico results show the impact of the residues directly in the cleavage bond, and their neighboring residues on the insertion efficiency of the loop into the thrombin cleavage cleft. Moreover, the in vitro analysis shows that the sequences flanking the Arg1708 cleavage site seem to be the most close to optimal residues for achieving the maximal proteolytic activation and profactor activity of FVIII. The residues flanking the scissile bonds of FVIIII affect the cleavage rates and modulate the profactor activation. We were able to provide insights into the mechanisms of the specificity of thrombin for the P1 cleavage sites of FVIII. Thus, the P4-P2´ residues surrounding Arg1708 of FVIII have the highest impact on rates of thrombin proteolysis which contributes to thrombin activation of the profactor and eventually to the thrombin generation potential.
Hemophilia A (HA) is a
F8
gene mutational disorder resulting in deficiency or dysfunctional FVIII protein. However, surprisingly, in few cases, HA is manifested even without mutations in
F8
. To ...understand this anomaly, we recently sequenced microRNAs (miRNAs) of two patients with mild and moderate HA with no
F8
gene mutations and selected two highly expressing miRNAs, miR-374b-5p and miR-30c-5p, from the pool to explain the FVIII deficiency that could be mediated by miRNA-based
F8
/FVIII suppression. In this report, an established orthogonal
in vivo
RNA-affinity purification approach was utilized to directly identify a group of
F8
-interacting miRNAs and we tested them for
F8
/FVIII suppression. From this pool, two miRNAs, miR-19b-3p and miR-186-5p, were found to be upregulated in a severe HA patient with a mutation in the
F8
coding sequence and two HA patients without mutations in the
F8
coding sequence were selected to demonstrate their role in
F8
gene expression regulation in mammalian cells. Overall, these results provide further evidence for the hypothesis that by targeting the 3′UTR of
F8
, miRNAs can modulate FVIII protein levels. This mechanism could either be the primary cause of HA in patients who lack
F8
mutations or control the severity of the disease in patients with
F8
mutations.
Familial multiple coagulation factor deficiencies (FMCFDs) are a group of inherited hemostatic disorders with the simultaneous reduction of plasma activity of at least two coagulation factors. As ...consequence, the type and severity of symptoms and the management of bleeding/thrombotic episodes vary among patients. The aim of this study was to identify the underlying genetic defect in patients with FMCFDs.
Activity levels were collected from the largest cohort of laboratory-diagnosed FMCFD patients described so far. Genetic analysis was performed using next-generation sequencing.
In total, 52 FMCFDs resulted from coincidental co-inheritance of single-factor deficiencies. All coagulation factors (except factor XII (FXII)) were involved in different combinations. Factor VII (FVII) deficiency showed the highest prevalence. The second group summarized 21 patients with FMCFDs due to a single-gene defect resulting in combined FV/FVIII deficiency or vitamin K-dependent coagulation factor deficiency. In the third group, nine patients with a combined deficiency of FVII and FX caused by the partial deletion of chromosome 13 were identified. The majority of patients exhibited bleeding symptoms while thrombotic events were uncommon.
FMCFDs are heritable abnormalities of hemostasis with a very low population frequency rendering them orphan diseases. A combination of comprehensive screening of residual activities and molecular genetic analysis could avoid under- and misdiagnosis.
Hemophilia A and hemophilia B are rare congenital, recessive X-linked disorders caused by lack or deficiency of clotting factor VIII (FVIII) or IX (FIX), respectively. The severity of the disease ...depends on the reduction of coagulation FVIII or FIX activity levels, which is determined by the type of the pathogenic variants in the genes encoding the two factors (
and
, respectively). Molecular genetic analysis is widely applied in inherited bleeding disorders. The outcome of genetic analysis allows genetic counseling of affected families and helps find a link between the genotype and the phenotype. Genetic analysis in hemophilia has tremendously improved in the last decades. Many new techniques and modifications as well as analysis softwares became available, which made the genetic analysis and interpretation of the data faster and more accurate. Advances in genetic variant detection strategies facilitate identification of the causal variants in up to 97% of patients. In this review, we discuss the milestones in genetic analysis of hemophilia and highlight the importance of identification of the causative genetic variants for genetic counseling and particularly for the interpretation of the clinical presentation of hemophilia patients.
Antibodies against factor (F)VIII are a major complication in the treatment of patients with severe hemophilia A. The Nijmegen-Bethesda assay (NBA) is the gold standard for detection of neutralizing ...antibodies (inhibitors), whereas both inhibitors and nonneutralizing antibodies can be detected by immunoassays such as enzyme-linked immunosorbent assay (ELISA) and multiplex bead-based assays.
Evaluation of an in-house Luminex bead-based assay (LumiTope) compared with a commercially available ELISA and NBA.
The LumiTope method comprised full-length and B-domain–deleted FVIII as well as 9 purified FVIII single or multidomains. The respective proteins were coupled to magnetic beads to detect domain-specific immunoglobulin (IgG; IgG1-4) anti-FVIII antibodies in a large cohort of patients with hemophilia A with and without inhibitors.
Overall, LumiTope assay had a high sensitivity (94.9%) and specificity (91.2%), particularly in patients with low-titer inhibitors compared with ELISA (sensitivity of 72.2% vs 27.7%). IgG4 was the most abundant IgG subclass in NBA-positive patients. NBA-positive and -negative patients showed different domain profiles. Patients with genetic variants in the heavy chain predominantly exhibited antibodies specific to this chain, while those with a light-chain variant showed a more diverse distribution of antibody specificities. Patients with an intron 22 inversion resembled those with a light-chain defect, with a majority of antibodies targeting the light chain.
LumiTope assay provides a sensitive and specific method for not only detection but also domain specification of anti-FVIII-antibodies. Implementation of bead-based assays could improve antibody detection, profiling, and comparability of results and complement NBA.
About one-third of patients with non-severe haemophilia A (HA) show a discrepancy of factor (F)VIII activity (FVIII:C) measured by one-stage (FVIII:C1st), two-stage assays or the chromogenic method ...(FVIII:Cchr). The aim of the study was to characterise the mutation profile in patients with FVIII:C assay discrepancies. FVIII:C discrepancy was considered significant if the calculated ratio between FVIII:C1st and FVIII:Cchr was ≤ 0.6. In 16 patients FVIII:C1st was higher than FVIII:Cchr. The reversephenomenon was observed in 83 patients. Genetic analysis revealed 23 different missense mutations of which 17 were novel. Most mutations, exhibiting a higher FVIII:C1st were localised in the A1-A2-A3 interface. The majority of mutations associated with FVIII:Cchr>FVIII:C1st discrepancy were located close to or within the thrombin cleavage sites, FIX or vWF binding sites. Our data show a correlation between FVIII:C and thrombin generation testing with a clear differentiation between patients with haemophilia and normal controls. However, in the subgroup of FVIII:C1st>FVIII:Cchr discrepancy, the endogenous thrombin potential and peak thrombin parameters were similar to non-discrepant haemophilia patients, while in the inverse discrepancy FVIII:Cchr>FVIII:C1st, these variables showed values close to that of the normal control group. Certain hereditary F8 missense mutations cause discrepancy in FVIII:C as measured with different assays. This can lead to failure in diagnosing HA or incorrectly classifying the severity of the disease. Therefore, we recommend that initial diagnosis of non-severe HA phenotypes should be based on results of both FVIII:C1st and FVIII:Cchr assays.
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