Recombinant factor VIII (rFVIII) concentrates differ due to cell lines, culture conditions, presence of the B domain and authorized potency assays. This study characterizes three commercially available rFVIII concentrates: a second-generation full length (A), a third-generation full length (B) and a third-generation B domain-deleted (BDD) product (C). rFVIII concentrates were characterized for FVIII activity (FVIII:C) by one-stage clotting and chromogenic assays, FVIII antigen (FVIII:Ag), thrombin activation profile and FXa-generation assay. The rFVIII concentrates exhibited significant differences with regard to FVIII:C, FVIII:Ag and thrombin activation profile. Product A had significantly greater FVIII:C and FVIII:Ag relative to the measured values of products B and C. In addition, product A demonstrated faster and more complete activation by thrombin than the two others. BDD product C had the slowest measured thrombin activation rate. Product A exhibited a greater in vitro FXa generation than products B and C. We found no differences in FXa generation among all three products when FXa generation was normalized for FVIII:Ag. The greater FVIII:C and FVIII:Ag values for product A compared with that for products B and C are due to application of different authorized potency assays (one-stage assay for A vs. chromogenic assay for B and C). The variation in thrombin activation profiles may arise from differences in cell line-dependent posttranslational modifications of the various recombinant proteins.
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.
SummaryHaemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII (FVIII). It is caused by FVIII gene intron 22 inversion (Inv22) in approximately 45% and by intron 1 inversion (Inv1) in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The aim of this study was to analyze the presence of these mutations in 25 HA Costa Rican families. Patients, methods: We studied 34 HA patients and 110 unrelated obligate members and possible carriers for the presence of Inv22or Inv1. Standard analyses of the factor VIII gene were used incl. Southern blot and long-range polymerase chain reaction for inversion analysis. Results: We found altered Inv22 restriction profiles in 21 patients and 37 carriers. It was found type 1 and type 2 of the inversion of Inv22. During the screening for Inv1 among the HA patient, who were Inv22 negative, we did not found this mutation. Discussion: Our data highlight the importance of the analysis of Inv22 for their association with development of inhibitors in the HA patients and we are continuous searching of Inv1 mutation. This knowledge represents a step for genetic counseling and prevention of the inhibitor development.
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