Risk of bleeding and thrombosis in inherited qualitative fibrinogen disorders.

OBJECTIVES: Inherited dysfibrinogenemia is a rare disorder, for which clinical studies related to the risk of bleeding or thrombosis and the type of causative mutation are scanty. MATERIALS AND METHODS: We analyzed the laboratory, clinical, and genotypic features of 50 patients with inherited dysfibrinogenemia belonging to 19 unrelated families. RESULTS: In all the index cases, fibrinogen activity by Clauss method was below the normal range, while it was observed in 57.9% only by PT-derived method. In three families, hypodysfibrinogenemia was evident, associated with three novel mutations (Ter492Gln in FGB, Cys365Asp, and Leu370Phe in FGG). Three additional novel mutations were also identified (Arg114Lys in FGA, Ile131Thr and Trp234Arg in FGG). Bleeding symptoms assessed by ISTH-BAT scored at least 1 in 30% of patients and, significant bleeding symptoms were mainly present in female patients, especially associated with pregnancy. Two patients with FGB Arg44Cys suffered from venous thromboembolism, and two with FGA Arg35His had ischemic stroke at older age. CONCLUSIONS: This study confirms the heterogeneity of clinical features in inherited dysfibrinogenemia, due to the wide spectrum of the causative mutations. Larger multicenter studies are needed to assess the definitive correlation of some mutations with bleeding or thrombosis.

Congenital hypofibrinogenemia associated with novel homozygous fibrinogen Aα and heterozygous Bß chain mutations.

We report the molecular characterisation of two novel cases of inherited hypofibrinogenemia. After sequencing all coding regions and intron-exon boundaries of the three fibrinogen genes (FGA, FGB, and FGG), two different novel mutations were found, one homozygous and one heterozygous. The first patient, with a mild bleeding history and mild discrepancy between functional and immunological fibrinogen, showed a novel homozygous nonsense mutation in exon 5 of FGA (p.Trp373*, p.Trp354* according to the mature protein) caused by a G>A transition at nucleotide position 1,119. The resulting truncation in the Aα chain is likely to reduce the efficiency of fibrinogen assembly and secretion. The second patient, referred after ischemic stroke (functional fibrinogen 77mg/dL), had a novel heterozygous splicing mutation in intron 5 of FGB (IVS5+2T>A or c.832+2T>A), which we demonstrated to cause either exon 5 skipping or the inclusion of 75bp belonging to intron 5. Neither splicing defect alters the reading frame: one results in a 38-residue deletion and the other in a 25-residue insertion in the D domain of fibrinogen Bß chain. This report confirms that genetically determined partial deficiencies of fibrinogen with levels greater than 50mg/dL are rarely associated with significant bleeding symptoms and that homozygous null mutations removing a significant portion of the Aα chain may be associated with mild fibrinogen deficiency.

A novel heterozygous missense mutation (His127Arg) in a family with inherited cross-reacting material positive factor XI deficiency.

Factor XI (FXI) deficiency is an autosomal inherited coagulation disorder, characterized by an inconsistent bleeding tendency, mainly associated with injury or surgery. Although most of the F11 gene mutations cause a true quantitative deficiency of FXI (cross-reacting material-negative, CRM-), very few variants characterized by a qualitative abnormality resulting in a discrepant FXI activity/FXI antigen ratio (CRM positive, CRM+) have been reported. We describe here a novel CRM+ mutation (His127Arg) identified in an asymptomatic woman from Indonesia and in her two sons.

Ribosome readthrough accounts for secreted full-length factor IX in hemophilia B patients with nonsense mutations.

We investigated the spontaneous ribosome readthrough, virtually unexplored in genes encoding secreted proteins, over coagulation F9 nonsense mutations. Expression of recombinant factor IX (FIX) in eukaryotic cells demonstrated appreciable levels of secreted FIX molecules for the mutations p.R162* (5 ± 0.3% of rFIX-wt antigen levels), p.R294* (3.1 ± 1.1%) and p.R298* (2.5 ± 0.7%), but not for the p.L103*. Western blotting revealed a large proportion of truncated molecules, which correlated with small amounts of full-length FIX (rFIX-162*, ∼0.5%; rFIX-294*; and rFIX-298*, ∼0.2%). Western blotting of plasma from FIX deficient (Hemophilia B) patients revealed traces of full-length FIX for the p.R294* and p.R298* mutations, but not for the p.L103* mutation that triggered major FIX mRNA decay. The detection of full-length proteins has clinical implication, particularly for post-therapeutic immunological complications in Hemophilia. Data in patients' plasma and in vitro, obtained in the proper protein context, support a ribosome readthrough gradient, consistent with its predicted determinants of efficiency.

Factor XI gene mutations in factor XI deficient patients of the Czech Republic.

Factor XI (FXI) deficiency is an autosomal inherited coagulation disorder characterized by bleeding symptoms mainly associated with injury or surgery. Although most of the FXI gene mutations in Ashkenazi Jews are represented by the Glu117stop or Phe283Leu mutations, considerable genetic heterogeneity has been reported in other populations. We report here the genotypic characterization of four families with severe inherited FXI deficiency from the Czech Republic. Seven different gene mutations (three novel) were identified, thus, excluding the existence of a major founder effect in this population. Interestingly, both Glu117stop and Phe283Leu were detected once, further demonstrating the occurrence of these mutations also outside the Jewish populations. In conclusion, we confirm that FXI deficiency in non-Jewish populations is because of different gene mutations; however, the presence of the Glu117stop and Phe283Leu mutations suggests that genetic testing in FXI-deficient patients can start with these two point mutations.

The cytotoxic T-lymphocyte response against a poorly immunogenic mammary adenocarcinoma is focused on a single immunodominant class I epitope derived from the gp70 Env product of an endogenous retrovirus.

The TS/A mouse mammary adenocarcinoma is a poorly immunogenic tumor widely used in preclinical models of cancer immunotherapy. CTLs have often been indicated as important in TS/A tumor destruction, but their generation in this model has been rarely studied, nor have their precise target(s) been identified. We hypothesized that the gp70 Env product of an endogenous murine leukemia virus could be a target antigen for TS/A-specific CTLs and investigated this possibility in four different TS/A cell lines engineered with the genes that encode IFN-alpha, IFN-gamma, interleukin-4, and B7.1, respectively. All tumor cell lines expressed gp70, albeit at different levels, as demonstrated by reverse transcription-PCR analysis. Transfected tumor cells exhibited a delayed growth in vivo, and partial tumor regression. Spleen cells from mice that displayed tumor regression had high percentages of CD8(+) T cells that were specifically stained with L(d) tetramers loaded with gp70(423-431), the antigenic epitope of gp70 protein. Mixed leukocyte-peptide and mixed leukocyte-tumor cultures, set up by stimulating splenocytes with the immunogenic peptide and with transfected TS/A tumor cells, respectively, resulted in similar large increases in tetramer-reactive CD8(+) T cells and showed high lytic activity specific for gp70(423-431). Finally, in a Cold Target Inhibition assay, lytic activity of a mixed leukocyte-tumor culture was inhibited in an overlapping fashion by both the TS/A line used for restimulation and 293L(d) cells loaded with gp70(423-431) peptide, but not by 293L(d) cells pulsed with an irrelevant H-2 L(d) epitope, thus demonstrating that all or most of the cytotoxic activity was directed exclusively against this antigenic epitope.