Recipient ADAMTS13 Single-Nucleotide Polymorphism Predicts Relapse after Unrelated Bone Marrow Transplantation for Hematologic Malignancy.

Relapse remains a major obstacle to the survival of patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation. A disintegrin-like and metalloprotease with a thrombospondin type 1 motif (ADMATS13), which cleaves von Willebrand factor multimers into less active fragments, is encoded by the ADAMTS13 gene and has a functional single-nucleotide polymorphism (SNP) rs2285489 (C > T). We retrospectively examined whether ADAMTS13 rs2285489 affected the transplant outcomes in a cohort of 281 patients who underwent unrelated human leukocyte antigen (HLA)-matched bone marrow transplantation for hematologic malignancies. The recipient ADAMTS13 C/C genotype, which putatively has low inducibility, was associated with an increased relapse rate (hazard ratio [HR], 3.12; 95% confidence interval [CI], 1.25⁻7.77; P = 0.015), resulting in a lower disease-free survival rate in the patients with a recipient C/C genotype (HR, 1.64; 95% CI, 1.01⁻2.67; P = 0.045). Therefore, ADAMTS13 rs2285489 genotyping in transplant recipients may be a useful tool for evaluating pretransplantation risks.

Thrombosis Prediction Based on Reference Ranges of Coagulation-Related Markers in Different Stages of Pregnancy.

INTRODUCTION: Careful monitoring of the hypercoagulable state is required during pregnancy. However, coagulation and fibrinolysis markers are not fully utilized because there are no reference values reflective of coagulation and fibrinolysis dynamics during pregnancy, which differ from the nonpregnant state. METHODS: Changes in antithrombin (AT), fibrinogen (Fbg), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), soluble fibrin (SF), D-dimer (DD), and protein S (PS) were investigated in healthy pregnant women, and reference ranges in the early, mid, late, and end stages of pregnancy were established. RESULTS: The AT was essentially constant throughout pregnancy. The Fbg, F1+2, TAT, and DD increased significantly as pregnancy progressed. In contrast, SF did not show a significant increase throughout the entire pregnancy period. Total PS antigen and total PS activity showed a corresponding decrease from early gestation. When test data in 3 cases in which deep vein thrombosis or intrauterine fetal death occurred during pregnancy were compared to the established reference ranges, all of the cases had multiple markers with values that exceeded the reference ranges. CONCLUSION: Establishing reference ranges for each week could potentially make it possible to evaluate abnormalities of the coagulation and fibrinolysis systems during pregnancy. Of note, SF might be a useful marker that reflects thrombus formation during pregnancy. Larger-scale studies will be required to establish reference ranges for every gestational week.

Gene analysis of six cases of congenital protein S deficiency and functional analysis of protein S mutations (A139V, C449F, R451Q, C475F, A525V and D599TfsTer13).

Congenital deficiency of protein S (PS), an anticoagulant factor, leads to venous thrombosis, with onset predominantly beginning in adolescence. In the present study, gene analysis of six unrelated Japanese families diagnosed with congenital PS deficiency identified five missense mutations in the PROS1 gene - c.757C>T (Ala139Val; A139V), c.1346 G>T (Cys449Phe; C449F), c.1352G>A (Arg451Gln; R451Q), c.1424G>T (Cys475Phe; C475F) and c.1574C>T (Ala525Val; A525V) - and one frameshift mutation, c.2135delA (Asp599ThrfsTer13; D599TfsTer13). C449F, R451Q, A525V and D599TfsTer13 are novel mutations. Results from ELISA to measure PS antigen levels in culture supernatant showed that the A139V variant was similar to wild-type, but other variants showed reductions when compared with wild-type. Results from pulse-chase analysis confirmed that the A139V variant exhibited secretion equivalent to wild-type, but for the other variants, there was no extracellular secretion, and it had nearly all been degraded inside the cell within six hours. Results from pulse-chase analysis using proteasome inhibitors also showed that intracellular degradation of mutant protein was inhibited. Activity of the A139V variant was decreased to 71% of wild-type, and the phospholipid binding capacity fell to as low as 45%. These results suggest that although the A139V variant has normal secretion, it has abnormal phospholipid binding capacity, and therefore causes type II PS deficiency, in which PS activity is decreased. It is also thought that with the other variants, misfolding due to amino acid mutations causes nearly all PS to be degraded intracellularly, therefore leading to type I PS deficiency.

A donor thrombomodulin gene variation predicts graft-versus-host disease development and mortality after bone marrow transplantation.

Thrombomodulin, encoded by the THBD gene, is a critical regulator of coagulation and innate immunity. Its gene variant (rs3176123, 2729A>C) in the 3' untranslated region has been reported to be associated with vasculopathies. The present study analyzed the impact of THBD variation on transplant outcomes in a cohort of 317 patients who underwent unrelated HLA-matched bone marrow transplantation (BMT) for hematologic malignancies through the Japan Marrow Donor Program. The donor A/C or C/C genotype vs. the donor A/A genotype resulted in a lower incidence of grades II-IV acute graft-versus-host disease [GVHD; hazard ratio (HR) 0.66; 95 % confidence interval (CI) 0.44-0.99; P = 0.05] according to a multivariate analysis. In patients with grades II-IV acute GVHD, the donor A/C or C/C genotype vs. the donor A/A genotype was associated with significantly better overall survival rates (HR 0.45; 95 % CI 0.21-0.99, P = 0.05), while this effect was absent in other patients. A functional analysis using lymphocytes obtained from healthy individuals revealed that the 2729C allele has a higher level of THBD mRNA than the 2729A allele. These findings suggest the functional relevance of the rs3176123 variation and indicate that higher thrombomodulin expression by individuals with the 2729C allele likely accounts for their decreased risk for acute GVHD development and subsequent mortality.

Antithrombin deficiency in three Japanese families: one novel and two reported point mutations in the antithrombin gene.

INTRODUCTION: Inherited antithrombin (AT) deficiency is associated with a predisposition to familial venous thromboembolic disease. We analyzed the AT gene in three unrelated patients with an AT deficiency who developed thrombosis. MATERIALS AND METHODS: We analyzed the SERPINC1 gene in three patients. Additionally, we expressed the three mutants in the COS-1 cells and compared their secretion rates and levels of AT activity with those of the wild-type (WT). RESULTS: We identified three distinct heterozygous mutations of c.2534C>T: p.56Arginine → Cysteine (R56C), c.13398C>A: p.459Alanine → Aspartic acid (A459D) and c.2703C>G: p.112 Proline → Arginine (P112R). In the in vitro expression experiments, the AT antigen levels in the conditioned media (CM) of the R56C mutant were nearly equal to those of WT. In contrast, the AT antigen levels in the CM of the A459D and P112R mutants were significantly decreased. The AT activity of R56C was decreased in association with a shorter incubation time in a FXa inhibition assay and a thrombin inhibition-based activity test. However, the AT activity of R56C was comparable to that of WT when the incubation time was increased. CONCLUSIONS: We concluded that the R56C mutant is responsible for type II HBS deficiency. We considered that the A459D and P112R mutants can be classified as belonging to the type I AT deficiency.