Familial macrothrombocytopenia due to a double mutation in cis in the alpha-actinin 1 gene (ACTN1), previously considered to be chronic immune thrombocytopenic purpura.

Congenital thrombocytopenia can easily be misdiagnosed as immune thrombocytopenic purpura, as is illustrated by this case of a woman and her two children. Doubts arose when steroid/IVIG therapy failed in the mother and the thrombocytopenia in the children persisted. By means of next-generation sequencing, two missense variants in cis in the ACTN1 gene of the affected family members were identified, both of unknown significance. We conclude, after further analysis of these mutations with, among others, in silico prediction tools, that the thrombocytopenia has a genetic cause, in particular the ACTN1 mutations, and is not immune mediated.

SERPINC1 gene mutations in antithrombin deficiency.

Existing evidence suggests that in most cases antithrombin deficiency can be explained by mutations in its gene, SERPINC1. We investigated the molecular background of antithrombin deficiency in a single centre family cohort study. We included a total of 21 families comprising 15 original probands and sixty-six relatives, 6 of who were surrogate probands for the genetic analysis. Antithrombin activity and antigen levels were measured. The heparin-antithrombin binding ratio assay was used to distinguish between the different subtypes of type II antithrombin deficiency. SERPINC1 mutations were detected by direct sequencing of all 7 exons and regulatory regions, and multiplex ligation-dependent probe amplification. Eighty-six per cent of the families had a detrimental SERPINC1 gene mutation that segregated in the family. We detected 13 different SERPINC1 gene mutations of which 5 were novel. Among all these mutations, 44% was associated with type I deficiency, whereas the remainder was associated with type II heparin binding site (11%), type II pleiotropic effect (33%), type II reactive site (6%) or had the antithrombin Cambridge II mutation (6%). The current study reports several novel SERPINC1 mutations, thereby adding to our knowledge of the molecular background of antithrombin deficiency. Finally, our results point out the importance of future research outside the conventional SERPINC1 gene approach.

Factor 11 single-nucleotide variants in women with heavy menstrual bleeding.

In a previous study it was shown that lower factor XI (FXI) levels in women with heavy menstrual bleeding (HMB). Our aim was to determine the single-nucleotide variants (SNVs) in the F11 gene in women with HMB. In addition, an extensive literature search was performed to determine the clinical significance of each SNV. Patients referred for HMB (PBAC-score >100) were included. With direct sequencing analysis of all 15 exons and flanking introns of the F11 gene, 29 different non-structural SNVs were detected in 49 patients with HMB. Interestingly, most of these SNVs have previously been associated with venous thrombosis instead of bleeding. These findings have not helped to elucidate the molecular basis of HMB. They also question the specificity of previously reported F11 variations in patients with thrombosis. More studies are needed to explain the lower FXI levels seen in patients with HMB. IMPACT STATEMENT Women with mild deficiencies of factor XI (FXI) (< 70%) are prone to excessive bleeding during menstruation. Bleeding manifestations are not well correlated with plasma FXI levels and bleeding episodes can vary widely among patients with similar low FXI levels. In a previous study we showed that women with heavy menstrual bleeding (HMB) had normal, but on average, lower levels of FXI than controls. In light of these findings, we performed F11 gene analysis to determine the single-nucleotide variants (SNVs) in women with HMB and performed an extensive literature search to determine the clinical significance of each SNV. By direct sequencing analysis of the F11 gene we found 29 different non-structural SNVs in 49 women with heavy menstrual bleeding. Remarkably, a number of these SNVs have previously been implicated in thrombosis. These findings have not helped to elucidate the molecular basis of lower FXI levels in HMB. They also question the specificity of previously reported F11 variations in patients with thrombosis. More studies are needed to explain the lower FXI levels seen in patients with HMB.

Clonal hematopoiesis in patients with stem cell mobilization failure: a nested case-control study.

Inadequate mobilization of peripheral blood progenitor cells (PBPCs) is a limiting factor to proceeding with autologous hematopoietic cell transplantation (auto-HCT). To assess the impact of clonal hematopoiesis (CH) on mobilization failure of PBPC for auto-HCT, we investigated the characteristics of poor mobilizers (with a total PBPC collection <2 × 106 CD34+ cells per kg) in a consecutive single-center cohort of 776 patients. Targeted error-corrected next-generation sequencing of 28 genes was performed in a nested case-control cohort of 90 poor mobilizers and 89 matched controls. CH was detected in 48 out of 179 patients (27%), with most patients carrying a single mutation. The presence of CH (detected at variant allele frequency [VAF] ≥ 1%) did not associate with poor mobilization potential (31% vs 22% in controls, odds ratio, 1.55; 95% confidence interval, 0.76-3.23; P = .238). PPM1D mutations were detected more often in poor mobilizers (P = .005). In addition, TP53 mutations in this cohort were detected exclusively in patients with poor mobilization potential (P = .06). The incidence of therapy-related myeloid neoplasms (t-MN) was higher among patients with mobilization failure (P = .014). Although poor mobilizers experienced worse overall survival (P = .019), this was not affected by the presence of CH. We conclude that CH at low VAF (1%-10%) is common at the time of stem cell mobilization. TP53 mutations and PPM1D mutations are associated with poor mobilization potential and their role in subsequent development of t-MN in these individuals should be established.

TEMPORARY REMOVAL: Direct oral anticoagulants in antithrombin deficiency: initial experience in a single center.

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Clinical relevance of decreased free protein S levels: results from a retrospective family cohort study involving 1143 relatives.

Conflicting data have been reported on the risk for venous thrombosis in subjects with low free protein S levels. We performed a post-hoc analysis in a single-center retrospective thrombophilic family cohort, to define the optimal free protein S level that can identify subjects at risk for venous thrombosis. Relatives (1143) were analyzed. Relatives with venous thrombosis (mean age 39 years) had lower free protein S levels than relatives without venous thrombosis (P < .001), which was most pronounced in the lowest quartile. Only relatives with free protein S levels less than the 5th percentile (< 41 IU/dL) or less than the 2.5th percentile (< 33 IU/dL) were at higher risk of first venous thrombosis compared with the upper quartile (> 91 IU/dL); annual incidence 1.20% (95% confidence interval [CI], 0.72-1.87) and 1.81% (95% CI, 1.01-2.99), respectively; adjusted hazard ratios 5.6, (95% CI, 2.7-11.5) and 11.3 (95% CI, 5.4-23.6). Recurrence rates were 12.12% (95 CI, 5.23-23.88) and 12.73% (95% CI, 5.12-26.22) per year; adjusted hazard ratios were 3.0 (95% CI, 1.03-8.5) and 3.4 (95% CI, 1.1-10.3). In conclusion, free protein S level can identify young subjects at risk for venous thrombosis in thrombophilic families, although the cutoff level lies far below the normal range in healthy volunteers.

Erythrocytosis in the general population: clinical characteristics and association with clonal hematopoiesis.

Erythrocytosis is a common reason for referral to hematology services and is usually secondary in origin. The aim of this study was to assess clinical characteristics and clonal hematopoiesis (CH) in individuals with erythrocytosis in the population-based Lifelines cohort (n = 147 167). Erythrocytosis was defined using strict (World Health Organization [WHO] 2008/British Committee for Standards in Hematology) and wide (WHO 2016) criteria. Individuals with erythrocytosis (strict criteria) and concurrent leukocytosis and/or thrombocytosis were 1:2 matched with individuals with isolated erythrocytosis and analyzed for somatic mutations indicative of CH (≥5% variant allele frequency). One hundred eighty five males (0.3%) and 223 females (0.3%) met the strict criteria, whereas 4868 males (7.6%) and 309 females (0.4%) met the wide criteria. Erythrocytosis, only when defined using strict criteria, was associated with cardiovascular morbidity (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.2-2.6), cardiovascular mortality (hazard ratio [HR], 2.2; 95% CI, 1.0-4.6), and all-cause mortality (HR, 1.7; 95% CI, 1.2-2.6), independent of conventional risk factors. Mutations were detected in 51 of 133 (38%) evaluable individuals, with comparable frequencies between individuals with and without concurrent cytosis. The JAK2 V617F mutation was observed in 7 of 133 (5.3%) individuals, all having concurrent cytosis. The prevalence of mutations in BCOR/BCORL1 (16%) was high, suggesting aberrant epigenetic regulation. Erythrocytosis with CH was associated with cardiovascular morbidity (OR, 9.1; 95% CI, 1.2-68.4) in a multivariable model. Our data indicate that only when defined using strict criteria erythrocytosis is associated with cardiovascular morbidity (especially in the presence of CH), cardiovascular mortality, and all-cause mortality.

Monitoring of heparins in antithrombin-deficient patients.

INTRODUCTION: Heparins exert their anticoagulant effect through activation of antithrombin. Whether antithrombin deficiency leads to clinically relevantly reduced anti-Xa activity of heparins is unknown. We investigated the relation between antithrombin deficiency and anti-Xa activity measurements of plasma samples spiked with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). MATERIALS AND METHODS: Plasma samples from 34 antithrombin-deficient subjects and 17 family controls were spiked with UFH and LMWH (nadroparin) aimed to correspond with an anti-Xa activity of 0.8 IU/mL. Antithrombin, ß-antithrombin and anti-Xa activities were measured. RESULTS: Mean anti-Xa activity with LWMH was 0.55 IU/mL (0.30-0.74) (recovery 69%, 38-93%) in antithrombin-deficient subjects and 0.82 (0.71-0.89) IU/mL in controls (recovery 103%, 89-111%). Expected anti-Xa measurements after LMWH spiking were found in 17/17 non-deficient subjects and in 8/34 antithrombin-deficient subjects. Anti-Xa measurements in the expected range (0.6-1.0 IU/mL) after UFH spiking were found in 17/17 non-deficient subjects and in 1/22 antithrombin-deficient subjects. Antithrombin activity correlated with anti-Xa activity of UFH (R = 0.77) and LMWH (R = 0.66). Mixing studies of pooled normal plasma and antithrombin-deficient plasma showed that anti-Xa recovery was linearly reduced with antithrombin activity decreasing below 100%. CONCLUSIONS: Reduced antithrombin activity causes significantly reduced anti-Xa levels. Standard LWMH- or UFH-doses are likely to lead to under treatment in antithrombin-deficient individuals.

PROS1 analysis in 87 pedigrees with hereditary protein S deficiency demonstrates striking genotype-phenotype associations.

Hereditary protein S (PS) deficiency predisposes to venous thrombosis. Previously, we demonstrated a difference in risk of venous thrombosis between PS deficiency type I and type III. We used direct sequencing, multiplex ligation-dependent probe amplification (MLPA), and linkage analysis to study whether this difference could be explained by molecular heterogeneity. The study contained two sets of families with PS deficiency type I (cohort 1; 35 probands, 155 relatives) or type III (cohort 2; 52 probands, 241 relatives). In cohort 1, a mixed type I/type III PS-deficient phenotype was observed in 66% of the pedigrees. A total of 34 probands carried a mutant PROS1 allele, compared to one proband in cohort 2 (P<10(-10)). The proband's mutation was identified in all type I, but only in 57% of type III PS deficient relatives. MLPA-analysis in the mutation negative families did not reveal PROS1 deletions or insertions. Linkage analysis in 16 families showed cosegregation of PROS1 markers in the family with type I deficiency, but not in the 15 families with type III deficiency. The genotype-phenotype associations point to differences in genetic architecture. Whereas PS deficiency type I is a monogenic disease due to PROS1 allelic heterozygosity, PS deficiency type III is most likely a more complex or heterogeneous disorder.

ß-Antithrombin, subtype of antithrombin deficiency and the risk of venous thromboembolism in hereditary antithrombin deficiency: A family cohort study.

Hereditary antithrombin deficiency is associated with a high incidence of venous thromboembolism (VTE), but VTE risk differs between families. Beta-antithrombin is reported to be the most active isoform of antithrombin in vivo. Whether ß-antithrombin activity and subtypes in antithrombin deficiency have impact on VTE risk has not been investigated outside the proband setting. We performed a retrospective family cohort study to investigate whether subtypes of antithrombin deficiency or ß-antithrombin levels are associated with the risk of first or recurrent VTE. Eighty-one subjects from 21 families were included, of which 52 were antithrombin deficient. Βeta-antithrombin levels were decreased in most type I and type IIPE subjects, but normal levels were found in all subtypes of antithrombin deficiency. The annual incidence of VTE in antithrombin-deficient family members was 1.24%, 95%CI: 0.72-1.99%, in low ß-antithrombin 1.36% (95%CI: 0.76-2.25%) and in normal ß-antithrombin 0.79% (95%CI: 0.10-2.77). The annual incidence of recurrence in family members was 3.1% (95%CI: 0.9-7.1%). Duration of anticoagulation had an impact on recurrence risk: In family members annual recurrence with fixed duration was 10% (95%CI: 2.1-29.2%), with indefinite duration 1.5% (95%CI: 0.2-5.4%), p < 0.05. Beta-antithrombin levels were not associated with the risk for first or recurrent VTE in antithrombin deficient subjects. CONCLUSIONS: In this high-risk antithrombin-deficient population, both subjects with low and normal plasma ß-antithrombin activity had high risks of first and recurrent VTE. This puts the importance of ß-antithrombin into question. Long-term anticoagulation is warranted in antithrombin-deficient VTE patients.

High protein S activity due to C4b-binding protein deficiency in a 34-year-old Surinamese female with ischemic retinopathy.

In this study, we present the first case of a 34-year-old Surinamese female with ischemic retinopathy and increased free protein S due to C4BP deficiency. Possibly, the low PS/C4BP complex level has increased the risk of arterial thrombosis in our patient.

Identification of a novel PROS1 c.1113T-->GG frameshift mutation in a family with mixed type I/type III protein S deficiency.

We report a family with type I and type III protein S (PS) deficiency, which showed to be phenotypic variants of the same genetic disease. Direct sequencing analysis of the PROS1 gene was performed to establish the genotype. The ratio of protein C antigen and total PS antigen levels (protein C/S ratio) was used to classify subjects at risk of venous thromboembolism. All PS deficient subjects had increased protein C/S ratios as well as a novel PROS1 c.1113T-->GG frameshift mutation.

Understanding the functional difference between growth arrest-specific protein 6 and protein S: an evolutionary approach.

Although protein S (PROS1) and growth arrest-specific protein 6 (GAS6) proteins are homologous with a high degree of structural similarity, they are functionally different. The objectives of this study were to identify the evolutionary origins from which these functional differences arose. Bioinformatics methods were used to estimate the evolutionary divergence time and to detect the amino acid residues under functional divergence between GAS6 and PROS1. The properties of these residues were analysed in the light of their three-dimensional structures, such as their stability effects, the identification of electrostatic patches and the identification potential protein-protein interaction. The divergence between GAS6 and PROS1 probably occurred during the whole-genome duplications in vertebrates. A total of 78 amino acid sites were identified to be under functional divergence. One of these sites, Asn463, is involved in N-glycosylation in GAS6, but is mutated in PROS1, preventing this post-translational modification. Sites experiencing functional divergence tend to express a greater diversity of stabilizing/destabilizing effects than sites that do not experience such functional divergence. Three electrostatic patches in the LG1/LG2 domains were found to differ between GAS6 and PROS1. Finally, a surface responsible for protein-protein interactions was identified. These results may help researchers to analyse disease-causing mutations in the light of evolutionary and structural constraints, and link genetic pathology to clinical phenotypes.

Low cut-off values increase diagnostic performance of protein S assays.

Conflicting data have been reported on the accuracy of protein S (PS) assays for detection of hereditary PS deficiency. In this study we assessed the diagnostic performance of two total PS antigen assays, four free PS assays and three PS activity assays in a group of 28 heterozygous carriers of mutations in PROS1 and 165 control subjects. Several control groups were formed, one of healthy volunteers and - because PS levels are influenced by oral contraception and pregnancy, and assays measuring PS activity may be influenced by the presence of the factor V Leiden mutation -, we also investigated the influences of these factors. All nine PS assays detected significantly reduced PS levels in subjects with a PROS1 mutation. Eight out of nine PS assays showed a 100% sensitivity and 100% specificity to detect heterozygous carriers of mutations in PROS1 with values far below the lower limit of the reference values obtained from healthy volunteers. Low specificities were found in subjects with a factor V Leiden mutation and in pregnant women. At lower cut-off levels, equal to the highest PS value found in heterozygous carriers of mutations in PROS1, the specificity considerably increased in these subjects. When using low cut-off levels equal to the highest PS value found in heterozygous carriers of mutations in PROS1, ensuring 100% sensitivity, the specificity in all study groups increases considerably, by which misclassification can be maximally avoided.

Associations between high factor VIII and low free protein S levels with traditional arterial thrombotic risk factors and their risk on arterial thrombosis: results from a retrospective family cohort study.

INTRODUCTION: Whether high factor (F)VIII and low free protein S levels are risk factors for arterial thrombosis is unclarified. MATERIAL AND METHODS: In a post-hoc analysis of a single-centre retrospective family cohort, we determined if these two proteins could increase the risk of arterial thrombosis. In total, 1399 relatives were analysed. RESULTS: Annual incidence in relatives with high FVIII levels was 0.29% (95%CI, 0.22-0.38) compared to 0.13% (95%CI, 0.09-0.19) in relatives with normal FVIII levels. In relatives with low free protein S levels, this risk was 0.26% (95%CI, 0.16-0.40), compared to 0.14% (95%CI, 0.10-0.20) in relatives with normal free protein S levels. Mean FVIII levels adjusted for age and sex were 11 IU/dL, 18 IU/dL, and 21 IU/dL higher in relatives with hypertension, diabetes mellitus, and obesity as compared to relatives without these arterial thrombotic risk factors. Moreover, a dose response relation between increasing FVIII and body mass index was found. None of these associations were shown for free protein S. CONCLUSIONS: High FVIII and low free protein S levels seemed to be mild risk factors for arterial thrombosis. High FVIII levels were particularly observed in relatives with traditional arterial thrombotic risk factors. Free protein S levels were not influenced by these thrombotic risk factors. This assumes that low free protein S levels were genetically determined.