Targeted sequencing to identify novel genetic risk factors for deep vein thrombosis: a study of 734 genes.

Essentials Deep vein thrombosis (DVT) has a large unknown genetic component. We sequenced coding areas of 734 hemostasis-related genes in 899 DVT patients and 599 controls. Variants in F5, FGA-FGG, CYP4V2-KLKB1-F11, and ABO were associated with DVT risk. Associations in KLKB1 and F5 suggest a more complex genetic architecture than previously thought. SUMMARY: Background Although several genetic risk factors for deep vein thrombosis (DVT) are known, almost all related to hemostasis, a large genetic component remains unexplained. Objectives To identify novel genetic determinants by using targeted DNA sequencing. Patients/Methods We included 899 DVT patients and 599 controls from three case-control studies (DVT-Milan, Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis [MEGA], and the Thrombophilia, Hypercoagulability and Environmental Risks in Venous Thromboembolism [THE-VTE] study) for sequencing of the coding regions of 734 genes involved in hemostasis or related pathways. We performed single-variant association tests for common variants (minor allele frequency [MAF] ≥ 1%) and gene-based tests for rare variants (MAF ≤ 1%), accounting for multiple testing by use of the false discovery rate (FDR). Results Sixty-two of 3617 common variants were associated with DVT risk (FDR < 0.10). Most of these mapped to F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11. The lead variant at F5 was rs6672595 (odds ratio [OR] 1.58, 95% confidence interval [CI] 1.29-1.92), in moderate linkage with the known variant rs4524. Reciprocal conditional analyses suggested that intronic variation might drive this association. We also observed a secondary association at the F11 region: missense KLKB1 variant rs3733402 remained associated conditional on known variants rs2039614 and rs2289252 (OR 1.36, 95% CI 1.10-1.69). Two novel variant associations were observed, in CBS and MASP1, but these were not replicated in the meta-analysis data from the International Network against Thrombosis (INVENT) consortium. There was no support for a burden of rare variants contributing to DVT risk (FDR > 0.2). Conclusions We confirmed associations between DVT and common variants in F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11, and observed secondary signals in F5 and CYP4V2-KLKB1-F11 that warrant replication and fine-mapping in larger studies.

Thrombotic microangiopathy without renal involvement: two novel mutations in complement-regulator genes.

UNLABELLED: ESSENTIALS: The differential diagnosis among thrombotic microangiopathies (TMAs) is challenging. We studied a case of TMA with neurologic symptoms, no renal impairment and normal ADAMTS-13 levels. Two novel mutations in complement factor I and thrombomodulin genes were identified. Complement-regulator genes can be involved in TMAs with normal ADAMTS-13 regardless of renal damage. BACKGROUND: Thrombotic microangiopathies (TMAs) often represent a challenge for clinicians, because clinical, laboratory, and even genetic features are not always sufficient to distinguish among different TMAs. OBJECTIVES: The aim of this study was to investigate the pathogenetic mechanisms underlying an acute case of TMA with features of both thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). PATIENTS/METHODS: We report the case of a 49-year-old woman who developed an acute TMA with neurologic involvement and no renal impairment. ADAMTS-13, von Willebrand factor, and complement-system biochemical characterization was performed on acute phase samples. Exome sequencing and direct Sanger sequencing of previously aHUS-associated genes were performed. The functional consequences of the thrombomodulin (THBD) mutation were investigated by in vitro expression studies. RESULTS: Despite a clinical diagnosis of TTP, the patient had normal ADAMTS-13 levels and increased VWF antigen levels with ultra-large von Willebrand factor multimers. C3, C4, and complement factors H and I (CFI) were normal. Molecular analysis confirmed two novel heterozygous mutations in CFI (c.805G>A, p.G269S) and THBD (c.1103C>T, p.P368L), and in vitro expression studies showed a reduction in the generation of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) caused by mutated THBD. This proinflammatory condition, associated with the p.G269S mutation in CFI, probably leads to a complement-mediated endothelial activation, with a relevant prothrombotic potential in case of transient environmental triggers. CONCLUSIONS: This study identified the first case of acute TMA without renal involvement but with neurological damage carrying two novel mutations in complement-regulator genes, highlighting the possible role of the complement system as a common pathogenetic mechanism in TMAs.

Plasma ADAMTS-13 levels and the risk of myocardial infarction: an individual patient data meta-analysis.

BACKGROUND: Low ADAMTS-13 levels have been repeatedly associated with an increased risk of ischemic stroke, but results concerning the risk of myocardial infarction are inconclusive. OBJECTIVES: To perform an individual patient data meta-analysis from observational studies investigating the association between ADAMTS-13 levels and myocardial infarction. METHODS: A one-step meta-analytic approach with random treatment effects was used to estimate pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) adjusted for confounding. Analyses were based on dichotomous exposures, with the 5th and 1st percentiles of ADAMTS-13 antigen levels as cut-off values. Quartile analyses, with the highest quartile as a reference category, were used to assess a graded association between levels and risk ('dose' relationship). Additionally, we assessed the risk of the combined presence of low ADAMTS-13 and high von Willebrand factor (VWF) levels. RESULTS: Five studies were included, yielding individual data on 1501 cases and 2258 controls (mean age of 49 years). Low ADAMTS-13 levels were associated with myocardial infarction risk, with an OR of 1.89 (95% CI 1.15-3.12) for values below the 5th percentile versus above, and an OR of 4.21 (95% CI 1.73-10.21) for values below the 1st percentile versus above. Risk appeared to be restricted to these extreme levels, as there was no graded association between ADAMTS-13 levels and myocardial infarction risk over quartiles. Finally, there was only a minor synergistic effect for the combination of low ADAMTS-13 and high VWF levels. CONCLUSIONS: Low ADAMTS-13 levels are associated with an increased risk of myocardial infarction.

FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients.

Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

Measurement and prevalence of circulating ADAMTS13-specific immune complexes in autoimmune thrombotic thrombocytopenic purpura.

BACKGROUND: The formation of ADAMTS13-specific circulating immune complexes (CICs) may be a pathophysiologic mechanism in autoimmune thrombotic thrombocytopenic purpura (TTP), but has not been systematically investigated. OBJECTIVES: (a) To develop an assay for ADAMTS13-specific CICs; (b) to evaluate their prevalence in autoimmune TTP; and (c) to assess their association with ADAMTS13-related measurements and clinical features in autoimmune TTP patients. PATIENTS/METHODS: We developed and validated an ELISA method for ADAMTS13-specific CICs. ADAMTS13-specific CICs were searched for in 55 patients with autoimmune TTP from the Milan TTP Registry (URL:http://www.ttpdatabase.org/) and 28 controls. The associations between ADAMTS13-specific CIC levels and ADAMTS13 activity, antigen, anti-ADAMTS13 IgGs and acute TTP clinical features were assessed by multivariate linear regression. RESULTS: Intra- and inter-assay coefficients of variation of the new test were 5.3 and 9.6%. In 36 patients with severe ADAMTS13 deficiency and anti-ADAMTS13 autoantibodies, the prevalence of ADAMTS13-specific CICs was 47% (n = 17; 95% confidence interval [CI], 32-63%). ADAMTS13-specific CICs were detected also in seven of 19 (37%; 95% CI, 19-59%) patients with reduced ADAMTS13 activity, but apparently negative anti-ADAMTS13 autoantibodies. ADAMTS13-specific CICs were not associated with ADAMTS13 activity, antigen or anti-ADAMTS13 IgGs. In patients with acute TTP, increasing levels of ADAMTS13-specific CICs were associated with a higher number of plasma-exchange procedures required to attain remission (per 0.1 increase in normalized OD values, beta, 2.9; 95% CI, -0.7 to 6.5). CONCLUSIONS: Approximately one to two-thirds of patients with autoimmune TTP display ADAMTS13-specific CICs. A thorough investigation of the prognostic relevance of ADAMTS13-specific CIC levels in autoimmune TTP is warranted.

Raised haematocrit concentration and the risk of death and vascular complications after major surgery.

BACKGROUND: Preoperative anaemia is associated with adverse postoperative outcomes. Data on raised preoperative haematocrit concentration are limited. This study aimed to evaluate the effect of raised haematocrit on 30-day postoperative mortality and vascular events in patients undergoing major surgery. METHODS: This was a cohort study using the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database. Thirty-day mortality and vascular events, demographics and perioperative risk factors were obtained for adults undergoing major surgery. The adjusted effect of raised (over 0·50) compared with normal (0·41-0·50, American Medical Association reference range) preoperative haematocrit concentration on postoperative outcomes was assessed. Separate sex-specific analyses were also conducted, using haematocrit concentration thresholds commonly used in the diagnosis and management of apparent or absolute erythrocytosis. RESULTS: Some 3961 (2·0 per cent) of 197 469 patients had a raised haematocrit concentration before surgery. After adjustment, the 30-day postoperative mortality rate was higher in patients with raised haematocrit than in those without (odds ratio (OR) 2·23, 95 per cent confidence interval 1·77 to 2·80). Thirty-day rates of deep vein thrombosis (OR 1·95, 1·44 to 2·64) and pulmonary embolism (OR 1·79, 1·17 to 2·73), but not myocardial infarction or stroke, were also higher in patients with a raised haematocrit concentration. The effect on mortality was noted beyond the haematocrit thresholds of 0·48 in women and 0·52 in men; the effect estimates were considerably higher for values exceeding 0·54. Values between 0·41 and 0·45 were not associated with increased mortality risk. Similar observations were noted for venous thrombosis, although with apparent sex differences. CONCLUSION: A raised haematocrit concentration was associated with an increased risk of 30-day mortality and venous thrombosis following major surgery.

Next-generation sequencing study finds an excess of rare, coding single-nucleotide variants of ADAMTS13 in patients with deep vein thrombosis.

BACKGROUND: The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Rare single-nucleotide variants (SNVs) of the coding areas of hemostatic genes may explain part of this missing heritability. The ADAMTS13 and VWF genes encode two interconnected proteins with fundamental hemostatic functions, the disruption of which may result in thrombosis. OBJECTIVES: To study the distribution and burden of rare coding SNVs of ADAMTS13 and VWF found by sequencing in cases and controls of DVT. PATIENTS/METHODS: The protein-coding areas of 186 hemostatic/proinflammatory genes were sequenced by next-generation technology in 94 thrombophilia-negative patients with DVT and 98 controls. Gene-specific information on ADAMTS13 and VWF was used to study the association between DVT and rare coding SNVs of the two genes. RESULTS: More than 70 billion base pairs of raw sequence data were produced to sequence the 700-kb target area with a median redundancy of × 45 in 192 individuals. Most of the 4366 SNVs identified were rare and non-synonymous, indicating pathogenetic potential. Rare (frequency of < 1%) and low-frequency (< 5%) coding SNVs of ADAMTS13 were associated with DVT (prevalence 17% vs. 4%; odds ratio [OR] 4.8 and 95% confidence interval [CI] 1.6-15.0 for rare coding; prevalence 36% vs. 23%, OR 1.9 and 95% CI 1.0-3.5 for low-frequency coding). Patients with rare coding SNVs of ADAMTS13 had lower plasma levels of ADAMTS-13 activity than patients without them. SNVs of VWF were not associated with DVT. CONCLUSIONS: We found an excess of rare coding SNVs of the ADAMTS13 gene in patients with DVT.

ADAMTS-13 activity and autoantibodies classes and subclasses as prognostic predictors in acquired thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare life-threatening disease. Of surviving patients, 45% develops an exacerbation or a late recurrence. Severe ADAMTS-13 deficiency, both during the acute episode and remission, is a well-established predictor of recurrence. The predictive value of anti-ADAMTS-13 antibodies, their inhibitory activity and Ig class subtype for disease recurrence is still to be established. OBJECTIVES: To analyze ADAMTS-13-related biomarkers (ADAMTS-13 and anti-ADAMTS-13 immunoglobulins, classes and subclasses) and their potential relationship with prognosis. PATIENTS/METHODS: In 115 patients with TTP, we assessed the association between levels of these biomarkers and the severity of acute episodes; we analysed also the hazard ratio (HR) and 95% confidence interval (CI) of recurrence in association with biomarkers levels retrieved at the previous acute episode or during remission, using Cox regression models. RESULTS: During the acute phase, higher IgA, IgG1 and IgG3 titers showed the strongest association with acute episode severity. In the survival analyzes, the only biomarker significantly associated with a high hazard of recurrence after an acute episode was the presence of IgG. Conversly, low ADAMTS-13 activity or antigen levels (<10%), the presence of ADAMTS-13 inhibitor or IgG during remission were all significantly associated with a higher hazard of recurrence. CONCLUSIONS: Both the Ig class and subclass are of predictive value for acute episode severity in patients with TTP. Although markers that could predict the risk of recurrence in the acute phase are limited, a thorough assessment of ADAMTS-13-related parameters during remission is warranted.

Platelet reactive conformation and multimeric pattern of von Willebrand factor in acquired thrombotic thrombocytopenic purpura during acute disease and remission.

BACKGROUND: Binding of von Willebrand factor (VWF) multimers of ultra-large size to platelets is considered the triggering mechanism of microvascular thrombosis in thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To assess the potential of VWF-related measurements as markers of disease activity and severity in TTP. METHODS: VWF antigen (VWF:Ag), platelet glycoprotein-Ib-α binding-conformation (GPIb-α/BC) and multimeric pattern were investigated in 74 patients with acquired TTP during acute disease, remission or both and 73 healthy controls. In patients with both acute and remission samples available, VWF ristocetin co-factor activity (VWF:RCo) and collagen binding (VWF:CB) were also measured. The relationships of study measurements with the presence of acute disease and remission and with markers of disease severity were assessed. RESULTS: VWF:Ag and VWF-GPIb-α/BC were higher in TTP patients than controls (P < 0.001 and 0.004). However, there was no statistically significant difference in VWF-GPIb-α/BC between samples obtained during acute TTP and remission. Larger VWF multimers were frequently lacking in acute TTP patients, who displayed ultra-large multimers at remission. The degree of loss of larger VWF multimers correlated with the degree of abnormality of hemoglobin, platelet counts and serum lactate dehydrogenase (LDH) and was associated with low levels of both VWF:RCo/Ag and VWF:CB/Ag ratios. CONCLUSIONS: In TTP the platelet-binding conformation of VWF is not exclusively present in acute disease, nor is it associated with its clinical and laboratory severity. The loss of larger VWF multimers, accompanied by low VWF:RCo/Ag and VWF:CB/Ag ratio values, represents an index of disease activity and severity of acute TTP in patients with severe ADAMTS-13 deficiency.

Non-invasive tool for foetal sex determination in early gestational age.

Free foetal DNA in maternal blood during early pregnancy is an ideal source of foetal genetic material for non-invasive prenatal diagnosis. The aim of this study was to evaluate the use of free foetal DNA analysis at early gestational age as pretest for the detection of specific Y-chromosome sequences in maternal plasma of women who are carriers of X-linked disorders, such as haemophilia. Real-time quantitative PCR analysis of maternal plasma was performed for the detection of the SRY or DYS14 sequence. A group of 208 pregnant women, at different gestational periods from 4 to 12 weeks, were tested to identify the optimal period to obtain an adequate amount of foetal DNA for prenatal diagnosis. Foetal gender was determined in 181 pregnant women sampled throughout pregnancy. Pregnancy outcome and foetal gender were confirmed using karyotyping, ultrasonography or after birth. The sensitivity, which was low between 4th and 7th week (mean 73%), increased significantly after 7+1th weeks of gestation (mean 94%). The latter sensitivity after 7+1th week of gestation is associated to a high specificity (100%), with an overall accuracy of 96% for foetal gender determination. This analysis demonstrates that foetal gender determination in maternal plasma is reliable after the 9th week of gestation and it can be used, in association with ultrasonography, for screening to determine the need for chorionic villus sampling for prenatal diagnosis of X-linked disorders, such as haemophilia.

ADAMTS-13 assays in thrombotic thrombocytopenic purpura.

ADAMTS-13, the thirteenth member of the ADAMTS (A Disintegrin And Metalloprotease with Thrombo-Spondin 1 repeats) family, is the plasma metalloprotease responsible for regulating the multimeric structure of VWF. In congenital or acquired deficiency it is actively involved in the pathophysiology of thrombotic thrombocytopenic purpura (TTP), a rare but life threatening disease characterized by microangiopathic haemolytic anaemia and consumptive thrombocytopenia leading to disseminated microvascular thrombosis and variable signs and symptoms of organ ischemia and damage. In the last few years, a number of in house and commercial laboratory assays for ADAMTS-13 and its autoantibodies have been developed. The features and clinical utility of ADAMTS-13 assays are summarized in this review.