Joseph P. Miletich

Absence of thrombosis in subjects with heterozygous protein C deficiency.

Protein C deficiency has been thought to be associated with an increased risk of venous thrombosis. To establish a normal range of values, we used a two-site monoclonal-antibody assay to measure protein C levels in 699 healthy adults. The distribution was log normal; 95 percent of the values ranged from 70 to 140 percent of the overall mean (4.03 micrograms per milliliter). Two subjects had levels more than 3.5 SD below the mean (34 and 50 percent), consistent with heterozygous deficiency. We also screened 4723 other blood donors and found 8 additional unrelated subjects with levels from 33 to 51 percent of normal. Autosomal inheritance of heterozygous protein C deficiency was confirmed in them by a detailed study of four families. Levels from 55 to 65 percent of normal are consistent with either heterozygous deficiency or the lower end of the distribution of normal values, and were found in 79 of 5422 subjects when the two groups were combined. None of the subjects had any history of venous thrombosis. We conclude that heterozygous deficiency of protein C has a prevalence of 1 in 200 to 300, a figure consistent with the known number of homozygous infants recently identified, and that levels consistent with heterozygous deficiency are found in 1 in 60 healthy adults but are not detectably associated with a risk of thrombosis.

Association between a literature-based genetic risk score and cardiovascular events in women.

CONTEXT While multiple genetic markers associated with cardiovascular disease have been identified by genome-wide association studies, their aggregate effect on risk beyond traditional factors is uncertain, particularly among women. OBJECTIVE To test the predictive ability of a literature-based genetic risk score for cardiovascular disease. DESIGN, SETTING, AND PARTICIPANTS Prospective cohort of 19,313 initially healthy white women in the Womens Genome Health Study followed up over a median of 12.3 years (interquartile range, 11.6-12.8 years). Genetic risk scores were constructed from the National Human Genome Research Institutes catalog of genome-wide association study results published between 2005 and June 2009. MAIN OUTCOME MEASURE Incident myocardial infarction, stroke, arterial revascularization, and cardiovascular death. RESULTS A total of 101 single nucleotide polymorphisms reported to be associated with cardiovascular disease or at least 1 intermediate cardiovascular disease phenotype at a published P value of less than 10(-7) were identified and risk alleles were added to create a genetic risk score. During follow-up, 777 cardiovascular disease events occurred (199 myocardial infarctions, 203 strokes, 63 cardiovascular deaths, 312 revascularizations). After adjustment for age, the genetic risk score had a hazard ratio (HR) for cardiovascular disease of 1.02 per risk allele (95% confidence interval [CI], 1.00-1.03/risk allele; P = .006). This corresponds to an absolute cardiovascular disease risk of 3% over 10 years in the lowest tertile of genetic risk (73-99 risk alleles) and 3.7% in the highest tertile (106-125 risk alleles). However, after adjustment for traditional factors, the genetic risk score did not improve discrimination or reclassification (change in c index from Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults [ATP III] risk score, 0; net reclassification improvement, 0.5%; [P = .24]). The genetic risk score was not associated with cardiovascular disease risk (ATP III-adjusted HR/allele, 1.00; 95% CI, 0.99-1.01). In contrast, self-reported family history remained significantly associated with cardiovascular disease in multivariable models. CONCLUSION After adjustment for traditional cardiovascular risk factors, a genetic risk score comprising 101 single nucleotide polymorphisms was not significantly associated with the incidence of total cardiovascular disease.

Loci Related to Metabolic-Syndrome Pathways Including LEPR,HNF1A, IL6R, and GCKR Associate with Plasma C-Reactive Protein: The Women's Genome Health Study

Although elevated levels of C-reactive protein (CRP) independently predict increased risk of development of metabolic syndrome, diabetes, myocardial infarction, and stroke, comprehensive analysis of the influence of genetic variation on CRP is not available. To address this issue, we performed a genome-wide association study among 6345 apparently healthy women in which we evaluated 336,108 SNPs as potential determinants of plasma CRP concentration. Overall, seven loci that associate with plasma CRP at levels achieving genome-wide statistical significance were found (range of p values for lead SNPs within the seven loci: 1.9 x 10(-)(8) to 6.2 x 10(-)(28)). Two of these loci (GCKR and HNF1A) are suspected or known to be associated with maturity-onset diabetes of the young, one is a gene-desert region on 12q23.2, and the remaining four loci are in or near the leptin receptor protein gene, the apolipoprotein E gene, the interleukin-6 receptor protein gene, or the CRP gene itself. The protein products of six of these seven loci are directly involved in metabolic syndrome, insulin resistance, beta cell function, weight homeostasis, and/or premature atherothrombosis. Thus, common variation in several genes involved in metabolic and inflammatory regulation have significant effects on CRP levels, consistent with CRPs identification as a useful biomarker of risk for incident vascular disease and diabetes.

Forty-three loci associated with plasma lipoprotein size, concentration, and cholesterol content in genome-wide analysis.

While conventional LDL-C, HDL-C, and triglyceride measurements reflect aggregate properties of plasma lipoprotein fractions, NMR-based measurements more accurately reflect lipoprotein particle concentrations according to class (LDL, HDL, and VLDL) and particle size (small, medium, and large). The concentrations of these lipoprotein sub-fractions may be related to risk of cardiovascular disease and related metabolic disorders. We performed a genome-wide association study of 17 lipoprotein measures determined by NMR together with LDL-C, HDL-C, triglycerides, ApoA1, and ApoB in 17,296 women from the Womens Genome Health Study (WGHS). Among 36 loci with genome-wide significance (P<5×10−8) in primary and secondary analysis, ten (PCCB/STAG1 (3q22.3), GMPR/MYLIP (6p22.3), BTNL2 (6p21.32), KLF14 (7q32.2), 8p23.1, JMJD1C (10q21.3), SBF2 (11p15.4), 12q23.2, CCDC92/DNAH10/ZNF664 (12q24.31.B), and WIPI1 (17q24.2)) have not been reported in prior genome-wide association studies for plasma lipid concentration. Associations with mean lipoprotein particle size but not cholesterol content were found for LDL at four loci (7q11.23, LPL (8p21.3), 12q24.31.B, and LIPG (18q21.1)) and for HDL at one locus (GCKR (2p23.3)). In addition, genetic determinants of total IDL and total VLDL concentration were found at many loci, most strongly at LIPC (15q22.1) and APOC-APOE complex (19q13.32), respectively. Associations at seven more loci previously known for effects on conventional plasma lipid measures reveal additional genetic influences on lipoprotein profiles and bring the total number of loci to 43. Thus, genome-wide associations identified novel loci involved with lipoprotein metabolism—including loci that affect the NMR-based measures of concentration or size of LDL, HDL, and VLDL particles—all characteristics of lipoprotein profiles that may impact disease risk but are not available by conventional assay.

G20210A Mutation in Prothrombin Gene and Risk of Myocardial Infarction, Stroke, and Venous Thrombosis in a Large Cohort of US Men

BACKGROUND A single base pair mutation in the prothrombin gene has recently been identified that is associated with increased prothrombin levels. Whether this mutation increases the risks of arterial and venous thrombosis among healthy individuals is controversial. METHODS AND RESULTS In a prospective cohort of 14 916 men, we determined the prevalence of the G20210A prothrombin gene variant in 833 men who subsequently developed myocardial infarction, stroke, or venous thrombosis (cases) and in 1774 age- and smoking status-matched men who remained free of thrombosis during a 10-year follow-up (control subjects). Gene sequencing was used to confirm mutation status in a subgroup of participants. Overall, carrier rates for the G20210A mutation were similar among case and control subjects; the relative risk of developing any thrombotic event in association with the 20210A allele was 1.05 (95% CI, 0.7 to 1.6; P=0.8). We observed no evidence of association between mutation and myocardial infarction (RR=0.8, P=0.4) or stroke (RR=1.1, P=0.8). For venous thrombosis, a modest nonsignificant increase in risk was observed (RR=1.7, P=0.08) that was smaller in magnitude than that associated with factor V Leiden (RR=3.0, P<0. 001). Nine individuals carried both the prothrombin mutation and factor V Leiden (5 controls and 4 cases). One individual, a control subject, was homozygous for the prothrombin mutation. CONCLUSIONS In a large cohort of US men, the G20210A prothrombin gene variant was not associated with increased risk of myocardial infarction or stroke. For venous thrombosis, risk estimates associated with the G20210A mutation were smaller in magnitude than risk estimates associated with factor V Leiden.

Factor V Leiden Mutation as a Risk Factor for Recurrent Pregnancy Loss

The cause of recurrent pregnancy loss is in most cases unknown, although hypercoagulability and placental infarction have been proposed as etiologic factors [1]. Data in support of this hypothesis include the observation of placental thrombi in patients with the antiphospholipid syndrome and reports of hypofibrinolysis and deficiencies of coagulation factor XII in patients with recurrent fetal loss. An inherited defect in anticoagulation known as resistance to activated protein C has recently been described [2, 3]. In most cases, resistance to activated protein C results from a point mutation in the gene coding for coagulation factor V [4, 5]. This mutation, factor V Leiden, is associated with threefold to fivefold increased risks for first episodes [6, 7] and recurrent episodes [8] of venous thromboembolism. Moreover, risks for thromboembolism are markedly increased among persons with the factor V Leiden mutation who also have concomitant risk factors for thrombosis, such as hyperhomocystinemia, oral contraceptive use, older age, and deficiencies of protein C and protein S [9-12]. Whether the hypercoagulable state associated with factor V Leiden results in increased risk for recurrent spontaneous abortion is uncertain [13-15]. We therefore determined the prevalence of factor V Leiden mutation in a consecutive series of 113 women referred for evaluation of recurrent spontaneous abortion (case-patients) and 437 post-menopausal women with at least one successful pregnancy and no history of pregnancy loss (controls). We limited our investigation to white women to avoid the potential for bias and confounding by ethnicity [16]. We also evaluated the prevalence of factor V Leiden mutation in an additional group of 387 postmenopausal women who reported at least one pregnancy loss. Methods Case-patients were consecutive white women referred for evaluation at the Recurrent Miscarriage Clinic at Brigham and Womens Hospital, Boston, Massachusetts, between 1 July 1995 and 1 June 1996. Eligibility criteria were a history of three or more spontaneous abortions and no parental chromosomal abnormality. Of 141 women screened, 28 were excluded because they had had fewer than three spontaneous abortions before the 24th week of gestation. Thus, 113 case-patients were available for genetic analysis; none had a history of venous thromboembolism. Controls were white women who provided a blood specimen before randomization into the Womens Health Study, a primary prevention trial being done in postmenopausal female health professionals [17]. These women reported no history of myocardial infarction, stroke, or cancer, and they completed a brief questionnaire that solicited demographic characteristics, medical history, and lifestyle information (for example, age at which menstrual periods began, number of pregnancies that reached term, and number of pregnancies that lasted less than 24 weeks). Of the women who reported at least one successful pregnancy and no history of pregnancy loss, 437 were randomly chosen as controls. None had a history of venous thromboembolism. All women underwent analysis for factor V Leiden mutation. Testing involved 1) second-generation screening for resistance to activated protein C that used factor V-deficient plasma and 2) genetic confirmation with polymerase chain reaction techniques for all borderline and low-value results [7, 16]. Case-patients also underwent screening for anticardiolipin antibodies, antiphosphatidylserine antibodies, and lupus anticoagulant. We also determined mutation status among the 387 white participants in the Womens Health Study who reported one or more pregnancy losses. This group included 294 women with one or two pregnancy losses and 93 women with three or more pregnancy losses. Thus, we evaluated a total of 206 women with three or more losses. The odds ratio was used as a measure of the strength of the association, and the chi-square statistic was used to test the significance of any difference in the prevalence of factor V Leiden mutation between case-patients and controls. All P values were two-tailed, and 95% CIs were computed. Results Case-patients and controls had similar mean (4.6 compared with 4.0) and median (4.0 compared with 4.0) numbers of pregnancies. The number of viable deliveries was greater for controls (mean, 3.0; median, 3.0 [range, 1 to 6]) than for case-patients (mean, 0.5; median, 0.0 [range, 0 to 5]). The mean number of pregnancy losses among case-patients was 3.9 (median, 3.0 [range, 3 to 11]); of these women, 46 (40.7%) had at least one successful pregnancy. Of the 437 controls, 16 (3.7%) carried the factor V Leiden mutation (Table 1). In contrast, 9 of 113 case-patients (8.0%) carried the mutation (odds ratio, 2.3 [95% CI, 1.0 to 5.2]; P = 0.050). In the subgroup of 67 case-patients with no history of successful pregnancy, 6 (9.0%) carried the factor V Leiden mutation (odds ratio, 2.6 [CI, 1.0 to 6.7]; P = 0.048). In contrast, in the subgroup of 46 case-patients who had had at least one successful pregnancy, 2 (4.3%) carried the mutation (odds ratio, 1.2; P > 0.2). None of the case-patients who carried the mutation tested positive for anticardiolipin or antiphosphatidylserine antibodies, had a positive result on testing for lupus anticoagulant, or had another known coagulation abnormality; 4 case-patients without the mutation had at least one of these abnormalities. Mean age at menarche did not vary according to factor V Leiden mutation status (12.6 compared with 12.5 years). Table 1. Odds Ratios for Recurrent Pregnancy Loss Associated with Factor V Leiden Mutation* We further evaluated the prevalence of the factor V Leiden mutation among an additional 387 post-menopausal women participating in the Womens Health Study who reported one or more pregnancy losses. Compared with women who had no history of pregnancy loss (prevalence of mutation, 3.7%), the prevalence of mutation was 5.4% (16 of 294) among those with one to two losses, 7.2% (6 of 83) among those with three to five losses, and 10% (1 of 10) among those with six or more losses. The prevalence of the factor V Leiden mutation for the 93 women in this group who had three or more losses (7.5%) was almost identical to that seen in case-patients. Thus, the overall prevalence of the factor V Leiden mutation among all 206 women evaluated who had recurrent pregnancy losses was 7.8% (odds ratio, 2.2 [CI, 1.1 to 4.5]; P = 0.026). Among the Womens Health Study participants who had at least one successful pregnancy, age at the time of first pregnancy did not vary by mutation status (23.8 years compared with 24.3 years). One Womens Health Study participant was homozygous for factor V Leiden mutation and had had three pregnancy losses and two successful pregnancies. Discussion Our data are compatible with the hypothesis that the factor V Leiden mutation, the most common inherited predisposition to thrombosis, plays a role in some cases of unexplained recurrent pregnancy loss. The possibility that placental thrombosis is an etiologic factor in some cases of recurrent spontaneous abortion has previously been raised in relation to patients with antiphospholipid syndromes and those with deficiencies of coagulation factor XII and intrinsic fibrinolysis. These abnormalities, however, are infrequent. In contrast, factor V Leiden mutation is common; thus, the possibility that this mutation contributes to the hypercoagulability of pregnancy raises issues of pathophysiologic and clinical interest. In this regard, it has been suggested that de novo resistance to activated protein C can develop during pregnancy among persons without the factor V Leiden mutation [15, 18] and that these persons may have an increased risk for pregnancy-related venous thromboembolism [18-21]. Furthermore, one report described a patient in whom the magnitude of resistance to activated protein C due to factor V Leiden mutation increased during pregnancy [15]. The risk estimates in our study are remarkably similar to those reported by Preston and colleagues [13], who found that the factor V Leiden mutation was associated with a 2.0-fold increase in risk for third-trimester pregnancy loss associated with intrauterine death. Similarly, Rai and colleagues [14] have reported an increased prevalence of resistance to activated protein C among women with second-trimester pregnancy loss. Two recent case series also suggest that the prevalence of factor V Leiden mutation is increased among women with several pregnancy losses [22, 23]. Despite the consistency of these observations, potential limitations of our study merit consideration. For example, it is possible that the case-patients, who were selected from a recurrent-miscarriage clinic, might have caused an overestimate of the true prevalence of the factor V Leiden mutation among women with several pregnancy losses. We believe that this possibility is unlikely, however, because 93 of the 387 additional women evaluated from the Womens Health Study reported a history of three or more pregnancy losses; in addition, the prevalence of factor V Leiden mutation in this group was almost identical to that in the case-patients. Furthermore, the sample size in the control group was large, greatly reducing the possibility that chance could have led to an underestimation of prevalence. Moreover, the prevalence of the factor V Leiden mutation in our control group is similar to that reported in population-based studies of the mutation [3, 5-710, 16]. We further believe that the comparison of mutation rates between case-patients selected from a hospital-based setting and controls derived from a population-based setting is unlikely to create any major biases because the exposure is genetic rather than acquired. Moreover, the potential for confounding by sex and ethnicity is unlikely because these factors were directly controlled for in the study design. Finally, by limiting controls to postmenopausal women,

Polymorphism in the CETP gene region, HDL cholesterol, and risk of future myocardial infarction: Genomewide analysis among 18 245 initially healthy women from the Women's Genome Health Study.

Background—Recent trial data have challenged the hypothesis that cholesteryl ester transfer protein (CETP) and high-density lipoprotein cholesterol (HDL-C) have causal roles in atherothrombosis. One method to evaluate this issue is to examine whether polymorphisms in the CETP gene that impact on HDL-C levels also impact on the future development of myocardial infarction. Methods and Results—In a prospective cohort of 18 245 initially healthy American women, we examined over 350 000 singe-nucleotide polymorphisms (SNPs) first to identify loci associated with HDL-C and then to evaluate whether significant SNPs within these loci also impact on rates of incident myocardial infarction during an average 10-year follow-up period. Nine loci on 9 chromosomes had 1 or more SNPs associated with HDL-C at genome-wide statistical significance (P<5×10−8). However, only SNPs near or in the CETP gene at 16q13 were associated with both HDL-C and risk of incident myocardial infarction (198 events). For example, SNP rs708272 in the CETP gene was associated with a per-allele increase in HDL-C levels of 3.1 mg/dL and a concordant 24% lower risk of future myocardial infarction (age-adjusted hazard ratio, 0.76; 95% CI, 0.62 to 0.94), consistent with recent meta-analysis. Independent and again concordant effects on HDL-C and incident myocardial infarction were also observed at the CETP locus for rs4329913 and rs7202364. Adjustment for HDL-C attenuated but did not eliminate these effects. Conclusion—In this prospective cohort of initially healthy women, SNPs at the CETP locus impact on future risk of myocardial infarction, supporting a causal role for CETP in atherothrombosis, possibly through an HDL-C mediated pathway.

Novel Association of ABO Histo-blood Group Antigen with Soluble ICAM-1: Results of a Genome-wide Association Study of 6,578 Women

While circulating levels of soluble Intercellular Adhesion Molecule 1 (sICAM-1) have been associated with diverse conditions including myocardial infarction, stroke, malaria, and diabetes, comprehensive analysis of the common genetic determinants of sICAM-1 is not available. In a genome-wide association study conducted among 6,578 participants in the Womens Genome Health Study, we find that three SNPs at the ICAM1 (19p13.2) locus (rs1799969, rs5498 and rs281437) are non-redundantly associated with plasma sICAM-1 concentrations at a genome-wide significance level (P<5×10−8), thus extending prior results from linkage and candidate gene studies. We also find that a single SNP (rs507666, P = 5.1×10−29) at the ABO (9q34.2) locus is highly correlated with sICAM-1 concentrations. The novel association at the ABO locus provides evidence for a previously unknown regulatory role of histo-blood group antigens in inflammatory adhesion processes.

Importance of factor Xa in determining the procoagulant activity of whole-blood clots.

The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.

The synthesis of sulfated dextran beads for isolation of human plasma coagulation factors II, IX, and X

Abstract We have prepared a new matrix for the chromatography of coagulation factors using bead-polymerized dextran (Sephadex) sulfated by anhydrous reaction with chlorosulfonic acid. The material is a useful adsorbent for isolation of human plasma coagulation Factors II, IX, and X. Good recoveries (70, 40, and 50% for Factors II, IX, and X, respectively) of proteins homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and >99% free of contaminating activities can be obtained from human plasma in 2 days. This new chromatographic material greatly simplifies the isolation of these coagulation factors.