Klaus Juul

Factor V Leiden and the Risk for Venous Thromboembolism in the Adult Danish Population

Context Estimates of risk for venous thromboembolism associated with factor V Leiden vary. Contribution This population-based cohort study found that heterozygotes and homozygotes for factor V Leiden had about 3 and 18 times higher risks for venous thromboembolism than noncarriers. Absolute 10-year risks for thromboembolism were 0.7% and 3% among heterozygotes and homozygotes younger than 40 years of age who did not smoke and were not overweight. The 10-year risks in heterozygotes and homozygotes older than age 60 years who smoked and were overweight were 10% and 51%. Implications Risks for thromboembolism associated with factor V Leiden are important but probably lower than previously reported. The Editors Venous thromboembolism is associated with more than 300000 hospitalizations and 50000 deaths every year in the United States alone (1). Factor V Leiden is the most frequent hereditary risk factor for venous thromboembolism; it is present in 1% to 7% of white persons and almost never affects black and Asian persons (1, 2). The reported excess risk for venous thromboembolism associated with factor V Leiden (or its phenotype, resistance to activated protein C) varies considerably among studies: Compared with noncarriers, odds ratios between 3 and 16 have been reported for heterozygotes (3-10), while a single study reported an odds ratio of 79 for homozygotes (4). Most of these studies are casecontrol studies (3, 4, 6-10), and no population-based prospective study has been published. Because odds ratios from casecontrol studies may overestimate risk in healthy factor V Leiden heterozygotes and homozygotes, a study to estimate such risks in the adult population at large is warranted. Our main purpose was to estimate hazard ratios for venous thromboembolism in factor V Leiden heterozygotes and homozygotes in the adult Danish population. We also investigated whether factor V Leiden increased hazard ratios for primary and secondary thromboembolic events equally and whether hazard ratios for deep venous thrombosis and pulmonary embolism differed. Finally, we estimated absolute risks for venous thromboembolism according to factor V Leiden genotype that depended on the presence or absence of other thromboembolic risk factors. For these purposes, we performed genotyping on 9253 individuals from the adult Danish population who were participants in the Copenhagen City Heart Study (11). Methods Study Design The Copenhagen City Heart Study is a prospective cardiovascular study of individuals randomly selected according to the Central-Population-Register code to reflect the adult Danish population at large. Those invited were stratified into 5-year age groups ranging from 20 to 95 years; 35- to 70-year-old persons were emphasized. In 19761978, 19329 individuals were invited, of whom 74% (14223) participated. In 19811983, the original cohort supplemented with 500 20- to 25-year-olds was invited to participate; 70% (12698) participated. Finally, in 19911994 the cohort was further supplemented with 3000 20- to 49-year-old persons, and of those invited 61% (10135) participated. More than 99% of participants were white persons of Danish descent. Of the 10135 participants who attended the 19911994 examination, 9259 gave blood for DNA analyses. Of these, 9253 underwent genotyping for factor V Leiden as previously described (11, 12). We did not exclude participants with a thromboembolic event before entry into the Copenhagen City Heart Study. Of the 9253 participants who were analyzed for factor V Leiden, all attended 1 examination, 80% attended 2 examinations, and 77% attended 3 examinations. Examinations included a self-administered questionnaire, a phys ical examination, and blood samples. Clinical and demographic data as well as data on nonresponders have been published previously (13-15). At some time during follow-up, 555 women used oral contraceptives and 1125 used postmenopausal hormone replacement therapy. No women experienced a thromboembolic event while using oral contraceptives, but 11 women had an event while using postmenopausal hormone replacement therapy. We followed all individuals from baseline until the occurrence of a venous thromboembolic event or until censoring. Baseline was defined as the date at which the participant was first selected for inclusion into the Copenhagen City Heart Study. For 7166 participants, baseline was the 19761978 examination, for 277 it was the 19811983 examination, and for 1810 it was the 19911994 examination. We terminated any further follow-up on 31 December 1999 because this was the last date on which complete diagnostic information on end points was obtained. Seventy-six participants emigrated during follow-up and were therefore censored at the emigration date. Four participants who could not be traced were censored at the date on which they were lost. Consequently, follow-up is more than 99% complete. Median follow-up time was 23 years (range, 0.04 to 23 years). We gathered information on incident cases of deep venous thrombosis (International Classification of Diseases, 8th revision [ICD-8], codes 451.00, 451.08, 451.09, 451.90, 451.92, 671.01671.09 and ICD, 10th revision [ICD-10], codes I80.1, I80.2, I80.3, O22.3, O87.1) and pulmonary embolism (ICD-8 codes 450.99, 673.99 and ICD-10 codes I26.0, I26.9, O88.2) until 31 December 1999 from the Danish National Hospital Discharge Register and from the Danish National Register of Causes of Death. We classified venous thromboembolic events as secondary if they occurred 1) in a participant with a history of cancer (n = 27) or with incident cancer within 5 years of the thromboembolic event (n = 12), 2) during a hospitalization for a cause other than venous thromboembolism (n = 72), 3) within 12 months of hospitalization for fracture of the lower extremities or for a cerebrovascular event (n = 6), 4) during pregnancy or puerperium (n = 0), 5) during use of oral contraceptives (n = 0), or 6) during postmenopausal hormone replacement therapy (n = 11). We classified all other events as primary thromboembolic events. In analyses on isolated deep venous thrombosis, we excluded participants with pulmonary embolism. Venous thromboembolic events obtained by requesting hospital records on individuals registered with a venous thromboembolic event from 1980 through 2000 from the Danish National Hospital Discharge Register were validated by others: Among the 176 medical records from North Jutland County with a discharge ICD code for venous thromboembolism, 72% of cases met objective diagnostic criteria (Bjerregaard Larsen T. Personal communication). The diagnostic criteria used were ultrasonography or venography in the case of deep venous thrombosis and ventilationperfusion scintigraphy or pulmonary angiography in the case of pulmonary embolism. All hospitals in Denmark report to the Danish Hospital Discharge Register. Likewise, all death certificates in Denmark are registered in the Danish Register of Causes of Death. The physicians attending patients from the Copenhagen City Heart Study did not use a diagnostic protocol defined specifically by the Copenhagen City Heart Study but rather used Danish standard diagnostic practices, which included venography for the diagnosis of deep venous thrombosis and ventilationperfusion scintigraphy for the diagnosis of pulmonary embolism in the period 19761999. The Danish ethics committee for the City of Copenhagen and Frederiksberg approved the study (#100.2039/91). All participants gave written informed consent. Statistical Analysis We analyzed data using the Stata statistical software package, version 8.0 (Stata Corp., College Station, Texas). We made 2-group comparisons using the Pearson chi-square test, Student t-test, or MannWhitney U-test. A 2-sided P value less than 0.05 was considered statistically significant. We present plots of cumulative incidence (NelsonAalen estimate) as a function of age, and we tested differences between factor V Leiden genotypes for significance by using the log-rank test. When left truncation (that is, delayed entry) was used with age as the time scale, Cox proportional hazards models estimated hazard ratios for venous thromboembolism. This means that differences in age are automatically adjusted for. The final Cox regression model included the following covariates, which were forced into all models: factor V Leiden genotype, sex, body mass index (<25, 25 to 30, and >30 kg/m2), smoking status (smoker or nonsmoker), previous myocardial infarction, leisure time physical activity (<2 hours per week, 2 to 4 hours of light exercise per week, 2 to 4 hours of demanding exercise per week, or >4 hours of exercise per week), use of oral contraceptives, use of postmenopausal hormone replacement therapy, menopausal status, and year of entry. We constructed 95% CIs using bootstrap estimation (10000 replications) with the 2.5th and 97.5th percentiles of the generated hazard ratio distribution as the lower and upper limits. The baseline value for each covariate, as well as values from subsequent examinations, was used. Interaction between factor V Leiden genotype and other covariates in the models was tested for statistical significance by using the likelihood ratio test to compare the model with and without the 2-factor interaction term. Proportionality of hazards over time for the individual covariate was assessed by plotting ln(ln(survival)) versus ln(analysis time). Suspicion of nonparallel lines was further tested by using Schoenfeld residuals. We detected no violations of the proportional hazards assumption. To investigate whether factor V Leiden increases the risk for deep venous thrombosis rather than for pulmonary embolism and also increases the risk for primary thromboembolism rather than for secondary events, we performed Cox regression on participants with venous thromboembolism. The end points in these models were deep venous thrombosis or primary events, and the same covariates

Genetically Reduced Antioxidative Protection and Increased Ischemic Heart Disease Risk. The Copenhagen City Heart Study

Background—Extracellular superoxide dismutase (EC-SOD) is an antioxidative enzyme found in high concentrations in the arterial wall. Two to three percent of all people in Denmark carry an R213G substitution, which increases plasma concentration 10-fold. This may reduce arterial wall EC-SOD concentrations, increase intimal LDL oxidation, and therefore may accelerate atherogenesis. Our primary hypothesis was that EC-SOD-R213G predisposes to ischemic heart disease (IHD). The secondary hypothesis was that EC-SOD-R213G offers predictive ability with respect to IHD beyond that offered by measurements of plasma EC-SOD and autoantibodies against oxidized LDL (oxLDL). Methods and Results—The primary hypothesis was tested in a prospective, population-based study of 9188 participants from The Copenhagen City Heart Study with 956 incident IHD events during 23 years of follow-up and retested cross-sectionally with independent case populations of patients with IHD (n=943) or ischemic cerebrovascular disease (ICVD) (n=617). Case populations were compared with unmatched IHD/ICVD-free control subjects from The Copenhagen City Heart Study (n=7992). The secondary hypothesis was tested by using a nested case-control study comparing patients with IHD (n=956) with age- and gender-matched control subjects (n=956). Age- and gender-adjusted relative risk for IHD in heterozygotes (n=221, 2.4%) versus noncarriers (n=8965, 97.6%) was 1.5 (95% CI, 1.1 to 2.1). Retesting confirmed this: Age- and gender-adjusted odds ratios for IHD was 1.4 (1.0 to 2.0) and for ICVD 1.7 (1.1 to 2.7). Additional adjustment for plasma EC-SOD produced an odds ratio for IHD in heterozygotes versus noncarriers of 9.2 (1.2 to 72), whereas adjustment for autoantibodies against oxLDL produced an odds ratio of 2.5 (1.2 to 5.3). Conclusions—Heterozygosity for EC-SOD-R213G is associated with increased IHD risk. Genotyping offers predictive ability with respect to IHD beyond that offered by plasma EC-SOD and autoantibodies against oxLDL.

Superoxide Dismutase 3 Polymorphism Associated with Reduced Lung Function in Two Large Populations

RATIONALE Superoxide dismutase (SOD) 3 inhibits oxidative fragmentation of lung matrix components collagen I, hyaluronan, and heparan sulfate. Inherited change in SOD3 expression or function could affect lung matrix homeostasis and influence pulmonary function. OBJECTIVES To identify novel SOD3 polymorphisms that are associated with lung function or chronic obstructive pulmonary disease (COPD). METHODS Resequencing of 182 individuals identified two novel polymorphisms, E1 (rs8192287) and I1 (rs8192288), in a conserved region of the SOD3 gene of potential relationship to lung function. We next genotyped 9,093 individuals from the Copenhagen City Heart Study for the polymorphisms and recorded spirometry, and admissions and deaths due to COPD during 26-year follow-up. Finally, we validated our findings in a cross-sectional analysis of 35,635 individuals from the Copenhagen General Population Study. MEASUREMENTS AND MAIN RESULTS Genotyping the Copenhagen City Heart Study identified 35 E1/I1 homozygotes, 1,050 heterozygotes, and 8,008 noncarriers (Hardy-Weinberg equilibrium: P = 0.93). Using quadruple lung function measurements, we found that E1/I1 homozygotes had 7% lower FVC % predicted (P = 0.006) and 4% lower FEV(1) % predicted (P = 0.12) compared with noncarriers. In the Copenhagen General Population Study, E1/I1 homozygotes also had lower FVC % predicted than noncarriers (P = 0.03), confirming an association between E1/I1 genotype and reduced lung function. E1/I1 homozygotes had adjusted hazard ratios for COPD hospitalization and COPD mortality of 2.5 (95% confidence interval, 1.0-5.9) and 3.7 (95% confidence interval, 0.9-15), respectively; the results were independent of influence from the R213G allele of the SOD3 gene. CONCLUSIONS We identified two novel polymorphisms in a conserved region of the SOD3 gene and show that individuals that are homozygous for these polymorphisms have reduced FVC % predicted in two large, population-based studies.

Oxidation of Plasma Low-Density Lipoprotein Accelerates Its Accumulation and Degradation in the Arterial Wall In Vivo

BACKGROUND The aim of the present study was to investigate whether oxidized LDL (ox-LDL) in the arterial intima could be derived from LDL already oxidized in plasma. METHODS AND RESULTS Rabbits received an intravenous injection of 125I-labeled normal LDL (N-LDL) mixed with 131I-labeled LDL that had been mildly oxidized through exposure to Cu2+. The aortic accumulation of undegraded labeled LDL was expressed as plasma equivalents and cakulated as radioactivity in the intima/inner media (cpm/cm2) divided by the time-averaged concentration of radioactivity in plasma (cpm/nL): for the thoracic aorta, the accumulation of undegraded ox-LDL in the intima/ inner media exceeded that of undegraded N-LDL by 286% (n = 6, P < .04), 863% (n = 7, P < .02), and 364% (n = 8, P < .01) after 1, 3, and 24 hours of exposure, respectively. There was a strong positive association between the extent of oxidation and the excess accumulation of undegraded ox-LDL compared with N-LDL (thoracic aorta; 3 hours of exposure: r = .97, n = 14, P < .00001). To measure degradation of N-LDL and ox-LDL, 125I-LDL labeled with 131I-tyramine cellobiose was injected intravenously 24 hours before the aortic intima/inner media was removed: for the thoracic aorta, the accumulation of degradation products from ox-LDL (n = 6) exceeded that from N-LDL (n = 6) by 301% (P < .04). CONCLUSIONS The present data suggest a novel mechanism: mildly oxidized LDL may circulate in plasma for a period sufficiently long to enter, accumulate, and be degraded in the arterial intima in preference to N-LDL.

Platelet glycoprotein IIb/IIIa PlA2/PlA2homozygosity associated with risk of ischemic cardiovascular disease and myocardial infarction in young men ☆: The Copenhagen City Heart Study

Abstract Objectives We tested the hypothesis that platelet glycoprotein (GP) IIb/IIIa PlA2/PlA2homozygotes or PlA1/PlA2heterozygotes versus PlA1/PlA1noncarriers have increased risk of ischemic cardiovascular disease and myocardial infarction (MI), stratified for age and gender. Background The GP IIb/IIIa PlA1/PlA2polymorphism influences aggregation of platelets; however, an association between ischemic cardiovascular disease and heterozygosity remains controversial, and association with homozygosity is largely unexplored. Methods We genotyped the participants of the Copenhagen City Heart Study, a prospective cardiovascular investigation of the Danish general population (n = 9,149, 22-year follow-up) and assessed the risk of ischemic cardiovascular disease in heterozygotes or homozygotes versus noncarriers. Results Of the participants, 70.0%, 27.3%, and 2.7% were noncarriers, heterozygotes, or homozygotes, respectively. Incidence of ischemic cardiovascular disease was 167 and 103 per 10,000 person-years in homozygous and noncarrier men (log-rank: p = 0.006), whereas this difference was not observed in women (p = 0.33) (genotype·gender interaction: p = 0.03). In homozygous versus noncarrier men 50 years at entry, age-adjusted relative risks (RRs) of ischemic cardiovascular disease were 3.6 (1.4 to 9.0), 2.4 (1.3 to 4.6), and 1.0 (0.6 to 1.8), respectively (age·genotype interaction in men: p = 0.04); equivalent multifactorially adjusted RRs were 3.0 (1.1 to 8.0), 2.0 (1.0 to 3.9), and 1.0 (0.6 to 1.8), respectively. The corresponding age-adjusted RR values of MI in men were 5.2 (1.5 to 18), 3.5 (1.6 to 7.5), and 0.5 (0.1 to 1.5), respectively (age·genotype interaction in men: p = 0.002); equivalent multifactorially adjusted RRs were 3.8 (1.0 to 15), 3.1 (1.4 to 6.9), and 0.5 (0.2 to 1.5), respectively. Conclusions PlA2/PlA2homozygosity is associated with a three-fold and four-fold risk of ischemic cardiovascular disease and MI in young men.

Quality of life and cognitive function in Fontan patients; a population-based study.

BACKGROUND After the Fontan procedure patients are at risk for reduced quality of life (QoL) and cognitive function. We aimed to assess these important factors in Danish Fontan patients and to compare the results with a group of healthy controls. METHODS All Fontan patients living in Denmark were identified and invited to participate. QoL was evaluated using the Pediatric Quality of Life Inventory (PedsQL) version 4.0 generic core module in patients <16 years and the Short Form 36 questionnaire (SF-36) in patients ≥16 years. Cognitive function was evaluated in all patients ≥6 years using the Quick Test of Cognitive Speed. To evaluate if QoL correlated with exercise capacity, patients performed a symptom-limited bicycle test. RESULTS 158 of 179 eligible patients (88%) consented to participate. Median age was 13.9 years (IQR: 10.2-19.3). PedsQL scores increased with age but were significantly lower among patients than among controls. SF-36 physical scores were significantly lower in patients compared to controls while psychosocial scores were similar. Cognitive speed was significantly reduced in patients at all ages compared to controls. No significant difference in PedsQL-/SF-36 scores or cognitive speed was found between hypoplastic left heart syndrome (HLHS) and non-HLHS Fontan patient. PedsQL-/SF-36 scores in patients ≥10 years correlated significantly to cognitive speed but not to peak exercise capacity. CONCLUSION QoL is reduced in Fontan children compared to their healthy counterparts whereas in patients ≥16 years only physical, but not psychosocial QoL is reduced. Cognitive speed was significantly lower in patients at all ages compared to controls.

Univentricular hearts in Denmark 1977 to 2009: incidence and survival.

BACKGROUND The incidence of children born with functional univentricular heart (UVH) and their prognosis presumably changed substantially in recent years. This is due to introduction of fetal echocardiography and potential termination of pregnancy (TOP) when UVH is diagnosed (UVH TOP), and to improvements in treatment. We aimed to explore changes in incidence, to estimate changes in survival, and to describe predictors of mortality in UVH patients. METHODS Using a population-based design we identified all UVH cases in Denmark from 1977 to 2009. RESULTS 703 UVH live births and 106 UVH TOP were identified. A dramatic decrease in birth incidence of UVH patients and a corresponding increase in UVH TOP was observed in recent years. Mean incidence rate of UVH (live births and UVH TOP) was 0.39 per 1000 births. In adjusted analysis survival improved significantly from birth era 1977-1989 to 1990-1999 (HR 2.65, 95% confidence interval (CI), 2.06-3.42) but not significantly from 1990-1999 to 2000-2009 (HR 0.77, 95% CI, 0.57-1.05). In the birth era 2000-2009, the lowest five-year survival was seen with hypoplastic left heart syndrome (HLHS) (18.8%), whereas the best survival was seen with tricuspid atresia (79.8%). Adjusted risk of death was 7.3 times higher in the HLHS group compared to the tricuspid atresia group (95% CI, 3.94-13.47). CONCLUSIONS This study demonstrates a dramatic decrease in birth incidence of UVH patients most probably due to a corresponding increase in UVH TOP. Despite survival improved after introduction of Fontan surgery, survival has not improved significantly during the last 20years.

Prothrombin and risk of venous thromboembolism, ischemic heart disease and ischemic cerebrovascular disease in the general population.

OBJECTIVE We tested the hypotheses that Prothrombin G20210A heterozygosity associate with increased risk of venous thromboembolism (VTE), ischemic heart disease (IHD), and ischemic cerebrovascular disease (ICVD) in the general population and re-tested risk of IHD and ICVD in two case-control studies. METHODS 9231 individuals from the Danish general population were followed for VTE (VTE=DVT+PE), deep venous thrombosis (DVT), pulmonary embolism (PE), IHD, myocardial infarction (MI), ICVD, and ischemic stroke (IS) for a median of 24 years. Case-control studies included 2461 IHD cases and 867 ICVD cases. RESULTS In the general population, Prothrombin G20210A heterozygotes had1.3 (95% CI:0.6-2.9) fold risk for VTE, 0.6 (0.2-2.0) for DVT, 1.7(0.6-4.8) for PE, 1.5(1.1-2.1) for IHD, 1.7(1.1-2.7) for MI, 1.1(0.6-1.9) for ICVD, and 1.1(0.5-2.1) for IS compared to non-carriers. Double heterozygotes for Prothrombin G20210A and Factor V Leiden versus double non-carriers had a multifactorially adjusted hazard ratio for IHD of 6.0(2.0-19). In case-control studies, multifactorially adjusted odds ratios for Prothrombin G20210A heterozygotes versus non-carriers were 2.0(1.1-3.4) for IHD, 2.0(1.0-3.8) for MI, 1.4(0.7-3.1) for ICVD, and 2.1(0.8-5.4) for IS. CONCLUSION Prothrombin G20210A heterozygosity alone and in combination with Factor V Leiden R506Q heterozygosity predicts 1.5 and 6.0 fold risk of IHD compared to non-carriers.

Arrhythmia and exercise intolerance in Fontan patients: current status and future burden.

BACKGROUND Long-term survival after the Fontan procedure shows excellent results but is associated with a persistent risk of arrhythmias and exercise intolerance. We aimed to analyze the current burden of clinically relevant arrhythmia and severe exercise intolerance in Danish Fontan patients and furthermore, to estimate the future burden from analysis of mortality and the current burden related to age. METHODS All Danish citizens with Fontan completion from 1981 to 2009 were identified (n=235). Surviving patients performed exercise test, Holter monitoring, echocardiography, pulmonary function test, and blood sampling and medical history was retrieved from medical records. RESULTS Twenty-six (11%) patients died or had heart transplantation (HTx) after a mean (± SD) post-Fontan follow-up of 8.3 ± 5.7 years. Excluding perioperative deaths (n=8), a linear probability of HTx-free survival was observed and estimated to 99.1% per year. Prevalence of clinically relevant arrhythmia and severe exercise intolerance increased significantly with age and was found in 32% and 85% of patients ≥ 20 years, respectively. Thus, from survival data and logistic regression models the future prevalence of patients, clinically relevant arrhythmia and severe exercise intolerance were estimated, revealing a considerable augmentation. Furthermore, resting and maximum cardiac index, resting stroke volume index and pulmonary diffusing capacity decreased significantly with age while diastolic and systolic ventricular function was unchanged. CONCLUSIONS The prevalence of clinically relevant arrhythmia and severe exercise intolerance increased significantly with age in Danish Fontan patients. The future Fontan burden was estimated showing an increase in the prevalence of older patients, clinically relevant arrhythmia, and severe exercise intolerance.

Influence of Factor V Leiden on susceptibility to and outcome from critical illness: a genetic association study.

IntroductionDisturbance of the pro-coagulatant and anti-coagulant balance is associated with a poor outcome from critical illness. The objective of this study is to determine whether the Factor V Leiden (FVL) mutation is associated with susceptibility to or death from critical illness.MethodsA genetic association study involving four case cohorts comprising two Gram negative sepsis, one invasive pneumococcal disease and one intensive care unit cohort with a total of 1,249 patients. Controls were derived from a population-based cohort study (N = 8,147). DNA from patients and controls was genotyped for the FVL mutation.ResultsWhen all patients were investigated together no significant difference in the frequency of FVL mutation was observed compared with controls (odds ratio (OR), 1.03; 95% confidence interval (CI), 0.83 to 1.29). However, when stratified among patients admitted to intensive care (N = 237), susceptibility and the likelihood of long-term death was influenced by the FVL mutation. In adjusted logistic regression analysis, FVL carriers had an increased risk of ICU admission compared to non-carriers (OR 1.62; 95% CI, 1.08 to 2.42). In adjusted Cox regression analysis, FVL carriers were at increased risk of long-term death compared to non-carriers (relative risk 1.78; 95% CI, 1.13 to 2.81). FVL carrier status did not predict either susceptibility to or outcome from Gram negative, Escherichia coli or Streptococcus pneumoniae sepsis.ConclusionsOverall, the FVL mutation did not appear to increase the risk of admission due to severe invasive infections. Nevertheless, in the subgroup of patients admitted to intensive care an increased risk and a poorer long-term outcome for individuals with critical illness were observed for FVL mutation carriers.