ORIGINAL INVESTIGATION. Factor V Leiden Homozygosity, Dyspnea, and Reduced Pulmonary Function

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1 ORIGINAL INVESTIGATION Factor V Leiden Homozygosity, Dyspnea, and Reduced Pulmonary Function Klaus Juul, MD; Anne Tybjærg-Hansen, MD, DMSc; Jann Mortensen, MD, DMSc; Peter Lange, MD, DMSc; Jørgen Vestbo, MD, DMSc; Børge G. Nordestgaard, MD, DMSc Background: Factor V Leiden homozygosity predisposes patients to deep venous thrombosis and major pulmonary thromboembolism. Consequently, factor V Leiden homozygosity could, via unrecognized repeated minor pulmonary thromboemboli, cause chronic pulmonary disease. We tested the hypothesis that factor V Leiden homozygosity is associated with pulmonary symptoms and signs. Methods: We studied a general population sample of 9253 individuals from the Copenhagen City Heart Study who were examined in Of these, 6475 participants were also examined in and/or End points were dyspnea and lung function. Results: Among 2 factor V Leiden homozygotes, a mean±sd of 32% ±11% had severe dyspnea compared with 6%±.3% of 8534 noncarriers ( 2 test; P.1). The corresponding adjusted odds ratio for severe dyspnea was 5.4 (95% confidence interval, ). During followup, forced expiratory volume in 1 second and forced vital capacity were 5% to 1% lower in homozygotes vs noncarriers (analysis of variance; P=.3 and P=.3). The annual mean±sd loss of forced expiratory volume in 1 second and forced vital capacity was 39±8 ml/y and 35±8 ml/y in homozygotes vs 21±1 ml/y and 15±1 ml/y in noncarriers (t test; P=.3 and P=.4), respectively. Factor V Leiden heterozygosity (n=699) did not influence pulmonary symptoms and signs. Conclusion: We demonstrate a previously unrecognized clinical presentation of factor V Leiden homozygosity with severe dyspnea and decreased pulmonary function. Arch Intern Med. 25;165: Author Affiliations: Department of Clinical Biochemistry, Herlev University Hospital, Herlev, Denmark (Drs Juul and Nordestgaard); Departments of Clinical Biochemistry (Dr Tybjærg- Hansen) and Clinical Physiology and Nuclear Medicine (Dr Mortensen), Rigshospitalet, Copenhagen, Denmark; Department of Respiratory Medicine, Hvidovre University Hospital, Hvidovre, Denmark (Dr Lange); North West Lung Center, Wythenshawe Hospital, Manchester, England (Dr Vestbo); and The Copenhagen City Heart Study, Bispebjerg University Hospital, Copenhagen (Drs Tybjærg- Hansen, Lange, and Nordestgaard). PULMONARY THROMBOEMBOlism secondary to deep venous thrombosis has a heterogeneous clinical presentation, and most cases are never clinically recognized. 1 Repeated pulmonary thromboemboli are known to cause chronic pulmonary disease, the cardinal symptom of which is dyspnea. Whether hereditary thrombophilia, possibly via unrecognized repeated minor pulmonary thromboemboli, is also associated with chronic pulmonary symptoms and signs is currently unknown. The most frequent and well-established hereditary prothrombotic risk factor is a single G-to-A substitution in the coagulation factor V gene in nucleotide position 1691 (factor V Leiden), which in the heterozygous and homozygous states is present in 8.% and.2% of white individuals, respectively. 2 This point mutation, which reduces coagulation factor V breakdown by activated protein C, increases the risk of deep venous thrombosis and pulmonary thromboembolism, particularly in the homozygous state. 3 Consequently, factor V Leiden homozygosity could, via unrecognized repeated minor pulmonary thromboemboli, cause chronic pulmonary disease. We tested the hypothesis that factor V Leiden homozygosity is associated with pulmonary symptoms and signs. To test this hypothesis, we genotyped 9253 individuals sampled from the Danish general population (The Copenhagen City Heart Study) and compared factor V Leiden homozygotes with noncarriers with respect to dyspnea and pulmonary function. METHODS PARTICIPANTS The Copenhagen City Heart Study is a prospective cardiopulmonary study of 2- to 93-year-old Danes of both sexes sampled from the general population in and reexamined in and Informed consent was obtained from all participants. The Ethics Committee of Copenhagen and Frederiksberg approved the study (study No /91). 232

2 Table 1. Characteristics of Participants* Factor V Leiden Genotype Characteristic Noncarriers Heterozygotes Homozygotes Women/men, No. 4713/ /32 (P =.42) 1/1 (P =.64) Age at study entry, mean ± SD, y 45 ± ± 12 (P =.52) 47 ±8(P =.54) Smoking before entry, mean ± SD, pack-years 13 ± ± 16 (P =.46) 13 ± 15 (P =.82) Smoking during follow-up, mean ± SD, g/d 11 ± 1 11 ± 1 (P =.2) 1 ±9(P =.79) Body mass index, mean ± SD 25 ± 4 25 ±4(P =.87) 24 ±3(P =.87) Exposure to occupational dust, mean ± SD, % 19 ±.4 19 ± 1.5 (P =.68) 3 ± 11 (P =.2) *Values in parentheses represent P values comparing homozygotes or heterozygotes with noncarriers by Pearson 2 test (sex and exposure to occupational dust) or Mann-Whitney test (age at study entry, body mass index, and smoking before entry or during follow-up). Body mass index is calculated as weight in kilograms divided by the square of height in meters. MEASUREMENT OF OUTCOMES At each examination, participants completed a questionnaire on risk factors for cardiopulmonary disease and details of dyspnea, the severity of which was graded according to a slightly modified version of the British Medical Research Council questionnaire s breathlessness scale, grades II to IV. Light (grade II), moderate (grade III), and severe (grade IV) dyspnea grades were defined as confirmatory answers to the following questions, respectively: Do you get shortness of breath when you walk at an ordinary pace on a level road alongside someone your age? Do you have to stop once in a while in order to catch your breath when walking at your own pace? and Do you get shortness of breath during morning toilette or when you get dressed? 9 At the and examinations, determinations of forced expiratory volume in 1 second (FEV 1 ) and forced vital capacity (FVC) were performed using an electronic spirometer (model N 43; Monaghan, Littleton, Colo), whereas at the examination, a dry wedge spirometer was used (Vitalograph, Maidenhead, England). Each spirometry procedure was performed in triplicate, and results were accepted only if the variation between 2 of these was less than 5%. The highest measurements of FEV 1 and FVC were used as absolute values (in milliliters) or as a percentage of predicted values (percent predicted). Algorithms for the calculation of the percent predicted FEV 1 and FVC were made using multiple linear regression, with age and height as covariates in men and women separately in a subsample of never smokers. 1 By using algorithms for each set of FEV 1 and FVC measurements (from the , , and examinations, respectively) to calculate the percent predicted FEV 1 and FVC, systematic errors introduced by change of spirometer between examinations were accounted for. Despite this, the possibility still exists that the absolute level of FEV 1 and FVC could have changed slightly from the 2 first examinations using the original electronic spirometer vs the last examination using the dry wedge spirometer, which potentially could have affected the measurement of change in FEV 1 and FVC over time. However, because this is true for individuals with all 3 genotypes (factor V Leiden noncarriers, heterozygotes, and homozygotes), it is unlikely that change of spirometers between examinations would affect the observed differences between genotypes that span all 3 examinations. Therefore, we do not believe that the change of spirometer between examinations could have affected the conclusions of this article. MEASUREMENTS OF PREDICTOR VARIABLES Blood was drawn for DNA extraction at the examination. All participants were genotyped for factor V Leiden as previously described. 11 Exposure to occupational dust was defined as a confirmatory answer to the question, Have you for long periods been exposed to occupational dust or welding fumes? Diagnoses of deep venous thrombosis and pulmonary embolism were gathered from the Danish National Hospital Discharge Register and the Danish Register of Causes of Death until January 2 (International Classification of Diseases, Eighth Revision [ICD-8], codes 45.99, , 451., 451.8, 451.9, 451.9, , and and International Classification of Diseases, 1th Revision [ICD-1], codes I26., I26.9, I8.1, I8.2, I8.3, O22.3, O87.1, and O88.2). STATISTICAL ANALYSES Data were analyzed using the Stata statistical software package, version 8. (Stata Corp, College Station, Tex). We used the Pearson 2 test, Mann-Whitney test, unconditional logistic regression, t test, 1-way analysis of variance (ANOVA), and repeated-measures ANOVA. Logistic regression analyses were performed with forced entry of covariates. Age, body mass index (square root transformation), FEV 1, FVC, and FEV 1 /FVC were introduced as continuous covariates. Pack-years of cigarette smoking was categorized (, 1, 1-2, and 2 packyears) because linearity on the logit scale could not be achieved with this continuous covariate. Two-sided P.5 was considered statistically significant. Data are presented as mean±sd. RESULTS Factor V Leiden genotype was not associated with any of the variables listed in Table 1. The genotype distribution did not differ from that predicted by the Hardy- Weinberg equilibrium (P=.17), and the frequency of factor V Leiden heterozygotes and homozygotes did not change as a function of age (Wald test; P=.78 and P=.85). DYSPNEA Dyspnea was more frequent among homozygotes than among noncarriers (Table 2); 26% ±1%, 2.% ±9.2%, and 32% ±11% of homozygotes complained of light, moderate, or severe dyspnea compared with 11.3%±.3%, 8.1%±.3%, and 6.4%±.3% of noncarriers ( 2 test; P=.4, P=.5, and P.1, respectively). Unadjusted odds ratios (ORs) for light, moderate, and severe dyspnea in homozygotes relative to noncarriers were 2.8 (95% confidence interval [CI], ), 2.8 (95% CI,.9-8.5), and 233

3 Table 2. Dyspnea by Factor V Leiden Genotype Dyspnea Type Noncarriers, Mean ± SD, % (n = 8534) Mean ± SD, % Heterozygotes (n = 699) Homozygotes (n = 2) P Value* Crude OR (95% CI) Adjusted OR (95% CI) Mean ± SD, % P Value* Crude OR (95% CI) Adjusted OR (95% CI) Light dyspnea 11.3 ± ± (.8-1.3) 1. (.7-1.2) 26.3 ± (1.-7.8) 2.1 (.7-6.4) Moderate dyspnea 8.1 ± ± (.8-1.3) 1. (.7-1.3) 2. ± (.9-8.5) 2.2 (.7-7.4) Severe dyspnea 6.4 ± ± (.8-1.4) 1. (.7-1.4) 31.6 ± ( ) 5.4 ( ) Abbreviations: CI, confidence interval; OR, odds ratio. *The 2 test vs noncarriers. Unadjusted ORs with 95% CIs vs noncarriers by unconditional logistic regression. The ORs with 95% CIs vs noncarriers by unconditional logistic regression adjusted for sex, age, smoking (pack-years), exposure to occupational dust, and body mass index. Answered yes to the question, Do you get shortness of breath when you walk at an ordinary pace on a level road alongside someone your age? Answered yes to the question, Do you have to stop once in a while in order to catch your breath when walking at your own pace? Answered yes to the question, Do you get shortness of breath during morning toilette or when you get dressed? Patients, % Noncarriers Heterozygotes Homozygotes A FEV 1, % Predicted Noncarriers Heterozygotes Homozygotes Age, y B 1 Figure 1. Prevalence of dyspnea by age group. Error bars indicate SDs; lines, regression lines. The P value by Wald testing for equality of logistic regression coefficients for age was.73 for heterozygotes vs noncarriers and.3 for homozygotes vs noncarriers. 6.7 (95% CI, ), respectively. After adjustment for sex, age, smoking, exposure to occupational dust, and body mass index, the association between homozygosity and light and moderate dyspnea was attenuated; however, it remained significant for severe dyspnea with an OR of 5.4 (95% CI, ). In a third logistic regression model, including additional adjustment for FEV 1, FVC, and FEV 1 /FVC, an OR for severe dyspnea of 4.2 (95% CI, ) was observed. With increasing age, the frequency of dyspnea (moderate and severe combined) increased in noncarriers (Wald test; P.1), heterozygotes (P.1), and homozygotes (P.1). The increase with age was more pronounced among homozygotes than among noncarriers (P=.3), as shown in Figure 1. The prevalence of dyspnea among heterozygotes did not differ from that in noncarriers (Table 2 and Figure 1). LUNG FUNCTION During follow-up, FEV 1 was consistently 5% to 1% lower among homozygotes than among noncarriers (repeated-measures ANOVA; P =.3), as shown in FVC, % Predicted Examination Figure 2. Forced expiratory volume in 1 second (FEV 1 ) and forced vital capacity (FVC) as percentage of predicted values by factor V Leiden genotype. Bars represent means±sds after adjustment for smoking. P values by repeated-measures analysis of variance adjusted for smoking were.99 (FEV 1 ) and.42 (FVC) for heterozygotes vs noncarriers and.3 (FEV 1 ) and.3 (FVC) for homozygotes vs noncarriers. Figure 2. Likewise, FVC was consistently lower among homozygotes than among noncarriers (P=.3). Annual decline in FEV 1 and FVC among homozygotes was 39±8 ml/y and 35±8 ml/y compared with 21±1 ml/y and 15±1 ml/y among noncarriers (t test; P =.3 and P =.4; Figure 3). No differences were observed between factor V Leiden heterozygotes and noncarriers on any of the spirometric measurements (Figure 2 and Figure 3). 234

4 FACTOR V LEIDEN HOMOZYGOTES Individual characteristics for the 2 factor V Leiden homozygotes at the examination are presented in Table 3. Notably, pulmonary embolism had been diagnosed in only 1 of the 2 homozygotes and deep venous thrombosis in 3. COMMENT This study demonstrates for the first time, to our knowledge, that factor V Leiden homozygosity is associated with A FEV 1, ml Noncarriers Heterozygotes Homozygotes P =.79 P =.3 B FVC, ml P =.64 P =.4 Figure 3. Annual change in forced expiratory volume in 1 second (FEV 1 ) (A) and forced vital capacity (FVC) (B) by factor V Leiden genotype. Bars represent means±sds. P values by analysis of variance with identical adjustment are shown. a 5-fold risk of severe dyspnea. Furthermore, homozygotes had lower FEV 1 and FVC alongside an accelerated decrease in FEV 1 and FVC. Factor V Leiden heterozygosity did not influence pulmonary symptoms and signs. The observation of lower pulmonary function in homozygotes compared with noncarriers was unexpected because most patients with repeated pulmonary thromboemboli have normal spirometry results. 12 To investigate if dyspnea among factor V Leiden homozygotes was related to the decreased ventilatory capacity, in addition to adjusting for age, sex, exposure to occupational dust, smoking, and body mass index, we adjusted for FEV 1, FVC, and FEV 1 /FVC ratio. These adjustments, which resulted in merely trivial changes in the estimated OR for dyspnea, led us to believe that factor V Leiden causes dyspnea not only through reduced pulmonary function but also by some other mechanism. A possible explanation for the observed increased frequency of dyspnea among factor V Leiden homozygotes is that these individuals experience repeated peripheral venous thrombi that dislodge and result in small, clinically unrecognized pulmonary thromboemboli. An alternative explanation for the observed findings is confounding by some other unmeasured factor. However, for such a factor to confound our results, it must be associated with both dyspnea and factor V Leiden. We are not aware of such a factor. Thirty percent of factor V Leiden homozygotes had been exposed to occupational dust compared with 19% of noncarriers, a difference that was not significant. To confidently exclude that our conclusions were confounded by exposure to occupational dust, we repeated analyses after excluding individuals ex- Table 3. Individual Characteristics of Factor V Leiden Homozygotes in FEV 1 FVC Patient No./ Sex/Age, y* DVT/PE Dyspnea Volume, ml % Predicted, ml Volume, ml % Predicted, ml FEV 1 /FVC 1/F/32 No No /F/38 No No /F/43 No No /F/45 No Yes /F/5 No No ND ND.82 6/F/52 No Yes /F/55 No No /F/56 No No ND ND.82 9/F/58 PE No /F/59 No No /M/35 No No ND ND.77 12/M/36 No No /M/39 No Yes /M/41 No Yes /M/46 No No /M/46 No No /M/47 No No ND ND.9 18/M/51 DVT Yes /M/53 DVT Yes /M/6 DVT Yes Abbreviations: DVT, deep venous thrombosis; FEV 1, forced expiratory volume in 1 second; FVC, forced vital capacity; ND, not determined; PE, pulmonary thromboembolism. *Age at entry. Answered yes to at least 1 of 3 questions on light, moderate, or severe dyspnea. At the examination. Annual change in FEV 1 and FVC during 16-year follow-up. 235

5 posed to occupational dust; after this exclusion, FEV 1 and FVC were still lower among homozygotes vs noncarriers (P=.4 and P=.3). Likewise, annual loss of FEV 1 and FVC was lower among homozygotes than among noncarriers (P=.5 and P=.3), whereas the OR for severe dyspnea in factor V Leiden homozygotes vs noncarriers was 9. (95% CI, ). Previous studies have suggested that factor V Leiden increases risk of deep venous thrombosis rather than pulmonary embolism. However, this apparent differential effect of factor V Leiden may not be real but could be a consequence of selection bias in studies of factor V Leiden and deep venous thrombosis. 18 In accordance with this, we could not demonstrate a difference in the excess risk of deep venous thrombosis and pulmonary embolism in The Copenhagen City Heart Study, which constitutes the largest prospective cardiopulmonary study performed so far on unselected individuals. 3 Finally, it should be recognized that previous studies on factor V Leiden and deep venous thrombosis or pulmonary embolism have investigated thromboembolic events that have been clinically detected. Therefore, even if factor V Leiden increases the risk of deep venous thrombosis more than that of pulmonary embolism, it does not contradict the findings of this study. We cannot exclude the possibility that severe pulmonary disease may have prevented some individuals from participating in The Copenhagen City Heart Study. However, we believe that a potential selection bias would not differ by genotype and that in the present study, selection bias against factor V Leiden homozygotes is of limited significance because (1) the factor V Leiden genotype distribution in this study is similar to that observed in 4188 Danish newborns 19 and (2) the relative genotype frequencies observed were not different from those predicted by the Hardy-Weinberg equilibrium. In conclusion, we demonstrate a previously unrecognized clinical presentation of factor V Leiden homozygosity with severe dyspnea and decreased pulmonary function. The fact that approximately.2% of white populations are factor V Leiden homozygotes and that 3% to 4% of these individuals may have severe dyspnea makes this a potentially important observation. Accepted for Publication: July 5, 24. Correspondence: Børge G. Nordestgaard, MD, DMSc, Department of Clinical Biochemistry, Herlev University Hospital, Herlev Ringvej 75, DK-273 Herlev, Denmark Financial Disclosure: None. Funding/Support: This study was supported by grants from The Danish Heart Foundation, Copenhagen; Chief Physician Johan Boserup and Lise Boserup s Fund, Haslev, Denmark; Lykfeldt s Fund, Hedehusene, Denmark; and Dagmar Marshall s Fund, Wedelborg s Fund, Lily Benthine Lund s Fund, The Beckett Fund, and P. Carl Petersen s Fund, Copenhagen. Disclaimer: The sponsors of the study are public or nonprofit organizations that support science in general. They had no role in gathering, analyzing, or interpreting the data and had no right to approve or disapprove of the submitted paper. The work of the authors was independent of the funders of this study. All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. 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