Asthma and COPD in cystic fibrosis intron-8 5T carriers. A population-based study

Background Carriers of cystic fibrosis intron-8 5T alleles with high exon-9 skipping could have increased annual lung function decline and increased risk for asthma or chronic obstructive pulmonary disease (COPD). Methods We genotyped 9131 individuals from the adult Danish population for cystic fibrosis 5T, 7T, 9T, and F508del alleles, and examined associations between 11 different genotype combinations, and annual FEV1 decline and risk of asthma or COPD. Results 5T heterozygotes vs. 7T homozygous controls had no increase in annual FEV1 decline, self-reported asthma, spirometry-defined COPD, or incidence of hospitalization from asthma or COPD. In 5T/7T heterozygotes vs. 7T homozygous controls we had 90% power to detect an increase in FEV1 decline of 8 ml, an odds ratio for self-reported asthma and spirometry-defined COPD of 1.9 and 1.7, and a hazard ratio for asthma and COPD hospitalization of 1.8 and 1.6, respectively. Both 5T homozygotes identified in the study showed evidence of asthma, while none of four 5T/F508del compound heterozygotes had severe pulmonary disease. 7T/9T individuals had annual decline in FEV1 of 19 ml compared with 21 ml in 7T homozygous controls (t-test:P = 0.03). 6.7% of 7T homozygotes without an F508del allele in the cystic fibrosis transmembrane conductance regulator gene reported asthma vs. 11% of 7T/9T individuals with an F508del allele (χ2:P = 0.01) and 40% of 7T homozygotes with an F508del allele (P = 0.04). 7T homozygotes with vs. without an F508del allele also had higher incidence of asthma hospitalization (log-rank:P = 0.003); unadjusted and adjusted equivalent hazard ratios for asthma hospitalization were 11 (95%CI:1.5–78) and 6.3 (0.84–47) in 7T homozygotes with vs. without an F508del allele. Conclusion Polythymidine 5T heterozygosity is not associated with pulmonary dysfunction or disease in the adult Caucasian population. Furthermore, our results support that F508del heterozygosity is associated with increased asthma risk independently of the 5T allele.


Background
Asthma and chronic obstructive pulmonary disease (COPD) are caused by complex interactions between environmental and genetic factors. A putative genetic risk factor for asthma and COPD is the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1][2][3]. This gene encodes a cAMP-regulated channel with chloride activity in pulmonary epithelia. When channel activities are absent, cystic fibrosis with life-threatening airways obstruction due to thickened secretions and secondary pulmonary infection develop [4]. The most common cause of cystic fibrosis is homozygosity for the phenylalanine-508 deletion (F508del), explaining about 70% of cystic fibrosis worldwide [4,5].
We hypothesised that carriers of the 5T allele have increased annual lung function decline and increased risk for asthma or COPD. To test this hypothesis, we geno-typed 9131 individuals from the adult Danish population for the 5T, 7T, and 9T alleles in the CFTR gene. We combined polythymidine and F508del genotypes [1], and examined associations between 11 different genotype combinations, and annual FEV 1 decline and risk of asthma or COPD. We also examined whether other common risk factors for lung disease or variations in the genes for mannose-binding lectin and α 1 -antitrypsin significantly add to risk of lung disease in 5T carriers.

Methods
Subjects participated in the 1976-78, 1981-83, and/or 1991-94 examination of the Copenhagen City Heart Study, a prospective epidemiological study initiated in 1976-78 [38]. Participants aged 20 years and above were selected randomly after age stratification into 5-year age groups from among residents of Copenhagen. Of the 17180 individuals invited, 10135 participated, 9259 gave blood, and 9131 were genotyped for the polythymidine tract variants of the cystic fibrosis conductance membrane regulator (CFTR) gene. Details of study procedures and some characteristics of non-responders are described elsewhere [38,39]. More than 99% were Whites of Danish descent. All participants gave written informed consent, and Herlev University Hospital and the ethics committee for Copenhagen and Frederiksberg approved the study (# 100.2039/91).
Participants filled out a self-administered questionnaire, which was validated by the participant and an investigator on the day of attendance. Participants reported on longterm occupational exposure to dust or welding fumes, pulmonary symptoms (dyspnea, wheezing, bringing up phlegm), familial predisposition to asthma (having at least one sibling with asthma), smoking habits (current smoker, ex-smoker, never-smoker), type of smoking and daily tobacco consumption. An estimate of life-time tobacco exposure (in packyears) was calculated as: daily tobacco consumption (g) times duration of smoking (years) divided by 20 (g/pack). If at least once during the study period participants aswered "Yes" to the question "Do you suffer from asthma?", we recorded they had selfreported asthma. Medication for asthma / bronchitis was "Yes" to the question "Do you daily take medication for asthma / bronchitis?" Additional information on hospitalizations due to asthma (ICD8: 493; ICD10: J45-46) and COPD (ICD8: 491-492; ICD10: J41-44) was drawn from the Danish National Hospital Discharge Register from May 1st 1976 through December 31st 2000. We confirmed in the Danish National Hospital Discharge Register covering all hospital discharges in Denmark, that no participants in the sample were ever hospitalized for cystic fibrosis.
Forced expiratory volume in one second (FEV 1 ) and forced vital capacity (FVC) were measured with an electronic spirometer (model N403, Monaghan, Littleton, Colo.) at the 1976-78 and 1981-83 examinations and with a dry wedge spirometer (Vitalograph, Maidenhead, UK) at the 1991-94 examination. At each examination, three sets of values were obtained, and as a criterion for correct performance of the procedure, at least two measurements of FEV 1 and FVC differing by less than 5% had to be produced. The highest set of FEV 1 and FVC were used in the analyses as percentage of predicted value using internally derived reference values based on a subsample of healthy never smokers [40]. Annual decline in FEV 1 (ml/year) was calculated as FEV 1 (ml) obtained at the latest measurement minus the FEV 1 value obtained at the first measurement, times 365.25 divided by the number of days between the two measurements (in years -1 ). Spirometry defined COPD was FEV 1 <80% predicted and FEV 1 /FVC<0.7, excluding self-reported asthma [41].
Linkage disequilibrium between the 9T and F508del alleles was tested by the linkage utility program "EH" http:// linkage.rockefeller.edu, which estimates allele and haplotype frequencies with and without allelic association. The linkage disequilibrium coefficient D was calculated as D = P 22 -p 2 q 2 , where P 22 is the observed frequency of the 9T/ F508del haplotype, p 2 is the frequency of the F508del allele in the general population and q 2 is the population frequency of the 9T allele. The degree of linkage disequilibrium was expressed as D' = D/D max × 100%.
Statistical analysis was performed with SPSS; for power calculations, NCSS-PASS and StatMate were used. P < 0.05 on a two-sided test was considered significant. Pearson's χ 2 -test or analysis of variance (ANOVA) was used for overall comparisons between several genotypes; Pearson's or Fisher's Exact χ 2 -test were used for post-hoc two-genotype comparisons. The most common genotype combination in the population, 7T homozygosity without F508del, was used as reference group for statistical comparisons. We evaluated asthma and COPD prevalences between genotypes using unadjusted and adjusted logistic regression with Wald's test as a measure of significance; the adjusted model included gender, age at study entry (deciles), and packyears at study entry (never smokers and deciles). We evaluated asthma and COPD incidences between geno- types using the log-rank test [42][43][44]. Unadjusted and adjusted Cox regression with forced entry examined time to disease by using hazard ratios (relative risks) and 95% confidence intervals; the adjusted model included gender, age at study entry (deciles), tobacco use during follow-up (never smokers and deciles), and FEV 1 % predicted at study entry (deciles). We tested possible interactions between the 5T/7T genotype and smoking habits, longterm occupational exposure to dust or welding fumes, familial predisposition to asthma, α 1 -antitrypsin MS genotype, α 1 -antitrypsin MZ genotype, or mannose-binding lectin deficiency in predicting FEV 1 at study entry in ANCOVA models.

Results
Characteristics of participants are given in Table 1

Annual decline in FEV 1
Annual decline in FEV 1 did not differ between 5T heterozygotes or homozygotes vs. 7T homozygous controls (Fig.  1). 7T/9T individuals had annual decline in FEV 1 of 19 ml compared with 21 ml in 7T homozygous controls (t-test: P = 0.03; Fig. 1 Incidence of hospitalization from asthma during 24 years follow-up did not differ between 5T heterozygotes or homozygotes versus 7T homozygous controls (Table 2). However, incidence of asthma hospitalization was increased in 7T homozygotes with F508del compared with 7T homozygous controls ( Table 2). Unadjusted and after adjusting for gender, age at study entry, tobacco consumption, and FEV 1 % predicted at study entry, the hazard ratio for asthma hospitalization was 11 (1.5-78) and 6.3 (0.84-47) in 7T homozygotes with F508del vs. 7T homozygous controls. None of the other genotype combinations differed from 7T homozygous controls ( Table 2). The analysis had 90% power to detect a hazard ratio for

Chronic obstructive pulmonary disease (COPD)
Prevalence of spirometry defined COPD did not differ between 5T heterozygotes or homozygotes vs. 7T homozygous controls (Ps ≥ 0.22) and did not differ between genotypes overall (χ 2 : P = 0.51) (data not depicted). Unadjusted and adjusted odds ratios for spirometry defined COPD did not differ between genotypes (Fig. 3) Incidence of hospitalization from COPD during 24 years follow-up was reduced in 5T/7T individuals vs. 7T homozygous controls (Table 3). Unadjusted and after adjusting for gender, age at study entry, tobacco consumption and FEV 1 % predicted at study entry, the hazard ratio for COPD was 0.47 (0.23-0.95) and 0.49 (0.23-1.0) in 5T/7T individuals vs. 7T homozygous controls (Table 3).

5T homozygotes and 5T/F508del compound heterozygotes
One of two 5T homozygous smokers reported having asthma and took daily medication for respiratory disease ( Table 4). The other homozygous individual showed evidence of airway obstruction with reversibility and was referred for further examination and treatment of asthma. None of four 5T/F508del compound heterozygotes had clinical signs of severe pulmonary disease (Table 4).

Discussion
This study shows that polythymidine 5T heterozygosity is not associated with increased annual decline in FEV 1 or risk of asthma or COPD in the adult Caucasian population; these results are independent of age, gender, tobacco smoking, and other potential confounders. Interestingly, however, both 5T homozygotes showed evidence of asthma. Furthermore, our results support that F508del heterozygosity is associated with increased asthma risk independently of the 5T allele.
Because 1 in 26 carries a 5T allele in this population, it is indeed important that 5T heterozygosity does not increase risk of obstructive lung disease in the population at-large. It appears that the 5T allele causes lung disease only in very rare circumstances [9][10][11][12][13][14], leaving the average heterozygous individual unaffected by obstructive lung disease. Previous results suggest that penetrance of pulmonary Odds ratios for self-reported asthma by intron-8 polythymi-dine tract and F508del genotype Figure 2 Odds ratios for self-reported asthma by intron-8 polythymidine tract and F508del genotype. 7T homozygotes without F508del was used as reference group. The adjusted model included gender, age at study entry, and packyears at study entry. Error bars are 95% confidence intervals. Self-reported asthma = "Yes" at least once during the study period to the question "Do you suffer from asthma?".
manifestations in 5T carriers might depend on the length of an adjacent TG repeat [46,47]. This could be particularly relevant for 5T homozygotes and compound heterozygotes. In 5T heterozygotes, however, longer TG repeats seem less likely to affect risk of pulmonary disease. This is because 5T heterozygosity was not associated with risk of lung disease in this study although predicted TG12 and TG13 allele frequency in 5T carriers in our population was 31% [47]. Other additional genetic variations have also been shown to influence exon-9 skipping in 5T carriers, but to a lesser degree than the TG repeat.
Because all 5T/F508del compound heterozygotes were free from severe pulmonary disease, the 5T allele did not appear to explain our previous results [1,6] suggesting that F508del heterozygosity may be overrepresented among asthmatics. A few recent studies also support this observation [2,19,48], while others have found no [20,21,49] or negative associations [50]. In the present analyses, 7T/9T and 7T/7T individuals with F508del heterozygosity had higher prevalences of self-reported asthma, and 7T/7T individuals with F508del heterozygosity also had higher incidence of hospitalization from asthma. F508del heterozygosity was only associated with increased asthma risk in individuals without the 5T allele, indicating that our previous observations are independent of influence from this allele. In addition, both 5T homozygotes showed evidence of asthma supporting the hypothesis that CFTR variations may be associated with asthma [2,19].
To identify factors in the population that significantly add to risk of lung disease in 5T heterozygotes, we tested for Odds ratios for spirometry defined COPD by intron-8 poly-thymidine tract and F508del genotype Figure 3 Odds ratios for spirometry defined COPD by intron-8 polythymidine tract and F508del genotype. 7T homozygotes without F508del was used as reference group. The adjusted model included gender, age at study entry, and packyears at study entry. Error bars are 95% confidence intervals. COPD = FEV 1 <80% predicted and FEV 1 /FVC<0.7, excluding self-reported asthma. interactions between 5T/7T genotype and potential risk factors for lung disease, but found no significant interactions. Garred [35] and coworkers found a worse prognosis in cystic fibrosis patients with MBL deficiency. We were not able to extend this finding, since lung function in 5T or F508del heterozygotes was not reduced by MBL deficiency. Previous studies by Mahadeva [36] and Frangolias [37] showed that pulmonary disease severity in cystic fibrosis patients were unaffected by α 1 -antitrypsin S and Z alleles. In line with this, we also observed no increased risk for pulmonary dysfunction in 5T carriers with α 1 -antitrypsin MS or MZ genotypes.
In the present study, bias caused by investigators' knowledge of disease or risk-factor status seems unlikely, because we selected from a general population and genotyped our sample without knowledge of disease status or lung function test results. Selection bias is possible if severe lung disease in some individuals with 5T genotypes prevented them from participating in our study; however, expected and observed numbers of these genotypes according to the Hardy-Weinberg equilibrium were similar. The 2.7% frequency of F508del heterozygosity found in this study is in accordance with the 2.9% frequency of F508del heterozygosity observed in another previous study of the Danish population [51]. Annual decline in FEV 1 was reduced in 7T/9T individuals and incidence of COPD hospitalization was reduced in 5T/7T individuals. If correction for multiple comparisons was performed, these significant findings become nonsignificant. There-  *Number of TG repeats adjacent to the polythymidine tract included. † FEV 1 30 minutes after inhalation of 0.5 mg terbutaline minus FEV 1 at 0 minutes divided by FEV 1 at 0 minutes times 100%; only individuals with FEV 1 /FVC<0.7 were tested for FEV 1 reversibility. ‡ "Yes" to "Do you suffer from asthma?" ¶ "Yes" to "Do you daily take medication for asthma / bronchitis?" **Hospitalizations from asthma (ICD8: 493; ICD10: J45-46) and COPD (ICD8: 491-492; ICD10: J41-J44) were drawn from the Danish National Discharge Register from 1976 through 2000.
fore, and because reduced COPD risk in 5T/7T individuals is less biologically plausible, the findings are likely due to chance alone rather than representing real phenomena. Misclassification of genotypes is unlikely, because diagnoses were confirmed by sequencing a subsample of different poly-T variants.

Conclusion
Polythymidine 5T heterozygosity was not associated with increased annual decline in FEV 1 or risk of asthma or COPD in adults in this population-based study; however, both 5T homozygotes showed evidence of asthma. Furthermore, our results also support that F508del heterozygosity may be associated with increased asthma risk independently of the 5T allele.