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End-Organ Dysfunction in Cystic Fibrosis

Association with Angiotensin I Converting Enzyme and Cytokine Gene Polymorphisms

Academic Unit of Child Health, Booth Hall Children's Hospital, and School of Biological Sciences, University of Manchester; Department of Clinical Genetics, Royal Manchester Children's Hospital; and Bradbury Cystic Fibrosis Unit, Wythenshawe Hospital, Manchester, United Kingdom

Correspondence and requests for reprints should be addressed to Peter D. Arkwright, M.D., Senior Lecturer in Paediatric Immunology, Booth Hall Children's Hospital, Charlestown Road, Manchester M9 7AA, UK. E-mail: peter_arkwright{at}lineone.net

	ABSTRACT

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The clinical course of patients with cystic fibrosis (CF) with functionally similar mutations in the CF transmembrane conductance regulator gene is variable and must therefore relate to secondary genetic and environmental factors. We examined the hypothesis that polymorphisms of certain inflammatory mediator and regulatory genes affect clinical outcome by influencing the degree of end-organ damage. By studying the possible association between clinical outcome and angiotensin I-converting enzyme (ACE) and cytokine genotypes by amplification refractory mutation system-polymerase chain reaction, using stored DNA from 261 white patients with CF, we found that ultrasound features of cirrhosis occurred more frequently in patients with the high-producer (DD) rather than the low-producer (II) ACE genotype (odds ratio [95% confidence interval], 3.7 [1.2 to 12]). Moreover, significant pulmonary dysfunction (age at which FEV₁ < 50%) was associated with the high-producer ACE genotype (2.3 [1.2 to 4.5]) and transforming growth factor–ß₁ genotype (2.6 [1.0 to 6.8]) as well as with age at first colonization with Pseudomonas aeruginosa (9.1 [1.1 to 72]). We conclude that the high-producer ACE genotype predicts patients with CF who have an increased chance of developing portal hypertension; and high-producer ACE and TGF-ß₁ genotypes are secondary genetic factors contributing to pulmonary dysfunction in these patients.

Key Words: angiotensin I converting enzyme • cirrhosis • cystic fibrosis • lung • transforming growth factor–ß₁

The variable clinical course of patients with cystic fibrosis can be explained only partially by defined genetic (e.g., nature of the mutation in the cystic fibrosis transmembrane conductance regulator [CFTR] gene), environmental (e.g., cross-infection with Burkholderia cepacia), and social factors (e.g., noncompliance with treatment) (1–4). For example, among patients with a similar CFTR genotype, only a minority of patients develop clinical evidence of advanced fibrosis in the liver, which manifests as cirrhosis. Similarly, whereas more than 90% of patients with CF have antenatal fibrosis of the pancreas leading to exocrine pancreatic failure, only a small proportion go on to develop endocrine pancreatic failure manifesting as insulin-dependent diabetes mellitus. The development of respiratory impairment and failure also varies significantly from patient to patient with identical CFTR genotypes. Lung damage may result from mucus plugging, which directly triggers inflammation and dysfunction (5, 6), and also viral and bacterial infections, which accelerate the rate of lung damage (7). Differences in the severity of inflammation in the lung and fibrosis in the liver and pancreas are likely to account for the differences in outcome observed in clinical practice.

The aim of this study was to examine the hypothesis that patients with genotypes associated with high rates of production of certain proinflammatory/fibrotic mediators (transforming growth factor-ß₁ [TGF-ß₁], tumor necrosis factor- [TNF-], interferon- [IFN-], and angiotensin I converting enzyme [ACE]) (8–11) are more likely to develop severe organ damage.

Functional gene polymorphisms of two groups of inflammatory mediators were examined. First, the profibrotic cytokine TGF-ß₁ and an enzyme that activates the latent cytokine, ACE (12–14), were examined, the hypothesis being that patients with CF with inherently higher rates of production of active TGF-ß₁ would develop more severe organ fibrosis. In support of this hypothesis, previous animal and human studies have shown that higher TGF-ß₁ production (15–20) and a high-producer TGF-ß₁ genotype (polymorphism at position +915) (18) are associated with the development of significantly more lung fibrosis in response to a number of inflammatory triggers such as radiation, chemotherapy, and lung transplantation. In a previous study of 171 patients with CF who were homozygous for the F508 CFTR gene mutation, we found a significant association between TGF-ß₁ genotype (polymorphism at position +869) and rate of deterioration in lung function (FEV₁ less than 50%) (odds ratio [95% confidence interval], 1.7 [1.1 to 2.7]) (21).

Second, inflammatory cytokines that augment (TNF-, IFN-) or suppress (interleukin-10 [IL-10], TGF-ß₁) the inflammatory response of phagocytes and lymphocytes were studied, as these cytokines may not only influence the severity of tissue inflammation, but also host immunity to viral and bacterial pathogens known to adversely affect lung function (22, 23).

	METHODS

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Patients
The Department of Clinical Genetics at the Royal Manchester Children's Hospital (Manchester, UK) has DNA stored from all patients with CF in the northwest region of the United Kingdom referred for genetic mutation testing over the last two decades. At the time of sample collection for CFTR gene analysis, informed consent for analysis of other genetic factors, which would aid in the better understanding and treatment of patients with CF was obtained and approved by the local ethics committee. Data from 261 patients who attended one of the three major CF units in Manchester (Royal Manchester Children's Hospital, Booth Hall Children's Hospital, and Wythenshawe Hospital) were examined. Only patients with F508 homozygous mutations, or compound mutations from Classes I, II, and III in the CFTR gene, known to compose the severe end of the clinical spectrum, were included in this study (24). Patients less than 4 years old were excluded, as no lung function information was available. Clinical information was obtained from hospital notes and physiotherapy records in which formal lung function tests were regularly recorded. The age at which FEV₁ decreased below 50% was defined as the age at which a persistent fall in FEV₁ below 50% of predicted with no subsequent improvement was noted. A "rapid deterioration in lung function" was defined as a deterioration in FEV₁ from more than 75% to less than 50% of expected within a 3-year period. The age at which patients were first colonized with Pseudomonas aeruginosa was defined as the age at which the first of at least three consecutive sputum-positive cultures was noted. In this study, patients were classified as having "cirrhosis" if on ultrasound scan they had features of abnormal liver echogenicity as well as significant splenomegaly (25, 26). The patients with diabetes were all insulin-dependent.

Amplification Refractory Mutation System-Polymerase Chain Reaction Assays
Archived DNA used in this study had been extracted from 5 ml of whole blood by standard methods. Individual samples were genotyped for TGF-ß₁ (positions +869 and +915) (10), IL-10 (position –592) (9), TNF- (position -308) (10), and IFN- (position +874) (11) by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) methodology, using primers already described. A polymorphism in ACE, consisting of the presence or absence of a 250-bp DNA fragment, was screened by a restriction fragment length polymorphism assay described previously (27).

Serum ACE Measurements
Serum ACE concentration was measured in 38 patients of the total cohort. These patients were selected on the basis of ACE genotype to make sure that sufficient numbers with low- and high-producer genotypes were represented. Additional consent was obtained from these patients and approved by the local ethics committee. Serum ACE was measured in duplicate, using a colorimetric assay kit (Buehlmann Laboratories, Allschwil, Switzerland). This kit measured the cleavage of a synthetic substrate into an amino acid derivative and a dipeptide. The kinetics of this cleavage reaction were measured by following the decrease in absorbance at 340 nm.

Statistical Analyses
The SPSS statistical package (version 10.1 for Windows; SPSS, Chicago, IL) was used for most statistical analyses. Survival analysis was performed with Kaplan–Meier statistics. Multivariate analysis and odds ratio (95% confidence internal) (OR [95% CI]) were calculated by Cox regression for continuous variables and binary logistic regression for discrete variables. Bivariate correlation was determined with the Pearson correlation coefficient. The Hardy–Weinberg equilibrium was calculated according to a standard formula (28).

	RESULTS

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Demographic Characteristics of the Study Population
A total of 261 white patients, of whom 134 (51%) were male with a median age of 22 years (range, 4 to 54 years), were studied. Sixty-seven (26%) of the patients had died, and 125 (48%) had significant respiratory impairment with FEV₁ less than 50% of expected. A total of 232 (90%) patients were colonized with P. aeruginosa and 56 (22%) were colonized with B. cepacia. All patients colonized with B. cepacia were also colonized with P. aeruginosa. Thirty-six (15%) of the patients had ultrasound features of cirrhosis and portal hypertension. Forty-two (16%) of the patients had insulin-dependent diabetes mellitus.

A total of 194 (74%) patients were homozygous for the F508 CFTR gene mutation and the remainder had other Class I to III CFTR mutations. Univariate Kaplan–Meier survival curves and ages at which lung function deteriorated to FEV₁ less than 50% were not significantly different in patients who were homozygous for the F508 CFTR gene mutation and those that had other CFTR mutations (p = 0.5).

Gene Polymorphism Frequencies
The relative frequencies of polymorphisms of TGF-ß₁ (positions +869 and +915), ACE (position I/D), IFN- (position +874), TNF- (position -308), and IL-10 (position –592) in this cohort of patients with CF are shown in Table 1 and compared with published frequencies of these polymorphisms in a normal population. There were no significant differences between the frequencies of these alleles and genotypes in our CF cohort and control subjects. All gene polymorphism frequencies examined conformed to the Hardy–Weinberg equilibrium, indicating no bias in sampling in this population, or problems with genotyping of the stored samples.

View larger version (10K): [in this window] [in a new window]   Figure 1. Serum ACE levels (IU/ml) of patients according to ACE genotype.

Respiratory Dysfunction
Age at colonization with P. aeruginosa was the major factor predicting age at which FEV₁ fell below 50% of predicted (r = 0.66; p < 0.0001; n = 110). On univariate analysis, none of the other variables studied were significantly associated with age at which FEV₁ fell below 50% of predicted (data not shown). However, on multivariate analysis, F508 homozygozity and high-producer DD ACE genotype were associated with a more severe clinical course than were other genotypes in terms of an earlier age at which significant pulmonary dysfunction occurred (Table 3).

Sixty-one patients (23%) had a rapid deterioration in lung function over 1 to 3 years, rather than a gradual decline in lung function over a longer time frame. The major factor associated with this rapid decline in lung function was colonization by B. cepacia (OR [95% CI], 3.1 [1.6 to 6.2]; p = 0.001). Twenty-five of the 61 patients (41%) were colonized with B. cepacia, and 8 patients (13%) had multiantibiotic-resistant P. aeruginosa. Multivariate analysis did not show an association between any of the cytokine polymorphisms tested, that is, TGF-ß₁ (positions +869 and +915), IFN- (position +874), TNF- (position -308), and IL-10 (position -592), and accelerated decline in lung function (data not shown).

Age at Acquisition of P. aeruginosa and B. cepacia in Pulmonary Secretions
The possibility that the gene polymorphisms influenced host resistance and thus age at lung colonization with P. aeruginosa or B. cepacia was also examined. Multivariate analysis by Cox regression demonstrated that none of the inflammatory cytokine polymorphisms, that is, TGF-ß₁ (positions +869 and +915), IFN- (position +874), TNF- (position -308), or IL-10 (position -592), were associated with differences in age at which sputum was initially positive for either P. aeruginosa or B. cepacia (data not shown). There was also no difference in age at acquisition of P. aeruginosa based on ACE genotype (p = 0.9).

Mortality
As expected, the major factor predicting a patient's age of death was the age at which significant deterioration in lung function occurred (FEV₁ less than 50% predicted; r = 0.72; p < 0.0001, n = 55). Using multivariate Cox regression analysis, no association between any of the gene polymorphisms studied and the age of death of the patient could be demonstrated. Patients with cirrhosis or diabetes did not die at a significantly younger or older age.

	DISCUSSION

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The interplay between genetic and environmental factors leading to tissue damage in CF is complex, especially in the lung, where infection might overshadow the effects of secondary genetic factors. In this study, we therefore first examined the possible association between functional polymorphisms (TGF-ß₁, TNF-, IFN-, IL-10, and ACE) (8–11, 27) and the development of liver and pancreatic disease in CF, where infection is not thought to play a role in the pathogenesis, before studying the possible association between these cytokine gene polymorphisms and pulmonary dysfunction.

A novel finding of this study is that significantly more patients with the high-producer DD ACE genotype had ultrasound features of portal hypertension than did patients expressing the low-producer II ACE genotype (p = 0.02). Independent evidence of an association between ACE genotype and liver disease comes from a study of patients with alcoholic cirrhosis, which showed similar trends (29). Although high levels of TGF-ß₁ have previously been associated with increased liver fibrosis in autoimmune hepatitis (30), and in animal models of liver and pancreatic fibrosis (31, 32), in this study we found no association between TGF-ß₁ genotype and either the development of ultrasound evidence of portal hypertension or insulin-dependent diabetes.

The TGF-ß₁ gene controls the release of the latent cytokine, which subsequently needs to be activated by one of a number of enzymes including angiotensin II (14). Thus a high-producer TGF-ß₁ genotype might cause organ fibrosis only if coexpressed with the high-producer genotype for one of the TGF-ß₁ activator enzymes. The absence of an association between TGF-ß₁ polymorphisms and liver disease, even when ACE polymorphisms are taken into account by multivariate analysis, suggests that ACE might cause portal hypertension predominantly through non–TGF-ß₁–mediated pathways. Excessive renin–angiotensin system activity in cirrhosis is known to cause increased vascular resistance and fluid retention (33), and it is possible that augmentation of renin–angiotensin system activity because of higher ACE levels might exacerbate clinical disease by affecting vascular reactivity. Further studies are required to address this possibility. From the therapeutic viewpoint, it would be interesting to investigate whether patients with CF who are symptomatic because of sequelae of cirrhosis benefit from treatment with ACE inhibitors (34).

The second part of this study examined the possible association between cytokine polymorphisms and the age at which significant deteriorations in lung function occurred. Unlike previous studies, this study also took into account the likely confounding effect of infection on this association. As would be expected, we found that the age at colonization with Pseudomonas aeruginosa was the major factor determining the age at which significant deterioration in lung function occurred, and colonization with B. cepacia was the major factor associated with more rapid pulmonary deterioration. Significant differences in age at which FEV₁ decreased to less than 50% were associated with position +915 TGF-ß₁ and I/D ACE genotypes, providing further support to the hypothesis that factors that would tend to increase the activity of profibrotic mediators lead to more severe pulmonary disease and that inherited genotypes may be either additive (ACE and TGF-ß) or balance each other out (TGF-ß and CFTR genotype). Previous animal and human studies suggest that ACE inhibitors may prevent pulmonary fibrosis both in vitro (35) and in vivo (36). On the basis of the results of these previous studies and our present findings, further work is warranted to determine whether ACE inhibitors may alleviate pulmonary symptoms in patients with CF.

The data from this study suggest that inherited variation in cytokine genes controlling production of TNF-, IFN-, IL-10, and TGF-ß₁ does not significantly influence the age at which lungs of patients with CF are colonized with bacterial pathogens. Polymorphisms in mannose-binding protein lectin have previously been associated with a higher rate of colonization with B. cepacia, a lower mean FEV₁, and a younger age of death in patients with CF (37). Thus, our findings do not rule out the possible role of other host immune mechanisms in determining the propensity to bacterial infections in the CF lung. The potential interaction between mannose-binding protein lectin polymorphisms and other secondary genetic factors described in this study deserves further investigation.

There is growing evidence that local changes in the airways related to CFTR dysfunction directly increase the propensity to bacterial colonization (7, 38). Although the study population is homogeneous, consisting solely of white patients with CF, only 74% of patients were homozygous for the F508 deletion. The remaining 67 of 261 patients had 26 other compound CFTR mutations from Classes I, II, and III, known to be at the severe end of the clinical spectrum (24). Although using univariate Kaplan–Meier curves, the rate of decline of lung function and the proportion of patients with cirrhosis were not significantly different; multivariate analysis did show significant differences in the rate of pulmonary dysfunction, suggesting that CFTR genotype may interact with a number of other factors, including the propensity to infection as discussed above, to determine clinical outcome.

In addition to the previously described link between mannose-binding lectin polymorphisms and pulmonary dysfunction and survival in CF, other secondary genetic factors may contribute to the clinical course of patients with CF (reviewed in Action and Wilmott [39]). Gene polymorphisms in factors controlling the activity of inflammatory mediators, such as (1) glutathione-S-transferase, an important antioxidant, (2) the antiprotease ₁-antitrypsin, and (3) neuronal nitric oxide synthase, an enzyme that controls the production of free radicals, have all been linked to variation in the clinical course of CF. Many of these studies are small, with 50 to 100 patients in the cohorts, and larger studies are required to confirm these findings. There are also data suggesting that specific HLA Class II genotypes may modify pulmonary and hepatic phenotypes of CF. The mechanisms linking HLA to clinical disease in CF remain unclear and also require further investigation.

In summary, this study uncovers a novel association between functional polymorphisms in the ACE gene and the development of portal hypertension, which may have implications for the treatment of this complication of CF. It also illustrates the complexity of interactions between factors, especially polymorphisms in profibrotic factors such as TGF-ß₁ and ACE and infection, in determining pulmonary dysfunction in patients with CF, and thus the potential pitfalls in examining the relevance of a single factor in isolation in this multifaceted disease.

	Acknowledgments

 
The authors thank Dr. G. Hambleton, Professor T. J. David, and Dr. L. Patel, who work in the CF units in Manchester, for allowing the study of their patients; and Professor T. J. David and Dr. M. A. Turner for useful comments and discussion.

	FOOTNOTES

 
Supported by grant PJ480 from the Cystic Fibrosis Trust (UK).

Received in original form April 24, 2002; accepted in final form September 3, 2002

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