INTRODUCTION

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Cystic fibrosis (CF) occurs with mutation in the CF transmembrane conductance regulator (CFTR) gene. Sixty-six percent of the CFTR gene alleles contain a 3-bp deletion in the CFTR gene which results in the loss of a phenylalanine residue at position 508 (F508 CFTR gene allele) (15). Therefore, about 44% of CF patients are homozygous for the F508 CFTR gene allele. Survival in CF is limited by progressive obstructive pulmonary disease secondary to abnormal secretions and injury from chronic infection and inflammation. The predominant pathogen for CF patients is mucoid Pseudomonas aeruginosa (MPA), and infection with this pathogen is associated with more severe pulmonary disease (9, 16). While F508 and other CFTR gene alleles (e.g., W1282X, and G551D) can be categorized as severe with respect to the level of exocrine pancreatic dysfunction (20), correlations of genotype with the severity of pulmonary disease have been less clear. Many analyses of cross-sectional data covarying measures of pulmonary function with the number of F508 CFTR gene alleles failed to demonstrate a dose effect of this allele on pulmonary disease severity (1, 3, 8, 17, 22, 34). Other evidence, however, suggests that the CFTR gene genotype may influence pulmonary phenotype (4, 5, 14, 18, 23, 25, 35).

The observation that mild pulmonary disease can exist in some patients homozygous for the F508 CFTR gene allele suggests that other genetic and environmental factors must modify the pulmonary phenotype in CF (2, 33). Given the impact of chronic MPA infection, factors that modify the time of onset or the persistence of infection may affect long-term outcome. One such factor may be a CF patient's immunologic status. An immune response composed of opsonic antibodies specific to the mucoid exopolysaccharide (MEP) coat of MPA was associated with the absence of MPA infection in older CF patients, and these antibodies were protective against infection in animal models (30, 31). Tosi et al. (37) also found naturally occurring opsonic antibodies of undefined antigenic specificity present in CF patients prior to infection with a decline in opsonic activity following MPA infection. The effect of genotype on MPA infection or pulmonary status was not considered in these studies of CF patients.

The identification of factors that explain the observed variability in pulmonary outcome could be valuable both in furthering our understanding of CF pathophysiology and in accelerating evaluation of the efficacy of new therapies. Recently, Corey et al. (5) presented rates of decline in pulmonary function based on a mixed-model statistical approach for 363 CF patients analyzed according to age, sex, CFTR gene genotype, and pancreatic status. However, neither infection nor immunologic status to MPA was incorporated into their model. We therefore performed a multivariate assessment of known risk factors (5, 9), modeling age, MPA infection status, immune response to MPA, CFTR gene genotype, pancreatic supplement requirement, and gender to weigh and combine this information for a prediction of differences in pulmonary outcome. Assessment of longitudinal analysis, in addition to cross-sectional analysis, of pulmonary function measurements was chosen because of the failure of prior cross-sectional studies to detect an impact of genotype on pulmonary outcome in CF patients and because of the recently demonstrated value of longitudinal analysis (5).

(This work was presented in part at the Ninth Annual North American Cystic Fibrosis Conference, Dallas, Tex., October 1995.)

	MATERIALS AND METHODS

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Study population and sample collection. Of the 425 CF patients monitored at Children's Hospital, Boston, Massachusetts, in 1993, 263 were at least 12 years of age, 87% of whom were infected with MPA. A matched-case control study was performed on CF patients (sweat chloride levels, >60 meq/liter) age 12 and over, with MPA infection as the exposure. Infected subjects grew MPA from two or more sputum cultures. Thirty-four of the 263 CF patients were uninfected subjects with sputum cultures that grew only normal bacterial throat flora, Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae, or non-MPA. None had evidence of persistent MPA infection on routine biannual cultures. Patients colonized only with non-MPA maintain pulmonary function levels comparable to those of CF patients carrying only normal throat bacteria (9). Blood for antibody assessment and genotypes were obtained from 26 of the 34 eligible uninfected patients. Twenty-seven infected subjects with both genotype and serum available were matched on gender and age to the qualifying uninfected subjects (Fig. 1). The infected subjects were derived from the intersection of two subpopulations: (i) the 76% of clinic patients age 12 or older who had blood drawn for antibody assessment during an outpatient visit and (ii) a sample selected by random number generation who had participated in a genotyping study (32).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of subject selection.

Genotype analysis. Genomic DNA isolated from each subject was evaluated for the presence of any of twelve CFTR gene mutations (F508, G551D, G542X, 621+1GT, I507, 1717-1 GA, R117H, N1303K, W1282X, R560T, R553X, and 3849+10kb CT) by one of three standard assays (10, 11, 32).

Antibody determinations. As a measure of exposure to and/or infection with MPA, titers of total opsonic antibody to MPA were determined on blinded serum samples by using a well-established opsonophagocytic killing assay (30). Aliquots (100 µl) of heat-inactivated (56°C, 30 min) serum samples were diluted and mixed with equal volumes containing 2 × 10⁶ CFU of MPA strain 2192, 2 × 10⁶ peripheral blood leukocytes obtained from healthy donors and prepared by dextran sedimentation, and a 1:15 dilution of intact human serum as a complement source. Tubes were incubated at 37°C with end-over-end rotation for 90 min, after which the surviving CFU of MPA were counted. The overall opsonic antibody titer to MPA was the reciprocal of the highest serum dilution mediating killing of >50% of the CFU of MPA.

Pulmonary function measurements. Forced expiratory volume (FEV₁) was determined by standard spirometry, and absolute volumes were converted to a percentage of the predicted volume expected for a healthy individual of the same age, gender, and height, on the basis of the regression equations developed by Knudson (19). For each subject, all percent predicted values, including those obtained prior to antibody testing, were included in the analysis. All values obtained subsequent to the initiation of DNase (Pulmozyme; Genentech, Inc., South San Francisco, Calif.) clinical trials were excluded. The time interval between evaluation points varied for each subject.

Statistical analysis. Mean values for continuous variables were compared by the independent-sample t test. The analysis of proportional data was performed by using Fisher's exact test (36). Logistic regression was used to estimate the probability of infection with MPA at a given level of MSOA, with the likelihood ratio chi-square test used to assess significance (12). All P values were two sided, with a P value of <0.05 considered statistically significant, unless otherwise stated.

	RESULTS

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Study population characteristics. CF patients 12 years old and older were chosen because this is the point at which 87% of patients in the overall clinic population had acquired MPA infection. Thus, it is likely that uninfected CF patients 12 years old and older represent those individuals whose lack of infection is not due to an environmental factor, such as not having been sufficiently exposed to MPA. After matching for age and gender, there were no significant differences in subject characteristics between MPA-infected and uninfected groups in terms of age at enrollment, sweat chloride level, or requirement for pancreatic supplementation (Table 1). The frequency of the F508 CFTR gene allele was higher among infected subjects (P = 0.05, one-sided Fisher's exact test) than among uninfected subjects. When infected and uninfected subjects were stratified by the count of F508 CFTR gene alleles in a subject's genotype, the distribution differed significantly, with more subjects in the MPA-infected group being homozygous for the F508 CFTR gene allele (P = 0.03). The relative odds of MPA infection in subjects homozygous for the F508 CFTR gene allele were 4.67 (95% confidence interval, 1.75 to 12.44; P = 0.01) compared to subjects heterozygous for this allele.

Characteristics of FEV₁ determinations in infected and uninfected subjects. The ages (years) at the first FEV₁ determination were similar in the MPA-infected and uninfected subjects (mean ± standard deviation [SD], 12.2 ± 5.5 versus 11.4 ± 7.1, respectively [P = 0.66]), as were the durations (years) of follow-up in pulmonary function measurements (mean ± SD, 13.1 ± 4.5 versus 10.8 ± 4.5, respectively [P = 0.07]). The mean of the first percent predicted FEV₁ was lower among infected subjects (mean ± SD, 80% ± 22% versus 95% ± 17% [P < 0.01]) than among uninfected subjects, likely reflecting the effect on pulmonary function of MPA infection prior to age 12 in infected subjects. To assess potential selection bias in the 76% of infected clinic patients 12 years old and older who provided blood samples, we compared the first percent predicted FEV₁ used in the analysis from this group to data from the 1997 Cystic Fibrosis Foundation patient registry report and found that the mean ± SD of the percent predicted FEV₁ in infected study subjects (80% ± 22%) was virtually the same as that in the national sample (age 13; mean ± SD, 79.1% ± 22.4% [n = 680]) (6). Follow-up for at least 6 years occurred in 25 infected (93%) and 22 uninfected (85%) subjects. MPA-infected subjects had over twice as many data points as uninfected subjects (mean number of FEV₁ measurements per subject, 44 ± 27 versus 19 ± 13, respectively [P < 0.001]). Eighty-five percent of infected subjects had at least 16 FEV₁ measurements, and 85% of uninfected subjects had at least 7 FEV₁ measurements.

Antibody measurements and relationship to pulmonary function. Forty of the 53 subjects had measurable serum opsonic activity that mediated killing of MPA without regard to the antigenic specificity of the antibodies, indicating exposure and/or infection with P. aeruginosa. However, when the opsonic antibodies were classified according to their specificity for the P. aeruginosa MEP antigen (i.e., MSOA), the mean (± standard error [SE]) MSOA level was dramatically lower in infected subjects than in uninfected subjects that were homozygous for the F508 CFTR gene mutation (6.4% ± 2.5% [n = 18] versus 71.3% ± 6.8% [n = 9], respectively [P < 0.001]).

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View larger version (12K): [in this window] [in a new window]   FIG. 2.   Relationship between the percentage of serum opsonic antibodies to MPA that encompass the MSOA and the estimated probability of MPA infection.

Correlations of clinical parameters and pulmonary status determined by cross-sectional outcome analysis. The ages (years) at which the most recent percent predicted FEV₁ was obtained were comparable for MPA-infected and uninfected subjects (mean ± SD, 18.0 ± 8.0 versus 20.7 ± 6.8, respectively [P = 0.20, t test]), but the mean of the percent predicted FEV₁ was lower among MPA-infected subjects than uninfected subjects (mean ± SE, 61% ± 5% versus 87% ± 5%, respectively [P < 0.0001, t test]). When the most recent percent predicted FEV₁ for infected and uninfected subjects was regressed on age, analysis of covariance indicated that there was no difference in the estimated rate of decline of percent predicted FEV₁ between these groups (P = 0.82), but there was a difference () between groups in the constants (y intercepts,  = 21.81; SE = 6.13; P < 0.001, t test).

Correlations of clinical parameters and pulmonary status determined by longitudinal analysis. We therefore sought to develop a model to estimate percent predicted FEV₁ in CF patients by incorporating the factors shown to impact pulmonary function in the cross-sectional analysis, along with genotype and the immunologic status toward MPA that could also be expected to affect pulmonary function. In the statistical analysis with a mixed model, the main effects modeled were age, gender, infection status, number of F508 CFTR gene alleles, pancreatic supplement requirement, and overall titer of opsonic antibody to MPA. The terms in the final mixed model were overall opsonic antibody titer (likelihood ratio test, 6.64; P < 0.01), age by infection status (likelihood ratio test, 31.13; P < 0.0001), age by gender (likelihood ratio test, 8.38; P < 0.01), and number of F508 CFTR gene alleles by gender (likelihood ratio test, 27.17; P < 0.0001). From the mixed-model analysis, specific equations were generated for the estimation of percent predicted FEV₁ in CF patients with evidence of exposure to P. aeruginosa as determined by the presence of an opsonic antibody of any specificity, classifying the patients by MPA infection status, gender, and genotype (Table 2).

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Comparison of the slopes for the annual rate of decline in percent predicted FEV1 among CF patients homozygous for the F508 CFTR gene allele that had a titer of opsonic antibody to MPA of 5. Bars represent means of slope of annual rate of decline, and error bars indicate the standard errors. IF, infected female; IM, infected male; UF, uninfected female; UM, uninfected male; , difference between means; SE, standard error of difference between means.

	DISCUSSION

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This study demonstrates that pulmonary function in CF, as reflected by percent predicted FEV₁ measurements, can be modeled with an appropriate statistical analysis. Factors we identified as being statistically significant in the model included infection by MPA, the presence of a high MSOA, the subject's gender, and the subject's genotype. The first three factors have all been previously determined to be independent factors associated with pulmonary disease in CF (9, 30). However, the manner in which these independent factors interact to cause the loss in pulmonary function in CF, and the magnitude of the impact of these different factors, was revealed only by using the mixed-model approach. Importantly, our findings on the rate of decline in pulmonary function based on age and gender are very similar to those recently reported by Corey et al. (5), who also used a mixed-model analysis of multiple factors, but they did not include infection or immunologic status as main terms in their model. While the number of patients we studied (n = 53) was considerably fewer than the number (n = 363) studied by Corey et al. (5), the comparability of the results from the two mixed-model analyses indicates that the number of patients we studied, along with the large number of spirometric measurements (n = 1,680) used, was sufficient to model the effects of the CFTR gene genotype, gender, and immunologic status on rates of pulmonary decline in CF patients.

The mixed-model longitudinal analysis incorporates the effects of multiple characteristics and their combinations which influence the trajectory of change over time. The complexity of the factors that influence pulmonary function in CF, including interactions of infection and immune status and gender-specific effects of mutations in the CFTR gene, may have obscured previous attempts to use cross-sectional analysis to associate genotype, as an isolated factor, with pulmonary status in CF patients. Our results indicate that under these complex circumstances genotype by itself may be more of a determinant of susceptibility to infection, whereas infection has more of a direct effect on pulmonary status.

One example of this relationship was seen among nearly one-half of 26 older, uninfected subjects lacking any antibody to MPA. These subjects had a higher percent predicted FEV₁ than those with any type of detectable opsonic antibody. Of the 13 subjects lacking any opsonic activity to MPA, only one was infected and this was the only patient in this group that was homozygous for the F508 CFTR gene allele. Since all the serum samples were screened against a single MPA strain which produces both a MEP antigen and additional, conserved P. aeruginosa cell surface antigens (29, 30), it is possible that this single infected patient carried a rare strain lacking shared epitopes that are targets of an opsonic antibody. The other 12 patients lacking any opsonic antibody were compound heterozygotes, and 10 of these had at least one CFTR gene allele that was not among the 75% of the most common alleles we screened for. The lack of MPA infection in these 12 subjects may have resulted from CFTR gene alleles that cause altered chloride ion secretion properties, leading to elevated sweat chloride levels and a diagnosis of CF, that still confer natural resistance to MPA infection comparable to that of humans without CF, who rarely, if ever, have high levels of MSOA (30).

A potential mechanism to explain these findings has been proposed by Pier and colleagues, who demonstrated that the wild-type CFTR protein is a receptor for P. aeruginosa involved in airway epithelial cell internalization of this organism leading to bacterial clearance from the lung epithelium (27, 28). The absence of a CFTR in epithelial cells, as occurs with F508 CFTR gene alleles and other alleles, has been proposed to prevent normal clearance of P. aeruginosa from the airway epithelium. Among the CF patients with no opsonic activity to MPA and no infection, most of whom carried at least one rare (<0.2% frequency) CFTR gene allele, it is possible that the rare allele encoded a protein with the wild-type property needed for P. aeruginosa clearance from the airway epithelium but diminished chloride ion conductance. Because the CFTR gene genotype is associated with both pulmonary function and infection status, it may have its greatest effect on pulmonary function indirectly by modifying infection status. The CF subjects lacking both MPA infection and a common CFTR gene allele likely maintain resistance to MPA infection that is comparable to that in individuals with wild-type CFTR gene alleles.

Our study reinforces results from previous reports demonstrating that lung infection with MPA is a key factor accelerating the decline in pulmonary function of CF patients (9, 14, 16). Better lung function is found among CF patients lacking MPA infection, and the presence of nonmucoid P. aeruginosa in the absence of MPA does not compromise lung function any differently in these patients than in those without any detectable P. aeruginosa in their sputum cultures (9, 26). High MSOA levels, an apparent specific immune resistance mechanism that has previously been related to lack of MPA infection in older, nongenotyped CF patients (30), was also shown in this study to independently modify the probability of MPA infection within a group of CF patients with a homogeneous CFTR gene genotype (F508/F508). Thus, host immune status is a determinant of the development of pulmonary pathology due to MPA infection and is unlikely to be affected by the CFTR gene locus.

Interestingly, when we grouped all patients with an opsonic antibody to MPA together, regardless of the antigenic specificity, the presence of an opsonic antibody to MEP (i.e., a high level of MSOA) was not found to be a main effect in the final statistical model. The finding that the presence of an opsonic antibody of any specificity to MPA was a factor to include in the final model likely reflects the association between this measurement and infection; almost all CF patients infected with MPA have a measurable opsonic antibody to this organism. The lack of an effect of a high level of MSOA in the final model was attributed to the small number of subjects with this phenotype. However, the fact that a high MSOA level independently predicted MPA infection and the infection had a major impact upon pulmonary status indicates that MSOA likely impacts the equations derived in the mixed model by its ability to categorize CF patients as either infected or uninfected with MPA.

The study was limited by several factors. We excluded patients under 12 years of age because the age-related rate of acquisition of MPA infection in CF patients indicates that some subjects under age 12 are too young to have been sufficiently exposed to become infected (6). Since 87% of our study population 12 years old and older was infected with MPA, this likely indicates an age where exposure or other age-related, nonimmunologic, and nongenetic factors impacting infection will be minimal. Indeed, as long as infection status is a major component for predicting pulmonary function in CF, it is unlikely that any model could be derived that would be applicable to patients under the specific age where nearly 90% of patients that will become infected have become infected. In addition, the frequency of severe non-F508 CFTR gene mutations may vary geographically (7), indicating that differing results among genotype-phenotype studies (1, 3, 8, 14, 17, 22, 23, 34, 35) could be partially explained by an increased representation of unidentified mutations that more or less adversely impact the respiratory system. It is also clear that greater confidence in our results will follow once they are validated with a larger number of patients. However, the equations we derived for effects of gender and infection status on pulmonary status using 53 patients and 1,680 spirometric measurements were very similar to those of Corey et al. (5), who analyzed 363 patients and 9,362 pulmonary function tests. Overall, both studies showed the value of mixed-model analysis for predicting pulmonary outcomes in CF, and we have extended the work of Corey et al. (5) to include infection and immune status in the mixed-model equations for predicting pulmonary outcome.