Main

CF is the most common lethal, autosomal recessive inherited disorder in the Caucasian population, caused by mutations within the CFTR gene that consists of 27 exons spanning 250 kb of genomic DNA on chromosome 7 (1–3). The CFTR protein consists of 1480 amino acids arranged in repeated motif structure and of 12 membrane-spanning regions (TM1 to TM12), two ATP-binding domains (NBF1 and NBF2), and a highly polarized domain named the R domain, which is thought to have a regulatory function (2). The CFTR protein is allocated in the apical membrane of normal epithelial cells and it forms a chloride channel regulated by cAMP. So far, more than 750 different CFTR mutations have been identified (CF Genetic Analysis Consortium, personal communication), and the information on mutations causing disease may provide an important insight into the CFTR function. Defects in the CFTR gene cause abnormal chloride concentrations across the apical membrane of epithelial cells in the airways, pancreas, intestine, sweat glands, and vas deferens, resulting in progressive lung disease, pancreatic dysfunction, elevated sweat electrolytes, and male infertility (4–7). CF is characterized by wide variations in clinical expression, and patients are diagnosed with various modes of presentation from birth to adulthood and with considerable variability in the severity and rate of disease progression.

The cloning of the CFTR gene and the identification of its many mutations has promoted extensive research into the association between genotype and phenotype. Several studies showed that there are mutations, like the ΔF508 (the most common mutation worldwide), which are associated with a severe disease presentation (8–10), and there are mutations associated with a milder phenotype (11,12). Regarding genotype-phenotype association, there are variables influencing the clinical conditions and hence the phenotypic expression which are crucial. Treatment schedules undoubtedly influence the clinical course, at least in its severity. Although progressive lung disease is the most common cause of mortality in CF, there is great variability in the age of onset and the severity of lung disease in different age groups (13). Therefore, the most suitable moment to study genotype-phenotype association, independent from therapeutic influences, is the time at diagnosis. Although infants with CF have no detectable lung disease at birth (14–16), a significant proportion presents with respiratory infections already within the first year of life. Thus, it is likely that disease-associated intermittent inflammation on the respiratory surface occurs early in life. This question is very important because the lung can usually maintain its normal architecture after acute neutrophil-dominated inflammation (such as acute bronchitis or pneumonia), but chronic neutrophil inflammation is typically associated with chronic derangement of the fragile respiratory tissues (17–20). The question is, which functional characteristics of lung involvement can already be detected at this stage of disease to define adequate treatment. The purpose of our study was to characterize clinical involvement in terms of clinical findings (weight gain, relative underweight, chest x-ray score), laboratory data (sweat electrolytes, stool fat excretion), and lung function (infant wholebody plethysmography) in relation to the three most common mutations of our population (ΔF508, 3905insT, R553X) in infants with CF at time of diagnosis before the initiation of any treatment.

SUBJECTS AND METHODS

Subjects. The subjects were 60 patients (33 females, 27 males) diagnosed early as having CF at a mean age of 7.2 months (range: 0.8-23.8 months). The diagnosis of CF was made using standard criteria, including two sweat chloride and sweat sodium tests (> 60 mmol/L). Three infants were diagnosed prenatally because of a known family history of cystic fibrosis and the remainder because of their clinical presentation. Eleven infants presented with meconium ileus defined as a failure to pass meconium within 24 h or more after birth and requiring surgical intervention. In addition to the sweat test, the diagnostic procedure included a clinical evaluation for age, length, weight, relative underweight, weight gain, chest roentgenograms, evaluated as described by Chrispin and Norman (21), lung function test by infant whole-body plethysmography (22–24), throat swab cultures (25), and stool fat excretion (26). The fecal fat balance was evaluated by collecting stool samples pooled over a 3-d period and analyzing stool fat content according to the method of van der Kamer et al. (27). At the time of diagnosis none of the subjects received inhalation treatment, chest physiotherapy, or dietary supplements. This study was approved by the human subjects review committee at the hospital.

Measurements of relative underweight and weight gain. Weight and length were measured by experienced nurses, using a standard measuring board or a Harpenden stadiometer, in the morning before feeding in accordance with recommended procedures (28). The relative underweight was calculated in relation to age and body length (29), expressed as SD-S relative to a healthy reference population of Switzerland (28). The SD-S is equal to a z score (30), indicating the number of standard deviations by which an infant's length and weight deviate from the reference population. The reference data (28) are very similar to those given by the US National Health and Nutrition Examination surveys (4,30,31). Based on these data, weight gain was computed for 1-, 2-, and 3-month intervals (31), as well as weight increments since birth. The latter were taken as values predicted to express weight gain from birth until the time of diagnosis in percentages.

Measurements of infant lung function. TGV, as a measure of development of static lung volumes, and sGaw, as a measure of airway mechanics, were measured in an infant whole-body plethysmograph (Jaeger Würzburg, Germany) by using standard techniques (22,24,32–34). All measurements were done 15 min after feeding and after the infants had been sedated with chloral hydrate (80-100 mg/kg bodyweight). During the plethysmographic measurements, pulse oximeter monitoring of heart rate and oxygen saturation (Biox III, Omeda) was performed. The plethysmographic technique is based on the mathematical application of Boyle's law. Technical details are given elsewhere (24,33). TGV was taken from at least five separate periods of airway occlusion, measured at the end of a normal expiration (end-expiratory resting level) after the shutter was closed to occlude the airway during two to three respiratory efforts. For measurement of airway mechanics, the baby breathed from a circuit containing air under BTPS conditions (23). A closed body-box pressure/flow loop and vertical angle of the zero-flow points (end-inspiration and end-expiration, respectively) on the box-pressure/volume loop were taken as evidence of satisfactory BTPS conditions (32). sGaw was calculated from the angle drawn through the 50% maximum inspiratory and expiratory flow points of the pressure-flow loop (35,36) as a mean of at least 10 breaths technically satisfactory recorded. All measurements (TGV, sGaw, respectively) were corrected for the dead space of the plethysmograph (30 mL) and the instrumental resistance (0.18 kPa·L-1·s), respectively. All patients completed the test procedure within 15 min without waking.

To present individual values of TGV and sGaw, numerically independent from sex, age, and height, lung function data were expressed as SD-S, based on regression equations for both, the TGV and the sGaw were taken from a total of 111 healthy infants (37). The SD-S is the SD score equal to the number of SD, a measured value of the TGV, sGaw, respectively, is away from the regression line. Therefore, the SD-S is the number of standard errors of prediction by which the TGV or sGaw values differed from the prediction (37,38), taking into account variability in the population.

Genotyping. Genomic DNA was extracted from peripheral-blood lymphocytes anticoagulated in EDTA by standard methods as described previously (39). CF mutations were detected using the SSCP technique first described by Orita et al. (40) and modified for gel composition, electrophoresis conditions, and staining procedures by Liechti-Gallati et al. (41). Identification of the mutations was followed by fluorescence sequencing of double-stranded polymerase chain reaction products using a 373A system (ABI, Foster City, CA). Genotyping of the 60 patients detected the ΔF508 deletion in 80% of the 120 CF chromosomes. Thirty-one patients (51.7%) were found to be homozygous for ΔF508, 20 patients(33.3%) were compound heterozygous for ΔF508 and G85E, R347P, 1199delG, R553X, G542X, 1717-1G→A, R560S, 3905insT, and N1303K, respectively, and 9 patients (15.0%) did not bear the ΔF508 mutation on either chromosome.

Statistical evaluation. Analysis of variance was used to assess the significance of differences among group means, with Bonferroni's adjustment of the p value for t tests between group means using a nominal significance level of p = 0.05. χ2 analysis was used to compare differences among group proportions.

RESULTS

Clinical aspects. The biometric data and clinical findings of the 60 infants with CF, subdivided into clinical groups according to initial clinical findings at the time of diagnosis, are presented in Table 1. The mean age at diagnosis was 7.2 months (range: 0.8-24.2 months). Including the 3 cases with positive family history, 32 of the 60 cases (53.3%) were diagnosed before the age of 6 months, 47 (78.3%) before the age of 12 months, but in the remaining cases diagnosis was delayed up to 24.4 months. Apart from 3 patients (5.0%) with positive family history, 11 (18.3%) presented with meconium ileus, 18 (30.0%) had gastrointestinal symptoms (diarrhea, failure to thrive) without respiratory symptoms, 11 (18.3%) had respiratory tract symptoms (cough, tachypnea, or wheezing), and 17 (28.4%) presented with both respiratory and gastrointestinal symptoms. Infants presenting with gastrointestinal symptoms were diagnosed significantly earlier (4.9 ± 4.0 months) than those presenting with pulmonary symptoms (11.2 ± 6.4 months; p = 0.003). Weight gain, expressed as percent predicted, was best in the three patients with initial pulmonary symptoms (77.1 ± 21.6% pred.) and worst in the group presenting with gastrointestinal symptoms (55.5 ± 19.7% pred.; p = 0.01). Other clinical parameters such as relative underweight, x-ray score, clinical score, and stool fat excretion were not significantly different between the symptom groups. However, the symptom groups are significantly associated with sGaw (p < 0.001). A significantly higher degree of bronchial obstruction was found in the clinical group initially diagnosed because of pulmonary symptoms (sGaw -7.6 ± 2.6 SD-S) in comparison with infants presenting gastrointestinal symptoms (sGaw -3.6 ± 2.6; p < 0.005) or infants showing both pulmonary and gastrointestinal symptoms at the time of diagnosis (sGaw -4.6 ± 3.7; p = 0.01).

Table 1 Biometric data and clinical finding collected at the time of diagnosis within different clinical and symptomatic groups
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Bacteriology. Throat swab cultures (not available in 13.3%) were negative in 27 cases (45.0%). Five percent of the patients swab cultures showed growth for Haemophilus influenzae, 25% were positive for Pseudomonas aeruginosa alone, and in 11.7% a combination of Staphylococci aeruginosa, H. influenzae, and Klebsiella was found at the time of diagnosis. Whereas the symptomatology at diagnosis (respiratory or gastrointestinal symptoms, or both) was not influenced by the genotype, positive throat swab cultures were found in the 3905insT group (7 of 9) at a significantly higher frequency than in the R553X group (2 of 8; p < 0.05).

Genotype-phenotype association. The biometric data and clinical findings of the 60 infants with CF, subdivided into groups of genotypes, are given in Table 2. No significant differences were found for the age at diagnosis, length, relative underweight, clinical score, and stool fat excretion between the genetic groups. However, weight gain was significantly better for the R553X mutation (85.9 ± 10.8% pred.) compared with the 3905insT mutation (57.8 ± 19.0% pred.; p = 0.005) as well as the homozygotes for ΔF508 (62.5 ± 20.6% pred.; p = 0.005). The R553X mutation was also expressed by lowest sweat test sodium (69.9 ± 10.2 mmol/L) when compared with the 3905insT mutation (85.7 ± 16.1 mmol/L; p = 0.015) and the homozygotes for ΔF508 (84.6 ± 12.6; p = 0.012). Concerning sweat chloride concentrations and, in accordance with previously published data (42), the miscellaneous group presented with lowest mean values (101.2 ± 6.1 mmol/L), significantly different to the frameshift 3905insT mutation (117.7 ± 15.3; p = 0.022). Included in the miscellaneous group were two patients with compound heterozygotes splice site mutation 1717-1G→A showing lowest (87.3 ± 26.5 mmol/L) sweat chloride concentrations (42).

Table 2 Biometric data and clinical finding collected at the time of diagnosis within different ethnic groups
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The lung function data at the time of diagnosis, subdivided into four functional groups (33,34), are given in Table 3, and the interrelation between the degree of pulmonary hyperinflation (TGV) and the degree of bronchial obstruction (sGaw) is shown in Figure 1. Only five infants (8.3%) presented as functionally normal. Three of the five were ΔF508 homozygotes, one was ΔF508/R553X compound heterozygote, and one presented with genotype ΔF508/1717-1G→A. In 41 infants with CF (68.3%), pulmonary hyperinflation was found, and especially patients with the 3905insT mutation (8 of 9) were found to be hyperinflated. The degree of pulmonary hyperinflation (Fig. 1) differentiates between the frameshift mutation 3905insT with a mean TGV of 8.4 ± 4.9 and the nonsense mutation R553X with a mean TGV of 1.8 ± 4.1 SD-S (p < 0.005). Figure 2 shows that pulmonary hyperinflation occurred more severely in the first months of life (TGV: upper panel), and that 3905insT is expressed early in life with highest TGV. It appears therefore, that differences regarding lung function within the genetic groups are closely related to pulmonary hyperinflation. There were no differences in airway mechanics between the groups measured and estimated by sGaw.

Table 3 Lung function data measured at the time of diagnosis subdivided into four functional groups
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Figure 1
figure 1

Lung function data measured at the time of diagnosis within four genetic groups (ΔF508 homozygotes, R553X compound heterozygotes, 3905insT compound heterozygotes, and miscellaneous) given as box plot chart (top, bottom, and line through the middle of the box corresponding to 75th, 25th, and 50th percentile, respectively; whiskers extending to the 10th and 90th percentile, respectively; and the symbol representing the arithmetic mean).

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Figure 2
figure 2

Distribution of TGV as a measure of pulmonary hyperinflation and sGaw as a measure of airway mechanics in relation with age at the time of diagnosis within four genetic groups (ΔF508 homozygotes, R553X compound heterozygotes, 3905insT compound heterozygotes, and miscellaneous).

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DISCUSSION

The identification of the cystic fibrosis gene has provided a focus for studies that attempt to elucidate the basic defect and pathophysiology of the disease. This study is our first major approach toward the understanding of clinical findings and lung function data collected from cystic fibrosis infants in correlation with genotypes, mainly with ΔF508 homozygotes and two frequent compound heterozygotes, ΔF508/R553X and ΔF508/3905insT, respectively. We found that the well-known, extremely variable clinical expression of the disease is partly reflected by different genotypes; the weight gain from birth to the time of diagnosis and the degree of pulmonary hyperinflation at the time of diagnosis are differently influenced by the R553X and 3905insT mutation. Therefore, clinical and functional characteristics can be attributed at least in part to specific genotypes at the CFTR locus. In contrast with Beardsmore (40) but in agreement with Tepper et al. (43,44), this study confirms that only a minor part of infants with cystic fibrosis (8.3%) diagnosed early in life, have normal lung function. Furthermore, the study reveals that pulmonary symptoms are positively correlated only with the degree of airway obstruction occurring later in life than pulmonary hyperinflation. However, the pulmonary hyperinflation is the major functional characteristics that seems to be recognized clinically, not accurately. Finally, pulmonary hyperinflation is significantly associated with the frameshift mutation 3905insT concerning frequency, early onset, and degree of severity.

These findings are in agreement with a study in which we showed in a collective of 73 children and young adults with CF, 3.4 to 28.0 y of age, that onset of P. aeruginosa infection of all genetic subgroups was significantly later in ΔF508/R553X compound heterozygotes compared with ΔF508 homozygotes (13). Moreover, the 3905insT mutation presented in this study with an extremely early onset, high chest x-ray scores, and greatest relative underweight resulting in a more severe clinical form than all the other mutations. Therefore, the hypothesis that the presence of an altered CFTR protein may be more deleterious than its total absence (45,46) and the speculation that the combination of one ΔF508 deletion and one 3905insT mutation will probably result in two mutant CFTR proteins trapped in the endoplasmatic reticulum and therefore acting at incorrect cellular locations (46) is furthermore supported. The nonsense mutation R553X seems to decrease the influence of the ΔF508 deletion in ΔF508/R553X heterozygotes, causing a milder course and slowed progression confirming our previous findings (13). Again, the frameshift 3905insT compound heterozygotes tend to have more severe and earlier disease involvement, as reflected by a lower weight gain per age and more pronounced pulmonary hyperinflation. In contrast, the patients with R553X mutation were generally recognized to have a better W% pred. and a later and milder onset of abnormalities in lung function. It is still an open question whether the symptomatology is clearly linked with the functional disorders on one hand and/or the genetic type on the other hand. Nevertheless, infants presenting with gastrointestinal symptoms were diagnosed earlier than those presenting pulmonary symptoms. Moreover, infants presenting with a pulmonary hyperinflation without bronchial obstruction are mainly recognized with gastrointestinal symptoms, whereas only infants presenting a certain stage of bronchial obstruction showed respiratory symptoms. The latter finding may be linked to positive throat swab cultures, which were found in a significantly higher frequency in the 3905insT than in the R553X group. Concerning ΔF508 homozygotes, the wide variations in clinical and functional findings seem to indicate that the severity and progression of the disease may also be determined by other factors within and/or outside the CFTR gene (17,47).

Limits of the method.. The finding of only a few significant differences between the genetically defined subgroups may be due to the yet small number of patients with identical genotypes, although this study collects the highest number of infants with CF published so far. To prove any further correlations between genotype and phenotype, studies of larger patient populations are required-a need that is hindered by low frequencies of the remaining mutations and their markedly different geographic and/or ethnic distributions. Because in this kind of study recruitment of patients is limited to the time of diagnosis, a multicenter approach is needed. Moreover, the large size of the CFTR gene (6100 bp of coding region, 27 exons) as well as the multiplicity of mutations complicate the design of an adequate technique for mutation detection. Therefore, we have established appropriate conditions providing a rapid and reliable method for an efficient mutation screening of all the 27 exons (including exon/intron boundaries) of the CFTR gene (41). In addition, the technical approach of the plethysmographic measurements of thoracic gas volume and specific airways conductance has to be considered. Standard techniques have been used taking care of several technical prerequisites. These are an accurate transmission of pressure between alveoli and airway opening, isothermal conditions within the lung, absence of airflow within the lung during airway occlusion, and negligible effect of abdominal gas. Infants breathed through a face mask which may have provided a degree of support to the face, not available to older children using the mouthpieces, none of the subjects was observed to puff out the cheeks during airway occlusion and therefore loss of pressure transmission in the upper airway was discounted in both groups. Chloral hydrate has been shown not to influence thoracic gas volume measurements in infants (48). Measurement of airways conductance was made during quiet breathing using a heated, humidified rebreathing system. The analysis was performed by computer. Expressing both values (TGV and sGaw) as SD-S, features a mode to present data, sex- and size-corrected, as weighted variables. All infants were, concerning age, height, and weight range, within the limits given by the population which serves for the computation of values predicted. Thus, no data were obtained by extrapolation. However, even under these circumstances, in which experimental data are compared with data from a reference population, an individual measurement may seem more extreme when assessed on an SD-S than one based on percent predicted. However, because there is not a linear standard error range around the prediction line, SD-S seems to be more accurate, especially when subjects with different age ranges are compared.

Notwithstanding the possibility that additional factors (notably recurrent viral infections and P. aeruginosa colonization) may contribute to the severity of the disease already established at the time of diagnosis, our data suggest that there is a correlation between genotype and phenotype in cystic fibrosis. The most striking example of this is the differentiation between the frameshift mutation 3905insT and the nonsense mutation R553X by the degree of pulmonary hyperinflation. The assessment of lung function and especially the state of pulmonary hyperinflation within the different genetic groups could serve as prognostic value for the course of the disease at the time of diagnosis. Such an evaluation however, should include measurements of estimates of end-expiratory resting level (either plethysmographically determined or by a gas dilution technique) and estimates of airway mechanics. As shown in asthmatic children this kind of functional abnormality is seldom associated with pulmonary symptoms unless it is linked with a certain degree of bronchial obstruction (25). The variability of pulmonary function in all the study groups indicates that additional factors affect the severity or progression of lung disease. Therefore, information on morbidity and prognosis should be used with caution when CF families are counseled. However, to improve the reliability of prognosis, infants with CF should be studied in clearly defined sequences during the first 2 y of life and thereafter in preschool age.