INTRODUCTION

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Inherited deficiency of surfactant protein-B (SP-B) is a fatal autosomal recessive disorder of lung cell metabolism that is characterized by rapidly progressive respiratory failure immediately after birth (1, 2). Lung transplantation is the only therapeutic intervention currently available (3). The most common null mutation observed in affected infants results from a net 2 base-pair insertion in codon 121 of the SP-B gene (121ins2). This mutation creates an Sful restriction enzyme site that can be used for screening amplified genomic DNA (4). Analysis of pulmonary surfactant composition and function suggests that absence of SP-B synthesis also disrupts other aspects of pulmonary surfactant metabolism (5). In contrast to the progressive, lethal respiratory failure of infants homozygous for 121ins2 SP-B deficiency, the obligate heterozygous parents have not reported respiratory illness from birth.

Genetically engineered murine lineages homozygous for a disruption of the SP-B gene exhibit a phenotype that is similar to infants homozygous for 121ins2: their lungs are airless and they die of respiratory failure within minutes of birth (9). Although heterozygous SP-B-deficient mice are phenotypically normal, adult and neonatal mice exhibit diminished lung compliance, with the adult mice also exhibiting mild air trapping with ex vivo measurements of lung function (10, 11).

Lung function of heterozygous SP-B humans has not been reported. To evaluate whether subclinical abnormalities in lung function may exist in persons related to these infants, we performed pulmonary function measurements in the family members of 121ins2 SP-B-deficient infants who were referred to the St. Louis Children's Hospital/Washington University Pediatric Lung Transplant Program.

	METHODS

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Subjects

Eleven Caucasian family members 7 to 44 yr of age of four homozygous 121ins2 SP-B-deficient infants and one heterozygous 121ins2 infant were screened for the 121ins2 mutation using restriction fragment analysis previously described (3). Nine subjects were heterozygous for the 121ins2 mutation (121/+), two subjects were homozygous normal (+/+). All were in good health and were free from respiratory symptoms at the time of the study. None had a history of asthma, respiratory distress as a newborn, emphysema, or pneumonia (information obtained by directed interview with the subjects and/or their parents). Two heterozygous family members were active cigarette smokers (20 and 40 pack-year histories) and one heterozygous family member had a remote history of smoking (0.1 pack-year) (Table 1).

Informed consent for evaluation was obtained from each parent for themselves and their minor children, when applicable. The study was approved by the Washington University Human Studies Committee.

Pulmonary Function Testing

Pulmonary function tests were performed with a Medgraphics System 1085 (Medical Graphics Corp., St. Paul, MN) according to American Thoracic Society standards (12). Spirometry was recorded before and after aerosolized albuterol administration; the prebronchodilator values were used for the data analysis. For spirometry, we used the predicted normal values of Knudson and colleagues (17). Lung volumes were measured by plethysmography immediately after bronchodilator inhalation, before maximum bronchodilator effect would be expected. Predicted normal values were those of the Intermountain Thoracic Society (18). Normal values for measurement of postbronchodilator airway resistance (Raw) were those of Gelb and colleagues (19). The diffusing capacity for carbon monoxide (DL_CO) was measured by the single-breath technique, and predicted normal values were those of Ayers and colleagues (20).

Maximal transpulmonary pressure (Ptp_max) and pressure-volume curves were generated using standard static esophageal balloon intermittent occlusion techniques (21). We calibrated equipment before all tests and confirmed balloon integrity. To obtain a constant volume history, three sets of full inspiration from FRC followed by slow exhalation were performed prior to the test set of breathing maneuvers. Patients performed a minimum of three maneuvers to obtain reproducible results.

Subjects were exercised on a stationary bicycle pedaling to 85% of their maximum predicted heart rate (220-age). Oxygenation was assessed by continuous pulse oximetry (Nellcor, Inc., Pleasanton, CA).

Data Analysis

Statistical comparisons were performed using Statview (Version 4.5; Abacus Concepts, Inc., Berkeley, CA) for Macintosh computers. The data were not normally distributed, and thus nonparametric statistical analyses were performed. Two-tailed one sample sign tests compared the subjects' percent predicted values with 100% of the predicted normal value. Wilcoxon's sign rank analyses compared spirometry measurements before and after bronchodilator administration (22).

	RESULTS

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For both the heterozygous and normal subjects, FVC and FEV₁/FVC did not change significantly with bronchodilator administration (Table 2). Although the increase in FEV₁ for the heterozygotes after bronchodilator administration was statistically significant, the magnitude of the change (median, 6%; range, 1 to 12%) was not clinically significant (23). As a group, FEV₁, FVC, RV, TLC, DL_CO, and Raw were all normal (Figure 1 and Table 3). For the 121/+ group, mean TLC (103 ± 3% of predicted) was statistically greater than 100% but of no clinical significance. Two subjects had increases in RV suggestive of air-trapping: one heterozygous and one normal subject, neither of whom smoked. These subjects, however, were asymptomatic. The 121/+ subject had normal TLC, RV/TLC, FEV₁, and flow-volume loops before and after bronchodilator administration, thereby suggesting an absence of airflow obstruction. Most of the normal subject's pulmonary function values were above the "normal" range, but he, too, had normal FEV₁ and flow-volume loops. Oxygen saturation remained normal before and after exercise for all subjects tested. Lung elastic recoil pressure (Ptp_max), the coefficient of retraction, and expiratory static compliance were normal in the seven subjects studied (24) (Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Individual values for percent predicted values of selected lung function studies from heterozygous (closed symbols) and normal (open symbols) family members of SP-B-deficient infants. The dashed lines indicate 100% and the arbitrary upper (120%) and lower (80%) bounds of the "normal" percent predicted range. Mean ± SD are represented by the error bars. Individual symbols are as in Table 1. Values for FEV1 were obtained before bronchodilator administration.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Individual values for maximum transpulmonary pressure and coefficient of retraction for the 121/+ and +/+ family members. Symbols are the same as in Figure 1 and Table 1. Values are normal (24).

	DISCUSSION

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Heterozygous family members of 121ins2 SP-B-deficient infants are free from pulmonary symptomatology by history and have normal lung function. Adult heterozygous SP-B-deficient mice are also phenotypically normal, but they exhibit mild air-trapping and diminished lung compliance (10, 11). One of the nine heterozygous 121ins2 subjects had elevated RV, which implies a similarity to the heterozygous mice. However, normal spirometry, TLC, and RV/TLC in this subject suggest an absence of airflow obstruction. In contrast to the mice, all subjects had normal lung compliance.

Methodologic and biologic issues may explain the differences between our pulmonary function results in humans and those in mice. First, the in vivo measurements of larger human lungs may not be as sensitive to subtle changes in lung volume and compliance as the ex vivo measurements in mice. Second, it is possible that the targeted disruption of the murine SP-B gene results in a more profound disturbance of surfactant metabolism than does the human 121ins2 mutation. Alternatively, the diverse genetic backgrounds of the 121ins2 families may provide adaptive responses not present in the more homogenous genetic background of the murine lineages.

Although the observations in mice suggest the possibility of susceptibility to pulmonary dysfunction, none of our heterozygous subjects had any history of significant respiratory illness. Our population, though, may represent a selected subset of subjects who have been healthy. On the other hand, a subset of infants who are heterozygous for mutations of the SP-B gene may be susceptible to transient pulmonary dysfunction in the neonatal period and would not have survived in the preneonatal intensive care era (25). Prospective studies to identify 121ins2 heterozygotes and their phenotype are in progress. Longitudinal studies of these heterozygous persons will be important to determine the impact of advancing age and environmental exposures on lung function.