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Published ahead of print on August 28, 2003, doi:10.1164/rccm.200304-524OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 1216-1222, (2003)
© 2003 American Thoracic Society

Surfactant Protein A and B Genetic Variants in Respiratory Distress Syndrome in Singletons and Twins

Riitta Marttila, Ritva Haataja, Susan Guttentag and Mikko Hallman

Seinäjoki Central Hospital, Seinäjoki, and Department of Pediatrics and Biocenter Oulu, University of Oulu, Oulu, Finland; and Division of Neonatology, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Correspondence and requests for reprints should be addressed to Mikko Hallman, M.D., Ph.D., Department of Pediatrics and Biocenter Oulu, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland. E-mail: mhallman{at}cc.oulu.fi


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interactive genetic and environmental factors may influence the differentiation of surfactant and the risk of respiratory distress syndrome (RDS). DNA samples from 441 premature singleton infants and 480 twin or multiple infants were genotyped for surfactant-specific protein (SP)-A1, SP-A2, and SP-B exon 4 polymorphisms and intron 4 size variants in a homogeneous white population. Distributions of the SP-A and SP-B gene variants between RDS and no-RDS infants were determined alone and in combination. SP-A1 allele 6A2 (p = 0.009) and the homozygous genotype 6A2/6A2 (p = 0.003) were overrepresented in RDS of singletons when the SP-B exon 4 genotype was Thr/Thr, and underrepresented in RDS of multiples when the SP-B genotype was Ile/Thr (p = 0.012 for 6A2 and p = 0.03 for 6A2/6A2) or Thr/Thr (p = 0.12 for 6A2 and p = 0.018 for 6A2/6A2, respectively). The SP-A 6A2 allele in the SP-B Thr131 background predisposed the smallest singleton infants to RDS, whereas near-term multiples were protected from RDS. There was a continuous association between fetal mass and risk of RDS, defined by the SP-A and SP-B variants. Labeled lung explants with the Thr/Thr genotype showed proSP-B amino-terminal glycosylation, which was absent in Ile/Ile samples. Genetic and environmental variation may influence intracellular processing of surfactant complex and the susceptibility to RDS.

Key Words: fetal mass • interaction of genes and environment • surfactant protein polymorphism

Respiratory distress syndrome (RDS) in premature infant remains the major life-threatening neonatal disease affecting 1 to 2% of newborn infants despite antenatal steroid and surfactant treatments (1, 2). RDS is principally associated with developmental deficiency in synthesis, intracellular processing, and secretion of pulmonary surfactant, required to reduce surface tension at the air–liquid interface of the distal conducting airways and alveoli. Pulmonary surfactant consists of lipids and surfactant-specific proteins (SPs); these components each exhibit developmental regulation. Some SP-B and -C mRNAs are expressed early in the second trimester, and SP-A early in the third trimester, as studied in explants from the human lung (3). It is therefore understandable that factors influencing the expression of any or all of these components, particularly the degree of prematurity but also sex, ethnicity, inflammation, and genetic factors, influence the disease risk (4–9).

The polymorphisms of the SP-A1, SP-A2, and SP-B genes have been studied for associations with RDS (9–14). On the basis of our earlier observations in different sets of Finnish neonates, the allelic associations between SP-A or SP-B genes and RDS are not similar throughout the population. In a population of 684 premature infants (of whom 466 were singletons and 218 were born from multiple pregnancy), the SP-A1 allele 6A2 and the SP-A2 allele 1A0 were found to be SP-B Ile131Thr genotype-dependent high-risk alleles for RDS in very premature infants with a gestational age of less than 32 weeks (11). On the other hand, in a separate study population consisting only of premature twins of Finnish origin (200 twins from 100 pairs), the allelic associations were different (12). Whereas the SP-B Ile131Thr polymorphism was not directly associated with the risk of RDS in a population consisting predominantly of singletons (68%) (11), the SP-B Ile131Thr allele was directly associated with RDS in the first-born, but not in the second-born, twins (12). The sample size did not warrant an evaluation of whether the SP-A and SP-B polymorphisms associate interactively with RDS in the twin study.

The conflicting observations in two separate subpopulations of the same ethnicity possibly arise from stratification and confounding by multiple birth. To date, it has remained unclear whether the incidence of RDS differs between birth weight- and gestational age-matched singletons and twins (15, 16), but it is lower in the first-born than the second-born twin (17). These observations have raised an interesting aspect regarding the role of factors involved in intrauterine environment in the complexity of genetic and environmental contribution to the risk of RDS. Here we evaluate for the first time the interaction of the SP-A and SP-B genetic variants as a genetic determinant of RDS separately in pure subpopulations of prematurely born (gestational age less than 37 weeks) singletons versus multiples. The SP-A1 and SP-A2 single-nucleotide polymorphisms, as well as the SP-B exon 4 Ile131Thr and intron 4 size variations, were studied for association with RDS, prematurity, and fetal size in a large ethnically homogeneous population.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Sample Collection and Study Population
The study population consisted of premature (less than 37 weeks of gestation) Finnish infants born from singleton and multiple gestations during the years 1987–2000. Altogether 636 blood samples were collected prospectively during 1997–2000 in the catchment areas of the Oulu and Tampere University Hospitals and Seinäjoki Central Hospital. In addition, 326 retrospectively recruited premature singletons and multiples born during 1987–1996, from whom buccal smear samples or a blood spot dried on a filter paper were available, were included in the study. The study protocol was approved by the ethics committees of the participating centers. The Finnish Ministry for Social Affairs and Health gave permission to conduct the study nationwide. Informed consent was obtained from the parents. Of the 480 multiples analyzed, 180 retrospectively recruited infants were part of the material reported in the study of the SP-B Ile131Thr polymorphism in twins (12).

Clinical data concerning gestational age, sex, birth order, and maternal and neonatal clinical histories were obtained from the medical records. The diagnosis of RDS was made on the basis of published criteria, as described (10). Surfactant was given in respiratory distress first, after the first chest radiograph established diffuse atelectasis and lung edema. None received exogenous surfactant at birth during the study period. The data describing the respiratory course were verified from documents in the medical records. Altogether 473 specimens from premature singleton infants and 489 from multiples (375 from twins, 98 from triplets, and 16 from quadruplets) were available for analysis.

Genotyping of SP-A and SP-B Polymorphisms and Definition of Twin Zygosity
Genomic DNA was extracted from fresh-frozen EDTA-anticoagulated whole blood specimens by conventional methods (10). When whole blood samples were not available, genotypes were determined using duplicate buccal smear swabs or blood spots dried on filter paper (10). Genotyping of the SP-A genes, SP-B exon 4 Ile131Thr, and intron 4 size variants was performed as described (11). Altogether 441 singletons and 480 multiples (184 sets of twins, 32 triplets, and 4 quadruples) were successfully genotyped for all the polymorphisms.

Twin zygosity was determined by using four highly informative tetranucleotide repeat markers, D7S3039, D15S1365, D17S976, and D21S11, in addition to genotyping the SP-A and SP-B polymorphisms.

Metabolic Labeling, Immunoprecipitation, Endoglycosidase Digestion, and Western Immunoblotting
Human fetal lung explants were cultured for 5 days in 10 nM dexamethasone, 0.1 mM 8-bromo-cyclic AMP, and 0.1 mM isobutylmethylxanthine to promote Type II cell differentiation and SP-B expression. After genotyping, homozygous Thr/Thr and Ile/Ile explants were starved in Met/Cys-free Dulbecco's modified Eagle's medium and then labeled for 30 minutes in Met/Cys-free Dulbecco's modified Eagle's medium supplemented with [35S]Met/Cys Express protein-labeling mix (200 µCi/ml; PerkinElmer Life Sciences, Boston, MA). Samples were collected at the end of the pulse and processed for immunoprecipitation as previously described (18). The proteins were immunoprecipitated with a rabbit polyclonal antibody to human SP-B and the products were separated on a 12% NuPAGE Bis-Tris gel with 2-(N-morpholino)ethanesulfonic acid buffer system (Invitrogen, Carlsbad, CA). SP-B fragments were visualized by Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For endoglycosidase digestion to remove Asn-linked oligosaccharides, 20-µg aliquots from unlabeled lung homogenates were incubated with 100 U of peptide N-glycosidase F (PNGase F; New England BioLabs, Beverly, MA) for 3 hours at 37°C. The digested samples were reduced and subjected to SDS-PAGE as described above, and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA). ProSP-B and the NH2-terminal cleavage fragment were detected with purified rabbit antiserum NFProx against SP-B Ser145-Leu160 (18) and enhanced chemiluminescence detection (ECL Plus Western blotting detection system; Amersham Biosciences, Piscataway, NJ).

Statistical Analysis
Comparisons of allele frequencies were performed by two-tailed {chi}2 tests. Allele distributions were compared by 2 x k tables and frequencies of individual alleles by 2 x 2 tables. Bonferroni corrections were made for multiple comparisons by multiplying the statistically significant p values by the number of pairwise comparisons being made. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by the Woolf logit method to assess the risk of RDS conferred by a particular allele and genotype. The Fisher exact test was used when the expected value was less than 5. The observed genotype frequencies were compared with the expected Hardy–Weinberg distributions by {chi}2 analyses. The homogeneity of the odds ratio (HOR) test was used to evaluate whether twinning or multiple birth was an effect modifier, and the data were thus analyzed separately for singletons and multiples (19). Multivariate logistic regression analyses were used to determine whether the homozygosity or heterozygosity of particular alleles/genotypes of SP-A explained the risk of RDS in the different SP-B Ile131Thr subgroups (i.e., Ile/Ile, Ile/Thr, and Thr/Thr). RDS was the dependent variable and the SP-B Ile131Thr genotype was an independent variable. Regression analyses were performed separately for the following two interdependent variables: prematurity (under or over 32 weeks) and antenatal steroid treatment (yes/no). Other confounders for RDS, that is, sex and birth order in multiple birth, were included in the analyses.

Statistical analyses were performed with SPSS for Windows (version 9.0; SPSS, Chicago, IL) (basic statistical calculations, logistic regression analysis), Arcus Quickstat (StatsDirect Software, Cheshire, UK) ({chi}2 tests and Fisher exact test), and StatXact-3 for Windows (Cytel Software, Cambridge, MA) (HOR test).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
According to the Finnish Birth Register the multiples represented 20–25% of all infants born premature. To facilitate the comparison between singletons and multiples, 276 of the multiples were recruited retrospectively. The profile of genetic susceptibility to RDS was similar in both the retrospectively and the prospectively recruited multiples, consistent with homogeneity of the population characteristics. DNA samples from 119 premature singleton infants with RDS, 322 without RDS, 245 multiples with RDS, and 235 multiples without RDS were successfully genotyped for SP-A and SP-B polymorphisms

The observed genotype frequencies did not deviate from the expected Hardy–Weinberg distributions (data not shown). The clinical characteristics of the study population are shown in Table 1 . Of the first- and second-born multiples, 30% were monozygotic. In 4% of the multiples, zygosity could not be determined. There was no detectable difference in the concordance rates of RDS between the monozygotic and dizygotic twins or multiples (first- and second-born included).


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  		TABLE 1. Clinical characteristics of infants of singleton and multiple gestations

 
Genetic Susceptibility to RDS in Singleton and Twin Infants
No significant differences in the allele distributions of the SP-A or SP-B genes were shown between the RDS and no-RDS infants when the study population was considered as a whole. However, the homogeneity of the odds ratio test (HOR test) indicated the presence of a profound effect modification due to multiple birth in the SP-A gene polymorphisms (p = 0.0002, 0.001, 0.0006, 0.0001, and 0.17 for the frequencies of alleles 6A2, 6A3, 1A0, genotype 6A2/6A2, and haplotype 6A2-1A0, respectively). The single-nucleotide polymorphism T/C, encoding an amino acid variation Ile131Thr in the last codon of SP-B gene exon 4, differed significantly between RDS and no-RDS only in the first-born infants from multiple gestations, the threonine allele being directly associated with RDS (p = 0.005, OR 1.72, 95% CI 1.18–2.52) or the isoleucine allele protecting against RDS (p = 0.005, OR 0.58, 95% CI 0.40–0.85), as previously described (12). In contrast to previous findings concerning other populations (13, 14), no differences between the RDS and no-RDS infants were found in the distributions of the intron 4 polymorphism in infants born from singleton or multiple gestations even after separate evaluation.

The distributions of both the SP-A1 and SP-A2 genes between RDS and no-RDS infants differed significantly when infants from singleton and multiple gestations were analyzed separately. This relationship was influenced by the SP-B Ile131Thr genotype for the most frequent SP-A1 allele 6A2 and the 6A2/6A2 genotype, respectively (Figures 1 and 2) .



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  		Figure 1. Interaction between the surfactant-specific protein (SP)-A and SP-B genes in predicting susceptibility to respiratory distress syndrome (RDS) in all premature infants from singleton and multiple gestations: frequencies of the SP-A1 allele 6A2 among 322 singletons and 235 multiples with no RDS (open columns) and 119 singletons and 245 multiples with RDS (hatched columns). The infants were divided into subgroups on the basis of their SP-B Ile131Thr genotype. The p values corrected for multiple comparisons, odds ratios (ORs), and 95% confidence intervals (CIs), which illustrate the association of the 6A2 allele with RDS, are as follows: in singletons with the SP-B genotype Thr/Thr, p = 0.009, OR 2.4, CI 1.3–4.6; in multiples with the Ile/Thr genotype, p = 0.012, OR 0.5, CI 0.4–0.8; and in multiples with the Thr/Thr genotype, p = 0.20. An asterisk (*) above a histogram indicates a significant difference.

 


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  		Figure 2. Interaction of the SP-A and SP-B genes in all premature infants born from singleton and multiple gestations: frequencies of the genotype 6A2/6A2 among 322 singleton and 235 multiple infants with no RDS (open columns) and 119 singleton and 245 multiple infants with RDS (hatched columns). The infants were divided into subgroups on the basis of their SP-B Ile131Thr genotype. The p values corrected for multiple comparisons, ORs), and 95% CIs, which illustrate the association of the 6A2/6A2 genotype with RDS in the different SP-B genotype groups, are as follows: in singletons with the SP-B Thr/Thr genotype, p = 0.006, OR 4.5, CI 1.8–11.3; in multiples with the Ile/Thr genotype, p = 0.05, OR 0.5, CI 0.3–0.9; and in multiples with the Thr/Thr genotype, p = 0.04, OR 0.4, CI 0.2–0.8. An asterisk (*) above a histogram indicates a significant difference.

 
Distributions of SP-A1 allele 6A3, SP-A2 allele 1A0, and SP-A haplotype 6A2-1A0 also differed between RDS and no-RDS infants. In singletons, allele 6A3 was underrepresented, whereas allele 1A0 and haplotype 6A2-1A0 were overrepresented in RDS when the genotype was Thr/Thr (frequencies for 6A3, 0.23 in RDS and 0.38 in no-RDS, p = 0.11; frequencies for 1A0, 0.69 in RDS and 0.51 in no-RDS, p = 0.033; and frequencies for 6A2-1A0, 0.68 in RDS and 0.47 in no-RDS, p = 0.011). In multiples, on the other hand, allele 6A3 was overrepresented, whereas allele 1A0 and haplotype 6A2-1A0 were underrepresented in RDS when the SP-B genotype was Ile/Thr (frequencies for 6A3, 0.34 in RDS and 0.22 in no-RDS, p = 0.015; frequencies for 1A0, 0.50 in RDS and 0.63 in no-RDS, p = 0.027; frequencies for 6A2 -1A0, 0.48 in RDS and 0.63 in no-RDS, p = 0.008). When the SP-B genotype was Thr/Thr in multiples, none of the other SP-A1 alleles or SP-A haplotypes was significantly associated with RDS (data not shown).

The data were analyzed separately for premature infants born before (very premature) and after 32 weeks of gestation (near term). Among the very premature singleton infants, the SP-B Ile131Thr genotype defined the cases with a significant association between SP-A allele 6A2 and RDS (Table 2) . No significant association was evident between the SP-A1 alleles and RDS among very premature multiples. By contrast, among near-term multiples, the presence of the SP-B genotype Ile/Thr or Thr/Thr defined the significant association between SP-A1 allele 6A2 and genotype 6A2/6A2 and RDS. In near-term singletons, SP-A and/or SP-B alleles/genotypes/haplotypes had no detectable association with RDS.


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  		TABLE 2. Frequencies of SP-A1 alleles 6A2 and 6A3 and genotype 6A2/6A2 in premature singletons and multiple infants with and without rds according to the sp-b ILE131thr genotype

 
Sex, birth order, or zygosity of multiples had no effect on the SP-A allele and genotype distributions with respect to RDS or on the associations between SP-A polymorphisms and RDS in the various SP-B Ile131Thr genotype subgroups. Nor were there any significant differences when the data were stratified according to birth year.

The results of logistic regression analyses supported the findings on allelic and genotype associations. They were used to determine whether the homozygosity or heterozygosity of different SP-A genotypes is associated with the risk of RDS in the SP-B Ile131Thr subgroups. In singletons, a homozygous 6A2/6A2 genotype increased the risk of RDS when the SP-B genotype was Thr/Thr (OR 3.8, 95% CI 1.4–10.2, p = 0.018). In multiples, on the other hand, a homozygous 6A2/6A2 genotype decreased the risk of RDS when the SP-B genotype was Ile/Thr or Thr/Thr (OR 0.42, 95% CI 0.22–0.80, p = 0.008). This was especially obvious in near-term multiples (OR 0.29, 95% CI 0.09–0.90, p = 0.032).

In a comparison of singleton and multiple pregnancies, the major SP-A allele and SP-B genotype had contrasting associations with the risk of RDS when defined on the basis of the length of pregnancy. Therefore, the pregnancies were analyzed on the basis of intrauterine size. The birth weight of singletons and the birth weight sum of multiples were considered. Among carriers of the SP-A 6A2 allele and the SP-B Thr genotypes, there was a continuous association between fetal weight and the risk of RDS (Figure 3) .



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  		Figure 3. Influence of the birth weight of singletons or the summarized weight of multiples on the interaction between the SP-A and SP-B genes in predicting susceptibility to RDS. Altogether 313 singleton and 344 twins or multiples with either the SP-B Ile/Thr or Thr/Thr genotype were analyzed. The birth weights and summarized birth weights were each categorized into two groups defined by the medians. Both premature singletons and multiples were categorized into two subgroups: below or above the median birth weight/summarized birth weight. Among the singletons, the median birth weight and the interquartiles were 2,220, 1,565, and 2,707 g, respectively. Among the multiples, the median birth weight and the interquartiles were 3,625, 2,920, and 4,605 g, respectively. Upward and downward arrows illustrate the increased and decreased risk of RDS associated with the SP-A allele 6A2. The ORs and 95% CIs, which illustrate the association of the 6A2 allele with RDS in the combined SP-B genotype Ile/Thr and Thr/Thr groups, are shown. They were, for singletons under median birth weight, as follows: OR 1.8, CI 1.1–2.9, p = 0.02; for singletons over median birth weight, OR 1.1, CI 0.6–2.1, p = 0.72; for multiples under median birth weight, OR 0.7, CI 0.5–1.2, p = 0.18; for multiples over median birth weight, OR 0.6, CI 0.4–1.0, p = 0.04. An asterisk (*) above a plot identifies a significant result. There were no significant associations with SP-A and RDS in the SP-B Ile/Ile group (data not shown).

 
Genotype-specific Glycosylation of ProSP-B
To begin to understand the biological basis for the role of the SP-B Ile131Thr polymorphism with RDS, we examined whether the Ile/Ile, Ile/Thr, and Thr/Thr genotypes influenced glycosylation of proSP-B in human fetal lung tissue. The molecular size of human proSP-B was indeed genotype specific, with the glycosylated Thr/Thr product migrating more slowly than the Ile/Ile product (Figure 4A) . Furthermore, the NH2-terminal fragment from Thr/Thr lung, but not from Ile/Ile lung, was sensitive to digestion with PNGase F, indicating the presence of Asn-linked glycosylation in proSP-B from explants representing SP-B Thr/Thr genotype (Figure 4B).



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  		Figure 4. Effect of genotype on mobility and glycosylation of proSP-B. (A) Representative PhosphorImage of proSP-B isolated by immunoprecipitation after 30 min of radiolabeling with [35S]Met/Cys (n = 3). As previously described, only proSP-B is visualized at this time point. The immunoprecipitated proSP-B product from the Thr/Thr sample migrates more slowly (42 kD) than the Ile/Ile product (39 kD). (B) Endoglycosidase treatment and immunoblotting of SP-B. Lung homogenates from Ile/Ile, Thr/Thr, and Ile/Thr genotypes were incubated with (+) or without (–) PNGase F and detected with NFProx antisera, which recognizes the full-length proSP-B and the NH2-terminal cleavage fragment. The NH2 terminus of the Thr allele product (21 kD) decreases in size to 17 kD after digestion with PNGase F, whereas the Ile allele product (17 kD) remains unchanged, indicating that the Thr peptide contains an oligosaccharide at Asn129 whereas the Ile peptide does not. The full-length proSP-B from both Ile (42 kD) and Thr (45 kD) allele products is sensitive to PNGase F because of glycosylation at their COOH terminus at Asn311. This is also evident as two bands in the untreated sample and as one smaller (39 kD) peptide in the PNGase F-treated heterozygous Ile/Thr sample. TT = Thr/Thr; II = Ile/Ile; IT = Ile/Thr; NIS = immunoprecipitation with nonimmune serum.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows that specific genotypes of SP-A and SP-B influence the susceptibility to RDS. The genetic associations were latent and intertwined with acquired factors, particularly with the degree of prematurity, birth order and twinning. Despite the lack of overall direct genetic influence on the risk of RDS in this population overrepresented by multiples, segments of the population had strong gene–gene and gene–environment interactions. According to the present results, the carrier of SP-B Thr131 was predisposed to genetic and environmental variation in the risk of RDS. In the following discussion, we propose that the associations between RDS and the SP-A and SP-B variants are due to gene–gene and gene–environment interactions that modify the synthesis or intracellular processing of surfactant proteins.

SP-B profoundly influences the intracellular processing, secretion, and pool size of surfactant (20). SP-A and SP-B have an interactive role in maintaining the surface activity in vitro, and they both are essential components of the highly surface active tubular myelin fraction (21, 22). According to a study of prematurely born rabbits, the surfactant proteins cooperatively improve lung function (23). In fetuses, SP-A levels in the amniotic fluid closely associate with the risk of RDS after premature birth (4). In addition, SP-A regulates surfactant uptake and secretion by Type II cells in vitro (24, 25) and protects against plasma-derived inhibitors decreasing the function of surfactant (26, 27). Specific changes in the expression levels of SP-A, confined to the major haplotype (6A2-1A0), have been observed in vitro (28), supporting the proposal that the expression levels of SP-A variants influence the risk of RDS. According to the present study, the risk of RDS, defined by the SP-A genotype among the carriers of the SP-B Thr allele, was restricted to the singleton carriers of SP-B Thr/Thr genotype, whereas the protection from the disease was evident in both heterozygous Ile/Thr and homozygous Thr/Thr subgroups, as shown in multiples (Figure 2). In another study of a population consisting of 78 black infants, the Ile/Ile genotype was associated with the risk of RDS interactively with SP-A genotypes, containing 6A3 alleles (29). In the previous study of premature singleton infants and some premature infants from multiple pregnancies, homozygous Thr/Thr defined the population having an association between SP-A genotype and RDS (11). An association between the SP-B Thr allele and an increased risk of chronic obstructive pulmonary disease was further reported by others (30). In other multifactorial diseases, as well, the interaction between genes and environmental factors appears to play an important role in determining the risk of an individual (31–33).

Substitution of isoleucine with threonine in the SP-B peptide at codon 131 creates a consensus sequence for amino-terminal glycosylation of proSP-B at Asn129. This glycosylation site appears to be phylogenetically confined to the human species (34). Pulse labeling of genotype-specific explants of the human fetal lung for proSP-B revealed amino-terminal glycosylation in the Thr/Thr genotype but not in the Ile/Ile genotype (Figure 4), as suggested by the preliminary data (35). The N-terminal glycosylation site is potentially important in the intracellular processing of proSP-B by Type II alveolar cells before secretion of the mature protein into the alveolar space (11). Thus far the proposed mechanism of intracellular interaction between glycosylated moieties of proSP-B and SP-A receptor have been postulated (11), but experimental data are lacking. The synthesis and processing of SP-B is essential for the proper function of Type II alveolar cells. Absence of SP-B expression leads to serious disturbance in the intracellular processing of the surfactant complex, secretion of abnormal surfactant exceptionally enriched with proSP-C, and fatal respiratory failure after birth (36). Additional experimental data on potential consequences of the difference in the glycosylation of the proSP-B allelic variants for processing of SP-B will be of high interest.

According to a popular theory the shared intrauterine environment will equalize the nongenetic disease risk between twins (37). This does not apply to RDS, however. There was no significant concordance difference between monozygotic and dizygotic twin pairs. The intrauterine environment is also different in multiple pregnancy compared with singleton pregnancy (e.g., excessive distension of the uterine wall, fetal crowding). The first-born twin has a higher susceptibility to ascending infections than the second-born, but at the same time is less prone to RDS (17). The SP-B Ile131Thr polymorphism serves as a discordance factor in twin and multiple pregnancies ([12], and present study). In multiple pregnancies the incidence of preterm births approaches 50%, whereas in singleton pregnancies prematurity is uncommon. In very premature singleton infants, genetic predisposition to RDS caused by the major SP-A 6A2 allele exerts a minor selection disadvantage, because all very premature infants used to die whether or not they developed RDS. In contrast, in near-term multiple births the 6A2 allele may have served as a survival factor during evolution as it protects from RDS. The same SP-A genotype also protected from severe airway diseases in early infancy (38, 39).

According to the present study the risk of RDS, defined by the interaction of SP-B and SP-A alleles, was associated with fetal mass. The major SP-A allele and specific SP-B genotypes showed a continuous relationship with intrauterine fetal mass when both singletons and multiples were included (Figure 3). Thus the difference in susceptibility to RDS in premature singletons and multiples may depend on the size of the conceptus. However, the length of gestation is still likely to be an environmental factor influencing genetic susceptibility, although the underlying mechanisms remain speculative. Many hormones and growth factors correlate with uterine distension and the onset of labor (40). Several of them additionally influence the differentiation of the surfactant system and the risk of RDS (41, 42). A single hormone or cytokine may influence the expression of SP-A or SP-B in very immature lung, whereas the same agonist may have an opposing effect on the same gene toward term (42–44). For instance, corticosteroid given before premature birth decreases the risk of RDS. In theory, the effect of antenatal glucocorticoid could be confined to specific SP-A genotype(s); this possibility is supported by the finding that certain SP-A gene variants expressed in bronchiolar Clara cells show differences in responsiveness to corticosteroid (45). However, the present study failed to confirm the association between antenatal glucocorticoid responsiveness and SP-A alleles/genotypes in the SP-B Ile131Thr subgroups. Furthermore, the different SP-A1/SP-A2 variant combinations show a difference in their ability to stimulate tumor necrosis factor-{alpha} production (46), which is known to strongly suppress the expression of SP-B and SP-A in vitro (47, 48).

We found that the common genetic variants of the two surfactant proteins interactively, intertwined with environmental factors, influence the susceptibility to RDS. In addition, we point out the potential utility of organ cultures from human fetal lung as a tool to investigate the genotype-specific differences in the maturation differentiation of the surfactant system. Continued investigations into the molecular functions of these genetic variants could eventually contribute toward more effective, individualized prevention of respiratory failure and its serious consequences.


   Acknowledgments
 
The authors thank Dr. Martti Virtanen (National Research and Development Center for Welfare and Health) for providing access to the Medical Birth Register; Drs. Vineta Fellman, Sami Ikonen, Pentti Kero, and Kirsti Heinonen (Helsinki, Tampere, Turku, and Kuopio University Hospitals) for enabling review of the clinical data and contacts with the families; Elsi Jokelainen, Mirkka Parviainen, Maarit Hännikäinen, and Marjatta Paloheimo for technical assistance; and Juha Turtinen, M.Sc., for statistical advice.


   FOOTNOTES
 
Supported by grants from the Finnish Academy and the Sigrid Juselius Foundation (M.H.) and by NHLBI 59959 (S.G.).

R.M. and R.H. contributed equally to the manuscript.

Conflict of Interest Statement: R.M. has no declared conflict of interest; R.H. has no declared conflict of interest; S.G. has no declared conflict of interest; M.H. has no declared conflict of interest.

Received in original form April 15, 2003; accepted in final form August 25, 2003


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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