Human Molecular Genetics, 2000, Vol. 9, No. 18 2751-2760
© 2000 Oxford University Press
Surfactant proteins A and B as interactive genetic determinants of neonatal respiratory distress syndrome
1Department of Paediatrics and Biocenter Oulu, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland and 2Laboratory of Developmental Immunology, Massachussetts General Hospital, Boston, MA, USA
Received 4 August 2000; Revised and Accepted 8 September 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Prematurity is the most important risk factor predisposing to neonatal respiratory distress syndrome (RDS). Genetic factors are likely to contribute to the risk of this complex disease. The present study was designed to investigate whether the surfactant protein B (SP-B) gene or interaction between the SP-A and SP-B genes has a role in the genetic susceptibility to RDS. The genotype analyses were performed on 684 prematurely born neonates, of whom 184 developed RDS. Of the two SP-B polymorphisms genotyped, the Ile131Thr variation affects a putative N-terminal N-linked glycosylation site of proSP-B and the length variation of intron 4 has previously been suggested to associate with RDS. Neither of the two SP-B polymorphisms associated directly with RDS or with prematurity. Instead, our data show that the previously identified association between SP-A alleles and RDS was dependent on the SP-B Ile131Thr genotype. On the basis of
2 and logistic regression analyses, the SP-A allele, haplotype and genotype distributions differed significantly between the RDS infants and controls only when the SP-B genotype was Thr/Thr. Among the infants born before 32 weeks of gestation and having the SP-B genotype Thr/Thr, the SP-A1 allele 6A2 was over-represented in RDS group compared with controls (P = 0.001, OR = 4.7, CI 1.8–12.2). In the same comparison, the SP-A1 allele 6A3 was under-represented in RDS (P = 0.001, OR = 0.2, CI 0.1–0.6). We propose that the SP-B Ile131Thr polymorphism is a determinant for certain SP-A alleles as factors causing genetic susceptibility to RDS (6A2, 1A0) or protection against it (6A3, 1A2). |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Neonatal respiratory distress syndrome (RDS) is the major cause of mortality and morbidity in premature infants. The disease is characterized by respiratory failure and deficient gas exchange. It is caused by a deficiency of pulmonary surfactant, a complex lipoprotein mixture produced by type II alveolar epithelial cells (1–4). The main function of the surfactant is to stabilize the alveoli throughout the respiratory cycle, thus preventing alveolar collapse at the end of expiration (5). Surfactant is composed of
90% phospholipids and 10% surfactant proteins (SP-A, SP-B, SP-C and SP-D) (6). The incidence of RDS has decreased significantly due to prophylactic antenatal glucocorticoid treatment (7). Prematurity is the most important factor predisposing to RDS and race and gender additionally contribute to the risk of the disease (8,9). Furthermore, as suggested on the basis of a twin study and some other epidemiologic reports, genetic factors are assumed to have a role in the aetiology of RDS (10–12). However, the specific genes underlying this susceptibility are incompletely known. The most promising candidates studied so far are the genes coding for the lung-specific protein components of the surfactant, especially those coding for SP-A and SP-B (13,14). On the whole, the aetiology of RDS is considered multifactorial and possibly multigenic.
The major surfactant protein, SP-A, is a hydrophilic collagen-like lectin that improves the surface activity of the surfactant complex and has a role in pulmonary host defence (15–17). Surprisingly, SP-A deficiency does not result in respiratory failure in newborn mice (18). The human SP-A is encoded by two highly homologous genes (SP-A1 and SP-A2) each spanning 5 kb in chromosome 10q22–q23 (19) and both genes are needed for fully functional mature SP-A protein (19–21). Several alleles that differ by a single amino acid have been identified for each SP-A gene (22–24). An association of SP-A alleles with the risk of RDS was recently reported among premature Finnish infants (25).
SP-B is a lung epithelial cell-specific secreted hydrophobic protein, which has an essential role in normal lung function (6,26,27). It accelerates the rate of adsorption and spreading of surfactant phospholipids into the air–liquid interface. SP-B mRNA appears as early as the 14th gestational week in the human fetal lung (28) and after 25 weeks of gestation SP-B mRNA and protein are co-localized in terminal airways and type II epithelial cells (27,29). In human neonates, hereditary SP-B deficiency caused by a frameshift mutation 121ins2 results in a fatal respiratory disease known as congenital alveolar proteinosis (30). Targeted disruption of the SP-B gene leads to neonatal death due to severe respiratory failure in homozygous SP-B–/– mice (31).
The human SP-B gene on chromosome 2p12–p11.2 consists of 11 exons and spans a region of 10 kb (32,33). The gene encodes a saposin-like glycoprotein precursor of 381 amino acids, which is proteolytically cleaved and processed to give rise to mature SP-B that is secreted to the epithelial lining of alveoli. SP-B is essential for the structural integrity of type II pneumocytes and for normal intracellular processing of the surfactant complex (31). The monomer of this hydrophobic dimeric protein contains 79 amino acids encoded by exons 6 and 7. The SP-B gene is known to be polymorphic (13,34,35). Length variation in intron 4 of the SP-B gene (SP-B 
i4) has been proposed to associate with RDS both independently (13) and in combination with an SP-A allele (14) in certain subgroups of infants. The studies on possible allelic association of the SP-B gene i4 polymorphism with RDS were complicated by racial differences in the allelic distributions and by the heterogeneity and small sample size of the study populations. Specifically, no difference was observed in SP-B i4 distributions between RDS cases and controls in a racially matched sample (36). On the basis of SP-B cDNA sequence data, the human genomic DNA sequence varies in the last codon of exon 4 (35). The position of this C/T nucleotide difference that leads to Ile131Thr variation in the SP-B precursor protein is 120 bp upstream of the i4 polymorphism.
The present study was undertaken to determine whether the SP-B gene associates with neonatal RDS in Finland. The two SP-B polymorphisms were evaluated for any association with RDS or prematurity in a large and racially homogenous population sample. Furthermore, the SP-A and SP-B genotype data were combined in order to examine whether these unlinked gene loci associate interactively with the risk of RDS.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
DNA samples from 184 prematurely born RDS infants and 500 premature control infants (Table 1) were available for genotype analysis of the SP-B Ile131Thr single nucleotide polymorphism, SP-B
i4 length polymorphism and SP-A1 and SP-A2 genotypes. As a reference population, 475 infants born at term were genotyped as well. Occasional failure in genotyping caused slight variation in the number of cases analysed.
|
SP-B Ile131Thr and
i4 polymorphism in the Finnish populationThe last codon of SP-B gene exon 4 showed a single nucleotide polymorphism T/C, encoding amino acid variation Ile131Thr. In term infants, the allele frequencies were 54% Ile and 46% Thr and the genotype frequencies were 29% Ile/Ile, 50% Ile/Thr and 21% Thr/Thr. None of the subgroups of premature infants in the study population differed significantly from these distributions.
SP-B intron 4 is composed of different short, 1–24 bp sequence motifs separated by 3–14 (CA)n repeats (Table 2) (32). Allelic variation results from deletion or insertion of one or several motif–(CA)n sequence stretches at conserved cut-points (13). The i4 alleles in the present study population are shown in Table 3. They differed from the previously published nucleotide sequences. There were minor variations in the number of (CA)n repeats, both within and between the variant classes. Instead of GAG, the motif 10 was always G. The insertion variants did not contain insertion motifs similar to those previously described, but consisted of various duplications of the preceding motifs. Altogether 10 length variant classes were found in the present study (Table 3). The most common i4 fragment (size 513 bp) was denoted as the invariant allele. The deletion variants of four different size classes were pooled together as the deletion allele, and the insertion variants of four different sizes were pooled together as the insertion allele. Additionally, one variant that occurred at a very low frequency consisted of a combination of an insertion and a deletion. The Finnish SP-B i4 allele frequencies were similar to those reported for Caucasians, but remarkably different from the frequencies seen in African-American and Nigerian populations (36).
|
|
Linkage disequilibrium between SP-B Ile131Thr and
i4 polymorphismsSP-B Ile131Thr–
i4 haplotype analysis revealed linkage disequilibrium between the two SP-B polymorphisms, as determined by deviation of the observed haplotype distribution from the expected haplotype distribution (P < 0.0001) (Table 4). This was evident as nearly complete linkage disequilibrium for the i4 variant alleles: the insertion variants always had T (encoding Ile) and 97% of the deletion variants had C (encoding Thr) in this position. The invariant i4 allele showed less linkage disequilibrium with the Ile131Thr polymorphism.
|
Lack of allelic or genotype association of SP-B Ile131Thr and
i4 polymorphisms with RDS or prematurityNeither of the SP-B polymorphisms differed in allele distribution between the RDS cases and premature controls or between the premature and term infants (Table 5). An analysis of the subgroups according to the known risk (severe prematurity, male sex) or protective (prenatal maternal glucocorticoid treatment) factors revealed no trends towards any allelic associations between SP-B and RDS and the observed genotype distributions did not deviate from the Hardy–Weinberg equilibrium in any of the subgroups studied (Table 5) (data not shown). The SP-B Ile131Thr–
i4 haplotype frequencies did not differ between the various infant subgroups, either. This indicates that the observed linkage disequilibrium between the two SP-B polymorphisms was not dependent on RDS status or the degree of prematurity.
|
Influence of SP-B genotypes on association between SP-A alleles and RDS
Several SP-A alleles (Table 6) are potential predisposers to (6A2, 1A0) or protectors from (6A3, 1A1, 1A2) RDS in very premature infants born before 32 weeks of gestation (25). The present study also showed differences in SP-A allele frequencies between the RDS and control infants born before 32 weeks of gestation, but not if the gestational age was higher (data not shown). When the infants who developed RDS despite antenatal corticosteroid therapy (i.e. infants unresponsive to steroid treatment and thus prone to RDS) were compared with those who were protected from RDS (no RDS despite severe prematurity and a lack of steroid prophylaxis), a difference was observed for both SP-A genes. The frequency of the SP-A1 allele 6A2 in the steroid, RDS group (n = 83) compared with the no-steroid, no-RDS group (n = 14) was 69 versus 36% (P = 0.0006; OR = 4.1, CI 1.8–9.4). Correspondingly, the SP-A1 allele 6A3 frequency was 19% in RDS versus 57% in controls (P < 0.0001; OR = 0.2, CI 0.1–0.4). The SP-A2 allele frequencies were different as well. In the steroid, RDS group, the 1A0 frequency was higher (P = 0.008) and the 1A1 frequency lower (0.022) than in the no-steroid, no-RDS group. In agreement with previous data (22), linkage disequilibrium between the SP-A1 and SP-A2 genes was evident in the present study (data not shown).
|
A report of another population sample suggested that the presence of any SP-B
i4 variant strengthened the association between the SP-A2 allele 1A0 and RDS (14). In the present population, SP-A allelic association with RDS was dependent on the SP-B Ile131Thr genotype (Table 7). However, the association was not dependent on the SP-B i4 deletion variant, despite the strong linkage disequilibrium between the Thr and i4 deletion alleles. Premature infants showed a significant difference in the SP-A1 and SP-A2 allele and haplotype frequencies between the RDS cases (n = 176) and controls (n = 491) if the SP-B genotype was Thr/Thr, but no significant difference if the genotype was Ile/Ile or Ile/Thr (Table 7).
|
The main outcome of the homogeneity of odds ratio (HOR) tests (P < 0.05) showed the presence of effect modification by the SP-B Ile131Thr genotype (HOR test P = 0.056, 0.014, 0.039, 0.27, 0.029 and 0.015 for frequencies of 6A2, 6A3, 1A0, 1A2, 6A2-1A0 and 6A3-1A2, respectively) and gestational age (P = 0.032, 0.043, 0.039, 0.56, 0.015 and 0.46 for frequencies of 6A2, 6A3, 1A0, 1A2, 6A2-1A0 and 6A3-1A2, respectively). Accordingly, the data were analysed separately for premature infants born before and after 32 weeks of gestation. Among the infants born before 32 weeks, the SP-B Ile131Thr genotype defined the cases with significant association between the SP-A alleles and RDS (Fig. 1). On the contrary, regardless of the SP-B Ile131Thr genotype, no association was evident between the SP-A alleles and RDS among the premature infants born after 32 weeks (Fig. 2). The exact test for homogeneity of odds ratios (HOR test) also showed a profound effect modification by the SP-B Ile131Thr genotype in very premature infants (P = 0.011, 0.006, 0.017, 0.15, 0.010 and 0.019 for frequencies of 6A2, 6A3, 1A0, 1A2, 6A2-1A0 and 6A3-1A2, respectively).
|
|
The SP-B Ile131Thr genotype-dependent association was further supported by a comparison with the homozygous genotype frequencies in the RDS infants and controls. The genotypes 6A2/6A2 and 1A0/1A0 were over-represented in RDS when the SP-B genotype was Thr/Thr (frequencies for 6A2/6A2, 54.5% in RDS versus 33.9% in controls and, for 1A0/1A0, 53.5 versus 33.0%; P = 0.019 and 0.020, respectively). For 6A3/6A3, a trend was observed (4.5% in RDS versus 14.7% in controls; P = 0.078). Among the infants born before 32 gestational weeks: for the SP-B genotype Thr/Thr, the 6A2/6A2 genotype frequencies were 65.4% in RDS versus 23.5% in controls (P = 0.007), the 6A3/6A3 genotype frequencies 3.8 versus 23.5% (P = 0.071) and the 1A0/1A0 genotype frequencies 68.0 versus 23.5% (P = 0.005). No significant differences were detected in the homozygous SP-A genotype frequencies between the RDS infants and controls when the SP-B genotype was Ile/Ile or Ile/Thr, or when gestational age was above 32 weeks (data not shown). Logistic regression models were used to determine whether the heterozygosity or homozygosity of SP-A alleles influences the odds of RDS. A homozygous 6A2/6A2 genotype increased and a homozygous 6A3/6A3 genotype decreased the odds of RDS, when SP-B genotype was Thr/Thr (Table 8). When the SP-B genotype was Ile/Ile or Ile/Thr, the homozygous SP-A genotypes had no detectable effects on the odds of RDS (Table 8). With the Ile/Ile genotype, the heterozygous SP-A 6A2/X genotype was unexpectedly associated with the odds of RDS (Table 8). This is possibly due to a non-significant trend towards over-representation of the allele 6A2 among RDS infants born after 32 weeks of gestation. This association disappeared when the regression analysis was performed only on infants born before 32 weeks of gestation, whereas the association of homozygous 6A2/6A2 and 6A3/6A3 genotypes in Thr/Thr group persisted (data not shown). Therefore, the association between the heterozygous SP-A 6A2/X genotype and RDS was probably due to type I error.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The present study was designed to determine whether the SP-B gene or the interaction between the SP-A and SP-B genes have roles in susceptibility to RDS in a racially homogenous population of 683 premature infants including 183 with RDS. This is the first population-based report of the SP-B polymorphism with Ile131Thr variation in exon 4 that affects a putative N-linked glycosylation site of proSP-B. The other SP-B polymorphism, i.e. the length variation of intron 4 (
i4), has been considered a genetic marker for RDS (13). In the present study, the association of the SP-A locus with RDS (25) was confirmed, whereas neither the SP-B Ile131Thr nor the SP-B i4 polymorphism associated directly with RDS or with prematurity. Instead of a direct association between the SP-B gene and RDS, the data show that the allele and haplotype association between SP-A genes and RDS was restricted to a subset of infants who carried the SP-B genotype Thr/Thr. Despite the linkage disequilibrium between the SP-B i4 and Ile131Thr polymorphisms, the SP-B i4 alleles or genotypes did not affect the association between SP-A and RDS. Among the infants born before 32 weeks of gestation and having the SP-B genotype Thr/Thr, the SP-A1–SP-A2 haplotype 6A2-1A0 was over-represented and the haplotype 6A3-1A2 under-represented in the RDS group compared with the controls. It was also evident that the associations were strongly dependent on the degree of prematurity. Only the very premature infants, i.e. those born before 32 weeks of gestation, had significant associations between the SP-A alleles and the risk of RDS. Because gestational age was an effect modifier, we propose that there are gestation age-dependent differences in the genetic background of RDS.
The present results are consistent with the previous study of the Finnish population indicating that the SP-A gene locus plays a role in the genetic predisposition to RDS in very premature infants (25). Substitution of isoleucine for threonine in the SP-B peptide at amino acid 131 results in the loss of a consensus sequence for glycosylation at Asn129. A new finding is that the variation in the SP-B gene affecting the potential N-linked N-terminal glycosylation of proSP-B plays a role in the genetic susceptibility to RDS after very premature birth. Alternatively, the SP-B Ile131Thr variation may be linked to an as yet unidentified variable genetic element that together with the SP-A allelic variants, has a co-operative biological role in the risk of RDS. The study of 107 white RDS infants suggested that there is a synergistic positive association between RDS and a combination of SP-B i4 and SP-A2 allele 1A0 (14). In the present study, i4 did not determine the association between the SP-A alleles and RDS. The i4 del/del genotype may be a determinant of the disease association, similar to the Thr/Thr genotype. However, because of the very low frequency of del/del, a considerably larger sample size is required to evaluate the gene–gene interaction in this particular subgroup. The lack of determination by the heterozygous i4 del/* genotype is presumably due to the weak linkage disequilibrium between the intron 4 invariant and Ile/Thr variation. On the basis of the linkage disequilibrium data, the origin of the Ile/Thr variation is likely to be more ancient than the mutation events giving rise to the intron 4 length variation. Instead of i4, the exon 4 Ile/Thr appears to be a determinant of the disease association.
Lamellar bodies (LBs) are alveolar type II cell-specific storage and secretory organelles responsible for the exocytosis of the surfactant complex. Lack of SP-B gene expression results in disturbance of LB morphogenesis, failure of SP-C processing (31,37,38) and fatal pulmonary alveolar proteinosis (39). The N-terminal propeptide is necessary and sufficient for intracellular trafficking of SP-B (40,41). Glycosylation of proSP-B at Asn129 in type II cells in vivo has not been resolved with certainty. NCI-H441-4 cells, a Clara cell-derived human adenocarcinoma cell line, have been used as a model to study SP-B processing and secretion. In this cell line, the SP-B genotype was heterozygous Ile/Thr (R. Haataja, unpublished data). In H441 cells, proSP-B is N-glycosylated C-terminally at Asn311, but apparently not N-terminally at Asn129 (42). On the other hand, there is evidence for glycosylation of both N- (Asn129) and C- (Asn311) terminal propeptides (32,39). Appropriate processing of proSP-B to mature SP-B is type II cell-specific (43). Post-translational regulatory mechanisms have a role in the expression of mature SP-B in type II cells and proSP-B processing in these cells is developmentally and hormonally regulated (44). Our finding that SP-B Ile131Thr variation is an interactive determinant of RDS susceptibility raises the possibility that the putative N-terminal glycosylation site is important in the folding, sorting or processing of proSP-B by type II alveolar epithelial cells. Although included in the N-terminal propeptide, this site is cleaved before the secretion of mature SP-B into the alveolar space, where it profoundly improves the surface activity of the surfactant phospholipids.
We propose two possible pathways for the SP-B genotype-dependent interaction between SP-B and SP-A. Differential folding of proSP-B N-glycosylation variants may affect SP-B secretion and thereby have an influence on the availability of extracellular mature SP-B. SP-A could also interact intracellularly with glycosylated proSP-B by its carbohydrate-recognition domain either directly or through its endoplasmic reticulum (ER)-resident receptor, calreticulin. Calreticulin is a soluble ER lectin, an important chaperone that facilitates proper folding of glycoproteins in the ER. It has the potential to act as a receptor for SP-A (45), another lectin that binds carbohydrates (46). All glycoproteins are presumed to interact transiently with ER lectins in mammalian cells (47). N-linked glycosylation is required for the correct folding of many glycoproteins and efficient interaction between the glycoprotein and the folding-assisting chaperone may be affected by the degree of glycosylation (48). Protein secretion can be regulated by variation of a single N-linked glycosylation site (49). Whether there are differences in the folding or sorting of proSP-B allelic variants and whether this would affect SP-A post-translationally remains to be studied.
SP-A acts in conjunction with SP-B in maintaining surface-active surfactant aggregates in vitro (50). SP-B and SP-A have a co-operative influence on the surface activity of the extracellular surfactant complex (51–53). The role of SP-A appears to be significant in the presence of surfactant inhibitors that are prevalent in very premature infants with RDS (54). Maintenance of the surface activity (i.e. low surface tension) is required for the prevention of generalized atelectasis, which is the principal pathophysiologic feature in RDS.
Analogous to the present study, interaction between angiotensin-converting enzyme (ACE) and angiotensin-II type 1 receptor (AT1R) gene polymorphisms was found to contribute to the risk of myocardial infarction (55) and early coronary disease (56), without a direct association between the AT1R polymorphism and the disease. In the near future, new interactions between unlinked genes are likely to be discovered in the aetiology of many other common diseases, such as cancer and osteoporosis.
In summary, we propose that interaction between the allelic SP-A and SP-B gene products are a genetic determinant of neonatal RDS. When the SP-B genotype is Thr/Thr, i.e. proSP-B is potentially glycosylated at Asn129, certain SP-A alleles can be regarded as factors causing genetic susceptibility (6A2, 1A0) to or protection (6A3, 1A2) against RDS. The mechanisms behind this gene–gene interaction remain to be elucidated.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Blood sample collection and study population
The samples were collected from Finnish prematurely born (<37 weeks of gestation) neonates (n = 707) during the years 1996–1999. The catchment areas were the University Central Hospitals in Oulu (n = 449) and Tampere (n = 149) and the Southern Ostrobothnia Central Hospital in Seinäjoki (n = 109). The geographic regions covered by these hospitals were parts of Northern and Central Finland. Additionally, umbilical cord blood specimens were obtained from 475 infants born at term during 2 months in 1998 in the Oulu University Central Hospital. The ethical committees of the participating centres approved the study. Informed consent was obtained from the parents of the infants.
The clinical data concerning sex, gestational age and maternal and neonatal clinical histories were obtained from the medical records. The diagnosis of RDS was made on the basis of the published clinical, radiographic and/or pathologic criteria, as described (25). None of the newborns had been treated with surfactant prophylactically. Exclusion criteria were intra-uterine blood transfusion and either parent not being Finnish in origin. Altogether 188 cord blood specimens from infants who developed RDS and 500 specimens from premature infants without RDS were available for analysis.
DNA samples
Genomic DNA was isolated from frozen EDTA-anticoagulated whole blood specimens (0.5–3 ml) using the Puregene DNA Isolation kit (Gentra Systems, Minneapolis, MN). An aliquot of the DNA solution was diluted to 50 ng/µl to be used for PCR amplification. When whole blood samples were not available (n = 70), genotypes were determined using a blood spot dried on a filter paper which was treated as described (25).
Genotyping of SP-B gene polymorphisms
For SP-B Ile131Thr genotyping, PCR amplification was performed using the forward primer SPBTaaF1, 5'-TGGGGGATTAGGGGTCAGTC-3', and the reverse primer SPBTaaR1, 5'-CCATGGGTGGGCACAGGGG-3' (PCR I), followed by nested PCR amplification with the forward primer SPBTaaF2, 5'-GGGGGATTAGGGGTCAGTCT-3', and the reverse primer SPBTaaR2, 5'-CATGGGTGGGCACAGGGGC-3' (PCR II), in 10 µl of reaction mixture containing 50 ng of genomic DNA, 0.2 µM dNTPs (Pharmacia, Peapack, NJ), 0.2 µM each primer, 1x GeneAmp PCR buffer (10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% w/v gelatine; Perkin Elmer, Rockville, MD) and 0.5 U of AmpliTaq Gold DNA polymerase (Perkin Elmer). The PCR cycles consisted of initial denaturation at 94°C for 10 min, followed by 25 (PCR I) or (PCR II) 32 cycles of 94°C for 1 min, 65°C for 40 s, 72°C for 1 min and a final extension at 72°C for 8 min (Thermal cycler PTC-200; MJ Research, Waltham, MA). The 290 bp PCR fragments were digested with the restriction enzyme TaaI (MBI Fermentas, Vilnius, Lithuania) and analysed on agarose gels. Genotyping of the SP-B i4 polymorphism was performed using a PCR fragment amplified by the forward primer SPBi4F, 5'-CTGGTCATCGACTACTTCCA-3', and the reverse primer SPBi4R, 5'-TGAAGGGCACGTAGTTTCCTA-3'. The conditions for genotyping have been described (25). The resulting 240–660 bp PCR fragments were analysed in ethidium bromide-stained LE or NuSieve GTG agarose gels. For sequencing of the SP-B alleles, the PCR fragments were ligated into pGEM-T Easy (Promega, Madison, WI) before analysis with the ABI Prism 377 DNA sequencing system.
SP-B haplotyping
SP-B i4–Thr131Ile haplotypes were determined on the basis of i4 and Thr131Ile genotypes. For double heterozygotes, the haplotypes were obtained by preferential amplification of the shorter i4 allele, followed by digestion with TaaI and analysis on agarose gels. The PCR conditions for haplotype verification were as follows: 15 µl of reaction mixture containing 50 ng of genomic DNA, 0.2 µM dNTPs, 0.2 µM primers SPBi4F and SPBi4R, 1x GeneAmp PCR buffer and 0.75 U of AmpliTaq Gold DNA polymerase. The PCR cycles were initial denaturation at 94°C for 10 min, followed by 32 cycles of 94°C for 10 s, 60°C for 30 s and a final extension at 72°C for 5 min.
Genotyping of SP-A gene polymorphisms
Genotyping of SP-A1 and SP-A2 genes was carried out as described (25). The PCR–cRFLP method based on converted PCR primers (22) was used to detect single nucleotide polymorphisms at codons 19, 50, 62, 133 and 219 for the SP-A1 gene and at codons 9, 91, 140 and 223 for the SP-A2 gene. Different combinations of polymorphisms at these sites distinguish between different alleles, which are denoted as 6An for SP-A1 and as 1An for SP-A2.
Statistics
Comparisons of allele frequencies were performed by two-tailed 2 tests. Allele distributions were compared by 2 x k tables and frequencies of individual alleles by 2 x 2 tables. To avoid type I errors in final conclusions, the multiple comparisons were corrected by multiplying the statistically significant P values by the number of pairwise comparisons being made. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the relative disease risk conferred by a particular allele, haplotype and genotype. Fisher’s exact test was used when the expected value was <5. The significance of linkage disequilibrium between SP-B polymorphisms was estimated using 2 analyses by comparing the observed and expected haplotype distributions. The expected SP-B haplotype distribution was calculated on the basis of the observed allele frequencies. The observed genotype frequencies were compared with the expected Hardy–Weinberg distributions by 2 analyses. The HOR test was used to evaluate whether the length of gestation or the SP-B genotype was an effect modifier, i.e. whether the risk of RDS by a given SP-A allele or haplotype is the same across all strata of gestational age or SP-B genotype and whether the data should be analysed separately in subgroups (57,58). Multiple logistic regression analyses were performed to investigate whether the heterozygosity or homozygosity of particular alleles explained the risk of RDS. ORs of allelic variables and confounders for RDS (gestational age at birth, antenatal maternal steroid treatment and gender) were included in the analyses with forced entry and were estimated using logistic regression models, where the presence or absence of RDS was the dependent variable.
The software used for computations included SPSS for Windows (for basic statistical calculations and logistic regression analysis), Arcus Quickstat (for 2 analysis and Fisher’s exact test) and StatXact for Windows (for HOR test). P values of <0.05 were considered to be statistically significant.
|
ACKNOWLEDGEMENTS |
|---|
The authors are grateful to Ms Maarit Hännikäinen and Ms Mirkka Parviainen for excellent technical assistance and to Juha Turtinen MSc for skilful advice regarding statistics. Drs Marja-Leena Pokela and Outi Tammela are acknowledged for the collection of blood specimens, Prof. Mikael Knip for the contribution of dried blood samples and Ms Eija Rautio for the collection of clinical data. The study was supported by grants from Biocenter Oulu (R.H., M.H.), the Academy of Finland (M.R., M.H.) and the Foundation for Paediatric Research (M.H., R.M. and M.R.).
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +358 8 315 5100; Fax: +358 8 315 5559; Email: mhallman@cc.oulu.fi
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Avery, M.E. and Mead, J. (1959) Surface properties in relation to atelectasis and hyaline membrane disease. Am. J. Dis. Child, 97, 517–523.
2 de Mello, D.E., Phelps, D.S., Patel, G., Floros, J. and Lagunoff, D. (1989) Expression of the 35 kDa and low molecular weight surfactant-associated proteins in the lungs of infants dying with respiratory distress syndrome. Am. J. Pathol., 134, 1285–1293.[Abstract]
3 de Mello, D.E., Heyman, S., Phelps, D.S. and Floros, J. (1993) Immunogold localization of SP-A in lungs of infants dying from respiratory distress syndrome. Am. J. Pathol., 142, 1631–1640.[Abstract]
4 Gluck, L., Kulovich, M.V., Borer Jr, R.C., Brenner, P.H., Anderson, G.G. and Spellacy, W.N. (1971) Diagnosis of the respiratory distress syndrome by amniocentesis. Am. J. Obstet. Gynecol., 109, 440–445.[Web of Science][Medline]
5 Hawgood, S. and Clements, J.A. (1990) Pulmonary surfactant and its apoproteins. J. Clin. Invest., 86, 1–6.
6 Pryhuber, G.S. (1998) Regulation and function of pulmonary surfactant protein B. Mol. Genet. Metab., 64, 217–228.[Web of Science][Medline]
7 Crowley, P.A. (1995) Antenatal corticosteroid therapy: a meta-analysis of the randomized trials, 1972 to 1994. Am. J. Obstet. Gynecol., 173, 322–335.[Web of Science][Medline]
8 Farrell, P.M. and Wood, R.E. (1976) Epidemiology of hyaline membrane disease in the United States: analysis of national mortality statistics. Pediatrics, 58, 167–176.
9 Richardson, D.K. and Torday, J.S. (1994) Racial differences in predictive value of the lecithin/sphingomyelin ratio. Am. J. Obstet. Gynecol., 170, 1273–1278.[Web of Science][Medline]
10 Graven, S.N. and Misenheimer, H.R. (1965) Respiratory distress syndrome and the high risk mother. Am. J. Dis. Child., 109, 489–494.
11 Lankenau, H.M. (1976) A genetic and statistical study of the respiratory distress syndrome. Eur. J. Pediatr., 123, 167–177.
12 Myrianthopoulos, N.C., Churchill, J.A. and Baszynski, A.J. (1971) Respiratory distress syndrome in twins. Acta Genet. Med. Gemellol., 20, 199–204.[Medline]
13 Floros, J., Veletza, S.V., Kotikalapudi, P., Krizkova, L., Karinch, A.M., Friedman, C., Buchter, S. and Marks, K. (1995) Dinucleotide repeats in the human surfactant protein-B gene and respiratory-distress syndrome. Biochem. J., 305, 583–590.
14 Kala, P., Ten Have, T., Nielsen, H., Dunn, M. and Floros, J. (1998) Association of pulmonary surfactant protein A (SP-A) gene and respiratory distress syndrome: interaction with SP-B. Pediatr. Res., 43, 169–177.[Web of Science][Medline]
15 Cockshutt, A.M., Weitz, J. and Possmayer, F. (1990) Pulmonary surfactant-associated protein A enhances the surface activity of lipid extract surfactant and reverses inhibition by blood proteins in vitro. Biochemistry, 29, 8424–8429.[Medline]
16 Hoppe, H.J. and Reid, K.B. (1994) Collectins—soluble proteins containing collagenous regions and lectin domains—and their roles in innate immunity. Protein Sci., 3, 1143–1158.[Web of Science][Medline]
17 Venkitaraman, A.R., Hall, S.B., Whitsett, J.A. and Notter, R.H. (1990) Enhancement of biophysical activity of lung surfactant extracts and phospholipid-apoprotein mixtures by surfactant protein A. Chem. Phys. Lipids, 56, 185–194.[Web of Science][Medline]
18 Korfhagen, T.R., Bruno, M.D., Ross, G.F., Huelsman, K.M., Ikegami, M., Jobe, A.H., Wert, S.E., Stripp, B.R., Morris, R.E., Glasser, S.W. et al. (1996) Altered surfactant function and structure in SP-A gene targeted mice. Proc. Natl Acad. Sci. USA, 93, 9594–9599.
19 Fisher, J.H., Kao, F.T., Jones, C., White, R.T., Benson, B.J. and Mason, R.J. (1987) The coding sequence for the 32 000-dalton pulmonary surfactant-associated protein A is located on chromosome 10 and identifies two separate restriction-fragment-length polymorphisms. Am. J. Hum. Genet., 40, 503–511.[Web of Science][Medline]
20 Bruns, G., Stroh, H., Veldman, G.M., Latt, S.A. and Floros, J. (1987) The 35 kDa pulmonary surfactant-associated protein is encoded on chromosome 10. Hum. Genet., 76, 58–62.[Web of Science][Medline]
21 Voss, T., Melchers, K., Scheirle, G. and Schafer, K.P. (1991) Structural comparison of recombinant pulmonary surfactant protein SP-A derived from two human coding sequences: implications for the chain composition of natural human SP-A. Am. J. Respir. Cell Mol. Biol., 4, 88–94.
22 DiAngelo, S., Lin, Z., Wang, G., Phillips, S., Rämet, M., Luo, J. and Floros, J. (1999) Novel, non-radioactive, simple and multiplex PCR-cRFLP methods for genotyping human SP-A and SP-D marker alleles. Dis. Markers, 15, 269–281.[Web of Science][Medline]
23 Floros, J., DiAngelo, S., Koptides, M., Karinch, A.M., Rogan, P.K., Nielsen, H., Spragg, R.G., Watterberg, K. and Deiter, G. (1996) Human SP-A locus: allele frequencies and linkage disequilibrium between the two surfactant protein A genes. Am. J. Respir. Cell Mol. Biol., 15, 489–498.[Abstract]
24 Krizkova, L., Sakthivel, R., Olowe, S.A., Rogan, P.K. and Floros, J. (1994) Human SP-A: genotype and single-strand conformation polymorphism analysis. Am. J. Physiol., 266, L519–L527.
25 Rämet, M., Haataja, R., Marttila, R., Floros, J. and Hallman, M. (2000) Association between the surfactant protein A (SP-A) gene locus and respiratory-distress syndrome in the Finnish population. Am. J. Hum. Genet., 66, 1569–1579.[Web of Science][Medline]
26 Suzuki, Y., Robertson, B., Fujita, Y. and Grossmann, G. (1988) Respiratory failure in mice caused by a hybridoma making antibodies to the 15 kDa surfactant apoprotein. Acta Anaesthesiol. Scand., 32, 283–289.[Web of Science][Medline]
27 Whitsett, J.A., Nogee, L.M., Weaver, T.E. and Horowitz, A.D. (1995) Human surfactant protein B: structure, function, regulation, and genetic disease. Physiol. Rev., 75, 749–757.
28 Liley, H.G., White, R.T., Warr, R.G., Benson, B.J., Hawgood, S. and Ballard, P.L. (1989) Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung. J. Clin. Invest., 83, 1191–1197.
29 Stahlman, M.T., Gray, M.E. and Whitsett, J.A. (1992) The ontogeny and distribution of surfactant protein B in human fetuses and newborns. J. Histochem. Cytochem., 40, 1471–1480.[Abstract]
30 Nogee, L.M., Garnier, G., Dietz, H.C., Singer, L., Murphy, A.M., deMello, D.E. and Colten, H.R. (1994) A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds. J. Clin. Invest., 93, 1860–1863.
31 Clark, J.C., Wert, S.E., Bachurski, C.J., Stahlman, M.T., Stripp, B.R., Weaver, T.E. and Whitsett, J.A. (1995) Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc. Natl Acad. Sci. USA, 92, 7794–7798.
32 Pilot-Matias, T.J., Kister, S.E., Fox, J.L., Kropp, K., Glasser, S.W. and Whitsett, J.A. (1989) Structure and organization of the gene encoding human pulmonary surfactant proteolipid SP-B. DNA, 8, 75–86.[Web of Science][Medline]
33 Vamvakopoulos, N.C., Modi, W.S. and Floros, J. (1995) Mapping the human pulmonary surfactant-associated protein B gene (SFTP3) to chromosome 2p12
p11.2. Cytogenet. Cell. Genet., 68, 8–10.[Web of Science][Medline]
34 Emrie, P.A., Jones, C., Hofmann, T. and Fisher, J.H. (1988) The coding sequence for the human 18 000 dalton hydrophobic pulmonary surfactant protein is located on chromosome 2 and identifies a restriction fragment length polymorphism. Somat. Cell Mol. Genet., 14, 105–110.[Web of Science][Medline]
35 Glasser, S.W., Korfhagen, T.R., Weaver, T., Pilot-Matias, T., Fox, J.L. and Whitsett, JA. (1987) cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe). Proc. Natl Acad. Sci. USA, 84, 4007–4011.
36 Veletza, S.V., Rogan, P.K., Ten Have, T., Olowe, S.A. and Floros, J. (1996) Racial differences in allelic distribution at the human pulmonary surfactant protein B gene locus (SP-B). Exp. Lung Res., 22, 489–494.[Web of Science][Medline]
37 Beers, M.F., Hamvas, A., Moxley, M.A., Gonzales, L.W., Guttentag, S.H., Solarin, K.O., Longmore, W.J., Nogee, L.M. and Ballard, P.L. (2000) Pulmonary surfactant metabolism in infants lacking surfactant protein B. Am. J. Respir. Cell Mol. Biol., 22, 380–391.
38 Vorbroker, D.K., Profitt, S.A., Nogee, L.M. and Whitsett, J.A. (1995) Aberrant processing of surfactant protein C in hereditary SP-B deficiency. Am. J. Physiol., 268, L647–L656.
39 Nogee, L.M., de Mello, D.E., Dehner, L.P. and Colten, H.R. (1993) Brief report: deficiency of pulmonary surfactant protein B in congenital alveolar proteinosis. N. Engl. J. Med., 328, 406–410.
40 Lin, S., Akinbi, H.T., Breslin, J.S. and Weaver, T.E. (1996) Structural requirements for targeting of surfactant protein B (SP-B) to secretory granules in vitro and in vivo. J. Biol. Chem., 271, 19689–19695.
41 Lin, S., Phillips, K.S., Wilder, M.R. and Weaver, T.E. (1996) Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B). Biochim. Biophys. Acta, 1312, 177–185.[Medline]
42 O’Reilly, M.A., Weaver, T.E., Pilot-Matias, T.J., Sarin, V.K., Gazdar, A.F. and Whitsett, J.A. (1989) In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors. Biochim. Biophys. Acta, 1011, 140–148.[Medline]
43 Hawgood, S., Latham, D., Borchelt, J., Damm, D., White, T., Benson, B. and Wright, J.R. (1993) Cell-specific posttranslational processing of the surfactant-associated protein SP-B. Am. J. Physiol., 264, L290–L299.
44 Guttentag, S.H., Beers, M.F., Bieler, B.M. and Ballard, P.L. (1998) Surfactant protein B processing in human fetal lung. Am. J. Physiol., 275, L559–L566.
45 Malhotra, R., Haurum, J., Thiel, S. and Sim, R.B. (1992) Interaction of C1q receptor with lung surfactant protein A. Eur. J. Immunol., 22, 1437–1445.[Web of Science][Medline]
46 Haagsman, H.P. (1998) Interactions of surfactant protein A with pathogens. Biochim. Biophys. Acta, 1408, 264–277.[Medline]
47 Parodi, A.J. (2000) Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem. J., 348, 1–13.
48 Zhang, Q. and Salter, R.D. (1998) Distinct patterns of folding and interactions with calnexin and calreticulin in human class I MHC proteins with altered N-glycosylation. J. Immunol., 160, 831–837.
49 Mehta, A., Lu, X., Block, T.M., Blumberg, B.S. and Dwek, R.A. (1997) Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion. Proc. Natl Acad. Sci. USA, 94, 1822–1827.
50 Veldhuizen, R.A., Hearn, S.A., Lewis, J.F. and Possmayer, F. (1994) Surface-area cycling of different surfactant preparations: SP-A and SP-B are essential for large-aggregate integrity. Biochem. J., 300, 519–524.
51 Hawgood, S., Benson, B.J., Schilling, J., Damm, D., Clements, J.A. and White, R.T. (1987) Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28–36 in surfactant lipid adsorption. Proc. Natl Acad. Sci. USA, 84, 66–70.
52 Hawgood, S., Derrick, M. and Poulain, F. (1998) Structure and properties of surfactant protein B. Biochim. Biophys. Acta, 1408, 150–160.[Medline]
53 Ogawa, A., Brown, C.L., Schlueter, M.A., Benson, B.J., Clements, J.A. and Hawgood, S. (1994) Lung function, surfactant apoprotein content, and level of PEEP in prematurely delivered rabbits. J. Appl. Physiol., 77, 1840–1849.
54 Hallman, M., Merritt, T.A., Akino, T. and Bry, K. (1991) Surfactant protein A, phosphatidylcholine, and surfactant inhibitors in epithelial lining fluid. Correlation with surface activity, severity of respiratory distress syndrome, and outcome in small premature infants. Am. Rev. Respir. Dis., 144, 1376–1384.[Web of Science][Medline]
55 Tiret, L., Bonnardeaux, A., Poirier, O., Ricard, S., Marques-Vidal, P., Evans, A., Arveiler, D., Luc, G., Kee, F. and Ducimetiere, P. (1994) Synergistic effects of angiotensin-converting enzyme and angiotensin-II type 1 receptor gene polymorphisms on risk of myocardial infarction. Lancet, 344, 910–913.[Web of Science][Medline]
56 Alvarez, R., Reguero, J.R., Batalla, A., Iglesias-Cubero, G., Cortina, A., Alvarez, V. and Coto, E. (1998) Angiotensin-converting enzyme and angiotensin II receptor 1 polymorphisms: association with early coronary disease. Cardiovasc. Res., 40, 375–379.
57 Fleiss, J.L. (1981) Statistical Methods for Rates and Proportions. John Wiley and Sons, New York, NY, pp. 160–187.
58 Mehta, C. and Patel, N. (1996) StatXact3 For Windows. Statistical Software for Exact Nonparametric Inference. User Manual. Cytel Software, Cambridge, MA, pp. 447–474.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. M. Pastva, J. R. Wright, and K. L. Williams Immunomodulatory Roles of Surfactant Proteins A and D: Implications in Lung Disease Proceedings of the ATS, July 1, 2007; 4(3): 252 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Garbrecht, T. J. Schmidt, Z. S. Krozowski, and J. M. Snyder 11beta-Hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites Am J Physiol Endocrinol Metab, April 1, 2006; 290(4): E653 - E660. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Clement and committee members Task force on chronic interstitial lung disease in immunocompetent children Eur. Respir. J., October 1, 2004; 24(4): 686 - 697. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rova, R. Haataja, R. Marttila, V. Ollikainen, O. Tammela, and M. Hallman Data mining and multiparameter analysis of lung surfactant protein genes in bronchopulmonary dysplasia Hum. Mol. Genet., June 1, 2004; 13(11): 1095 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Marttila, R. Haataja, S. Guttentag, and M. Hallman Surfactant Protein A and B Genetic Variants in Respiratory Distress Syndrome in Singletons and Twins Am. J. Respir. Crit. Care Med., November 15, 2003; 168(10): 1216 - 1222. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||