key: cord-0006394-kiujd1py
authors: Heidinger, Kathrin; König, Inke R.; Bohnert, Anette; Kleinsteiber, Anja; Hilgendorff, Anne; Gortner, Ludwig; Ziegler, Andreas; Chakraborty, Trinad; Bein, Gregor
title: Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced
date: 2005-02-08
journal: Immunogenetics
DOI: 10.1007/s00251-005-0775-5
sha: 597c4a14d2cae1912fbc50b443dbc6a86d6263a3
doc_id: 6394
cord_uid: kiujd1py

The collectin surfactant protein-D (SP-D) plays a significant role in innate immunity. Epidemiological studies described associations between single nucleotide polymorphisms (SNPs) of the human gene coding surfactant protein-D (SFTPD) and infectious pulmonary diseases. Studies on twins indicated very strong genetic dependence for serum levels of SP-D. The aim of this study was to determine the genetic influence of sequence variations within the SFTPD gene on the constitutional serum SP-D levels. We sequenced the 5′ untranslated region (5′UTR), the coding region and the 3′ region of the SFTPD gene of 32 randomly selected blood donors. Six validated SNPs were genotyped with sequence-specific probes (TaqMan 7000) in 290 German blood donors. Serum SP-D levels were analysed by ELISA, and the association of SFTPD haplotype estimates with the quantitative phenotype serum SP-D level was determined. One single SFTPD haplotype (allele frequency 13.53%) revealed a negative association with serum SP-D levels (P<0.0001). This was confirmed in a second prospectively collected group of blood donors (n=160, P=0.0034). The discovery of a frequent negative variant of the SFTPD gene provides a basis for genetic analysis of the function of SP-D in the resistance against pulmonary infections and inflammatory disorders in humans.

Surfactant protein-D (SP-D) is a member of the collagenous subfamily of calcium-dependent lectins (collectins) that includes pulmonary surfactant protein-A (SP-A) and the serum protein mannose-binding lectin (MBL) (Crouch 1998; Wright 1997; Eggleton and Reid 1999) . Strong indications prove that the collectins play a significant role in innate immunity.

SP-D is involved in the innate response to inhaled microorganisms and contributes to immune and inflammatory regulation within the lung. SP-D binds to microbial surfaces and induces effector mechanisms such as aggregation and virus neutralisation, attraction and activation of phagocytes and promotion of phagocytosis (Crouch 1999) .

SP-D is an M r 43,000 glycoprotein (Persson et al. 1989 ) synthesized and secreted by pulmonary alveolar type II cells and nonciliated airway cells (Wong et al. 1996) . However, recent results indicate that SP-D in humans is widely distributed on mucosal surfaces (Madsen et al. 2000) .

The gene for S-PD, SFTPD, is located on the long arm of Chromosome 10q22.2-23.1, as are the genes for SP-A and MBL. Four coding SNPs of SFTPD have been described previously (Crouch et al. 1993) . One of the SFTPD polymorphism resides in the N-terminal region at position 11, where a methionine is exchanged for a threonine (position 31 from transcription start site). The three other coding variants are located in the collagen-encoding region.

Up to this present study, allelic variations have not been associated with the levels of SP-D in bronchoalveolar lavage fluid or in serum. However, data of a recently published study on twins indicate strong genetic dependence of serum SP-D levels. Biometric model fitting estimated the heritability of the serum SP-D level to 0.91 (Husby et al. 2002) .

Polymorphic marker loci for SFTPD also have been characterized (DiAngelo et al. 1999) , and a recent study on patients with tuberculosis indicates that the SFTPD allele threonine 11 (position 31 from transcription start site) is associated with the susceptibility to this disease (Floros et al. 2000) . Interestingly, the opposite SFTPD allele coding for methionine 11 was associated with severe respiratory syncytial virus bronchiolitis in infants (Lahti et al. 2002) .

Discovering that SFTPD (−/−) mice spontaneously develop emphysema with increased metalloproteinase activity, as well as increased levels of oxidants in the absence of infections (Wert et al. 2000) , suggests that changes in the level of lung collectins may account for the pathogenesis of various diseases. It is likely that the decreased levels of SP-D, which have been observed in different clinical situations, increase the risk of infections . Clinically, serum SP-D levels have been used as biomarkers for lower respiratory tract infections, acute lung injury and type II cell hyperplasia as seen in pulmonary fibrosis and other forms of interstitial lung diseases (Takahashi et al. 2000; Asano et al. 2001; Ohnishi et al. 2002) .

In this study, we aimed at examining the association between SFTPD SNPs and estimated haplotypes found within a general Caucasoid population and serum levels of the SP-D protein. Here we report the discovery of one single SFTPD haplotype with a highly significant negative association with serum SP-D levels.

A total of 32 healthy voluntary blood donors were randomly selected for resequencing of the SFTPD gene, including the 5′ and 3′ flanking regions. Six validated SNPs were genotyped with sequence-specific probes (TaqMan 7000) in 290 German healthy blood donors without any signs of pulmonary dysfunctions (age 19-68 years, mean 34 years, 207 male, 83 female) after they gave their informed consent. To confirm associations, we analysed a second prospectively collected group of 160 blood donors (age 18-64 years, mean 34 years, 88 male, 72 female). The study protocol was approved by the Medical Ethics Committee of the University of Giessen.

Blood samples were drawn in K 2 -ethylenediaminetetraacetic acid tubes (Sarstedt, Nümbrecht, Germany). The samples were centrifugated at 800 g for 10 min. Plasma and buffy coat were separated immediately. Plasma was stored in aliquots at −70°C and was defrosted in a 37°C water bath before use. DNA was extracted from peripheral blood leukocytes, using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn., USA) according to the manufacturer's instructions. DNA was dissolved in DNA hydration solution and stored at −70°C until use.

DNA sequence analysis DNA sequencing of 800 bp upstream of the ATG-translation initiation codon, the complete coding region, and the 3′ region of the SFTPD gene was performed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA), using an ABI Prism Big Dye Terminator DNA sequencing kit according to the manufacturer's instructions. Sequence alignment and SNP validation were analysed using SeqScape software (ABI).

We selected six SNPs with an allele frequency exceeding 10% in the German population. The validated SNPs were genotyped with sequence-specific probes (ABI PRISM 7000 Sequence Detection System, TaqMan). Genotyping primers and probes are listed in Tables 1 and 2. PCR was carried out in a volume of 25 μl containing 20 ng genomic DNA, forward and reverse primers in final concentrations of 900 nM, fluorescence-labelled, sequence-specific minor groove binders probes in final concentrations of 200 nM, and MasterMix Buffer (containing polymerase, MgCl 2 , dNTPs including dUTP and Fluorescence emission was measured on an ABI PRISM 7000 Sequence Detection System at 60°C. Known DNA samples of each genotype and water samples were used as positive and negative controls for each run.

The concentrations of SP-D in sera were measured by a specific ELISA kit, using mAbs against SP-D 7C6 and horseradish peroxidase (HRP)-conjugated mAb 6B2 and recombinant SP-D as a standard. The commercially available test kit was used for the measurement of SP-D according to the manufacturer's instructions (Yamasa, Tokyo, Japan). The 96-microwell plate coated with SP-D Abs was incubated with 100 μl of 11-fold diluted plasma at 4°C for 24 h. The wells were then washed and incubated at 25°C for 2 h, with 100 μl 111-fold diluted HRP-conjugated anti-SP-D Ab. The wells were washed again, and 100 μl substrate mixture (11 ml tetramethylbenzidine solution + 50 μl 1% H 2 O 2 ) was added. Incubation was then performed at 25°C for 15 min. Finally, 100 μl 1 N sulphuric acid was added to terminate the peroxidase reaction, and the absorbance at 450 nm was measured.

The quantitative serum SP-D levels were calculated from a standard curve, employing serial dilutions of recombinant SP-D and an analytical sensitivity of 1.56 ng/ml.

For description, median SP-D serum levels were compared between men and women, using the two-sided exact Mann-Whitney U-test. To evaluate deviation from the Hardy-Weinberg equilibrium, observed and expected genotype frequencies were compared by a Monte Carlo simulationbased goodness-of-fit test.

To investigate the relation between the quantitative serum SP-D level and different genotypes, median, mean SP-D level and standard deviation (SD) are reported. Descriptive exact P-values from a Kruskal-Wallis test are also reported.

Linkage disequilibrium between pairs of polymorphic positions was estimated adopting a Malecot model as implemented in LDMAP (Maniatis et al. 2002) .

Frequencies of haplotypes were estimated with the expectation-maximization algorithm (Excoffier and Slatkin 1995) . Median values of SP-D were compared across the estimated haplotype frequencies, using the score test with simulated P-values from 10 6 replications (Schaid et al. 2002) .

We screened the 5′UTR, the coding region and the 3′ region of the SFTPD gene for possible sequence variations in 32 healthy volunteers and validated six known SNPs. One nucleotide substitution was characterized in the 5′UTR (−759A>G), another substitution was found in the exon 2 (92A>G), which leads to an amino acid exchange from methionine to threonine at position 31. Furthermore, a nucleotide substitution was characterized in exon 5 (4694C>T), leading to an amino acid exchange from threonine to alanine at position 180. The others are located in the 3′UTR (11208A>G; 11384A>G; 11466G>A). Additional SNPs were not identified. These six SNPs were genotyped with sequence-specific probes (TaqMan 7000) in at least 248 German blood donors and, four of the six SNPs in a second independent group of at least 158 German blood donors. We determined the allele frequencies of these polymorphisms as shown in Table 3 . The rate of homozygosity and heterozygosity for all polymorphism showed no deviation from the Hardy-Weinberg equilibrium, indicating the state of random assortment. Separate analysis for each gender indicated that male subjects showed higher serum SP-D levels than females (descriptive, P=0.024). The mean serum SP-D level for male subjects was 89.56 ng/ml (SD, 48.18 ng/ml; median, 79.17 ng/ml; n=207), the mean serum SP-D level for females was 73.15 ng/ml (SD, 33.06 ng/ml; median, 66.99 ng/ml; n=83). The second group again revealed a trend towards higher serum SP-D levels in males (mean serum SP-D levels, 78.36 ng/ml; SD, 41.92 ng/ml; median, 70.93 ng/ml; n=88) than in females (mean serum SP-D level, 68.20 ng/ml; SD, 33.88 ng/ml; median, 60.93 ng/ml; n=72).

Differences in median serum levels of SP-D for carriers of SFTPD polymorphisms are shown in Table 4 . There was an association between two of the six examined SNPs (92, A→G and 11208, A→G) and serum levels of SP-D in the first cohort showing that, under a model of dominant inheritance, carriers of the G allele at position 92 had lower levels of serum SP-D compared to those who were homozygous for the A allele (Mann-Whitney U-test, P=0.001). Prospective analysis of the second independent group of blood donors confirmed the initial observation (Mann-Whitney U-test, P=0.031). In addition, carriers of the G allele at position 11208 showed higher median values of SP-D in serum (Mann-Whitney U-test, P=0.0039).

A comparable association between this polymorphism and the serum SP-D levels was also observed in the second group (Mann-Whitney U-test, P=0.027).

Analysis of the linkage disequilibrium units among the six SFTPD SNPs revealed that the genetic region consists of a single haplotype block, with high levels of LD towards the end (Table 5) .

Nine putative human SFTPD haplotypes with a frequency of at least 1% were estimated, of which the four most common possessed a cumulative frequency of 74.47% (Table 6) .

The association of putative SFTPD haplotype estimates with the quantitative phenotype serum SP-D level was calculated using a score test assuming ordinally scaled values. Overall, SP-D serum levels differed across the estimated haplotypes (Monte Carlo, P<0.0001). One single SFTPD haplotype, SFTPD*03 (allele frequency, 13.53%), revealed a negative association with serum SP-D levels (Monte Carlo, P<0.0001) as shown in Table 6 . We attempted to confirm this observation by analysing a second independent group of blood donors (n=160). Analysis of this second cohort confirmed the initial observation of the negative association of the SFTPD haplotype SFTPD*03 with the serum SP-D levels (Monte Carlo, P=0.0034) ( Table 7) .

Currently, there is a growing interest on the role of SP-D in host defense against infections. This present study has been the first to elucidate the genetic basis for the variation in the SP-D serum protein level. Associations between polymorphism of the SFTPD gene and serum SP-D levels were identified as well as nine putative frequent human SFTPD haplotypes, including one single SFTPD haplotype, SFTPD*03 (allele frequency 13.53%), which revealed a negative association with serum SP-D levels (P<0.0001).

Mean serum levels of SP-D in this study were in accordance with serum SP-D levels of healthy subjects from Caucasian populations in previously published studies using the same commercially available SP-D ELISA (Greene et al. 2002; Janssen et al. 2003) . In the Japanese population, lower mean serum levels of SP-D were reported (Nagae et al. 1997; Takahashi et al. 2000; Ihn et al. 2002; Greene et al. 2002) . A direct comparison of the serum SP-D levels among ethnic groups could be a matter of particular interest.

However, higher mean serum SP-D levels were reported in healthy subjects of Caucasian origin, employing another not-commercially available ELISA (Husby et al. 2002; Leth-Larsen et. al. 2003) .

Polymorphisms of the SFTPD gene were associated with the risk of severe respiratory syncytial virus (RSV) bronchiolitis in susceptible infants (Lahti et al. 2002) . In this study from an ethnic homogenous Finnish population, the SFTPD allele coding for methionine 11 (position 31 from transcription start site) was associated with severe RSV bronchiolitis in infants (P=0.033). Floros et al. (2000) reported association of the opposite SFTPD allele coding for threonine 11 (position 31 from transcription start site) with tuberculosis within the Mexican population (P= 0.002). Different alleles appear to be protective in the two studies. One possible reason for these discrepant results is that false-positive results have occurred by chance (type I error) (Colhoun et al. 2003) . The P-value from the study of Floros et al. (2000) would have been near or above 0.05 if it had been corrected for multiple testing and there were no replication in either study. Thus, the P-values reported in both studies are far greater than the P-values needed to assure a low posterior error rate (Colhoun et al. 2003; Freimer and Sabatti 2004; Thomas and Clayton 2004; Wacholder et al. 2004 ).

To our knowledge, we demonstrated and validated for the first time that a specific haplotype is significantly associated with decreased serum SP-D levels.

Serum concentrations of SP-D are largely determined genetically as shown by a recently published study on twins: model fitting showed that the estimated heritability was 0.91 (95% CI 0.83-0.95) for SP-D with additive genetic and non-shared environmental factors (Husby et al. 2002) . Based on the genotypic data, we used an expectation-maximization algorithm to estimate the frequencies of putative SFTPD haplotypes (frequency >1%) and have suggested a nomenclature according to haplotype frequency. Four common putative haplotypes with a cumulative frequency of >74% were identified.

We were able to define one single SFTPD haplotype, SFTPD*03 (allele frequency 13.53%), which revealed a negative association with serum SP-D levels. We conclude that this is not due to a possible effect of the missense variation at position 92, which may affect binding of the mAbs that were used for ELISA, based quantification of serum SP-D levels, since the G at position 92 is also included in several haplotypes without association to the serum SP-D level. Moreover, we were able to confirm the association of the haplotype SFTPD*03 with low SP-D serum levels in a second independent cohort of blood donors.

Serum SP-D has been shown in clinical investigations as a promising biomarker for acute lung injury and type II cell hyperplasia as seen in pulmonary fibrosis and other forms of interstitial lung disease (Takahashi et al. 2000; Asano et al. 2001; Ohnishi et al. 2002; Greene et al. 2002) . The elevation of SP-D is related to the extent of the parenchymal lung disease and predicts survival in idiopathic pulmonary fibrosis (Greene et al. 2002) . Serum SP-D is also increased in patients with acute ARDS (Greene et al. 1990 ) and patients with clinical pneumonia (Leth-Larsen et al. 2003) . In these conditions, elevated SP-D serum levels were partly accounted to leakage from the alveolar space, since alveolar type II cells are regarded as the main cellular source of SP-D. Although the lung is the major site of SP-D expression, it is likely that the protein has more generalized roles in host defence and the acute response to infection and tissue injury (Crouch 2000) .

In patients with acute pneumonia, the SP-D serum concentration may increase up to 22-fold within a few days (Leth-Larsen et al. 2003) . It is likely that decreased serum levels of SP-D which have been observed in different clinical situations are associated with the risk of infections : temporal changes in serum SP-D concentrations occur during infections, and infections superimposed on other pulmonary disease states may radically interfere with the outcome of cross-sectional measurements of serum SP-D concentrations.

In conclusion, here we describe a frequent SFTPD haplotype that is associated with low serum SP-D levels.

Since the investigated SNPs belong to a single haplotype block, we did not identify the 'functional' SNP that may affect the serum protein level by allele specific regulation of transcription or other mechanisms (Knight 2003) .

However, the genetic discovery of a frequent negative variant of the SFTPD gene provides an excellent basis for analysis of the function of SP-D in the resistance against pulmonary infections and inflammatory disorders in humans.

Clinical significance of surfactant protein D as a serum marker for evaluating pulmonary fibrosis in patients with systemic sclerosis

Problems of reporting genetic associations with complex outcomes

Collectins and pulmonary host defense

Modulation of host-bacterial interactions by collectins

Surfactant protein-D and pulmonary host defense

Genomic organisation of human surfactant protein D (SP-D)

SP-D is encoded on chromosome 10q22.2-23.1

Novel, non-radioactive, simple and multiplex PCR-cRFLP methods for genotyping human SP-A and SP-D marker alleles

Lung surfactant proteins involved in innate immunity

Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population

Surfactant protein genetic marker alleles identify a subgroup of tuberculosis in a Mexican population

The use of pedigree, sib-pair and association studies of common diseases for genetic mapping and epidemiology

Serial changes in surfactantassociated proteins in lung and serum before and after onset of ARDS

Serum surfactant proteins A and D as biomarkers in idiopathic pulmonary fibrosis

Collectins and ficolins: humoral lectins of the innate immune defense

Heritability estimates for the constitutional levels of the collectins mannan-binding lectin and lung surfactant protein D. A study of unselected like-sexed mono-and dizygotic twins at the age of 6-9 years

Clinical significance of serum surfactant protein D (SP-D) in patients with polymyositis/ dermatomyositis: correlation with interstitial lung disease

Study of Clara cell 16, KL-6, and surfactant protein-D in serum as disease markers in pulmonary sarcoidosis

Functional implications of genetic variation in non-coding DNA for disease susceptibility and gene regulation

Surfactant protein D gene polymorphism associated with severe respiratory syncytial virus infection

Surfactant protein D (SP-D) serum levels in patients with communityacquired pneumonia, filled

Localization of lung surfactant protein D on mucosal surfaces in human tissue

The first linkage disequilibrium (LD) maps: delineation of hot and cold blocks by diplotype analysis

Enzyme-linked immunosorbent assay using F(ab') 2 fragment for the detection of human pulmonary surfactant protein D in sera

Comparative study of KL-6, surfactant protein A, surfactant protein D, and monocyte chemoattractant protein-1 as serum markers for interstitial lung diseases

Purification and biochemical characterization of CP4 (SP-D), collagenous surfactant-associated protein

Score tests for association between traits and haplotypes when linkage phase is ambiguous

Serum levels of surfactant proteins A and D are useful biomarkers for interstitial lung disease in patients with progressive systemic sclerosis

Betting odds and genetic associations

Assessing the probability that a positive report is false: an approach for molecular epidemiology studies

Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice

Localization and developmental expression of surfactant proteins D and A in the respiratory tract of the mouse

Immunomodulatory functions of surfactant

Acknowledgements The authors thank Ms. Gabriela Haley and Ms. Uta Schellenberg for excellent technical assistance. This work was supported by grants from the Bundesministerium fuer Bildung und Forschung, Germany (NGFN 01GS0111). The experiments performed comply with the current laws of Germany.