Background
Idiopathic interstitial pneumonias (IIPs) are a group of diffuse parenchymal lung diseases characterized by interstitial involvement resulting from various patterns of inflammation and fibrosis of unknown cause. The prevalence of IIPs has been generally reported as 5–20 per 100,000 persons [
1‐
4], with a recommendation that the diagnosis of IIPs be made according to clinical history, physical findings, chest radiographs, and/or lung function tests [
5,
6]. However, some patients may not complain of symptoms or present with abnormal chest radiographs and/or lung function tests, even though they already suffer from IIPs. Therefore, the availability of diagnostic tools that can discriminate patients with IIPs from healthy subjects (HS) at an early stage will be undoubtedly useful in clinical practice. In this regard, serum biomarkers draw particular interest because they are easy to obtain from patients.
Pulmonary surfactant protein A (SP-A) and SP-D are water-soluble proteins derived mainly from type II pneumocytes and belong to the collectin subgroup of the C-type lectin superfamily [
7]. Because one of the key histological feature of the lung affected with interstitial lung diseases (ILDs) involves injury and/or regeneration of Type II pneumocytes [
8], soluble proteins derived from Type II pneumocytes, such as SP-A, SP-D, and Krebs von den Lungen 6 (KL-6), have been studied as potential biomarkers for ILDs [
9‐
15]. These biomarkers can be useful for early detection of ILDs, predicting disease outcome, and monitoring the clinical course [
16‐
18]. On the basis of these findings, serum SP-A, SP-D, and KL-6 have been clinically approved by Japan’s Health Insurance Program as diagnostic markers for ILDs in 1999, and more than 2,000,000 samples of these biomarkers are now examined yearly in Japan. However, in most countries, assays for these biomarkers are limited to research and are currently unavailable for clinical practice.
We recently conducted an international study to measure the serum levels of KL-6 and analyze the rs4072037 genotypes of Mucin 1 (
MUC1) in German and Japanese cohorts that included patients with ILDs and healthy subjects. We demonstrated that the cutoff value of KL-6 that discriminated patients with ILDs from HS was significantly higher in the German than in the Japanese cohort because of differences in the distribution of rs4072037 genotypes between them [
19]. The correlations between rs4072037 genotypes and serum KL-6 levels have also been demonstrated in a Dutch cohort [
20]. Moreover, serum SP-D levels were found to be correlated with genetic polymorphisms of surfactant protein D (
SFTPD) [
21‐
23]. According to the International HapMap project [
24], the genotype distributions of some single-nucleotide polymorphisms (SNPs) in the
SFTPD gene differ according to ethnicities. We hypothesized, therefore, that differences exist in serum SP-D and/or SP-A levels between different ethnic populations. To test this hypothesis, we first measured serum SP-A and SP-D levels and compared them between German and Japanese cohorts that included patients with IIPs and healthy subjects. Next, we evaluated the correlations between serum SP-D levels and
SFTPD gene polymorphisms in the German and Japanese cohorts.
Discussion
In the present study, we demonstrated that serum SP-A and SP-D levels were significantly higher in patients with IIPs than in HS in both German and Japanese subjects. Moreover, we found that in patients with IIPs and HS, serum SP-D levels were significantly higher in the German than in the Japanese cohort, whereas there were no significant differences in serum SP-A levels between the two cohorts. The genotype distributions of SNPs in the SFTPD gene, which affect the serum SP-D levels, differed between the cohorts. Furthermore, multivariate analyses demonstrated that there were statistically independent correlations between serum SP-D levels and the rs721917, rs1998374, and rs2243639 genotypes of the SFTPD gene, regardless of ethnicity and presence of IIPs.
The significantly higher serum SP-A and SP-D levels in patients with IIPs compared with those of the German HS suggest their utility as diagnostic biomarkers for IIPs, even in the German population. Serum SP-A levels in the German patients with IIPs or HS were comparable to those in their Japanese counterparts (Figure
2A); however, the serum SP-D levels in German patients with IIPs or HS were significantly higher than those in their Japanese counterparts (Figure
2B). These results imply that serum SP-D levels are affected by the different ethnicity, whereas serum SP-A levels are not. In agreement with our present study results, serum SP-D levels in Caucasian HS were reported to be higher than those in Asian HS [
32].
To explain the differences in serum SP-D levels between the German and Japanese cohorts, we determined the relationship between serum SP-D levels and genotypic differences in the
SFTPD gene. We found that among the SNPs in
SFTPD gene, rs721917, rs1998374, and rs2243639, but not rs3088308, affected the serum SP-D levels (Figure
3), and the distributions of these polymorphisms were different between the German and Japanese cohorts. SP-A and SP-D molecules comprise an N-terminal domain (NTD), a collagen domain, an α-helical neck region, and a carbohydrate recognition domain (CRD; Figure
1) [
33]. SP-D polypeptide chains bind together through interpolypeptide disulfide bonds in the NTD to form oligomers, and the degree of oligomerization is affected by the genotypes of coding SNP rs721917 in the NTD. Thus, the T/T and C/C genotypes correlate with higher and lower-order oligomers, respectively [
33‐
36]. These structural variations of SP-Ds might affect their serum levels, and our results demonstrate that serum SP-D levels differed according to the rs721917 genotype (Figure
3A). Furthermore, we also found that serum SP-D levels were affected by the genotypes of rs1998374 and rs2243639, both of which are located in the collagen domain (Figures
1,
3B and C), but they were not affected by the genotypes of rs3088308, the coding SNP in the CRD (Figures
1 and
3D). These findings suggest the possibility that the collagen domain is also associated with the degree of oligomerization of the SP-D molecule.
To determine whether serum SP-D levels were independently correlated with
SFTPD gene polymorphisms and/or ethnicity, we performed multivariate regression analyses and found that the correlations between the genotypes of three SNPs in the
SFTPD gene and serum SP-D levels remained statistically significant in the multivariate models (Table
3). In contrast, the correlations between ethnicity and serum SP-D levels were insignificant (Table
3). These findings suggest that serum SP-D levels are more strongly affected by
SFTPD gene polymorphisms than by ethnicity. Therefore, the difference in serum SP-D levels that were observed between German and Japanese cohorts might be partially explained by the differences in the frequencies of
SFTPD gene polymorphisms between the cohorts.
In contrast, we found that the genotype distributions of four SNPs in the
SFTPD gene did not differ between patients with IIPs and HS in both cohorts (Table
2). SP-A and SP-D play important roles in surfactant-related functions and in host defense against inhaled pathogens [
37]. Moreover,
SFTPD gene polymorphisms, rs721917 in particular, have been reported to correlate with susceptibility to COPD, community-acquired pneumonia, ILDs, and lung cancer [
23,
38‐
41]. As discussed above, rs721917 is known to be associated with the degree of oligomerization of the SP-D molecules [
33‐
36]. This difference in oligomerization might affect the surfactant and/or host defense functions of SP-D and thus correlate with susceptibility to various respiratory diseases. In the present study, however, no significant correlation between
SFTPD gene polymorphisms and susceptibility to IIPs was demonstrated. We believe that a larger sample size of study is needed to determine the correlations between
SFTPD gene polymorphisms and susceptibility to IIPs.
We are aware that there are some limitations in this study. First, age, gender, and smoking status were significantly different between German HS and Japanese HS because the populations who undergo health checkup were different between Germany and Japan. We performed linear regression analysis to assess the interference between these factors and serum SP-D levels, and we found a significant correlation between age and serum SP-D levels. Thus, we included age into the multivariate analyses and confirmed that our results were significant and independent of age differences. Second, the number of subjects available for genomic analyses was relatively small. Third, only German and Japanese populations were studied. It remains unclear whether the findings of the present study can be applied to other ethnic groups such as African Americans.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YH drafted and finalized the manuscript, performed part of the serum measurement, extraction of DNA, genotyping, and statistical analyses. NH, NI, NK and UC conceived the study, and participated in its design and coordination and helped to draft and finalize the manuscript. ST performed part of the extraction of DNA and genotyping. FB, JG and SO recruited the study subjects, ascertained diagnosis, and helped to draft and finalize the manuscript. All authors read and approved the final manuscript.