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Rheumatology Advance Access originally published online on February 8, 2008
Rheumatology 2008 47(3):289-291; doi:10.1093/rheumatology/kem355
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Genetic polymorphisms in the surfactant proteins in systemic sclerosis in Japanese: T/T genotype at 1580 C/T (Thr131Ile) in the SP-B gene reduces the risk of interstitial lung disease

Y. Sumita1, T. Sugiura2, Y. Kawaguchi2, S. Baba2, M. Soejima2, Y. Murakawa1, M. Hara2 and N. Kamatani2

1Third Department of Internal Medicine, Faculty of Medicine, Shimane University, Shimane and 2Institute of Rheumatology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.

Correspondence to: Y. Kawaguchi, Institute of Rheumatology, Tokyo Women's Medical University School of Medicine, 10-22 Kawada-cho, Shinjuku-ku, Tokyo 162-0054, Japan. E-mail: y-kawa{at}ior.twmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. To investigate the association between single-nucleotide polymorphisms (SNPs) in the pulmonary surfactant protein (SP) genes and the presence or absence of interstitial lung disease (ILD) in SSc patients.

Methods. We studied 127 Japanese patients with SSc and 206 normal subjects. Investigated SNPs were C/T within amino acid (aa) 219, Arg219Trp in the SP-A1 gene (rs4253527), C/T within aa 131 (at nucleotide 1580) and Thr131Ile of the SP-B gene (rs1130866). Genotypes were determined by the TaqMan method.

Results. Genotype frequencies were not different between the SSc patients and normal controls for both loci. The patients were subsequently divided into two groups based on presence or absence of ILD. In the SNP in the SP-B gene, the frequency of the T/T genotype was significantly lower in the patients with ILD than in those without ILD. Limited in the patients who were positive for anti-Scl-70 antibody, the difference in the frequency of the T/T genotype between the ILD-positive and ILD-negative groups became more apparent. On the other hand, in the SNP in the SP-A1 gene, there was no significant skewing for a certain genotype.

Conclusion. In SSc, where massive fibrosis occurs, possession of the T/T genotype in the SP-B gene would reduce the risk of ILD in Japanese.

KEY WORDS: Systemic sclerosis, Interstitial lung disease, Genetic polymorphism, Surfactant protein


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
SSc is a generalized connective tissue disorder and affects at least three components: microcirculation, the immune system and fibroblasts. Fibroblasts obtained from SSc patients present an activated phenotype synthesizing increased amounts of collagen [1]. Although patients with SSc exhibit common symptoms, such as Raynaud's phenomenon, autoantibody production and skin fibrosis, the disease expression and prognosis are different in patients. SSc is clinically divided into two major subtypes: dcSSc and lcSSc [1]. The dcSSc subtype is associated with visceral fibrosis, which affects the lungs, heart and gastrointestinal tract with high frequency and is characterized by the possession of anti-Scl-70 (topoisomerase I) antibody. In lcSSc, the dominant clinical feature is vascular manifestation and the representative antibody is anti-centromere.

In visceral involvements of SSc, interstitial lung disease (ILD) is one of the most important complications because it occurs in over 50% of patients and is a major cause of death [2]. Although ILD has been associated with possession of anti-Scl-70 antibody [3] and disease subtype dcSSc, we speculated that some genetic factors other than clinical parameters might influence individual susceptibility to ILD in SSc patients.

Surfactant proteins (SPs), including SP-A, -B, -C and -D, are specifically produced from alveolar type II cells, contribute to lowering of the surface tension of the alveolus, and act as protective agents from pulmonary injury [4]. Deficiency of SPs or derangement of surfactant activity can cause severe respiratory disorders such as congenital alveolar proteinosis [5]. The SP genes are polymorphic [6], and some genetic variants, especially within the SP-A and -B genes, have been associated with individual variability in susceptibility to various pulmonary diseases [7, 8].

In this study, we investigated whether genetic polymorphisms in the SP-A and -B genes influenced the presence or absence of ILD in Japanese SSc patients. Investigated single-nucleotide polymorphisms (SNPs) were nucleotide (nt) 1580 C/T (Thr131Ile) in the SP-B gene and C/T within amino acid (aa) 219 (Arg219Trp) in the SP-A1 gene, which characterizes the 6A4 haplotype of the SP-A1 gene.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients
We studied 127 SSc patients (115 females, 12 males) who were followed at the Institute of Rheumatology, Tokyo Women's Medical University, and 206 healthy controls (150 females, 56 males) with their informed consent. All the subjects were Japanese, and all the patients met the American College of Rheumatology criteria for the diagnosis of SSc [9]. The clinical subtypes of dcSSc and lcSSc were defined according to the classification of LeRoy and Medsger [1]. ILD was diagnosed according to findings of high-resolution computed tomography of the chest.

The study was approved by the Genome-Ethics Committee of Tokyo Women's Medical University. All subjects gave their written informed consent before participating.

Determination of the SP-A and -B gene polymorphisms
Genotypes for both C/T within aa 219 of the SP-A1 gene (CGG/Arg to TGG/Trp change, rs4253527) and C/T at nt 1580 (within aa 131, ACT/Thr to ATT/Ile change, rs1130866) for the SP-B gene were determined by the TaqMan method using specific primers and fluorescently labelled probes designed by Applied Biosystems (Foster City, CA, USA). Primers for the SP-A1 and -B genes were as follows: SP-A1 forward 5'-TGGAGACTTCCGCTACTCAGA-3'; SP-A1 reverse 5'-CACTGCCCATCTGTGTACATCTC-3'; SP-B forward 5'-CCCTGGTCATCGACTACTTCCA-3' and SP-B reverse 5'-GCAGGAGGTGAGCTTGCA-3'. Probes for the SP-A1 and -B genes were as follows: SP-A1 allele C 5'-VIC-CTTTTCCCCGACCTGC-MGB-3'; SP-A1 allele T 5'-FAM-TTTTCCCCAACCTGC-MGB-3'; SP-B C allele 5'-VIC-CCCTCACAGTCTGGT-MGB-3' and SP-B T allele 5'-FAM-CCCTCACAATCTGGT-MGB-3'. The two SNPs were not genetically related (not in linkage disequilibrium).

Statistical analysis
To compare the allelic frequencies between two groups, Fisher's exact probability test was used. Differences were considered to be significant at P < 0.05. Odds ratios (ORs) for susceptibility to ILD in carriers of a specific genotype or allele were determined. The 95% CIs for ORs were also calculated.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Acknowledgements
 References
 
Characteristics of the study subjects
The mean age of the patients was 52.6 ± 13.3 yrs, and 115 of the 127 patients (90.6%) were female. Anti-nuclear antibody was detected in 96.1% of the patients. Anti-Scl-70 antibody was detected in 36.2% of the patients, while anti-centromere antibody was positive in 22.8% of the patients. Sixty-eight patients (53.5%) were defined as dcSSc, while the remaining 59 patients (46.5%) were lcSSc. The prevalence of ILD in the present study group was 62.2%.

Involvement of the SP-A1 and -B gene polymorphisms in the susceptibility to ILD in SSc patients
In healthy Japanese subjects (n = 206), the frequencies of C/C, C/T and T/T genotypes in the SP-A1 gene were 59.7, 36.4 and 3.9%, respectively, while those in SSc patients were 62.2, 32.3 and 5.5%, respectively. There were no differences in the genotype distributions between the two groups. In the SP-B gene, the frequencies of C/C, C/T and T/T genotypes were 47.6, 46.1 and 6.3%, respectively in healthy controls, while those in SSc patients were 53.5, 38.6 and 7.9%, respectively. There also were no differences in the distributions.

SSc patients were then divided into two categories, according to the presence or absence of ILD, and the frequencies of genotypes were compared. In the SP-A1 gene (Table 1), the distributions of three genotypes were significantly different (P = 0.015) between the two groups. However, this difference disappeared when the patients were sorted by the presence of ant-Scl-70 antibody or disease subtype of dcSSc (data not shown). There was no significant skewing for a certain genotype in the SP-A1 gene.


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  		TABLE 1. Genotype distribution of the SP-A1 gene in SSc patients with or without ILD

 
In the SP-B gene (Table 2), the distributions of three genotypes were also significantly different (P = 0.0091) between the two groups, with or without ILD. Furthermore, the frequency of the T/T genotype appeared to be significantly lower in the ILD-positive group compared with that in the ILD-negative group (2.5% vs 16.7%; P = 0.0062, OR = 0.13, 95% CI = 0.026, 0.64). Therefore the T/T genotype of the SNP in the SP-B gene was inversely associated with ILD. When the patients were sorted by the possession of anti-Scl-70 antibody (n = 46), none of the 32 patients with ILD had the T/T genotype, whereas 5 (35.7%) of the 14 patients without ILD carried the T/T genotype, and the difference was statistically significant (P = 0.0015, OR = 0.027, 95% CI = 0.0013, 0.53). Next, the patients were sorted by disease subset of dcSSc (n = 68), and the frequencies were compared. The frequency of T/T genotype was 2.2% in ILD-positive patients and 17.4% in ILD-negative patients. Although the difference between the two groups in dcSSc patients was statistically significant (P = 0.041), the OR of 0.11 would be controversial (95% CI = 0.011, 1.03).


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  		TABLE 2. Genotype distribution and allele frequency of the SP-B gene in SSc patients with or without ILD

 
Furthermore, in the anti-Scl-70 antibody-positive group, the frequency of the T allele was significantly lower in patients with ILD (16 of 64 chromosomes, 25%) than in those without ILD (14 of 28 chromosomes, 50%; P = 0.029, OR = 0.33, 95% CI = 0.13, 0.85) (Table 2).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we have demonstrated that the T/T genotype at nt 1580 in the SP-B gene was inversely correlated with the complication of ILD in SSc. Our findings suggest that the SP-B protein with the T/T genotype could protect against the development of ILD in patients with SSc. Interestingly, this phenomenon was statistically emphasized in the examination of SSc patients with anti-Scl-70 antibody. Being limited in anti-Scl-70 antibody-positive patients, not only the T/T genotype, but the T allele was also inversely correlated with susceptibility to ILD.

Possession of anti-Scl-70 antibody and disease subtype dcSSc had been associated with susceptibility to ILD in SSc, whereas anti-centromere antibody reduced the risk [10]. Several recent studies have indicated that genetic polymorphisms in the genes, such as IL-1{alpha} [11] or β [12], which are susceptible genes for SSc, influence the predisposition to ILD in SSc. This study showed that genetic polymorphisms that can modulate pulmonary defence also influence susceptibility to ILD in SSc. We sometimes encountered patients who had not been complicated with ILD for a long time, even though they had anti-Scl-70 antibody. Genetic polymorphisms in SPs might explain some parts of this discrepancy. Additionally, by logistic regression analysis by the stepwise method, both T/T genotype in the SP-B gene and possession of anti-centromere antibody were independently associated with the absence of ILD in this study.

This is a case–control study, and the sample size is relatively small. Furthermore, the frequency of T/T genotype in the SP-B gene, the protective genotype from ILD, is low in Japanese (6.3% in the healthy population). These factors would reduce the power of statistical analysis of this study. Another cohort study with a larger sample size or from a different ethnicity is required to overcome these limitations. Interestingly, our results were consistent with previous reports of other pulmonary diseases among different ethnicities [7, 8, 13]. The observation of the advantage of the T allele and disadvantage of the C allele was conserved among various lung diseases from different ethnicities.

SPs do not only have surface tension-lowering characteristics in the alveolus, but also influence the activity of alveolar macrophages, modulating local immune responses. For example, SPs suppress production of inflammatory cytokines from lipopolysaccharide-activated alveolar macrophages [14]. In SSc, alveolar macrophages are phenotypically altered and play an important role in the fibrotic process [15]. Therefore, genetic polymorphisms that alter protein function of SPs can influence the individual susceptibility to ILD in SSc. Because the SNP nt 1580 C/T at the end of exon 4 of the SP-B gene was associated with various lung diseases [7, 8, 13], functional meaning of the SNP has been studied. The C–T substitution causes a Thr–Ile change at aa 131, which can block potential N-linked glycosylation sites, Asn at aa 129 [16]. Wang et al. [17] showed that a stable transfectant with C allele was indeed glycosylated at Asn129-Gln-Thr131, while that of the T allele was not. N-linked glycosylation would interfere with SP-B processing, secretion and folding, resulting in modulating protein levels or functions of SP-B protein. In healthy men, however, there was no association between individual pulmonary function and genotype at nt 1580 C/T in the SP-B gene [18]. We speculate that the SNP in the SP-B gene influences immune response in the lung under certain disease conditions such as SSc.

There are considerable ethnic differences in allele frequency of nt 1580 C/T of the SP-B gene. The frequency of the C allele is reported to be ~0.3 in black subjects and around 0.5 in white and Hispanic subjects [19]. Our data showed that the frequency of the C allele was 0.73 (n = 203) in the Japanese population, higher than in other ethnicities. Taking the functional significance of the SNP into consideration, it is plausible that Japanese people are at a genetic disadvantage for pulmonary defence.

The human SP-A locus includes two linked functional genes, SP-A1 and -A2 [20]. The SP-A1 gene has five haplotypes (6A1, 6A2, 6A3, 6A4 and 6A5), all of which are composed of combinations of SNPs located in the coding sequences [6]. In idiopathic pulmonary fibrosis in adults, 6A4 was associated with disease risk in smokers [8]. Haplotype 6A4 is characterized by SNP within aa 219, and the Arg–Trp substitution is suggested to alter SP-A protein function [8]. In the present study group, although the genotype distributions of the SP-A1 gene were different between the patients with and without ILD, the contribution of a certain genotype to susceptibility to ILD was not determined.

In conclusion, carrying the T/T genotype at SP-B gene would provide a beneficial role, reducing the risk of ILD in Japanese SSc patients.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
References
 
Funding: This study was supported by a research grant from the Ministry of Health, Labor, and Welfare in Japan.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. LeRoy EC, Medsger TA Jr. Criteria for the classification of early systemic sclerosis. J Rheumatol (2001) 28:1573–6.[Abstract/Free Full Text]
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  7. Lin Z, Pearson C, Chinchilli V, et al. Polymorphisms of human SP-A, SP-B, and SP-D genes: association of SP-B Thr131Ile with ARDS. Clin Genet (2000) 58:181–91.[CrossRef][Web of Science][Medline]
  8. Selman M, Lin HM, Montano M, et al. Surfactant protein A and B genetic variants predispose to idiopathic pulmonary fibrosis. Hum Genet (2003) 113:542–50.[CrossRef][Web of Science][Medline]
  9. Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheumatol (1980) 23:581–90.[CrossRef]
  10. McNearney TA, Reveille JD, Fischbach M, et al. Pulmonary involvement in systemic sclerosis: associations with genetic, serologic, sociodemographic, and behavioral factors. Arthritis Rheumatol (2007) 57:318–26.[CrossRef]
  11. Kawaguchi Y, Tochimoto A, Ichikawa N, et al. Association of IL1A gene polymorphisms with susceptibility to and severity of systemic sclerosis in the Japanese population. Arthritis Rheumatol (2003) 48:186–92.[CrossRef]
  12. Beretta L, Bertolotti F, Cappiello F, et al. Interleukin-1 gene complex polymorphisms in systemic sclerosis patients with severe restrictive lung physiology. Hum Immunol (2007) 68:603–9.[CrossRef][Web of Science][Medline]
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  14. Raychaudhuri B, Abraham S, Bonfield TL, et al. Surfactant blocks lipopolysaccharide signaling by inhibiting both mitogen-activated protein and IkappaB kinases in human alveolar macrophages. Am J Respir Cell Mol Biol (2004) 30:228–32.[Abstract/Free Full Text]
  15. Hamilton RF Jr, Parsley E, Holian A. Alveolar macrophages from systemic sclerosis patients: evidence for IL-4-mediated phenotype changes. Am J Physiol Lung Cell Mol Physiol (2004) 286:L1202–9.[Abstract/Free Full Text]
  16. Jacobs KA, Phelps DS, Steinbrink R, et al. Isolation of a cDNA clone encoding a high molecular weight precursor to a 6-kDa pulmonary surfactant-associated protein. J Biol Chem (1987) 262:9808–11.[Abstract/Free Full Text]
  17. Wang G, Christensen ND, Wigdahl B, Guttentag SH, Floros J. Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease. Biochem J (2003) 369:179–84.[CrossRef][Web of Science][Medline]
  18. Raleigh SM, Davies BM, Cleal D, Ribbans WJ. No association between coding polymorphism within exon 4 of the human surfactant protein B gene and pulmonary function in healthy men. J Physiol Sci (2007) 57:199–202.[CrossRef][Web of Science][Medline]
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  20. Hoover RR, Floros J. Organization of the human SP-A and SP-D loci at 10q22-q23: physical and radiation hybrid mapping reveal gene order and orientation. Am J Respir Cell Mol Biol (1998) 18:353–62.[Abstract/Free Full Text]
Submitted 26 September 2007; revised version accepted 3 December 2007.
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This Article
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