Single-nucleotide polymorphisms in SMAD3 are associated with chronic obstructive pulmonary disease

Introduction

Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD); however, only approximately 15% of smokers develop clinically relevant airflow obstruction. ¹ Multiple studies in diverse populations have shown a large genetic contribution to the variability in pulmonary function and for the familial aggregation of COPD patients. ^2,3 As expected, segregation analysis suggests that multiple genes may be involved. At present, however, only a single gene, α1-antitrypsin, a potent inhibitor of inflammatory cell protease in the lung, has been unequivocally implicated in the development of COPD. ⁴ Recently, the associations between COPD and polymorphisms in several other genes have been studied, which include α1-antichymotrypsin, ⁵ microsomal epoxide hydrolase, ⁶ vitamin D-binding protein, ⁷ tumor necrosis factor-α, ⁸ SERPINE2 ⁹ and a-nicotinic acetylcholine receptor (CHRNA 3/5). ^10,11

Transforming growth factor-β (TGF-β) plays an important role in the pathogenesis of COPD. Signals from the activated TGF-β receptor complex are transduced to the nucleus of airway cells by SMAD proteins, which represent a family of transcription factors that have recently been implicated to play a major role as intracellular mediators of inflammation. The Smad family consists of the receptor-regulated Smads, a common pathway Smad, and inhibitory Smads. Receptor-regulated Smads are phosphorylated by the TGF-β type I receptor. They include Smad2 and Smad3, which are recognized by TGF-β and activin receptors, and Smads 1, 5, 8 and 9, which are recognized by bone morphogenetic protein receptors. Smad4 is a common pathway of Smad, which is also defined as co-operating Smad and is not phosphorylated by the TGF-β type I receptor. Inhibitory Smads (anti-Smads) include Smad6 and Smad7, which downregulate TGF-β signaling. To date, the Smads are the only TGF-β receptor substrates with a demonstrated ability to propagate signals, and with regard to the growing number of investigations of Smad-mediated effects in the airways, Smads may prove to be an important target for future development of new therapeutic strategies for asthma and COPD.

The human Smad3 gene has been assigned to chromosome 15q21–22 and spans over 5.7 kb with nine exons and eight introns. ^12–14 So far, data on the association between DNA polymorphisms in the Smad3 gene and COPD have not been reported. The current study was to screen for DNA sequence variants in the Smad3 gene in Chinese Hans – which account for 95% of the Chinese population – and to determine the association between the polymorphisms and COPD (Table 1).

To examine the interaction between a Smad3 gene polymorphism and COPD, we carried out a case-control study.

Materials and methods

Candidate single-nucleotide polymorphism selection and polymorphism genotyping

The single-nucleotide polymorphisms (SNPs) of Smad3 used for the test are shown in Figure 1.Their chromosomal location are illustrated in Figure 1. SNPs of Smad3 were extracted from the database of SNP (http://www.ncbi.nlm.nih.gov/SNP/index.html) (Figure 1).

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Figure 1

A schematic overview of the nine investigated single-nucleotide polymorphisms in the Smad3 gene

Results

Discussion

In COPD, a marked fibrosis of the airways is observed, which is partly mediated by TGF-β1 ¹⁸ that has been proposed to be a potent fibrogenic factor in numerous diseases. ¹⁹ The effects of the extensively studied TGF-β1 include fibroblast proliferation and an increased production and deposition of extracellular matrix proteins. Enzymes involved in extracellular matrix degradation and destruction can also be stimulated by TGF-β1. ^19–21 While much interest has focused on the TGF-β1-induced effects in COPD, little attention has been paid to the intracellular signal transduction pathways. The pathways of the TGF-β family include the family of transcription factors termed SMADs that are directly activated by the receptors. The most predominant member of the TGF superfamily in COPD, TGF-β1, mediates its effects by the regulatory SMAD2 and SMAD3 proteins, which are phosphorylated by the type I receptor after binding of TGF-β1. The phosphorylated SMADs bind the common SMAD4 and translocate into the nucleus. In contrast, the inhibitory SMAD6 and SMAD7 ^22,23 function as a negative feedback loop of the TGF-β-signal by either competing for SMAD4 or terminating the signal and therefore may represent important regulators of TGF-β1-induced effects.

The data above confirmed the central role of TGF-β and the Smad pathway for proceeding to progressive fibrosis. In these animals, the administration of interleukin-1β caused extensive inflammation and tissue damage in both the Smad3 null as well as wild-type mice. If anything, the Smad3 null mouse showed an even greater degree of inflammation. However, after 20 d, only the wild-type animal had progressed to fibrosis, whereas the Smad3 null mouse had no indication of fibrogenesis and was not markedly different from animals receiving only control vector. ²⁴ The Smad3 null mouse has been used to demonstrate that, in the absence of Smad3, the ALK5 receptor cannot transmit messages through to the nucleus and thus cannot upregulate matrix gene expression in either bleomycin- or TGF-β gene-mediated fibrotic stimulation. ^25,26 Taken together, these data indicate that only when there is an intact TGF-βRI signaling mechanism and intact Smad3 signaling can the progressive nature of fibrosis proceed.

In large genome-wide association studies on COPD, Dr Silverman and Spitz identified two SNPs at the CHRNA 3/5 locus, identified earlier as a risk factor for both lung cancer and nicotine dependence, to be associated with COPD. Smad3 plays an important role in the pathogenesis of COPD, and the CHRNA 3/5 gene and the Smad3 gene are both located in chromosome 15q. ^10,11 Taking these backgrounds into account, we hypothesize that SNPs in the Smad3 may render susceptibility to COPD, and thereby to contribute in predicting its occurrence during the course of the disease. Although introns were originally believed to be non-functional because they do not code for proteins, it has been suggested that some of these sequences do indeed have relevance. Introns have also been implicated in regulating gene expression and DNA–protein interactions; mutations in intron sequences may affect these functions. ²⁷ Otsuka et al. ²⁸ performed the tag SNP analysis using 111 atopic dermatitis (AD) families (384 members) in Japan. Thirty SNPs to cover the Smad3 gene region were chosen for genotyping using an Illumina GoldenGate assay. One intron SNP (rs4147358) was associated with AD in the pedigree disequilibrium test (PDT) and transmission disequilibrium test (TDT) analysis (significance level of P < 0.05). The rs4147358 was further genotyped in independent case-control samples and it was found that rs4147358 was associated with AD; the association was strengthened when the AD samples were limited to those of patients with IgE ≥250 IU/mL. In another case-control study, three intron SNPs of the Smad3 gene (rs2289790, rs2289791 and rs3825977) were found to be in association with diffuse cutaneous scleroderma in a UK Caucasian population. ²⁹ Just like COPD, the fibrosis induced by transforming growth factor ss-Smads signaling also plays a major role for pathogenesis of AD and diffuse cutaneous scleroderma. So in this study, we have investigated nine intron SNPs of the Samd3 gene in COPD patients and controls. And we only found rs28683050, as a known SNP in intron 1 of the Smad3 gene, has a significant difference in allelic or genotypic frequencies between the COPD patients and control subjects. Its frequencies of the TT genotype and T allele in the COPD patients were significantly higher than that in controls. Rs2289259 was not found to be in association with COPD, which was also not associated with AD in the PDT and TDT analysis. ²⁸ Contrary to the study by Pushpakom et al., ²⁹ rs3825977 was not associated with COPD in our study, and the different ethnic backgrounds may be one of the reasons.

In summary, we have performed a comprehensive investigation of Smad3 gene polymorphisms. The SNP showing significant differences may be useful for predicting COPD susceptibility or possible preventive intervention. No previous research reported the SNPs in the current study have a function, and the SNPs are not in spliced junction regions. The SMAD3 SNP (rs28683050) found to be associated with COPD in this study has no known function, and it is likely that the SNPs are in LD with other functional SNPs that determine disease susceptibility. In the current study, no extron SNP of SMAD3 was included. Considering they may also render susceptibility to COPD, further studies need to be conducted to clarify the association between extron SNPs of SMAD3 and COPD and the functional characterization of these SNPs.

Author contributions: TY, BY and FW participated in research design; TY, BY, XS and DL contributed to the performance of laboratory measurements; TY, BY, DX and TW were involved in data collection and analysis; and TY, BY, HF, SZ and FW wrote the manuscript. TY and BY contributed equally to this work.