MOLECULAR MEDICINE REPORTS 6: 631-638, 2012 Single-nucleotide polymorphisms in the TSPYL-4 and NT5DC1 genes are associated with susceptibility to chronic obstructive pulmonary disease YI GUO1*, YI GONG2*, GUOCHAO SHI1, KUN YANG1, CHUNMING PAN3, MIN LI1, QINGYUN LI1, QIJIAN CHENG1, RANRAN DAI1, LIANG FAN1 and HUANYING WAN1 1Department of Respiratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University (SJTU), School of Medicine; 2Department of Respiratory Medicine, Huashan Hospital, Fudan University; 3Ruijin Hospital, State Key Laboratory of Medical Genomics, Molecular Medicine Center, Shanghai Jiao Tong University (SJTU), School of Medicine, Shanghai 200025, P.R. China Received February 12, 2012; Accepted June 18, 2012 DOI: 10.3892/mmr.2012.964 Abstract. The risk of developing chronic obstructive pulmo- block. We constructed the TSPYL-4 and NT5DC1 haplo- nary disease (COPD) is partially determined by genetic types of the cases and controls, but no significant difference and environmental factors. Many published candidate gene between the two groups was found. rs3749893 A allele of studies show conflicting results due to ethnic differences TSPYL-4 and rs1052443 C allele of NT5DC1 were associated and sample sizes. The number of these studies carried out in with a protective effect against the deterioration of pulmonary Chinese populations is small. To investigate candidate genes function. In conclusion, TSPYL-4 and NT5DC1 gene poly- and haplotypes for susceptibility to COPD in a southern morphisms are associated with susceptibility to COPD and Han Chinese population, we performed genotyping of DNA pulmonary function. samples in 200 COPD patients and 250 control subjects by analyzing 54 single-nucleotide polymorphisms (SNPs) in Introduction 23 genes associated with the development of COPD and/or pulmonary function identified by genome-wide association Chronic obstructive pulmonary disease (COPD) is expected studies (GWAS). We also performed linkage disequilibrium to be the third leading cause of mortality and the fifth (LD) and haplotype analysis according to the results of geno- leading cause of morbidity by the year 2020 (1). The disease typing. The frequencies of the SNP [rs3749893 of testis-specific is mainly characterized by the presence of chronic airflow protein Y-encoded-like 4 (TSPYL-4) gene] G allele and SNP limitation that progresses slowly over a period of years and is [rs1052443 of 5'-nucleotidase domain containing 1 (NT5DC1) largely irreversible (2,3). In China, it is becoming an increas- gene] A allele were significantly higher in the cases studied ingly common problem. A survey of 20,245 participants in compared to the control subjects (P=0.032, P<0.05, OR=0.692, seven regions of China conducted in 2007 indicated that 95% CI 0.495-0.970; P=0.0205, P<0.05, OR=0.670, 95% CI the prevalence of COPD in adults aged over 40 years was 0.477-0.941, respectively). Results showed that two blocks of 8.2% (4). However, the disease remains under-recognized SNPs (rs1052443 and rs3749893; rs11155242 and rs6937121) and under-diagnosed, and we need to further understand the had sufficient precision to allow construction of a haplotype pathogenesis, particularly in the earlier mild and moderate stages of COPD. Although cigarette smoking is the major risk factor for COPD, only a minority (20%) of smokers develop the disease clinically (5). Hodge et al (6) revealed in their study that apoptosis of airway epithelial cells and inflam- Correspondence to: Professor Huanying Wan, Department of mation of the airway mucosa persisted even after smoking Respiratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University cessation in patients with COPD. (SJTU), School of Medicine, Shanghai 200025, P.R. China Evidence suggests that the risk of developing COPD is E-mail: [email protected] partially determined by genetic and environmental factors (7). *Contributed equally A number of candidate gene studies have therefore been carried out in recent years. Family studies and linkage analysis Key words: chronic obstructive pulmonary disease, single-nucleotide in early-onset COPD pedigrees have highlighted a genetic polymorphism, genome-wide association studies, genotype, predisposition (8-11), and genome-wide association studies testis-specific protein Y-encoded-like 4, 5'-nucleotidase domain (GWAS) for COPD or pulmonary function identified some containing 1 susceptibility loci (12-16), but with varying degrees of repro- ducibility. Conflicting results may be due to ethnic differences and sample sizes. 632 GUO et al: SNPs OF TSPYL-4 AND NT5DC1 IN COPD The number of these studies carried out in Chinese indi- showed deviation from the Hardy-Weinberg equilibrium viduals is small. Past candidate gene studies may focus on a (HWE): rs2070600, rs2395730, rs6830970, rs13147758, single gene or on a few genes in combination, with these genes rs17019336, rs2035901, rs10498230, rs6712954 and rs6734100. identified based on prior knowledge or suspected mechanisms Therefore, 24 SNPs were selected for the investigation. The of disease pathogenesis. Nonetheless, elucidating the genetics sequence information of these 24 SNPs is shown in Table II. of these disorders is severely hampered by genetic heteroge- neity, the low penetrance of individual disease alleles and the Genotyping by multiplex PCR. Genotyping was performed potential for gene-gene and gene-environment interactions. It by multiplex PCR, which was a variant of PCR enabling the is probable that groups of genes rather than single genes are simultaneous amplification of numerous targets of interest involved in disease development. in one reaction using more than one pair of primers (19). We The aim of the present study was to investigate candidate used Mass-array Assay Design 2.0 software to design multi- genes and haplotypes in susceptibility to COPD in a south Han plex primers: 1st-PCR primer, 2nd-PCR primer and UEP Chinese population. primer for each SNP; primers of the 24 SNPs are shown in Table II. Materials and methods Statistical analysis. P-values for genotype and allele frequen- Subjects. A total of 200 male COPD patients visiting the cies were obtained using the χ2 test with SPSS 13.0 software Department of Respiratory Disease of the Shanghai Ruijin (P<0.05). We excluded the SNPs in which MAF was <0.03. Hospital, China, between December 2008 and December 2009 The relative risk associated was estimated as an odds ratio (OR) were recruited. COPD was diagnosed according to the criteria with a 95% confidence interval (CI), as analyzed by the Woolf established by the NHLBI/WHO Global Initiative for COPD method. Each SNP was tested for deviation from HWE (http:// (GOLD) (17). Criteria were as follows: age ≥40 years; chronic ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). SNPs were respiratory symptoms and signs, such as cough and dyspnea; excluded from the analysis if they were out of HWE (P≤0.05). airflow limitation as indicated by forced expiratory volume Haplotype frequencies and linkage disequilibrium (LD) anal- in 1 sec (FEV1)/forced vital capacity (FVC) <70% and FEV1 ysis were evaluated using the Phase and Haploview software. reversibility after inhalation of 400 µg salbutamol to <12% of the pre-bronchodilator FEV1. Patients were excluded if they Results had a diagnosis of asthma, lung cancer or radiographic abnor- malities suggestive of other significant respiratory diseases, Study population characteristics. The study population char- such as bronchiectasis or pulmonary tuberculosis. acteristics for those subjects with successful genotyping are A total of 250 control male subjects were enrolled at the shown in Table III. Due to a lack of certain data, the case group General Health Check-up Center in Shanghai No. 10 Hospital comprised 160 subjects and the control group 177 subjects. The during the same period. Their characteristics were mentioned two groups were matched for age, gender and percentage of in a previous study (18). The cases and control subjects were smokers. FEV1 and FVC of the case group were significantly from an ethnic Chinese, southern Han population who resided decreased compared to the control group (P<0.05). in Shanghai City or the surrounding regions, and were matched for age, gender and smoking history. The study protocol was Result of genotyping. As a result of a quality control measure, approved by the medical ethics committee of Shanghai Ruijin a total of 24 SNPs were finally compared between the case Hospital, School of Medicine, Shanghai Jiaotong University, and control groups. The frequencies of the SNP [rs3749893 and all the participants gave written informed consent. of testis-specific protein Y encoded-like 4 (TSPYL-4) gene] G allele and SNP [rs1052443 of 5'-nucleotidase domain DNA extraction and genotyping of study samples. We collected containing 1 (NT5DC1) gene] A allele were significantly 4 ml of peripheral blood from each participant for DNA higher in the cases studies compared to the control subjects preparation. Genomic DNA was extracted using a Blood DNA (P=0.032, P<0.05, OR=0.692, 95% CI 0.495-0.970; P=0.0205, Extraction kit (Tiangen Biotech, Co. Ltd., Beijing, China). Any P<0.05, OR=0.670, 95% CI 0.477-0.941, respectively). The sample with a DNA concentration <10 ng/µl was excluded. details are shown in Table IV. In total, 54 single-nucleotide polymorphisms (SNPs) were found in 23 genes associated with the development of COPD Linkage disequilibrium and haplotype analysis. Using and/or pulmonary function, as identified by publications of Haploview, haplotype blocks were constructed separately previous GWAS and by searching the dbSNP database of NCBI according to the confidence interval method of Gabriel et al (Table I). Genotyping was performed using the Mass-Array™ (20) for the cases and controls. This method uses both an esti- Technology Platform of Sequenom Inc. (San Diego, CA, USA). mate of d' and a measure of its precision (confidence bounds) to As a result of a quality control measure, we excluded 30 SNPs: construct haplotype blocks (Fig.