LINC01414/LINC00824 genetic polymorphisms in association with the susceptibility of chronic obstructive pulmonary disease

Objective Chronic obstructive pulmonary disease (COPD) is a complicated multi-factor, multi-gene disease. Here, we aimed to assess the association of genetic polymorphisms in LINC01414/ LINC00824 and interactions with COPD susceptibility. Methods Three single nucleotide polymorphisms (SNPs) in LINC01414/LINC00824 was genotyped by Agena MassARRAY platform among 315 COPD patients and 314 controls. Logistic analysis adjusted by age and gender were applied to estimate the genetic contribution of selected SNPs to COPD susceptibility. Results LINC01414 rs699467 (OR = 0.73, 95% CI 0.56–0.94, p = 0.015) and LINC00824 rs7815944 (OR = 0.56, 95% CI 0.31–0.99, p = 0.046) might be protective factors for COPD occurrence, while LINC01414 rs298207 (OR = 2.88, 95% CI 1.31–6.31, p = 0.008) risk-allele was related to the increased risk of COPD in the whole population. Rs7815944 was associated with the reduced risk of COPD in the subjects aged > 70 years (OR = 0.29, p = 0.005). Rs6994670 (OR = 0.57, p = 0.007) contribute to a reduced COPD risk, while rs298207 (OR = 7.94, p = 0.009) was related to a higher susceptibility to COPD at age ≤ 70 years. Rs298207 (OR = 2.54, p = 0.043) and rs7815944 (OR = 0.43, p = 0.028) variants was associated COPD risk among males. Rs7815944 (OR = 0.16, p = 0.031) was related to the reduced susceptibility of COPD in former smokers. Moreover, the association between rs298207 genotype and COPD patients with dyspnea was found (OR = 0.50, p = 0.016), and rs7815944 was related to COPD patients with wheezing (OR = 0.22, p = 0.008). Conclusion Our finding provided further insights into LINC01414/LINC00824 polymorphisms at risk of COPD occurrence and accumulated evidence for the genetic susceptibility of COPD. Supplementary Information The online version contains supplementary material available at 10.1186/s12890-021-01579-3.


Introduction
Chronic obstructive pulmonary disease (COPD) is a severely disabling chronic lung disease. COPD is characterized by persistent airflow limitation of respiratory systems due to emphysema and obstructive bronchiolitis [1]. The airflow limitation is caused by the large exposure of lung to harmful particles or gases. At present, the high incidence of COPD exceeds 250 million, which is the third leading cause of death in the world, and it is estimated to cause 4 million deaths every year [2,3]. In China, COPD caused over 0.9 million deaths is related to several public health problems including pollution, an aging population, and smoking [4,5]. COPD is a complicated multi-factor, multi-gene disease. Several studies displayed that the occurrence of COPD is associated with various factors such as tobacco smoking, air pollution, pulmonary tuberculosis, occupational exposure and genetic factors [6]. Increasing evidence suggested that genetic polymorphisms exert an important role in COPD occurrence and development [7][8][9].
Long non-coding RNAs (lncRNAs) is one of the key members of ncRNA family, with greater than 200 nucleotides, participating in the regulators of genetic expression and regulation [10]. Recent study has demonstrated that lncRNAs could contribute to the pathogenesis of respiratory diseases, including COPD [11]. Abnormal expression or function of lncRNAs has been considered to be involved in the development and progression of COPD [12,13]. Recently, several studies reported some lncRNA gene polymorphisms to the susceptibility of COPD such as PVT1, MiR-146a, and nsv823469 [14,15]. Previously, abnormal expression of LINC00824 was associated with smoking [16]. However, the contribution of LINC01414/LINC00824 genetic polymorphisms to COPD predisposition remains unclear.  Here, we genotyped three polymorphisms in LINC01414/ LINC00824 to assess the genetic association of variants and interactions with COPD susceptibility among the Chinese Han population. Furthermore, the heterogeneity of relationship among subgroups (defined by age, gender and smoking status) and the correlation of selected polymorphisms with clinical symptoms of COPD patients were explored.

Study subjects
A total of 315 COPD patients and 314 healthy controls were enrolled in the present study from Hainan General Hospital. All subjects were ethnic Han Chinese population. COPD patients were diagnosed based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [17]. Patients with lung cancer, asthma, tuberculosis, interstitial fibrosis, bronchiectasis, and other respiratory diseases were excluded. Healthy controls who had no cancer history, respiratory diseases, inflammatory or immune diseases were recruited. We collected the demographic and clinical data of all subjects from the questionnaires and medical records. The study was approved by the medical ethics committee of Hainan General Hospital and was in the Declaration of Helsinki. All the subjects signed a written informed consent.

SNPs genotyping
Peripheral blood samples (5 mL) were collected from each subject into EDTA tubes. A commercially available DNA extraction Kits (GoldMag Co. Ltd, Xi'an, China) was used for the extraction of genomic DNA. Three single nucleotide polymorphisms (SNPs) including rs6994670 and rs298207 in LINC01414, rs7815944 in LINC00824 were selected based on the minor allele frequency (MAF) > 0.05 from 1000 Genomes Project database, Hardy-Weinberg equilibrium (HWE) > 0.05, and the calling rate > 98%. Agena MassARRAY platform (Agena, San Diego, CA, USA) performed the process of  genotyping. Primer design (Additional file 1: Table S1) and data management are performed based on corresponding supporting software. About 10% of subjects were repeatedly genotyped for quality control, and the results were consistent.

Statistical analysis
Sample t test or χ 2 test were used to evaluated the distribution of age and gender between COPD patients and healthy controls. HWE of selected SNPs in controls was detected by a goodness-of-fit χ 2 test. Logistic analysis adjusted by age and gender were applied to estimate the genetic contribution of selected SNPs to COPD susceptibility by calculating odds ratios (OR) and 95% confidence intervals (CI). Multifactor dimensionality reduction (MDR) analysis was used for analyze genegene interaction. Analysis of Variance (ANOVA) was used to evaluate the association between genotypes of LINC01414/LINC00824 variants and clinical characteristics of COPD patients. Data analyses were conducted using SPSS 20.0, PLINK 1.0.7, and MDR software. A p value < 0.05 was defined as statistical significance.

Stratification analysis for the genetic correlation by age, gender and smoking
We also evaluated the contribution of confounding factors (age, gender and smoking status) to the genetic relationship between selected polymorphisms and COPD risk, as listed in Tables 4 and 5. When stratified analysis by age (Table 4) In the stratified analysis by gender (Table 4), rs298207 and rs7815944 variants contributed to COPD risk in males. In which, rs298207-AA genotype was seen more frequent in COPD-patients compared with GG (OR = 2.53, p = 0.046) or GG-GA (OR = 2.54, p = 0.043) genotype among males. Rs7815944 was a protective factor for COPD susceptibility (OR = 0.43, p = 0.028; and OR = 0.45, p = 0.037) in males.
Stratified analysis by smoking status (Table 5), we found that rs7815944 was associated with a reduced susceptibility of COPD in the allele (OR = 0.45, p = 0.040), genotype (OR = 0.17, p = 0.044), recessive (OR = 0.16, p = 0.031) models among former smokers. However, no significant association of these SNPs with COPD risk in current smokers and never smokers was found.

Correlation of selected polymorphisms with clinical symptoms in COPD patients
The correlation of selected polymorphisms with clinical symptoms in COPD patients was also assessed, and the results was shown in Table 6. We found the association between rs298207 genotype and COPD patients with dyspnea (OR = 0.50, p = 0.016; and OR = 0.56, p = 0.029). Moreover, our result displayed that rs7815944 was related to COPD patients with wheezing under the allele (OR = 0.64, p = 0.021), genotype (OR = 0.22, p = 0.008), recessive (OR = 0.25, p = 0.014) and additive (OR = 0.59, p = 0.011) models.

MDR analysis for gene-gene interaction
MDR analysis was analyzed to evaluate the contribution of gene-gene interaction to COPD risk. Figure 1 revealed the additive effect between LINC01414 rs6994670-GG, LINC01414 rs298207-AA, LINC00824 rs7815944-GG towards COPD susceptibility. The interactions of these SNPs was displayed as the dendrogram and Fruchterman-Reingold (Fig. 2). Our results demonstrated that LINC01414 rs6994670 was the best onefactor model for COPD risk (testing accuracy = 0.5468, CVC = 10/10, p = 0.0188, Table 7). Furthermore, the twofactor model (LINC01414 rs6994670 and LINC00824 rs7815944) was found to be the best multi-loci model for COPD risk (testing accuracy = 0.5518, CVC = 10/10, p = 0.0037).

The association between selected variants and clinical characteristics of COPD patients
The association between LINC01414/ LINC00824 SNPs and clinical indicators in COPD patients was assessed, as displayed in Table 8. We found that the genotypes of LINC00824 rs7815944 was associated with respiratory rate of COPD patients (p = 0.022). However, no statistically association was observed on rs6994670 and rs298207 in LINC01414.

Discussion
In our study, rs699467 in LINC01414 and rs7815944 in LINC00824 might be protective factors for COPD occurrence, while LINC01414 rs298207 was associated with the increased risk of COPD in the whole population. Specially, age, gender, and smoking status might contributed to the association of these polymorphisms with COPD risk. Moreover, we found the association between rs298207 genotype and COPD patients with dyspnea, and rs7815944 was related to COPD patients with wheezing. Our findings firstly indicated that LINC01414/LINC00824 polymorphisms might play a role in the occurrence of COPD. LINC01414, located at chromosome 8q12.3, is a long intergenic non-protein coding RNA 1414. The function of LINC01414 has not been reported. LINC00824, located at chromosome 8q24.21, is also known as LINC01263. Genome-wide association studies reported that LINC00824 polymorphisms were associated with primary spontaneous pneumothorax and rheumatoid arthritis [18,19]. Here, we firstly found that rs699467 in LINC01414 and rs7815944 in LINC00824 might be protective factors for COPD occurrence, while LINC01414 rs298207 increased the risk of COPD in the whole population.
The occurrence of COPD is caused by combined effects of genetic background, gender, smoking and an aging population [20]. COPD is the leading causes of disability and death in older people, and significant sex difference can be observed, especially deaths in older men [21]. We also evaluated the contribution of confounding factors  (age and gender) to the genetic relationship between selected polymorphisms and COPD risk. Stratified analysis by age, rs7815944 was related to a reduced COPD risk in the subjects aged > 70 years. Rs6994670 was associated with the reduced risk of COPD, while rs298207 might have a higher susceptibility to COPD at age ≤ 70 years. In the stratified analysis by gender, rs298207 and rs7815944 variants were correlated with COPD risk in males.
Our results suggested that the genetic contribution of LINC01414/LINC00824 variants to COPD risk was gender-and age-specific. It is generally believed that smoking is the main risk factor for COPD development [22]. Previously, LINC00824 was higher expression in current smokers compared with former smokers [16]. Stratified analysis by smoking status, we found that rs7815944 was associated with the reduced susceptibility of COPD in former smokers but not current smokers. These hinted that LINC00824 might have an important role in the COPD pathogenesis. The potential function of rs7815944 is unknown. We speculate that the biological function of rs7815944 may be involved in affecting the expression of I LINC00824, which needed to be further studied. Several limitations in our study is unavoidable. First, the participants were Chinese Han population from a single center (the same hospital), therefore, the selection bias was inevitable and our results are not representative of other ethnic groups. Second, we only analyzed three SNPs in the LINC01414/LINC00824 gene, and other polymorphisms and other lncRNA genes were not considered. Third, the functional effects of LINC01414/ LINC00824 variants in the pathogenesis of COPD were not explored.

Conclusion
In summary, we found that LINC01414 rs699467 and LINC00824 rs7815944 were associated with lower prevalence of COPD, while LINC01414 rs298207 was associated with the increased risk of COPD in the Chinese Han population. Our finding provided further insights into LINC01414/LINC00824 polymorphisms at risk of COPD occurrence and accumulated evidence for the genetic susceptibility of COPD.