EPO rs1617640 A>C is a protective factor for chronic obstructive pulmonary disease: a case control study

Abstract


Introduction
Chronic obstructive pulmonary disease (COPD) is the most common pulmonary disease characterized by small airway lesions and chronic respiratory symptoms and continuous air ow restriction (1) .Its common pathological features include bronchiolitis and destruction of lung parenchyma (emphysema) (2) .COPD usually progresses slowly, which often leads to the neglect of early treatment and prevention (3) .Thus, when the disease is brought to the attention of patients, it has often progressed to the third or fourth stage and may be accompanied by complications (4) .It is estimated that 4.5 million deaths a year will be attributed to COPD by 2030 (1) , and with the progressive increase in the number of people developing COPD, it is on track to become the third leading cause of death worldwide (5) .Thus, COPD will also place a heavy burden on the global healthcare economy.
COPD is considered to be a process of chronic hypoxia and systemic chronic in ammation with complex pathogenesis.It is reported that environmental and genetic factors jointly affect the risk of COPD (6,7) .Smoking is a recognized risk factor for COPD, but this proportion in long-term smokers is only 10%-20%, and there is a considerable proportion of non-smoker people diagnosed with COPD, which indicates the importance of genetic factors to COPD (4) .With the development of genome-wide association analysis (GWAS), Pillai and colleagues rst found that the single nucleotide polymorphisms (SNPs) of CHRNA3/CHRNA5/IREB2 located on chromosome 15q25 were signi cantly associated with COPD (8) .Since then, GWAS at home and abroad have successively reported lots of genes in uencing susceptibility to COPD (9)(10)(11) .However, there is still largely unknown in the genetic etiology of COPD.
Erythropoietin (EPO) stimulated by Hypoxia is a recognized hematopoietic cytokine that can control the oxygen-carrying capacity of blood and enhance ventilatory function under hypoxic conditions (12)(13)(14) .EPO was observed to play antiin ammatory, anti-apoptosis and anti-oxidant/ brosis roles in the lung by interacting with in ammation, apoptosis, oxidative stress, or brosis-related pathways (15)(16)(17)(18)(19)(20) .Some studies indicated that the expression of EPO was positively correlated with the severity of COPD, while negatively correlated with the pulmonary function index FEV 1 (21)(22)(23) .The evidence hypothesized that EPO may take part in the occurrence and progress of COPD.
Therefore, we conducted a case-control study enrolled 1095 COPD patients and 1144 healthy controls in several hospitals in Guangdong to evaluate the association between EPO polymorphism (rs1617640 A > C, rs507392 A > G, rs564449 G > T) and COPD susceptibility.A total of 872 participants were recruited from a prevalence study performed in Gansu Province to verify the effect of EPO polymorphisms on lung function.Using the method developed by Gail and Bruzzi, we tted a 10-year absolute risk model for southern Chinese.

Study population
Han Chinese, who enrolled in a case-control study included 1095 COPD patients and 1144 healthy control in several hospitals in Guangdong Province (Songshan Lake Central Hospital of Dongguan, the Dongguan Binwan Central Hospital, and the Shenzhen Longhua District Central Hospital) during January 2015 to January 2019 (Demographic characteristics are shown in Table S1).Moreover, we recruited 872 participants from a prevalence study in Zhuoni County of Gannan Tibetan Autonomous Prefecture of Gansu to verify the effect of EPO polymorphisms on lung function (Demographic characteristics are shown in Table S2).All subjects received the lung function test and the respiratory health questionnaire for collecting their demographic characteristics and environmental exposure information (24) .We also obtained their 5ml peripheral blood for genotyping after they signed informed consent.
In addition, we used the electronic medical records of the above institutions from 2016 to 2020 to conduct a clinical cohort study to acquire the incidence of COPD.
Finally, this study was reviewed by the Ethics Committee of Guangzhou Medical University, Xi'an Jiaotong University and Gansu University of Chinese Medicine.

Copd's Diagnostic Criteria And Lung Function Test
According to the Global Initiative for Chronic obstructive pulmonary disease 2019 (25) , we diagnosed COPD if participants experienced respiratory symptoms like coughing, expectoration, dyspnea and wheezing in their daily lives, and the ratio of forced expiratory volume in 1 second (FEV 1 ) to forced vital capacity (FVC) after inhaling of 400µg salbutamol for half an hour was less than 70%.The lung function was measured by the EasyOne Spirometer (NDD Medizintechnik AG, Switzerland) following the instrument instructions.

Snps Selection And Genotyping
Potential risk SNPs are screened through multiple tools such as the NCBI database, Ensembl web tool, and SNPinfo website.
The selection criteria are as follows: SNPs were potential functional variations that belong to missense mutation, nonsense mutation, and splicing mutation in the coding region, or located in transcription factors binding region within 5 'UTR, or located in miRNA binding region within 3 'UTR; the minor allele frequencies (MAFs) were more than 0.05 in the Chinese population; tag SNPs.Combined with the results of screening and existing studies, the rs1617640 A > C, rs507392 A > G, and rs564449 G > T were selected for further analysis nally (26,27) .
Using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing) to extract DNA from the collected peripheral blood sample.TaqMan real-time polymerase chain reaction (PCR) was used for genotyping.Table S3 shows the details of primers and probes.Furthermore, in order to ensure the reliability of the PCR reaction, we not only set up a negative control on each plate, and 10% random samples repeat tests have been carried out.

Statistical analysis
The Chi-square test was used to assess the difference in demographic characteristics and the frequency distribution of genotypes between the case and control.After adjusting for age, sex, smoking, education, coal fuel exposure, and biofuel exposure, logistic regression analysis was used to evaluate the relationship between EPO polymorphism and susceptibility to COPD.Strati ed analysis was then performed by age, sex, smoking, education level, coal, and biofuel exposure.The consistency of ORs between the layers was tested by the Breslow-day test.The effect of genotypes on lung function was compared by the Kruskal-Wallis test.
The 10-year absolute risk was calculated by the method of Gail and Bruzzi (28)(29)(30)(31) .Based on a case-control study in Guangdong, the risk factors were screened by the backward stepwise logistic regression model, and the relative risk and populationattributable risk were estimated by IRAP 2.2.0.
IBM SPSS® Statistics 26.0 software was used to analyze the data.Odds ratio (OR) and 95% con dence interval (CI) were used as evaluation indexes.All analyses were two-sided with a signi cance level of 0.05.

Association analysis between EPO SNPs and COPD risk
We adjusted for sex, age, smoking status, education, coal as fuel and biomass as fuel at a signi cance level of 0.05.As shown in Table 1, EPO rs1617640 C allele signi cantly reduced COPD risk in southern Chinese (AC vs. AA: adjusted OR = 0.805, 95%CI = 0.669-0.969;AC + CC vs. AA: adjusted OR = 0.822, 95%CI = 0.689-0.980).Unfortunately, we could not observe the association between the other two SNPs (rs507392 A > G and rs564449 G > T) and the risk of COPD (P > 0.05).

Strati cation and interaction analysis between EPO rs1617640 A > C and COPD risk
The results of strati cation and interaction analysis were shown in Table 2. Compared with EPO rs1617640 AA genotypes, AC and CC genotypes remained protective in the strata of non-smokers, avoiding coal and biofuels (P < 0.05).The homogeneity test showed that there were no differences among the sublayers (P homo >0.05).Furthermore, there was no multiplicative interaction between the strati ed factors and rs1617640 A > C on the risk of COPD (P inter >0.05).

Incidence Of Copd Based On A Clinical Cohort
We calculated the incidence of COPD from a clinical cohort constructed by electronic medical records from 2016 to 2020 in several hospitals in Guangzhou, Shenzhen and Dongguan (see Table S4).The incidence of the 40-50 years old group was 1436.73/100,000person-years in males and 697.82/100,000 person-years in females.In the 50-60 years old group was 2972.24/100000person-years in males and 1321.07/100000person-years in females.In the 60-70 years group was 5795.21/100,000person-years in males and 2260.12/100,000person-years in females.

The 10-year absolute risk of COPD in southern Chinese
A backward stepwise logistic model was tted (Table 3), which nally retained four factors including education, smoking status, coal as fuels, and rs1617640 A > C for males, while three factors as smoking status, biomass as fuels, and rs1617640 A > C were retained for females.The population-attributable risk for them were 0.449 (0.258-0.641) and 0.262 (0.128-0.396) respectively.
Combined with the retained factors, tables were developed to show the 10-year absolute risk of COPD for men (Table 4) and women (Table 5) at different individual relative risks (30,31) .For example, a 45-year-old woman wants to know her risk of developing COPD in 10 years.It is known that the woman with rs1617640 AA genotype does not smoke, and has a history of biomass as fuels.The individual relative risk of this woman is 2.922 (r = RR non−smoking ×RR biomass as fuels ×RR rs1617640 AA =1×2.219×1.317).As demonstrated in Table 5, the 10-year absolute risk of the woman with COPD is about 0.142.

Discussion
To reveal the role of EPO polymorphism on COPD, we investigated the relationship between three SNPs of EPO (rs1617640 A > C, rs507392 A > G, and rs564449 G > T) and the risk of COPD in southern Chinese in this study.We found that the C allele of EPO rs1617640 A > C was signi cantly associated with a reduced risk of COPD and an increase in FEV 1 and FVC in Chinese.
Meanwhile, compared with EPO rs1617640 AA genotypes, AC and CC genotypes remained protective effects in the strata of non-smokers, avoiding coal and biofuels (P < 0.05).
EPO, a pleiotropic factor, can affects the occurrence and progression of many diseases (32,33) .EPO was reported that expressed in the lung to respond to Hypoxia by promoting erythropoiesis and neovascularization (13,(34)(35)(36) .Also, in the condition of acute lung injury, EPO could activate the proliferation of pulmonary endothelial cells and reduce the in ltration of in ammatory cells, which plays a key role in controlling pulmonary ventilation.Some studies have shown that the in ammatory response and hypoxemia caused by COPD change the expression of EPO.For example, COPD patients have up-regulated EPO expression due to increased in ammatory factors and decreased erythrocytes in the body (37,38) .Hypoxemia is more common in COPD patients, which can be corrected by elevating the expression of EPO to increase hemoglobin concentration (22,39,40) .Existing researches indicate that EPO may participate in the development of COPD.However, no study has explored whether EPO polymorphisms modify the susceptibility to COPD.
In this study, we rst discovered that the variation of EPO rs1617640 A > C was associated with COPD risk, and patients with AC/CC genotype had a reduced risk.For the function prediction of the SNPinfo website, we know that rs1617640 A > C is located in the upstream promoter region of EPO.The promoter SNP can cause abnormal gene expression by affecting the binding of transcription factors to templates (41) .In the present study, EPO rs1617640 polymorphism was shown to be associated with erythropoietin expression and level (42) , its CC genotype can promote the expression of erythropoietin (43) , which leads to the increase of Hb level, hematocrit and erythrocyte count (44) .In other words, EPO rs1617640 polymorphism can cause abnormal expression of EPO and affect the hematopoietic function of the body (44) .Moreover, we found a correlation between the EPO rs1617640A > C genetic variant and the level of pre-drug lung function indexes in both Guangzhou and Gannan.Similar to our results, Wang L found through a clinical study that serum EPO levels in COPD patients were signi cantly and negatively correlated with FEV 1 % (P < 0.05) (21) .EPO levels may, to some extent, re ect the condition and prognosis of COPD patients.
Therefore, we concluded that rs1617640 may affect the risk of COPD by in uencing the binding of transcription factors to DNA fragments and regulating the expression level of target genes.
Our clinical cohort found differences in the incidence of COPD and the mortality without COPD between men and women (see Table S4).This is consistent with the ndings of Liu (45) and Garcia Rodriguez Hence, we decided to t a 10-year absolute risk model for COPD in southern Chinese by sex.The factors reserved in the models of male and female were slightly different (see Table 3), which may be related to the reduction of sample size after strati cation.At the same time, the differences in the social division of labor and lifestyles between men and women may also lead to differences in risk factors (47,48) .In the model for the female, the population attributable risk was 0.262 (0.128-0.396), indicating that additional risk factors need to be considered to accurately predict the risk of COPD (49) .
There are some limitations in this study.First, this was a case-control study, and there was a certain degree of recall bias when obtaining environmental exposure information.Secondly, the participants in this study were mainly Han Chinese, which may restrict the extrapolation of conclusions to other populations.Moreover, this study clari ed the association between EPO polymorphism and COPD in epidemiological methods, which needed further veri cation by cellular and molecular experiments.

Conclusion
In conclusion, this study reported for the rst time that the EPO rs1617640A > C polymorphism was associated with COPD susceptibility, and the C allele was associated with better lung function in southern Chinese.On this basis, the study suggests that EPO rs1617640 A > C can be used to predict the 10-year absolute risk for COPD among southern Chinese as a genetic marker when combined with environmental factors.

Table 1
Frequency distributions of EPO gene polymorphisms and the associations with COPD susceptibility a ORs were adjusted for gender, age, smoking status, education level, coal or biomass as fuels by the logistic regression model.

Table 2
Strati cation analysis of the association between EPO rs1617640 A > C and COPD susceptibility P-value of test for the multiplicative interaction between EPAS1 rs13419896 genotypes and the factors.
OR, odds ratio; CI, con dence interval.aORs were adjusted for age, gender, education level, smoking status, coal or biomass as fuels by the logistic regression model.c

Table 3
Estimates relative risk based on case-control studies

Table 4
The 10-year absolute risk of COPD in southern China for men