Association analysis between chronic obstructive pulmonary disease and polymorphisms in circadian genes

Introduction

Chronobiology is a branch of biology that studies biological rhythms in living organisms. A typical cyclical phenomenon is the ability to respond to infectious disease cycles, and accumulating evidence has proven that various diseases such as cancer, cardiovascular disease, psychiatric disorders and neurodegenerative diseases are associated with chronic mismatch between our life style and biological clock (Badar, 2018; Touitou, Reinberg & Touitou, 2017; Lunn et al., 2017; Smolensky et al., 2016). Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal inflammation and decline of lung function. It is currently estimated as the fourth leading cause of death worldwide and is projected to be the third leading cause of death by 2020 (https://goldcopd.org/gold-reports/) (Yao et al., 2015). COPD symptoms exhibit circadian variation, with significantly reduced forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and peak expired flow at night (Borsboom et al., 1999; Goyal et al., 2020, 2019). In addition, the risk for intubation in the emergency department patients with COPD was found to be elevated during the night or early in the morning (Tsai, Brenner & Camargo, 2007). Evidence also shows that environmental risks of COPD, such as tobacco smoking, can disrupt the biological oscillations, leading to chronic inflammatory responses (Truong et al., 2016). The rhythmic activation and repression of some genes such as Clock Circadian Regulator (CLOCK), Aryl Hydrocarbon Receptor Nuclear Translocator Like (ARNTL, also known as BMAL1), Period Circadian Regulator (PER), Cryptochrome Circadian Regulator (CRY) and Nuclear Receptor Subfamily 1, group D, member 1 (NR1D1, also known as REV-ERBα) are recognized as key factors driving physiological circadian oscillations (Sundar et al., 2015a). Sundar et al. (2015b) investigated the circadian molecular clock in lung pathophysiology; their results showed that altered levels of REV-ERBα and RORα (Nuclear Receptor subfamily 1, group F, member 1 (NR1F1)) in mammal’s lung tissue was associated with heightened DNA damage response, cellular senescence and inflammation. Many physiological parameters are regulated by circadian clocks and circadian clock genes, but how these clock molecules influence the development of diseases and impact the genetic predisposition to disease risk are largely unknown. In recent years, positive relationships between single nucleotide polymorphisms (SNPs) in clock genes and diseases, such as cancer, diabetes and mood disorders, has been established, but not yet in COPD. Therefore, we chose to test the SNPs of circadian genes indicated by a search of the literature that might be involved in lung pathology to explore whether they may be linked to risk for COPD in the Chinese Han population.

Materials and Methods

Results

Discussion

In this case-control study, we investigated the potential association between nine SNPs from five different circadian genes and the COPD risk in the Chinese Han population. The overall result didn’t show any significant association between these SNPs and COPD risk or in the age, sex and smoking status stratification analysis.

The circadian oscillator is located in the anterior hypothalamus and controls circadian processes, though peripheral tissue, including the lung also shows cell autonomous oscillators, responsive to environmental stimuli (Sundar et al., 2015b). The core clock genes that drive circadian-related feedback loops include CLOCK, CRY1, CRY2, PER1, PER2 and PER3; of these CLOCK acts as a positive regulator, while the remaining five genes act as negative regulators (Lee et al., 2015). The heterodimer of the transcription factors CLOCK and BMAL1 regulates the transcription of PER and CRY genes via E-box sequences within their promoters. PER and CRY heterodimerize and block E-box-dependent transcriptional activity of the CLOCK: BMAL1 complex. The counteracting activity of REV-ERBs and ROR mediate BMAL1 expression and CHRONO, a transcriptional repressor that acts independently of the circadian transcriptional repressor such as CRY1 and CRY2, can directly interact with BMAL1 and negatively regulate circadian oscillations (Takahashi, 2015). An environmental exposome, such as cigaret smoking, could influence the lung cells and macrophages through activations of kinases that lead to posttranslational modification of molecular clock proteins (Sundar et al., 2015b). Theoretically, dysfunction of the clock-controlled genes in the bronchial epithelial cells can cause internal desynchrony, contributing largely to clock-dependent pathophysiology of chronic airway diseases (Sundar et al., 2015b). Animal studies have shown that after cigaret smoking or lipopolysaccharide inducement, cytokine or neutrophil levels were significantly higher in BMAL1 knockout mice compared with controls (Gibbs et al., 2014; Jaradat et al., 2006). In addition to alteration in the environmental stimuli-induced defense, theoretically circadian rhythm dysfunction could also influence the pulmonary function by changing timing signals through sympathetic and parasympathetic innervation (Buijs & Kalsbeek, 2001). Notably, genetic variants in clock genes have been reported to be associated with susceptibility to cancer, metabolic diseases, and psychotic and mood disorders (Gu et al., 2015). However, there are no published association studies evaluating the circadian gene polymorphism and chronic pulmonary disease, and this study is the first study to investigate any potential association between clock gene polymorphisms and COPD risk.

In this case-control study, overall analyses showed that no significant SNPs were found to be associated with COPD. However, in the stratification analysis, a weak positive association was observed in RORB rs3750420 in the male subjects, but this association became insignificant after adjusting for multiple comparisons. In our opinion, larger samples are needed to properly explore this hypothesis. Abundant researches have shown that the chronotype, which is the diurnal preferences that manifest in personal sleep-wake rhythms, differs in females and males as well as in young and elderly, and this difference, at some point, depends on the variants of “clock” genes (Roenneberg et al., 2007). The expression of circadian genes PER2, PER3 and ARNTL1 show differences with sex in human cerebral cortex and much earlier expression timing of these three genes in women than in men (Lim et al., 2013). Recent studies on psychiatric disorder also found a relationship between PER3 VNTR genotype and age at onset of bipolar disorder (Benedetti et al., 2008). The minor allele of CLOCK gene polymorphism 3111T/C was associated with higher susceptibility of Alzheimer’s disease only in APOE ε4 carriers (Yang et al., 2013). However, low significance may also be due to the relatively smaller numbers of subjects and variations in the patient population. Hence, a larger sample size of both groups should be considered in the future to further validate the results.

There are several limitations in our current study. Firstly, the SNPs we chose from genes involved in lung pathology were based on literature reporting positive relationship with other disorders rather than respiratory disease, per se, due to scarce evidence of association of circadian gene polymorphism with lung disease. Therefore, it is possible that we missed finding out potentially important lung-disease-related SNPs because of literature renewal lag or genotyping method limitations. Secondly, this study did not explore enough circadian rhythmic phenotypes, such as sleep patterns and pulmonary functions, which could also be linked to these SNPS. Circadian rest-activity rhythm disturbance, such as insomnia, dyspnea and nocturia, is commonly seen in COPD patients. Disparity from normal of the 24 h rest-activity rhythm of COPD subjects has been reported for the activity parameters of mean intradaily variability, interdaily stability, mean of 10 consecutive hours of highest activity, and mean of five consecutive hours of lowest activity. These measures were confirmed by previous studies to be significantly different in COPD patients compared with controls (Nunes et al., 2017), but whether they are associated with circadian gene polymorphisms remains unclear. As these tests were not available during this investigation, further prospective studies are needed to investigate the linkage of these measures to circadian gene polymorphisms and whether they are associated with COPD.

Conclusion

In conclusion, no overall relationship between circadian gene polymorphisms and COPD was detected in the Chinese Han population. A larger cohort investigation is required to gain evidence within each stratum to validate that whether the circadian gene polymorphisms are associated with COPD only in a sex or age stratification. Additionally, as the alternative allele frequency distribution varies among different ethnic groups, which would influence the susceptibility of the disease (Lee et al., 2003), a validation of these SNPs across the ethnicities may be need in the future.

Supplemental Information