Correlation of EYS polymorphisms with lumbar disc herniation risk among Han Chinese population

Abstract Background Lumbar disc herniation (LDH) is a common spinal disease in clinical practice. Once lumbar disc herniation occurs, it seriously reduces patient's quality of life. The EYS (eyes shut homolog) was discovered in recent years and it may be related to lumbar disc herniation. So we conducted a case–control study to explore the relationship between EYS polymorphism and lumbar disc herniation risk. Methods We selected 5 single‐nucleotide polymorphisms (SNPs) of EYS gene in a case–control study with 508 cases and 508 healthy controls to evaluate the relatedness by using genetic model, haplotype, and stratification analysis. Results We found that the minor alleles of rs62413038 (OR = 1.21, 95%CI: 1.01–1.43, p = .036) and rs9450607 (OR = 1.26, 95% CI: 1.05–1.53, p = .016) were associated with an increased risk of lumbar disc herniation in the allelic model analysis. In the genotypic model analysis, rs62413038 displayed a significantly increased risk of lumbar disc herniation in log‐additive models (OR = 1.20, 95% CI: 1.01–1.43, p = .039). While the rs9450607 was also obviously associated with an increased lumbar disc herniation risk in recessive (OR = 1.98, 95% CI: 1.24–3.13, p = .004) and log‐additive models (OR = 1.27, 95% CI: 1.05–1.55, p = .014). In addition, in the haplotype analyses of the SNPs, we found that the “CGGA” haplotype of rs1482456, rs9342097, rs9450607, and rs7757884 was associated with lumbar disc herniation. (OR = 0.52, 95% CI: 0.30–0.89, p = .017). Conclusion These results suggest that EYS polymorphism may be associated with lumbar disc herniation among Han Chinese population. It also opens up a new exploration direction for the etiology of lumbar disc herniation.


| INTRODUCTION
Lumber disc herniation (LDH) is a common degenerative disease of the lumbar spine, and is the main leading cause of spinal surgery in adults. Although it is not a malignant condition, it is considered a global health problem. It leads to chronic low back pain, negative impacts on physical and work activities, decreased quality of life, and psychological distress in affected individuals (Casa et al., 2016). Generally, LDH is a complex disease involving multifactorial interactions. In terms of current research, trauma, environmental and genetic factors are considered to be the main causes of disease. However the precise etiology and pathogenesis underlying of LDH are complex and still poorly understood (Mu, Ge, Zuo, Chen, & Huang, 2014).
Recently, it was suggested that the heritability of LDH is 52% to 68%, indicating that genetic factors might play a decisive role in the pathogenesis of LDH (MacGregor, Andrew, Sambrook, & Spector, 2004). To date, with the completion of high-quality sequencing of the human genome and a deeper understanding of disease mechanisms at the cellular and molecular level, the genes responsible for susceptibility to many complex diseases, including LDH, have been identified (Guo, Liu, Zhang, Guo, & Wu, 2011;Zhang, Sun, Liu, & Guo, 2008). Considering that LDH is a frequent disorder among adults, understanding its relationship to genetic factors can provide new prevention and intervention measures for individuals affected by the disease. With the identification of the polymorphisms of various LDH-related genes, we lay a foundation for elucidating the genetics of intervertebral disc herniation and also open a new door for us to study the etiology of LDH (Casa et al., 2016).
EYS is a new gene identified at the RP25 locus on chromosome 6q12 commonly mutated in autosomal recessive retinitis pigmentosa (Abd El-Aziz et al., 2008;Zelhof, Hardy, Becker, & Zuker, 2006). According to relevant research in an unselected birth cohort, they found locus 6q12 which the EYS located in was associated with nitric oxide levels. Moreover, we know that disc cells were able to release nitric oxide and nitric oxide might play an important role in the pathogenesis of disc degeneration through the induction of apoptosis of disc cells in situ (Fuchs et al., 2017(Fuchs et al., , 2011Kohyama, Saura, Doita, & Mizuno, 2000). Therefore, we hypothesize that mutations in the EYS (located in chromosome 6q12) cause problems in the regulation mechanism of the EYS in the intervertebral disc cells, leading to abnormal nitric oxide metabolism, followed by apoptosis of the intervertebral disc cells, which eventually leads to degeneration and herniation of the intervertebral disc (Janeczko, Janeczko, Chrzanowski, & Zielinski, 2014;Videman et al., 2009). Therefore, we infer that EYS polymorphism may affect individual LDH risk. We conducted a case-control study to evaluate the possible association of EYS polymorphisms at allele, genotype, and haplotype interface with the development of LDH among Han Chinese population and the results of this study will further confirm the role of EYS in the pathogenesis of LDH.

| Study participants
A case-control study involving of 508 LDH patients and 508 controls was conducted at The Second Affiliated Hospital of Inner Mongolia Medical University, Inner Mongolia, China. All cases were diagnosed with LDH according to the latest clinical guidelines. The inclusion criteria for cases group were: patients who had typical clinical symptoms and physical signs, patients' whose imaging examination showed prominent intervertebral disc. LDH symptoms were those described as follows:(a) lower back pain, (b) pain in the inferior lumbar part of the spine and regional typical sciatica; (c) difficulty in straight leg raising test and augmentation test; (d) the limited lumbar flexion range. Lumbar spine MRI confirmed the patients with LDH according to the Pfirrmann grading system. Patients with complicated blood diseases, autoimmune diseases, tumors, trauma, rheumatoid arthritis, and related lumbar spine disease containing lumbar spinal stenosis, spinal congenital dysplasia, intraspinal tumor, and spondylolisthesis were excluded from this study. The control subjects were recruited from the health checkup center of The Second Affiliated Hospital of Inner Mongolia Medical University, which they had visited for an annual health examination. These volunteers were not scanned by MRI and no history of sciatica and low back pain. Inclusion criteria of the control group were: (a) individuals had no family history of LDH; (b) individuals without spinal instability from trauma, scoliosis, spondylolisthesis, osteoarthritis, rheumatism and rheumatoid arthritis; (c) individuals without infections and any history of tumors. All studied individuals were Chinese Han subjects tracing back at least three generations. Demographic and related clinical data of the study population were collected by a face-to-face questionnaire and medical case record search. All of the participants were genetically unrelated ethnic Han Chinese from Inner Mongolia and provided written informed consent for their participation in the present study.

| Ethical compliance
This study was approved by the ethics committee of The Second Affiliated Hospital of Inner Mongolia Medical University. All subjects provided written informed consent before the collection of blood samples.

| DNA isolation
We used venipuncture blood vacutainer collection tubes to collect peripheral blood samples from each subject and then stored at −80°C for further use. We used the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag. Co. Ltd.) to extract genomic DNA from blood samples following the manufacturer's instructions. We assessed the purity and concentration of the extracted DNA using a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific) by absorbance measurements at 260 and 280 nm.

| SNP selection and genotyping
Five SNPs from EYS were chosen for analysis in this study and they were selected from the GWAS, NCBI dbSNP (http://www. ncbi.nlm.nih.gov/snp) and the 1000 Genomes Project databases (http://www.inter natio nalge nome.org/). All five SNPs had minor allele frequencies >5% in Han Chinese Beijing population. We used the Agena Bioscience Assay Design Suite V2.0 software (https ://agena cx.com/online-tools/ ) to design the primers of PCR amplification and extension of the five selected SNPs. These SNPs in EYS were genotyped in the case and control groups using the Agena MassARRAY platform with iPLEX gold chemistry (Agena Bioscience, San Diego, CA) according to the manufacturer's instructions. We used the Agena Bioscience TYPER software (version 4.0) to manage and analyze data.

| Statistical analysis
Data were analyzed using SPSS version 18.0 statistical software (SPSS Inc, Chicago, IL, United States). Allele frequency of each SNP in the control subjects was analyzed using χ 2 test to evaluate departure from Hardy-Weinberg equilibrium (HWE). The genotype and allele distributions for SNPs in LDH patients and control subjects were compared by χ 2 test. Under four different genetic models (codominant, dominant, recessive, and log-additive), we assessed the association between each SNP and the risk of LDH while adjusting for age and gender. Next, using PLINK software, version 1.07 (http://zzz.bwh.harva rd.edu/plink/ ld.shtml ), association intensity between each genotype and steroid-induced ONFH risk was estimated by odd ratios (OR) and the corresponding 95% confidence intervals (CIs) under four genetic models. All two-sided p values < .05 were considered statistically significant. Associations between SNPs and risk of LDH were tested in genetic models using SNP Stats software. The SHEsis software platform was used for the analyses of linkage disequilibrium and haplotype construction. Table 1 shows the volunteers' characteristics. A total of 508 cases of LDH (297 males and 211 females, mean ± Standard Deviation: 49.16 ± 14.90) and 508 healthy controls (297 males and 211 females, mean ± Standard Deviation: 48.49 ± 13.71) were enrolled in our study. There were no statistically significant differences on the gender and age distribution between the case and control groups.

| RESULTS
Five SNPs were selected for our study which included rs62413038, rs1482456, rs9342097, rs9450607, and rs7757884. The basic information of the SNPs including chromosomal position, minor allele frequency (MAF) of cases and controls, and Hardy-Weinberg equilibrium (HWE) test results are summarized in Table 2. All five SNPs were in Hardy-Weinberg equilibrium. (p > .05) ( Table 2).We compared the differences in frequency distributions of alleles between cases and controls by χ 2 test and found that Then we assumed that the minor allele of each SNP was a risk allele compared to the wild type allele and four multiple inheritance models (codominant, dominant, recessive, and log-additive models) were applied for analyzing the association between SNPs and LDH risk by logistic regression analysis adjusted for age and gender. (Table 3) In the genetic model analyses, the genotype G/T of rs62413038 was associated with an increased risk of LDH under the logadditive model (OR = 1.20, 95% CI: 0.94-1.61, p = .039). The genotype G/G-G/A of rs9450607 was also significantly associated with an increased LDH risk in recessive model (OR = 1.98, 95% CI: 1.24-3.13, p = .004) and log-additive model (OR = 1.27, 95% CI: 1.05-1.55, p = .014).

| DISCUSSION
As time goes, the idea that lumbar disc herniation was affected by genetic factors had been accepted by more and more people (Mayer et al., 2013). In the present case-control study, we investigated the associations between five SNPs of the EYS gene and the risk of LDH. We demonstrated that EYS genetic polymorphisms (rs62413038, rs9342097 and rs9450607) were associated with an increased risk of LDH among Han Chinese population. We found that sex and age differences may interact with EYS polymorphisms to affect the development of LDH. We also found that the "CGGA" haplotype of EYS was related to a 0.52-fold decrease in the risk of LDH.
In 2013, a meta-analysis of 4,600 subjects was performed based on new genetic variations associated with lumbar disc degeneration in Northern Europe. In this multivariate analysis, the locus rs11754641 on EYS was also included for association analysis. The final conclusion was that this site was associated with lumbar disc degeneration (Williams et al., 2013). From our research, we could observe that rs9450607 in EYS was found by the association with increased LDH risk and it was more significant among people over 49 years old. In addition, it also increased T A B L E 3 Genotypic model analysis of the relationship between SNPs and the risk of LDH 2.10-fold risk of LDH in males compared to females. This result demonstrated that rs9450607 of EYS was associated with the susceptibility to LDH in Han Chinese population and also the risk association of the polymorphisms which was gender-dependent.
Recently, Isackson et al indicated that the sequence similarity of the EGF-like repeats in the N-terminal region of the expression products of EYS in human eyes to the EGF-like repeats of Notch1. Certainly, proteins containing similar clusters of EGF-like domains in the Notch pathway have been shown to bind to each other. Thereby, it suggested that the expression products of EYS may play a role in the Notch signaling pathway (Isackson et al., 2011). Meanwhile Wang et al also found an increase in Notch signaling protein expression in human degenerated intervertebral discs, including the expression of Notch receptor and target genes in degenerated intervertebral discs. Coincidentally, they compared degenerated discs with nondegenerative disc samples and found increased Notch2 receptor expression in the nucleus pulposus of degenerated discs in middle age (Wang et al., 2013). From the above studies, we could find that EYS products may be related to disc degeneration or herniation through Notch pathway. Therefore, in combination with our experimental results, we propose for the first time that EYS polymorphism was associated with the risk of lumbar disc herniation in Han Chinese population (Risbud & Shapiro, 2014).
Although the results of our present study provide scientific evidence about EYS and LDH in the future studies, our study has some limitations. Firstly, the subjects of investigation were enrolled from the identical hospital and the control group did not undergo MRI examination. Therefore, selection biases could not rule out. Secondly, some potential confounding factors such as occupational exposures and physical activity were not included in our analysis and should be assessed in the future. Thirdly, our study does not include an analysis of biological functions yet, which will be crucial for elucidating the role of EYS in LDH (Liu et al., 2016;Zhu et al., 2018).

| CONCLUSION
In summary, we have confirmed for the first time that the association three SNPs (rs62413038, rs9342097, and rs9450607) of EYS gene with increased risk of LDH among Han Chinese population. While larger population-based studies and further functional researches are needed to confirm our results.

ACKNOWLEDGMENTS
We thank all authors for their contributions and support. We are also grateful to all participants for providing blood samples.