CHRNA5/CHRNA3 gene cluster is a risk factor for lumbar disc herniation: a case-control study

For this case-control association analysis of LDH, a total of 380 LDH patients and 400 unrelated healthy controls were recruited from Xi’an Honghui hospital. All participants lived in Xi’an area, and all participants were Han people. So, there are no regional and ethnic differences. The diagnosis of LDH required the following criteria: (1) patients who had a history of lumbar sprain and/or a history of chronic strain, (2) patients who had pain in the inferior lumbar part of the spine and regional sciatic nerve pain in the leg caused by bed rest, (3) patients with tenderness beside the lumbar spine that affects the leg or foot, (4) patients whose lumbar flexion range was obviously limited, (5) patients with positive results in the straight-leg raising test and augmentation test (Bragard’s sign), and (6) patients who had the following nerve injury symptoms: muscular atrophy, motor weakness, decreased sensation, and hyporeflexia. Meeting any one of (1)–(6) and then combined with patients with clinical manifestations of LDH in accordance with imaging findings, including computed radiography, computed tomography, and/or magnetic resonance imaging, is considered positive for LDH. Primary exclusion criteria included spinal and joint diseases such as trauma, spinal tumor, synovial cyst, inflammatory disease, scoliosis, osteoarthritis, spondylosis, and spondylolisthesis. Moreover, patients who have history of labor work or heavy smoking were also excluded. According to the Labor Protection Measures Standard for Heavy Labor Work (No. 103007971) and the National Standard “Classification of Physical Labor Intensity” (No. BG386983), we exclude labor worker by inquiring about the nature of their work and the intensity of their physical consumption. By referring to the literature [21], according to the smoking index is equal to the number of cigarettes per day multiplied by the number of years of smoking (365 day/year), the smoking index ≥ 60 pack years is defined as a heavy smoker, using this criterion to distinguish whether the patient is a severe smoker. Four hundred unrelated healthy controls were recruited from Xi’an Honghui hospital. Inclusion criteria of the control group were (1) good health as confirmed by physical examination, (2) no recent infections, (3) no history of tumors, and (4) history of lumbar sprain and/or chronic strain.

Ahsan et al. adopted a retrospective case-control study found that physical exertion, work stress, and daily work time of more than 8 h were highly correlated with the occurrence of LDH [22]. An HS and other studies found that smokers had a 50% increased risk of disc herniation compared with non-smokers [23]. It shows that heavy labor or smoking has a greater impact on lumbar disc herniation. So, we exclude labor worker or heavy smoker.

Fifteen tag single-nucleotide polymorphisms (SNPs) of CHRNA5 and CHRNA3 were selected from the NCBI SNP database (www.​ncbi.​nlm.​nih.​gov/​SNP) and the HapMap database (www.​hapmap.​org). All the SNPs selected had a minor allele frequency (MAF) of more than 0.05. And primers for 15 tag SNP typing were designed by Agena on-line design software (https://​agenacx.​com/​online-tools/​).

Genomic DNA was extracted from peripheral blood leukocytes of affected individuals and controls using standard protocols. The DNA quantity was evaluated by spectrometry (DU530UV/VIS spectrophotometer, Beckman Instruments, Fullerton, CA, USA). The SNP genotyping was performed on the Agena MassARRAY SNP genotyping platform (Agena Bioscience, San Diego, CA, USA) according to the manufacturer’s protocol.

Statistical analysis was performed using SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Hardy–Weinberg equilibrium was assessed by using a chi-square test. Measurement data were expressed as the mean ± standard deviation (SD). The difference between the two groups was compared using a t test. In a multivariate logistic regression model, we assessed the independent association between CHRNA3/CHRNA5 gene polymorphism, smoking, drinking, and risk of LDH. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to determine the relative risk of LDH. Haplotype blocks and the linkage disequilibrium (LD) patterns were estimated by using the Haploview program (version 4.2, Broad Institute of MIT, and Harvard, Cambridge, MA, USA). Linkage disequilibrium coefficients (D’ and r²) were calculated as described previously [24]. All tests were two-sided, and p < 0.05 indicated a significant difference.

The general characteristics of both groups are summarized in Table 1. A total of 780 Northwest Chinese subjects with Han ethnicity were recruited in this case-control study, comprising 380 patients with LDH (152 females and 228 males; mean age 50.43 ± 12.27 years) and 400 healthy controls (166 females and 134 males; mean age 50.79 ± 8.13 years). No significant differences in gender and age were found between the two groups. Of the 380 patients, 43 were smokers and 337 were non-smokers; 12 were drinking and 368 were non-drinking.

The basic information about all the SNPs including gene, band, position, alleles, and Hardy–Weinberg equilibrium (HWE) results are presented in Table 2. The results of the HWE showed that the genotype frequency distributions of CHRNA5/CHRNA3 in the case and control groups were in line with genetic balance (all p > 0.05), which showed that all of the 15 tag SNPs were at equilibrium and were representative. In the allele model, we found that there was no site affected by the genetic risk of lumbar disc herniation. Genetic models (genotype, dominant, recessive, and additive) and the genotype frequencies were used to further identify the associations between the SNPs and the risk of LDH. The results without stratification showed no association between SNPs and the risk of LDH (data were not shown).

In a multivariate logistic regression model, we assessed the independent association between CHRNA3/CHRNA5 gene polymorphism, smoking, drinking, and risk of LDH. We found that the individuals with rs8040868 CT genotype had a 0.46-fold higher risk of lumbar disc herniation than those with rs8040868 TT genotype, in the men group (OR = 0.46, 95% CI 0.25–0.84, p = 0.012) (Table 3). Also among women, rs8040868 CT + CC genotype still reduced the risk of lumbar disc herniation under the dominant model (OR = 0.50, 95% CI 0.28–0.89, p = 0.019) (Table 3).

Haplotypes were constructed on the basis of the genotype data from 15 SNPs using Haploview software (version 4.2). Linkage disequilibrium D’ value between SNPs and the reconstructed LD plots of the 15 SNPs are shown in Fig. 1. Two LD blocks were observed according to the confidence interval method [25] (D’ > 0.9 and r² > 0.8). The block contained rs667282, rs16969948, rs588765, rs6495306, rs17486278, rs680244, rs569207, and rs6927800; another includes rs3743077, rs1317286, and rs938682. Another block includes rs3743077, rs1317286, and rs938682. Compared with the CHRNA5 “TACAACCG” wild-type, the “TACACCCG” haplotype was found to be associated with a decreased risk of LDH (OR = 0.79, 95% CI 0.63–1.00, p = 0.047; Table 4), while, in the less than 50-year-old group, CHRNA5 “TACACCCG” increased the risk of LDH (OR = 1.46, 95% CI 1.01–2.13, p = 0.047; Table 4).