Association of the Polymorphism of TRAIL with the Risk and Severity of Lumbar Disc Degeneration in Chinese Han Population

Abstract

Background: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis of various normal and tumor cells. Apoptosis plays an important role in the development of lumbar disc degeneration (LDD). Aims: The aim of this study was to determine whether the 1595C/T polymorphism of the TRAIL gene is associated with the increased risk and severity of LDD in the Chinese Han population. Methods: This study consisted of 312 patients with LDD and 196 healthy controls. The grade of disc degeneration was determined according to Schneiderman's classification for magnetic resonance imaging (MRI). Results: The genotype frequency of the 1595C/T polymorphism was in agreement with the Hardy-Weinberg equilibrium (p=0.278). Patients with LDD had significantly lower frequencies of CT and TT genotypes compared with normal controls (p<0.001). In addition, a lower frequency of the T allele was found in patients with LDD than normal controls (p<0.001). Unconditional logistic regression analysis revealed that CT and TT genotypes were both significantly associated with a reduced risk of LDD compared with the CC genotype (p<0.001; odds ratio [OR] 0.327; 95% confidence interval [95% CI] 0.217-0.493 and p<0.001; OR 0.393; 95% CI 0.234-0.661, respectively). Furthermore, the T allele was significantly associated with a decreased risk of LDD compared with the C allele (p<0.001; OR 0.483; 95% CI 0.369-0.633). In addition, the CT and TT genotypes, as well as the T allele, were associated with lower degenerative grades of LDD compared with the CC genotype and the C allele, respectively (p=0.008 and p=0.001, respectively). Conclusion: The 1595C/T polymorphism of TRAIL may be adversely associated with the risk and severity of LDD in the Chinese Han population.

Introduction

Lumbar disc degeneration (LDD), the major cause of low back pain (LBP), is a major source of disability and has brought a huge medical and economical burden to society (Katz, 2006). The etiology of LDD is complex. Some environmental factors such as age, gender, physical loading, vehicular vibration, and smoking have been shown to play an important role in the mechanism of LDD (Higashino et al., 2007). On the other hand, genetic factors also contribute to the susceptibility of LDD. Some gene polymorphisms, such as aggrecan, insulin-like growth factor-1 receptor, interleukin 1, vitamin D receptor, and matrix metalloproteinase 3, have been demonstrated to be associated with LDD (Shang et al., 2012).

An imbalance between disc cell death and proliferation may result in disc degeneration (Martin et al., 2002). The apoptosis of disc cell number is an important cause of LDD (Zhao et al., 2006). Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the TNF/nerve growth factor superfamily. TRAIL induces apoptosis in susceptible cells by binding with equal affinity to two proapoptotic receptors, TRAIL-receptor 1 (TRAIL-R1, DR4) and TRAIL-receptor 2 (TRAIL-R2, DR5) (Pan et al., 1997). Three single-nucleotide polymorphisms (SNP) have been identified in the 3′ untranslated region at nucleotides 1525, 1588, and 1595 of the TRAIL gene (Gray et al., 2001). TRAIL expression in human intervertebral discs was found to be correlated with disc degeneration, indicating the possible role of TRAIL in the mechanism of LDD (Bertram et al., 2009).

This study aims to determine whether the 1595C/T polymorphism of the TRAIL gene is associated with the presence and severity of LDD in the Chinese Han population.

Materials and Methods

Patients

A total of 312 patients with LDD and 196 healthy controls were enrolled in this study.

The patients were all symptomatic with surgically or radiologically proven LDD. Exclusion criteria were synovial cysts, spondylolisthesis, spinal tumor, spondylosis, trauma, and inflammatory disease. The grade of disc degeneration was evaluated according to Schneiderman's classification for magnetic resonance imaging (MRI) (Schneiderman et al., 1987). Control subjects were recruited from healthy subjects with a medical checkup. They had no history of back problems and with negative MRI findings. Subjects with familial relation to any of the cases and a history of seeking medical attention for LBP were excluded. Controls were serially matched to the cases by age and gender. All the subjects were unrelated Han Chinese.

This study was approved by the research ethics committee of our hospital and was conducted in agreement with the Declaration of Helsinki. Written informed consent was obtained from the patients and healthy controls before their participation in this study.

DNA genotyping

Genomic DNA from all the subjects was extracted from peripheral blood leukocytes using a DNA Isolation Kit following the manufacturer's instructions (Qiagen, Venlo, Netherlands). The 1595C/T polymorphism of the TRAIL gene was examined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The primer sequences for these polymorphisms were as follows (Kikuchi et al., 2005): forward for 1595C/T polymorphism, 5′-TGAGCACTACAGCAAACATGA-3′; reverse for 1595C/T polymorphism, 5′-GCACCACTAAAAGATCGCAGT-3′. PCR was performed using Taq DNA polymerase (Life Technologies, Inc., Invitrogen, Carlsbad, CA) with a supplied buffer. For genotyping the 1595C/T polymorphism, PCR products were digested using the restriction enzyme RsaI (MBI Fermentas, Vilnius, Lithuania) and then separated by electrophoresis. After RsaI digestion of this PCR product, we detected two fragments having 59 and 332 bp (C allele) or three fragments having 59, 146, and 186 bp (T allele). To control for genotype failures, a 10.00% random sample of these study populations was regenotyped in a blinded manner. The results were found to be 100% consistent with the primary results of genotyping.

Statistical analyses

Statistical analyses were performed using SPSS version 16.0 software (SPSS, Inc., Chicago, IL). The characteristics of LDD patients and controls were compared with the chi square or Student's t-test according to the variable types. The Hardy-Weinberg equilibrium for the SNP in the control group was analyzed by a chi square test. The genotypes between the two groups were compared using the chi square test. The severity of disc degeneration of different genotypes among LDD patients was analyzed using chi square. Unconditional logistic regression was used to estimate the association between polymorphisms and risk of LDD development. All statistical tests were two sided, and p<0.05 for the differences was considered statistically significant.

Results

The characteristics between patients with LDD and normal controls

The characteristics of patients with LDD and healthy controls are displayed in Table 1. No significant differences in age and gender were found between the two groups.

Table 1.

The Characteristics Between Patients with Lumbar Disc Degeneration and Normal Controls

Characteristics	Patients with LDD	Normal controls	p-Value
N	312	196
Age (years)	49.54±8.87	48.65±8.12	0.256
Gender (male/female)	198/114	121/75	0.695

LDD, lumbar disc degeneration.

The 1595C/T polymorphism in patients with LDD and normal controls

Table 2 shows the genotype distribution of the 1595C/T polymorphism of the TRAIL gene in the LDD group and control group. The genotype frequency of the 1595C/T polymorphism was in agreement with the Hardy-Weinberg equilibrium (p=0.278). Lower frequencies of CT and TT genotypes were found in patients with LDD than in normal controls (p<0.001). In addition, the LDD group showed a lower frequency of the T allele compared with healthy subjects (p<0.001). Unconditional logistic regression analysis revealed that CT and TT genotypes were both reversely associated with the presence of LDD compared with the CC genotype (p<0.001; odds ratio [OR] 0.327; 95% confidence interval [95% CI] 0.217-0.493 and p<0.001; OR 0.393; 95% CI 0.234-0.661, respectively). Furthermore, the T allele was significantly associated with a decreased risk of LDD compared with the C allele (p<0.001; OR 0.483; 95% CI 0.369-0.633).

Table 2.

The Genotype and Allele Distributions of 1595C/T Polymorphism of TRAIL in Lumbar Disc Degeneration Group and Control Group

	LDD group		Control group
Genotype	n	%	n	%	p-Value	95% CI
CC	192	61.5	70	35.7	<0.001	1.00 (reference)
CT	79	25.3	88	44.9		0.327 (0.217-0.493)
TT	41	13.1	38	19.4		0.393 (0.234-0.661)
C	463	74.2	228	58.2	<0.001	1.00 (reference)
T	161	25.8	164	41.8		0.483 (0.369-0.633)

95% CI, 95% confidence interval; TRAIL, tumor necrosis factor (TNF)-related apoptosis-inducing ligand.

The association of the 1595C/T polymorphism with the severity of LDD

The association between the 1595C/T polymorphism and the severity of LDD is shown in Table 3. Patients with CT and TT genotypes had lower grades of disc degeneration than those with the CC genotype (p=0.008). In addition, lower frequencies of the T allele were found in higher grades of disc degeneration compared with the C allele (p=0.001).

Table 3.

The Association of Different Genotypes and Alleles of the 1595C/T Polymorphism of TRAIL with the Severity of LDD

Genotype	n	Grade 2 (%)	Grade 3 (%)	Grade 4 (%)	p-Value
CC	192	94 (49.0)	42 (21.9)	56 (29.2)	0.008
CT	79	48 (60.8)	22 (27.8)	9 (11.4)
TT	41	20 (48.8)	15 (36.6)	6 (14.6)
C	463	236 (51.0)	106 (22.9)	121 (26.1)	0.001
T	161	88 (54.7)	52 (32.3)	21 (13.0)

Discussion

Genetic factors are involved in the mechanism of the development of LDD. Many gene polymorphisms have been revealed to be correlated with the susceptibility for developing LDD in different ethnics and populations (Gologorsky and Chi, 2014). LDD caused by degeneration of intervertebral discs of the lumbar spine and concurrent disc herniation is a primary cause of LBP. LBP is a major source of disability and has a substantial impact on the cost of healthcare (Lefevre-Colau et al., 2009). Therefore, it is important to identify more susceptibility genes for LDD and then to assess the genetic susceptibility for LDD in an early stage. Subjects with susceptibility genes should establish primary prevention strategies for LDD, including avoiding or stopping the environmental and occupational risk factors of LDD.

Recent studies revealed the association of different polymorphisms in apoptosis-associated genes with the risk of LDD. Sun et al. (2011) reported that the Ex5+32 G/A polymorphism of the caspase 9 gene was associated with the presence of LDD in the Han population. −1263 GG genotype of −1263A/G polymorphism in the caspase 9 gene was found to be with a significant increased risk of discogenic LBP (Guo et al., 2011). In addition, the polymorphism of other apoptosis-related genes, such as FAS and FAS ligand (FASL) (Zhu et al., 2011), BCL-2 (Shang et al., 2012), and death receptor 4 (Tan et al., 2012), was shown to be associated with the susceptibility and severity of LDD in the Chinese Han population. The current results indicated the adverse association of the 1595C/T polymorphism of TRAIL with the presence and severity of LDD. This suggests that genetic polymorphism in apoptosis-associated genes may serve as a genetic risk marker for the presence and severity of LDD.

TRAIL has been shown to be involved in the pathogenesis of LDD. The degree of TRAIL immunostaining was correlated with the histopathologic degeneration score of human temporomandibular joint (TMJ)-degenerated discs, indicating that the disc cell loss due to the involvement of the TRAIL apoptotic pathway seems responsible for TMJ disc degeneration (Leonardi et al., 2011). Bertram et al. (2009) reported that the expression of TRAIL and its receptors DR4 and DR5 correlates with progression of degeneration in human intervertebral discs. In addition, diseased discs affected by disc displacement exhibited a much higher percentage of TRAIL- and DR5-positive cells, as well as stain intensity, compared with normal tissue (Leonardi et al., 2010). These results point to the important role in the mechanism of LDD. However, the exact mechanism of TRAIL in LDD development should be explained by further studies.

The limitations of these findings should be considered. First, the population size in the current study is relatively small. Our result should be validated in prospective studies in a larger population. Second, we did not perform population stratification in our study. All these limitations may lead to false-positive findings. Therefore, our data should be validated in prospective studies with a larger population size in other ethnic populations.

In conclusion, this study showed that the 1595C/T polymorphism of the TRAIL gene was adversely associated with the risk and severity of LDD in the Chinese Han population. This polymorphism may serve as a genetic marker of the susceptibility for LDD.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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