Research Note A preliminary association study of fat mass and obesity associated gene polymorphisms and degenerative disc disease in a Chinese Han population Journal of International Medical Research 2014, Vol. 42(1) 205 212! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: 10.1177/0300060513503761 imr.sagepub.com Lifeng Lao, Guibin Zhong, Xinfeng Li and Zude Liu Abstract Objective: To assess whether polymorphisms of the fat mass and obesity associated gene (FTO) are associated with the presence and severity of degenerative disc disease (DDD) in a Chinese Han population. Methods: In this case control study, patients with DDD and controls matched for age, sex and body mass index were genotyped for six single nucleotide polymorphisms (SNPs) of FTO. Disease severity was measured using the Japanese Orthopaedic Association score. Allelic, genotypic and genotype phenotype association analyses were performed. Results: A total of 80 patients with DDD and 80 controls were studied. All six SNPs were in Hardy Weinberg equilibrium. The frequencies of allele G and genotype G/G of the SNP rs11076008 were significantly associated with DDD after Bonferroni correction. No associations were shown between the SNPs studied and sex or disease severity. Conclusion: The SNP rs11076008 of FTO may play an important role in the development of DDD in a Chinese Han population. The G/G genotype and/or G allele may be a risk factor for DDD. These results suggest that FTO is a DDD predisposition gene and may support a close relationship between obesity and DDD. Keywords Fat mass and obesity associated gene, single nucleotide polymorphisms, degenerative disc disease, gene polymorphisms Date received: 4 July 2013; accepted: 20 July 2013 Department of Orthopaedic Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Corresponding author: Professor Zude Liu, Department of Orthopaedic Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, China. Email: renjispine@gmail.com
206 Journal of International Medical Research 42(1) Introduction Degenerative disc disease (DDD) is a major cause of low back pain and is an important source of disability, imposing a large medical and economical burden on society. 1 Several environmental risk factors, including physical loading, vehicular vibration, ageing and smoking, are thought to play key roles in the aetiology of DDD. 2 In addition, a number of gene polymorphisms, including genes coding for collagen IX, 3 aggrecan, 4 vitamin D receptor, 5 matrix metalloproteinase 2 6 and secreted protein acidic and rich in cysteine (SPARC)/osteonectin 7 have been demonstrated to be associated with DDD. Several independent large-scale genomewide association studies have consistently reported that the fat mass and obesity associated gene (FTO) is associated with obesity-related traits and obesity risk. 8 10 This association has been replicated in multiple studies in different populations. 11 15 Furthermore, experimental animal studies have provided direct functional evidence that FTO is a causal gene for obesity. 16,17 It has been demonstrated that body weight is closely related to the occurrence and development of DDD. 18,19 Although a number of genetic association studies have been performed for obesity-related genes in Chinese populations, 20 22 associations between FTO polymorphisms and DDD have not yet been investigated. The aim of the present study was to determine whether polymorphisms of FTO are associated with the presence and severity of DDD in a Chinese Han population. Patients and methods Patients Patients with DDD and healthy controls with a body mass index (BMI) <25 kg/m 2 from a Chinese Han population were included in the study. Patients with DDD attending the Department of Orthopaedic Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, between April 2010 and March 2012 were sequentially enrolled in the study. DDD was diagnosed on the basis of clinical symptoms and signs and magnetic resonance imaging findings. Patients with DDD had suffered from various periods of mild to severe low back pain; when severe, patients may have had referred pain in the buttock or the lower limbs. Patients had received nonoperative treatment, including activity modification, medication, physiotherapy and corticosteroid injections. Those with previous fracture of the spine, Bechterew s disease, or malignancies involving the spine or poliomyelitis were excluded from the study. Controls were recruited from healthy subjects attending for medical check-ups and were matched for age, sex and BMI to the patients. Controls were evaluated by computed tomography or magnetic resonance imaging to confirm the absence of DDD and had no medical history of low back pain, sciatica or DDD. Patients and controls were all assessed using the Japanese Orthopaedic Association (JOA) score. 23 The study was confined to participants with a BMI <25 kg/m 2, which is the cut-off value for overweight in a Chinese population, in order to control for the presence of obesity. Written or verbal informed consent was obtained from all study participants, and the study protocol was approved by the Ethics Committee of the Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. SNP selection and genotyping Based on genotype data from the International HapMap project (http:// www.hapmap.org), the following six tagging single nucleotide polymorphisms (SNPs) present in FTO were selected using
Lao et al. 207 Haploview 4.1 software (Broad Institute, Cambridge, MA, USA): rs9939609, rs12933928, rs1421085, rs11076008, rs7185783 and rs9923147. The minor allele frequency of all selected SNPs was >5%. All study participants provided a 5 ml venous blood sample for gene typing. The sample was collected in 2% ethylenediaminetetraacetic acid anticoagulant tubes and fully mixed to prevent clotting. DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer s instructions and stored at 80 C until used. Genotyping was performed using the SNPstream Õ system (Beckman Coulter, Brea, CA, USA), which employs a combination of a high-fidelity polymerase reaction, single base primer extension and microarray methods, according to the methods previously described by Bell et al. 24 Details of the six SNPs and the primer sequences used are given in Table 1. Statistical analyses Baseline demographic characteristics in patients and controls were compared using the Student s t-test. The goodness of fit of each SNP to the Hardy Weinberg equilibrium was assessed in controls using the 2 test. Allelic and genotypic association analyses were also performed using the 2 test. s that showed a significant relationship with DDD were further evaluated for genotype phenotype associations in patients, using the 2 test to compare genotype and sex and the Kruskal Wallis test to compare genotype and JOA score levels. All results were modified by Bonferroni correction for multiple comparisons. A P-value <0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS software version 19.0 (SPSS Inc., Chicago, IL, USA). Results Eighty patients with DDD aged 30 72 years and 80 healthy controls aged 28 69 years were included in the study. There were no statistical differences in age, sex, height, weight or BMI between the two groups; however, patients with DDD had a significantly lower JOA score than controls (P < 0.001) (Table 2). Table 1. Reference identification numbers and primer sequences for the six single nucleotide polymorphisms (SNPs) of the fat mass and obesity associated gene used in the study. SNP reference identification no. Allele 1 Allele 2 Primer sequence rs9939609 T A Forward: 5 0 -GGCAAATGAGCGTAGACTCC-3 0 Reverse: 5 0 -GAGCAGGACAGGAGAACAGG-3 0 rs12933928 A G Forward: 5 0 -CCTTCCGTGCCTTATGGTAA-3 0 Reverse: 5 0 -CAGGTGGTTCCAGATGGACT-3 0 rs1421085 A G Forward: 5 0 -GCCATCTTCCACACAAGGTT-3 0 Reverse: 5 0 -GAGTGGCAGCTGTTTTCCTC-3 0 rs11076008 G A Forward: 5 0 -CCTTCCGTGCCTTATGGTAA-3 0 Reverse: 5 0 -CAGGTGGTTCCAGATGGACT-3 0 rs7185783 C A Forward: 5 0 -GCAAACATCTGCCTACAGCA-3 0 Reverse: 5 0 -AGTTCACAGCCACGTTACCC-3 0 rs9923147 G A Forward: 5 0 -GCCATCTTCCACACAAGGTT-3 0 Reverse: 5 0 -GAGTGGCAGCTGTTTTCCTC-3 0
208 Journal of International Medical Research 42(1) Table 2. Baseline demographic characteristics of patients with degenerative disc disease (DDD) (n ¼ 80) and controls (n ¼ 80). Parameter Patients with DDD Controls Statistical significance a Age, years 44.9 9.4 44.2 5.8 NS Sex NS Male 44 (55.0) 45 (56.3) Female 36 (45.0) 35 (43.8) Height, cm 167.6 7.0 167.0 6.9 NS Weight, kg 62.9 10.2 63.2 10.8 NS BMI, kg/m 2 22.3 2.7 22.5 2.9 NS JOA score 21.1 3.0 29.0 0.0 P < 0.001 a Using t-test. Data presented as mean SD or number (%) of patients. BMI, body mass index; JOA, Japanese Orthopaedic Association; NS, no statistically significant between-group difference (P 0.05). Allelic and genotypic association analyses The allele distributions for all six SNPs studied met the Hardy Weinberg equilibrium in controls (P > 0.05, goodness-of-fit 2 test). As shown in Table 3, allele 2 (G) in the SNP rs12933928 (P ¼ 0.024), allele 1 (G) in the SNP rs11076008 (P ¼ 0.002) and allele 1 (G) in the SNP rs9923147 (P ¼ 0.040) were significantly more frequent in patients with DDD than in controls. After strict Bonferroni correction, the P-value for rs11076008 was 0.012. Differences in the allele frequencies between the two groups for the other three SNPs were not statistically significant (Table 3). When analysed for genotypic associations (Table 4), the allele 2/allele 2 (G/G) homozygote in the SNP rs12933928 (P ¼ 0.025) and the allele 1/allele 1 (G/G) homozygote in the SNP rs11076008 (P ¼ 0.005) were significantly more frequent in patients with DDD than in controls. After strict Bonferroni correction, the P-value for rs11076008 was 0.03. The results of the allelic and genotypic association analyses are summarized in Figure 1: this shows the statistically significant differences between patients and controls for rs12933928, rs11076008 and rs9923147 on allelic association analysis and for rs12933928 and rs11076008 on genotypic association analysis. Only the relationship between polymorphisms of rs11076008 and DDD was still significant after strict Bonferroni correction. phenotype association analyses phenotype associations were evaluated between the rs11076008 polymorphism and sex or JOA score in patients with DDD. No significant associations were seen between sex and genotype (data not shown) or between JOA score and genotype (Table 5). Discussion Degenerative disc disease is caused by degeneration of the intervertebral disc; symptoms include neck, waist, low back or leg pain. Epidemiological studies suggest that genetic factors play an important role in the occurrence and development of lumbar disc degeneration. 2 In 1998, Videman et al. 25 reported that vitamin D receptor gene polymorphism was associated
Lao et al. 209 Table 3. Allelic association analysis of single nucleotide polymorphisms (SNPs) in patients with degenerative disc disease (DDD) (n ¼ 80) and controls (n ¼ 80); total number of alleles was 160 per group. Patients with DDD Controls Statistical parameters SNP Allele 1 Allele 2 Allele 1 Allele 2 P-value a P-value after Bonferroni correction Odds ratio 95% confidence interval rs9939609 144 (90.0) 16 (10.0) 139 (86.9) 21 (13.1) NS NS 1.36 0.68, 2.71 rs12933928 97 (60.6) 63 (39.4) 116 (72.5) 44 (27.5) 0.024 NS 0.58 0.37, 0.94 rs1421085 145 (90.6) 15 (9.4) 139 (86.9) 21 (13.1) NS NS 1.46 0.72, 2.95 rs11076008 150 (93.8) 10 (6.3) 132 (82.5) 28 (17.5) 0.002 0.012 3.18 1.49, 6.80 rs7185783 98 (61.3) 62 (38.8) 103 (64.4) 57 (35.6) NS NS 0.88 0.56, 1.38 rs9923147 143 (89.4) 17 (10.6) 130 (81.3) 30 (18.8) 0.040 NS 1.94 1.02, 3.68 a Between patients and controls using 2 test. Data presented as number (%). NS, no statistically significant between-group difference (P 0.05). Table 4. Genotypic association analysis of single nucleotide polymorphisms (SNPs) in patients with degenerative disc disease (DDD) (n ¼ 80) and controls (n ¼ 80). Patients with DDD Controls Statistical parameters SNP 1/1 1/2 2/2 1/1 1/2 2/2 P-value a P-value after Bonferroni correction rs9939609 66 (82.5) 12 (15.0) 2 (2.5) 61 (76.3) 17 (21.3) 2 (2.5) NS NS rs12933928 32 (40.0) 33 (41.3) 15 (18.8) 40 (50.0) 36 (45.0) 4 (5.0) 0.025 NS rs1421085 66 (82.5) 13 (16.3) 1 (1.3) 61 (76.3) 17 (21.3) 2 (2.5) NS NS rs11076008 70 (87.5) 10 (12.5) 0 (0.0) 54 (67.5) 24 (30.0) 2 (2.5) 0.005 0.03 rs7185783 27 (33.8) 44 (55.0) 9 (11.3) 34 (42.5) 35 (43.8) 11 (13.8) NS NS rs9923147 64 (80.0) 15 (18.8) 1 (1.3) 52 (65.0) 26 (32.5) 2 (2.5) NS NS a Between patients and controls using 2 test. Data presented as number (%). NS, no statistically significant between-group difference (P 0.05). with DDD: this was the first confirmation of a relationship between genetic factors and DDD. Since then, the study of genetic factors associated with DDD has progressed, with the majority of researchers believing that DDD is a multifactorial disease, with genetic factors having a multiple gene effect. Genetic factors may affect intervertebral disc degeneration through a variety of mechanisms, including changes in the morphology and mechanical structure of the spine leading to intolerance to external force. In addition, genes may influence the biological processes of the intervertebral disc, such as the synthesis and destruction of the biochemical components of the disc, which may lead to accelerated disc degeneration. The gene FTO has become a focus for research since it was reported as a novel
210 Journal of International Medical Research 42(1) obesity-susceptibility gene by a number of genome-wide association studies and followup replication studies. 8 15 In a study by Liuke et al. 18 of 1 832 Finnish people followed up for 4 years using magnetic resonance imaging, body weight was closely related to the occurrence and development of DDD. A study by Hangai et al. 19 reached a similar conclusion that BMI was an important risk factor for DDD. In biomechanical studies, mechanical stress has been shown to lead to injury and degeneration of the intervertebral disc, supporting the role of body weight in the development of DDD. 26,27 Given the relationship between obesity and disc degeneration, the present study examined various SNPs of FTO for any association with the presence of DDD in a Chinese population, and demonstrated that the SNP rs11076008 was significantly associated with lumbar DDD. The biological mechanisms involved in the link between FTO polymorphism and DDD are unknown. Since FTO has a variety of functions, there may be other pathways leading to DDD in addition to an increase in BMI. In the present case control study, in which BMI was matched for, the FTO Figure 1. Summary of allelic and genotypic association analyses showing the log P-values for the differences in frequencies of various single nucleotide polymorphisms of the fat mass and obesity associated gene between patients with degenerative disc disease and controls. The horizontal line represents statistical significance. Table 5. Association of rs11076008 genotype with Japanese Orthopaedic Association (JOA) score (maximum score 29) in patients with degenerative disc disease (n ¼ 80). JOA score Mild (>25) Medium (25, 13) Severe (<12) G/G 16 46 8 A/G 3 5 2 A/A 0 0 0 Data presented as number of patients. There were no statistically significant between-group differences (P 0.05) using the Kruskal Wallis test.
Lao et al. 211 rs11076008 polymorphism (G/G) was significantly correlated with DDD after strict Bonferroni correction. FTO has nine exons and covers 400 kb on the long arm of chromosome 16. 8,9 It has been shown that some regions of the gene are highly conserved, with minor differences among different species of vertebrates. 28 In addition to its effect on BMI, FTO is also closely associated with human leptin levels and energy balance. 29 Genetic factors may have different effects in different populations, and the interaction between environmental factors and genetic factors makes the situation even more complex. It is therefore important to study genetic factors in large samples from different populations and to verify the reproducibility of such research results. Since DDD is a complex multifactorial disorder, it is important to develop a statistical model that includes both environmental and genetic factors. As yet there is limited understanding about the physiology of FTO, and the relationship between FTO polymorphism and the aetiology of DDD needs further clarification. Limitations of this study include the small sample size, lack of generalizability beyond the Chinese Han population, and the limited evaluation of clinical symptoms. Multicentre studies with a large sample population with further analysis of SNPs are necessary. In conclusion, the present study showed that the genetic variant rs11076008 of the gene FTO is associated with DDD and may play an important role in its development in Chinese Han population. These results indicate that the G/G genotype and/or the G allele of this SNP may be a risk factor for DDD. No associations were shown between the SNPs studied and sex or disease severity. The results of the present study suggest that FTO is a DDD predisposition gene and may support a close relationship between obesity and DDD. Declaration of conflicting interest The authors declare that there is no conflict of interest. Funding This research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors. References 1. Andersson GB. Epidemiological features of chronic low-back pain. Lancet 1999; 354: 581 585. 2. Deyo RA and Tsui-Wu YJ. Descriptive epidemiology of low-back pain and its related medical care in the United States. Spine (Phila Pa 1976) 1987; 12: 264 268. 3. Higashino K, Matsui Y, Yagi S, et al. The alpha2 type IX collagen tryptophan polymorphism is associated with the severity of disc degeneration in younger patients with herniated nucleus pulposus of the lumbar spine. Int Orthop 2007; 31: 107 111. 4. Mashayekhi F, Shafiee G, Kazemi M, et al. Lumbar disk degeneration disease and aggrecan gene polymorphism in northern Iran. Biochem Genet 2010; 48: 684 689. 5. Kawaguchi Y, Kanamori M, Ishihara H, et al. The association of lumbar disc disease with vitamin-d receptor gene polymorphism. J Bone Joint Surg Am 2002; 84-A: 2022 2028. 6. Dong DM, Yao M, Liu B, et al. Association between the 1306C/T polymorphism of matrix metalloproteinase-2 gene and lumbar disc disease in Chinese young adults. Eur Spine J 2007; 16: 1958 1961. 7. Gruber HE, Sage EH, Norton HJ, et al. Targeted deletion of the SPARC gene accelerates disc degeneration in the aging mouse. J Histochem Cytochem 2005; 53: 1131 1138. 8. Dina C, Meyre D, Gallina S, et al. Variation in FTO contributes to childhood obesity and severe adult obesity. Nat Genet 2007; 39: 724 726. 9. Frayling TM, Timpson NJ, Weedon MN, et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 2007; 316: 889 894.
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