Association Analysis Between Single Nucleotide Polymorphisms Of THBS1,2 Genes And Intervertebral Disc Degeneration In A Northern Chinese Han Population

ObjectivesMore than 80%of people all over the world will experience some form of low back pain in his life.The intervertebral disc degeneration(IDD)is one of the most common cause of low back pain.The main cause of IDD is genetic factors.It is of great significance to study the genetic factors that affect IDD.Genome research reveals a large number of gene polymorphisms related with IDD,such as genes coding collagen,proteoglycan,interleukins(ILs),apoptosis factors,vitamin D receptor(VDR),matrix metalloproteinases(MMPs)and other proteins.With our in-depth study and understanding of the genetic polymorphisms associated with IDD,we will be able to personalize and individualize patients with IDD.Therefore,the discovery of genetic polymorphisms associated with IDD susceptibility has important clinical application value.Thrombospondins(THBSs)is a set of genes coding the proteins which can combine with collagen and tissue glycoprotein.They participate in cell-to-cell and cell-to-matrix interactions during tissue development and repair.THBS2 gene and THBS1 gene are two genes those have similar structure and function in THBSs family.Bioinformatics methods have been used to analyze the differences in gene expression between normal nucleus pulposus of intervertebral disc and nucleus pulposus of IDD disc.It is found that the expression of THBS1 was down-regulated in degenerated intervertebral disc nucleus pulposus,which revealed that THBS1 might have an important role in the onset of IDD.The single nucleotide polymorphisms(SNPs)of THBS2 gene were also reported to be closely related to the development of lumbar intervertebral disc herniation and lumbar spinal stenosis.Summing up the above,the main problem of our research is to study the relation of polymorphisms of THBS1 gene and THBS2 gene and the onset of IDD in a northern Chinese Han population.It needs to be clear that whether the SNPs of the two genes can be used as potential IDD related molecular markers.With the results reported in literatures and the information of HapMap database(a directory of common genetic polymorphisms in the human genome),we chose the rs6422747 and rs6422748 of THBS2 gene and rs2228262 and rs2292305 of THBS1 gene as the main research objects of our research.MethodsAccording to the inclusion criteria and exclusion criteria of case group and control group,the main research objects of our research are patients diagnosed with IDD in Qilu Hospital of Shandong University(Qingdao)and healthy people who participate in the physical examination in Health Management Center from July 2015 to July 2016.The diagnosis of IDD was based on the results of MRI.MRI was performed using a 1.5 T scanner and a Synergy Spine Coil.T2-weighted fast spinecho sagittal and axial images were obtained(TR/TE:3500/120;slice thickness,4 mm;slice gap,0.4 mm).The level of IDD was assessed by evaluation standard of Schneiderman.All MR images were examined both clinically and radiologically to confirm the degree of IDD.The venous blood samples were extracted from the case group and control group participants.And blood genomic DNA was extracted.Polymerase chain reaction(PCR)was used to get the DNA fragments including the SNP loci.And DNA sequencing and restriction fragment length polymorphism(RFLP)were respectively used to get the SNP alleles and genotypes.At last the correlations between alleles/genotypes and clinical phenotype of IDD were analyzed.ResultsWe recruited 138 IDD patients(71[51.45%]female;mean±SD age=47.6±12.3 years;mean±SD body mass index[BMI]=22.5±2.7 kg/m2).There were 93 patients(67.39%)diagoned with lumbar disc herniation and 45 patients(32.61%)diagnosed with lumbar spinal stenosis.The control group consisted of 136 healthy volunteers(66[58.53%]female;mean±SD age=43.6± 15.3 years;mean±SD BMI=22.5±3.5 kg/m2).Firstly,DNA sequencing was used to detect the alleles and genotypes of rs6422747 and rs6422748 of THBS2 gene.Sequencing results showed that A/G alleles of rs6422747 were detected in both case group and control group.And all the three genotypes of AA,AG and GG were present.C/G alleles of rs6422748 were also detected in both case group and ontrol group.And all the three genotypes of CC,CG and GG were present.The frequency distribution of the two SNP in the case group and the control group was consistent with Hardy-Weinberg equilibrium(HWE).The results of correlation analysis between rs6422747 of THBS2 gene and IDD susceptibility showed that frequency distribution of allele A and G existed significant difference(p = 0.009)between case group and control group.People with G allele are more likely to develop IDD compared with people with allele A(OR = 1.261,95%CI:1.055?1.506).Based on the genotype analysis,the results were similar with the analysis of allele.In the case group AG genotype and GG genotype was significantly more than the proportion of the AA genotype,indicating that population with AG and GG genotype were more likely to develop IDD compared with AA genotype.Therefore,for rs6422747,the allele G might be a risk factor for IDD.The results of rs6422748 were similar to rs6422747.Between the case group and control group,the allele frequency distribution of alleles G and C exists significant difference(p = 0.012).The existence of G allele indicated higher IDD susceptibility(OR = 1.250,95%CI:1.047?1.493).The proportion of CG genotype and GG genotype in the case group was significantly higher than CC genotype,which indicated that the population with CG and GG genotypes was more susceptible to IDD than those with CC genotypes.Therefore,for rs6422748,the allele G is also a risk factor for IDD.The genotype frequency distribution of 2 SNPs was not related to the severity of IDD.The rs2228262 and rs2292305 of THBS1 gene were detected by PCR-RFLP method.The experimental results showed that for rs2228262,only AA genotype was detected in all the patients and the control group.AG and GG genotypes were not detected.For rs2292305 three genotypes of AA,AG and GG were all detected.Therefore,only the correlation analysis of rs2292305 was conducted.The frequency distribution of rs2292305 in the case group and control group was consistent with HWE.Between the case group and control group,the frequency distribution of allele A and G had no significant difference(p = 0.629,OR = 1.261,95%CI:1.055?1.506).The frequency distribution of genotypes had also no statistical difference.Therefore,the rs2292305 of THBS1 gene has nothing to do with IDD susceptibility.The correlation analysis between genotype frequency distribution and IDD severity showed that the genotype frequency distribution of rs2292305 was not related to the severity of IDD.Conclusions1.The rs6422747 and rs6422748 of THBS2 gene polymorphisms are closely related to IDD susceptibility,which may be the risk factors for IDD.2.The polymorphisms of rs6422747 and rs6422748 of THBS2 gene were not related to the severity of IDD.3.The rs2292305 polymorphism of THBS1 gene was not related to IDD susceptibility and severity.Significances1.The correlation between the SNPs of THBS2 gene(rs6422747 and rs6422748).and IDD susceptibility was revealed in a northern Chinese Han population.2.A new monitoring index for IDD diagnosis was found.3.Our study deepened the understanding of IDD mechanisms and provided a new research direction for individualized treatment of IDD patients.