Influence Of Vitamin D Receptor Gene Polymorphism And Its Levels Of Serum On Susceptibility Of Bone And Joint Tuberculosis

BACKGROUNDDespite the significant decline in tuberculosis,the burden of tuberculosis disease is still serious.World Healthy Organization said that about a third of people worldwide are infected with Mycobacterium tuberculosis,of which only 10%of people develop tuberculosis.In recent years,studies have found that,in addition to environmental and lifestyle factors,individual's genetic background is another factor in the occurrence of tuberculosis.It has been found that multiple gene locus are closely related to the incidence of tuberculosis,among which the vitamin D receptor(VDR)is one of the major candidate genes.The human VDR gene is located on the long arm of chromosome 12.Using restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP)showed that the gene sequence has several endonuclease sites,confirmed that the VDR gene has significant polymorphism.Up to now,no less than 25 VDR polymorphic locus have been found,including Apa I,Bsm I,Fok I and Taq I,which are the major sites for the regulation of bone metabolism.In addition,many of the VDR genes regulate the metabolism of vitamin D by a conservative endocrine pathway,so any changes in VDR protein or its function can affect the metabolism of vitamin D,which affects the susceptibility of bone and joint tuberculosis.The VDR gene polymorphism on susceptibility to pulmonary tuberculosis has been reported at home and abroad,but whether the VDR gene polymorphism has impact on susceptibility of bone and joint tuberculosis,the investigations are rare either in domestic and foreign.So far there is no study on the polymorphism of vitamin D receptor gene and the susceptibility to bone and joint tuberculosis in Guangdong Han population.Meanwhile,as a ligand of VDR,whether the serum concentration of vitamin D affects the susceptibility of bone and joint tuberculosis,the conclusions of different regions are not consistent.PURPOSE1.To investigate the association between vitamin D receptor gene polymorphism and bone and joint tuberculosis susceptibility in Guangdong Han nationality by detecting the vitamin D receptor gene polymorphism in bone and joint tuberculosis patients and non-bone and joint tuberculosis patients.2.To investigate the relationship between vitamin D serum concentration and the susceptibility of bone and joint tuberculosis in Guangdong Han nationality by detecting vitamin D serum concentration in bone and joint tuberculosis patients and non-bone and joint tuberculosis patients.MATERIALS AND METHODS1.133 patients with bone and joint tuberculosis in clinic from May 2015 to December 2016 were enrolled randomly to be tuberculosis group.130 volunteer were chosen as the control group from October 2015 to December.Preoperative blood test indicators and radiological imaging revealed bone and joint tuberculosis lesions,postoperative pathology showed signs of tuberculosis infection,isolation of Mycobacterium tuberculosis positive or positive acid fast stain lesions,included in the tuberculosis group.There are no difference in sex and age between two groups.2.2ml peripheral vein blood was drawn from each person with empty stomach in the early morning.Serum were prepared from 1ml venous blood and placed at-80 ?.Other 1ml of each one was extracted genomic DNA.3.Using polymerase chain reaction(PCR),gene cloning and DNA sequencing to analyze the vitamin D receptor polymorphism(rs7975232,rs1544410,rs2228570,rs731236).4.The serum 25-hydroxy vitamin D levels were determined by enzyme-linked immunosorbent assay(ELISA):All experimental procedures were carried out according to the instructions,including sealing plates,washers,pipetting,water baths,coloring,enzymes and OD values.5.SPSS software(version 20,SPSS,Chicago,IL)was used for statistical analysis.The measured data are expressed as mean � standard deviation(x�s),and the data of this study are in accord with the normal distribution.The enumeration data are expressed as frequency.Chi square test was used to compare categorical variables and enumeration data.Independent samples t test was used to compare continuous variables.Bilateral P<0,05 indicated that the difference was statistically significant.RESULT1.The expected values of each genotype were calculated according to Hardy-Weinberg's law of genetic equilibrium.The chi square test was similar to the observed value and the expected value,the P value was>0.05,indicating that the control group of samples were good representative came from a genetic balance group.2.The results showed that there was no significant difference between the 2 groups in age and gender(P>0.05).3.The genotype of the vitamin D receptor gene rs7975232(Apa I)was divided into wild type AA,mutant Aa and aa.Three genotypes were detected in the tuberculosis group and the control group,and the distribution in the two groups was statistically(P<0.05).The genetype AA was significantly overexpressed in case group.Compared with the Aa genotype,AA genotype is associated with greater risk of bone and joint tuberculosis.The odds ratios were 1.924 and 95%confidence intervals were 1.141-3.245.4.The genotypes of the vitamin D receptor gene rs1544410(Bsm ?)were divided into wild-type BB,mutant Bb and bb.In the tuberculosis group and the control group,bb genotype were not detected in the two groups.There was no significant difference in genotype BB and Ba in the two groups(X2 = 0.003,P>0.05).5.The genotype of vitamin D receptor gene rs2228570(fok ?)was divided into wild-type FF,mutant Ff,and ff.Three genotypes were detected in the tuberculosis group and the control group,and there was a statistically significant difference between the two groups(P<0.05).The genetype ff was remarkably lower in case group.Compared with the FF and Ff genotype,ff genotype is associated with lower risk of bone and joint tuberculosis.The odds ratios were 0.458,0.549 and 95%confidence intervals were 0.235?0.893,0.306?0.982 respectively.6.The genotype of the vitamin D receptor gene rs731236(Taq I)was divided into wild-type TT,mutant Tt,tt.In the tuberculosis group and the control group,tt genotype were not detected in the two groups.There was no significant difference between the two groups(X2=1.640,P>0.05).7.Compared with the control group,the concentration of 25-hydroxy vitamin D was lower in the tuberculosis group,and there was a statistically significant difference between the two groups(t=2.132,P<0.05).CONCLUSION1.The genotypes of rs7975232(Apa ?)and rs2228570(Fok ?)of the vitamin D receptor gene polymorphisms were different between the two groups,suggesting that the polymorphisms of the vitamin D receptor gene may be related to bone and joint tuberculosis.Among them,Apa I-AA may be the susceptible genetype while Fok I-ff may be the protective genetype of bone and joint tuberculosis.There was no significant difference in rs1544410(Bsm ?)and rs731236(Taq ?)genotype distribution between the two groups,suggesting that the polymorphism might not be associated with bone and joint tuberculosis.2.The concentration of 25-hydroxy vitamin D in serum of patients with tuberculosis was reduced,suggesting that patients with bone and joint tuberculosis may have a deficiency of serum 25-hydroxy vitamin D.Serum levels of 25-hydroxy vitamin D may play a role in the pathogenesis of bone and joint tuberculosis.