Association Of Tag SNPs Of Genes Related To Bone Metabolism With ONFH Risk And Clinical Phenotypes

Osteonecrosis of the femoral head (ONFH) is a complex diseases caused by theinteraction of genetic and environmental factors.The pathogenesis of ONFH hasbeen remained obscure.It is difficult to make a early diagnosis, and the patients withONFH eventually have to face the hip replacement.Since the incidence of ONFH hasbeen increased in recent years, it is significant to clarify the molecular etiology andpathogenesis of ONFN.During recent years, one of the most important developments of molecularpathogenesis of ONFH is to find the association the gene polymorphism withONFH risk, including in the fibrinolytic enzyme activation/inhibition series,angiogenesis series, lipid metabolism series, collagenⅡ type series, cytokinesseries, growth factors series etc.Those genes may be able to influence thepathogenesis of ONFH by the linkage disequilibrium, to be the united roles togetherwith the other genes, or to affect the expression of other susceptible genes during theonset and the development of ONFH.The research results of genetic molecularetiology clearly suggested that the development of ONFH involved multiple minorgenes and environmental factors.The information of gene polymorphism is huge,and the investigation of susceptible genes related to ONFH often needs to get largesamples and confirm the genetic association of ONFH with the clinical phenotype.At present, the main problems of the gene polymorphism research of ONFHinclude:①there were more scattered genes reports and less systemic andmultiple genes reports, which is disadvantaged to develop the molecularpathogenesis of ONFH caused by multiple minor genes;②there were the morereports only limited gene types other than the researches related to clinical phenotypeof ONFH, which is not useful to confirm the roles of SNPs in the development ofONFH;③there were more information only reported gene polymorphisms but theless information related to gene expression and its function, which is not enough todemonstrate the relationship between gene mutation and the pathogenesis of ONFH.Particularly important, the finding and development of signaling pathways on bonemetabolism make the chance to develop the molecular pathogenesis of ONFH butalmost no the report of the association of genes polymorphism on signaling pathwaysof bone metabolism with ONFH risk.Based on the molecular pathogenesis progress of ONFH and the bottleneckproblems,This investigation will respectively elaborate:(1)the association ofgenotyping, allele frequency, haploid type, the genes interaction with the ONFH risk; (2)the association of genotyping,allele frequency,haplotype of Tag SNPs with ONFHclinical phenotypes;(3) the difference of protein expression levels among the differentgenotyping and haplotype, and the effects of gene mutations on the expressionefficiency and function related to ONFH;(4) the association of genes polymorphismson signaling pathways of bone metabolism with ONFH risk and its clinicalphenotypes.Our main research results are as following:1.The genotyping、 allele frequency and haplotype of7tagSNPs fromSREBF-2,IGFBP3,SOX9gene in361cases of ONFH group and healthy controlsystem were completed by LDR techniques. It was showed that the frequency ofSOX9gene rs1042667(A/C)-rs12601701(A/G) A-A haplotype in patients withONFH were significantly lower than that in control group (P=0.03),which suggestedA-A haplotype as a protection type to decrease ONFH risk. The haplotype A-T-G ofSREBP2rs1052717（A/G）-rs2267439(C/T)-rs2267443(A/G） in patients with ONFHwas lower tendency than that in control group（P=0.063), while the G-T-A haplotypewas higher direction（P=0.076). The interaction results among the TagSNPs suggestedthat the interaction between the SREBP2rs1052717（A/G）and IGEBP3rs3110697(A/G)was related to increased ONFH risk（P=0.037).2.The serum protein expression of IGFBP3,IGF-1,SREBF2and SOX-9genewere detected by immune enzyme-linked technology. The result showed that serumprotein levels of IGFBP3as well as IGF-1in the patients with ONFH weresignificantly higher than that in control group（P=0.045, P=0.007）.The serumprotein level of SOX9in ONFH patient was most significantly lower than that incontrol group（P=0.000).The results not only firstly put forward evidence ofabnormal expression of IGFBP3, IGF1and SOX-9genes in the patients with ONFHbut also elaborated the function of the target genes, which suggested the scientificbasis on the effects of genetic polymorphism on gene function.3.In the polymorphism typing system of target genes, It was demonstrated thatthe serum TG level as well as LDL-c level of ONFH group were significantly higherthan that in control group (P=0.01,P=0.005）,while both the HDL-c level and Theratio of LDL-c/HDL-c of ONFH group were obviously lower than that in controlgroup（P=0.00,P=0.00）. In the Tag SNPs analysis system of key genes on bonemetabolism, the results synchronously proposed the abnormal evidence of serum lipid metabolism in patients with ONFH and again confirmed that the lipid metabolismdisorder was a key link in the pathogenesis of ONFH, suggesting an important role ofkey genes mutation and theirs function of lipid metabolism in the development ofONFH.4.The correlation analysis between TagSNPs genotyping and clinicalphenotypes of ONFH revealed that the association of SREBF-2rs2267439TT typewith the Ficat stages was a significant direction (P=0.076); in the IGFBP3rs2453839TT type or CT Type carriers, the Ficat IV patients were more than Ficat IIIpatients,while in the CC type carriers, Ficat IV patients were less than Ficat IIIpatients（P=0.017); in the SOX9gene rs12601701GG type or AG Type carriers，thepatients suffered double hips lesions were more than the patients suffered singlehips lesions, while in the AA type carriers，the patients suffered double hip lesionswere less than the patients suffered single hips lesions （P=0.009）,which proposedthat the scientific basis of the TagSNPs typing of IGFBP3, SOX9gene involved inclinical phenotypes of ONFH.5.The analysis between the haplotypes of SREBF-2,IGFBP3,SOX9genes andclinical phenotype of ONFH showed that the frequency of double hip lesions wassignificantly less than that of single hips lesions in the ONFH patients to carrySOX9rs1042667-rs12601701A-A or C-A phenotype（P=0.03,P=0.048)，while thefrequency of double hip lesions was obviously more than that of single hips lesions inthe ONFH patients to carry A-G haplotype(P=0.013). The results first proposed thatthe haplotypes.of SOX9gene was closely associated with the clinical phenotypes ofONFH.6.The analysis of genotype,allele frequencies,and haplotypes of TagSNPs ofGsk3β,Sfrp4gene on WNT signaling pathway was completed in377ONFH cases-control system by Mass Spectrometry technique. The results showed that in thecarriers of Sfrp4rs1052981CC genetype of ONFH patients, their onset age was earlierthan that of TC and TT type carriers(P=0.05);the frequency of Sfrp4C-A haplotype ofONFH group was lower tendency than that of control group (P=0.073); the malepatients of Gsk3β rs3755557TA carriers was higher trend than female patients of thegenetype carrier (P=0.067), which first proposed that the molecular genetics of Sfrp4 and Gsk3TagSNPs on Wnt signal pathway was related to clinical phenotype ofONFH.7.The analysis results of genotype, allele frequencies,and haplotype of10TagSNPs of OPG、Rank、Rankl、Traf6、Nfatc1on Rank/Rankl/OPG signal pathwayshowed that the Nfatc1rs9518CC genotype frequency of ONFH patients was higherdirection than that of control group（P=0.057) and the Ficat IV patients carryingNfatc1rs9518TC genotype was significantly higher than Ficat III patientst of the samegenetype（P=0.03); in the patients of OPGrs2073617TT genotype carrier, theproportion of idiopathic ONFH was significantly higher than that of steroids andalcoholic ONFH(P=0.02); in the TRAF6rs5030411(C/T)-rs5030416(C/A)C-Chaplotype carrier, the patients suffered double hips lesions were more tendency thanthe ones suffered single hip lesions（P=0.06）；the interaction of Rankl rs1054016andRankl rs9525641was associated with the ONFH risk（P=0.026), which first suggestedthat molecular targets of RANK/RANKL/OPG signiialing pathway associated withONFH risk.8.The analysis of genotypes, allele frequencies,and haplotypes of11TagSNPsof COL2A1,Runx-2,Osterix, PPARγ,IGFBP3genes on key transcription factors ofbone metabolism were investigated in377cases-control system by MassSpectrometry technique. The results showed that not only thegenotype frequency carrying PPAR γ2920502CC but also the allele frequencycarrying PPAR γrs2920502C of ONFH group were significantly higher than thatof control group (P=0.0005, P=0.0005). However, the allele frequency carryingPPARγ2920502G in ONFH group was significantly lower than that of the controlgroup (P=0.0005), first proposing the gene polymorphism of PPARγ was closelyrelated to ONFH risk. The results also revealed that genotype frequency ofIGFBP3rs2132572AA in ONFH group was significantly higher than that of thecontrol group (P=0.042), but in rs2132572GA carriers, the frequency suffered doublehips lesions was obviously lower than that suffered single hip lesions (P=0.039).In COL2A1rs2070739AA carrier, the onset age of ONFH was significantly olderthan that of GG and GA type carriers(P=0.008) and in COL2A1A-T-G-A haplotypecarriers, the proportion suffered double hips lesions was significantly lower than thatsuffered single hip lesions. These results also suggested that the gene polymorphism association of multiple transcription factors with the onset anddevelopment of ONFH and also proposed the multiple molecular targets related toONFH risk.9.The investigation also found that the interaction among TagSNPs of multipletranscription factors genes were significantly correlated to ONFH risk, incLuding inCOL2A1rs3809322vs COL2A1rs1859444（P=0.043)，COL2A1rs3803183vsCOL2A1rs3809322（P=0.041), COL2A1rs1859444vs Osterix rs4759113（P=0.013)，COL2A1rs2070739vs IGFBP3rs2132572（P=0.006)，COL2A1rs2070739vs IGFBP3rs2854744（P=0.002); COL2A1rs2070739vsIGFBP3rs2854746（P=0.006)，COL2A1rs3809322vs RUNX-2rs376319（P=0.032)，RUNX-2rs7751427tvsIGFBP3rs2854744(P=0.009）， RUNX-2rs7751427vsIGFBP3rs2854746（P=0.005). These results Indicate that the synergistic roles frommultiple transcription factors genes may increase ONFH risk and suggest that thecombined effects of multiple minor genes are the basic characteristics ofONFH molecular genetics.