The Study Of Susceptible Gene Relevant To Ossification In Ankylosing Spondylitis

PART1:THE ASSOCIATION OF LRP5 GENE WITH ANKYLOSING SPONDYLITISBackground:Ankylosing spondylitis is a common inflammatory rheumatic disease that affects the axial skeleton, causing characteristic inflammatory back pain, which can lead to structural and functional impairments and a decrease in quality of life. AS with incidence 0.3% usually starts when the patient is 20 to 30 years old, and is more common in males, with a sex ratio ranging from 2 to 5:1. It is not yet fully understood whether the natural course of the disease in women is similar to that in men. Genetic factors provide over 90% of the overall susceptibility to ankylosing spondylitis, with about half of the genetic contribution attributed to HLA-B27 and other major histocompatibility complex genes. Recent studies have shown the other genes within and/or outside the HLA-B27 gene contribute to pathogenic mechanism of AS.The area of Wnt signaling has expanded to encompass areas as diverse as embryogenesis, oncogenesis, haematology, stem cell research and now bone biology. The low-density lipoprotein receptor-related protein 5 (LRP5) functions as a cell membrane co-receptor for Wnt and plays an important role in the Wnt signaling pathway.The present study we performed to explore the association of AS with LRP5 was based on two hypotheses.1) LRP5 is involved in the variance of Bone Mass Density (BMD) and/or bone formation in AS.Loss-of-function and gain-of-function mutations in the human LRP5 gene are associated with osteoporosis-pseudoglioma syndrome (OPPG) and high bone mass (HBM) phenotypes, respectively. In addition, the LRP5 gene plays an important role in the regulation of BMD in the general population.It seems likely that syndesmophyte formation in the advanced stages of AS disease results in an increase in recorded BMD score and masks the actual underlying progressive demineralization of vertebral bodies. Importantly, there is a striking difference between cortical bone and trabecular bone in AS:whereas trabecular bone mass decreases and leads to vertebral osteoporosis and increased fracture risk in AS patients, specific sites of the cortical bone start to proliferate and expand.Based on above findings, the LRP5 regulates the BMD, and AS patients manifest not only osteoporosis but also pathological bone formation.2) LRP5 acts as a switch for inflammation and ossification.The Wnt signaling pathway plays a role in the pathogenesis of RA. Via induction of Osteoprotegerin (OPG) expression, Wnt-signaling proteins are able to block osteoclastogenesis. In human RA synovial tissue, TNF interferes with the Wnt/β-catenin pathway via induction of the Wnt-signaling inhibitor Dickkopf-related protein 1 (DKK1). Through increased induction of DKK1, the Wnt/p-catenin signaling is inhibited, which consequently leads to a decrease in production of OPG. Under physiological circumstances, OPG binds to RANKL and provides a balance between bone resorption and bone formation. In inflammatory arthritis, this balance is disturbed in favor of increased production of RANKL. We hypothesized that LRP5 would serve as a switch of inflammation/ossification, and play a pathologic role independent of DKK1 in AS. Firstly, LRP5 will cause increased induction of OPG, inhibiting bone resorbing of osteoclasts. Concurrently, LRP5 facilitated osteoblast proliferation and differentiation should be observed.Objective:The aim of this study was to investigate the association of LRP5 with AS in a Chinese Han population. Especially, to test whether there are mutations or novel polymorphisms within or flanking the gene of LRP5, including the promoter region(2000bp upstream of the first exon), exonic region, and exon-intron junctions.Methods:Subjects:The cohort consisted of 16 unrelated AS patients (12 men, average age 26.5±5.56 years) who were recruited for discovering mutation or novel polymorphisms in LRP5. All study subjects were of Han Chinese origin from Shandong Province, China. Diagnosis of AS was confirmed by a qualified rheumatologist,with all cases meeting the modified New York criteria for AS after a thorough physical examination and review of medical history. The study was approved by the relevant Ethical Committee, and written informed consent was obtained from each patient participating in the study.Gene sequencing:Samples of whole blood were obtained from AS patients. DNA was extracted using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA).We designed primers with primer3 software on line. After purification of PCR products, we sequenced the LRP5 gene, including the promoter region (2000bp upstream of the first exon), exonic region, and exon-intron junctions with an ABI3130 sequencer. Then SNPs were identified with PolyPhred. Result:By using PCR-based direct sequencing, a total of 24 SNPs including 3 novel SNPs were found in the promoter, exon regions and exon-intron junction of LRP5. The novel SNPs were identified with PolyPhred software.3 novel SNPs named LRP5SNP1 (c.-1596T>C), LRP5SNP2 (c.3764-30G>A) and LRP5SNP3 (c.4488+74G>A) are located in 5' flanking, intron 17 and intron 21, respectively. Furthermore, the minimus allele frequency (MAF) of LRP5SNP1, LRP5SNP2 and LRP5SNP3 were 6.2%,9.4% and 12.5%, respectively. The LRP5SNP3 polymorphism is located in intron 21, just 73bp downstream of exon 21 and 20bp downstream of rs2201466.No mutations were found.Conclusion:There may be no mutations in LRP5 in AS. For the three novel SNPs, a large sample size is required to verify the association of LRP5 with AS.PART2:THE STUDY OF ASSOCIATION BETWEEN GENES INVOLVED IN ENDOCHONDRAL OSSIFICATION AND ECTOPIC OSSIFICATION WITH ANKYLOSING SPONDYLITISBackground:Ankylosing spondylitis is a highly heritable, common rheumatic condition, primarily affecting the axial skeleton. The main symptom of AS is inflammatory spinal pain; with time, some patients develop ankylosis and spinal immobility. Pathological bone formation associated with AS are a major health issue and produce a significant economic and social burden; therefore, early recognition of individuals who may be susceptible to ectopic ossification can provide a substantial benefit.So far, the susceptible gene study of AS primarily lied in the field of inflammation and immunization, however, few experiment have been performed in the field of ossification which is a important cause of disability. TNF-a antagonists provide long-term control of the clinical symptoms and systemic inflammation in most patients with AS. But neither etanercept nor infliximab significantly slowed the radiological progression of AS over a 2-year period treatment. So, exploring the target point related to ossification will certainly be the subject of intense investigations in treatment of AS. Other than gene related to inflammation and immunization, is there ossification gene Involved in pathogenic mechanism of AS? And how would we identify candidate susceptibility genes related to ossification? We consider the following points:1). Ossification initiation depend on inflammation,but ossification development is independent of inflammation. Ossification would have it's own genetic background.2).The essence of ossification in AS is endochondral ossification. Historical studies have demonstrated that both endochondral and direct bone formation contribute to ankylosis in SpA.3). Signal pathway of endochondral ossification maladjustment and osteoblasts and osteoclasts loss coupling have been found in AS. several signaling pathways have been identified involved in the process of endochondral ossification, such as Wnt, BMP, hedgehog and matrix mineralization signaling pathways, and so on.Candidate gene selection strategy:1. By meta-analysis of the previous whole genome scan, we select the susceptibility genes of heterotopic ossification within susceptible region, including BMP6, RUNX2, IHH,COL11A2, ENPP1.2. Select important genes and transcription factor Involved in signaling pathways related to endochondral ossification, including LRP5, DKK1 in Wnt signal pathway; BMP6, BMP4, BMP2 in BMP signal pathway; DMP1, ENPP1, MGP in matrix mineralization pathway; IHH in hedgehog signal pathway; RUNX2, SOX9 transcription factor related to ossification)3. Select susceptibility genes of diseases related to ectopic ossification, including ACVR1, COL11A2.4. Select effective target for treatment of heterotopic ossification, including PTGS2.5. Select the genes with positive loci through Genome-wide scan of other races, including ANTXR2.In finally, we selected 15 candidate genes to study the association with AS.Objective:to explore the susceptible gene related to ectopic ossification of ankylosing spondylitis, in order to provide a new therapeutic target for the treatment of AS, inhibit the formation of pathological bone of AS and reduce disability of AS.Method:Participants:A total of 300 patients with AS and 180 ethnically matched healthy controls were enrolled for this study. All cases and controls were native Shandong Han Chinese population, ranging in age from 11 to 70 years, and AS cases satisfied the modified New York criteria after a thorough physical examination and review of medical history. The patients'pelvic and lumbosacral spine radiographs were read by a radiologist. Information was collected systematically and included sex, age of onset, HLA-B-27 status, Kyphosis and bamboo spine, radiology grading. Ethnically matched, unrelated healthy blood donors (n=180) recruited from the Ji'nan Blood Bank were included as controls. The study was approved by the relevant Ethical Committee, and written informed consent was obtained from each patient participating in the study.Genotyping:Peripheral blood from participants was collected and the genomic DNA was isolated. Genotype was identified by Illumina GoldenGate gene chip genetyping platform.SNP selection:To select the SNPs for this study, Tag SNPs covering selected genomic regions (including 3000 base pairs upstream of the first exon) were selected using Tagger in Haploview 4.0 and gave 100% coverage of all selected genes (r2>0.8). In the block. SNPs within promoter, coding region, splice locus, missense locus and nonsense locus were preferentially selected. The markers rs41494349. rs3736228, LRP5SNP1, LRP5SNP2, LRP5SNP3 and other markers related to ectopia ossifcation were forcibly included in the set of tagSNPs. In total,96 SNPs were finally selected.Statistical analysis:SNPs with absence rates> 0.1 were excluded, and an exact test for Hardy-Weinberg equilibrium was performed in controls. Difference in genotype and allele frequencies between patients and controls were compared using the Fisher chi-square test. The odds ratios (OR) with 95% confidence intervals (95%CI) were calculated to estimate the effect of different alleles. Allelic association tests were conducted for each marker using PLINK soft ware version 1.06, and genotype, haplotype analysis were performed using SHEsis on line program. P values of<0.05 were considered statistically significant.Results:1). Inclusion criteriaVia Inclusion criteria (Hardy-Weinberg equilibrium for controls, p>0.05, Minimum allele frequency>0.05, SNP callrate>0.9, SNP call freq>0.9), in final,296 patients,170 controls and 84 SNPs were selected.2). Quality controlTo test the accuracy of genotyping control quality, we regenotyped the randomly selected 10 samples, and the reproducibility was>96%.3). Association analysis①.Case-control association analysisA). Significant association in allele frequencies of 7 SNPs (rs 10019009 in DMP1, LRP5SNP3 in LRP5, rs16873348 in RUNX2, rs35565233 in RUNX2, rs6910759 in BMP6, rs3178250 in BMP2, rs6935458 in ENPP1) were found between AS patients and healthy controls.B). Significant association in genotype frequencies of 3 SNPs (rs 10019009 in DMP1, rs16873348 in RUNX2, rs35565233 in RUNX2) were found between AS patients and healthy controls.C). Significant association in haplotype frequencies of 9 genes (DMP1,RUNX2,BMP6,BMP2,ANTXR2,ACVR1,ENPP1,LRP5,PTGS2) were found between AS patients and healthy controls.②. Association analysis within the case groupA). Significant differences in allele frequencies of 6 SNPs (rs7595478 in ACVR1, rs7750470 in RUNX2, rs267180 in BMP6, rs267205 in BMP6, rs235768 in BMP2, rs6054512 in BMP2) were found between AS patients with and without bamboo spine.B). Significant differences in genotype frequencies of 5 SNP (rs7595478 in ACVR1, rs7750470 in RUNX2, rs267180 in BMP6, rs267205 in BMP6, rs6054512 in BMP2) were found between AS patients with and without bamboo spine.C). Significant differences in allele frequencies of 4 SNPs (rs2820339 in RUNX2, rs7750470 in RUNX2, rs 1266562 in RUNX2, rs6054512 in BMP2) were found between AS patients with and without kyphosis.D). Significant differences in allele frequencies of 4 SNPs (rs2820339 in RUNX2, rs7750470 in RUNX2, rs 1266562 in RUNX2, rs 1225930 in BMP6) were found between AS patients with and without Radiology standards 3-4 grade.E). Significant differences in allele frequencies of 2 SNPs (rs7592246 in IHH, rsl 358893 in BMP6,) were found between>20 and<20 years old of age of onset in AS patients.F). Significant differences in allele frequencies of 3 SNPs (rs4333130 in ANTXR2, rsl98354 in BMP6, rs686921 in LRP5) were found between male and female in AS patients.③. Association analysis within controls. significant differences in allele frequencies of 3 SNPs (rs686921 in LRP5, rs12651388 in ANTXR2, rs4236 in MGP) were found between male and female in controls.Conclusion:1. DMP1, ENPP1 may be involved in the pathogenesis of AS through the mechanism of matrix mineralization.2. Support the notion that Wnt signaling pathway is involved in the pathogenesis of AS.3. Through the BMP signaling pathway, RUNX2, BMP6, BMP2 may be involved in the pathogenesis of AS and the formation of heterotopic ossification.4. FOP and AS may have a similar mechanism of heterotopic ossification.5. Support the notion that COX-2 is involved in the pathogenesis of AS.6. IHH and BMP6 is related to early-onset of AS.7. ANTXR2, LRP5, BMP6 may be associated with male dominance of AS. PART3 THE STUDY OF FUNCTION FOR LRP5SNP3Objective:The LRP5SNP3 polymorphism is located in intron 21. just 73bp downstream of exon 21 and 20bp downstream of rs2201466. We hypothesized that LRP5SNP3 may play a pathological role through interference of splicing the exon-intron junction. Experimental purposes was to explore whether the effects of LRP5SNP3 on RNA splicing between intron and exon.Method:Splice-site prediction programs (SSPPs)Splice-site prediction programs (SSPPs) have been developed to predict the possible effect of a variant on RNA splicing.The following splice-site analysis tools were used:1. NetGene 2 (NG2).2. Automated Splice-Site Analysis (ASSA),3. MaxEntScan (MES).These programs are very useful to assess LRP5 variant that are likely to affect RNA splicing for further analysis.RT-PCRWe used Software primer5 to designe three pairs of primers. The first pair of primers was located in exon 3; the second pair of primers was located exon 20 and exon 22:the third primer was located exon 20 and exon 22. GAPDH was used as internal control. The total RNA of peripheral blood of patients and healthy people was extracted using commercial kits (two-step method for RT-PCR experiments).Results:1. Using Splice-site prediction programs (SSPPs), we didn't predict the possible effect of the LRP5SNP3 on RNA splicing.2. We did not find expression of LRP5 in the peripheral blood of patients and controls.Conclusion:1. The expression of LRP5 in the peripheral blood is relatively low, and the RNA of LRP5 is degraded in the extraction process.2. LRP5SNP3 may be involved in the pathogenesis of AS through other mechanisms, such as effect on expression of LRP5. PART4:experimental validation studies of rs10019009 within DMP1 gene associated with ankylosing spondylitisBackground:In the second part of the present study with300 patients and 180 controls, we found that the rs10019009 located DMP1 gene have a strong correlation with AS, p =0.001229, OR=0.64.95% CI [0.49-0.84].Dentin matrix acidic phosphoprotein is an extracellular matrix protein and a member of the small integrin binding ligand N-linked glycoprotein family. This protein, which is critical for proper mineralization of bone and dentin, is present in diverse cells of bone and tooth tissues. The protein contains a large number of acidic domains, multiple phosphorylation sites, a functional arg-gly-asp cell attachment sequence, and a DNA binding domain. In undifferentiated osteoblasts it is primarily a nuclear protein that regulates the expression of osteoblast-specific genes. During osteoblast maturation the protein becomes phosphorylated and is exported to the extracellular matrix, where it orchestrates mineralized matrix formation. Mutations in the gene are known to cause autosomal recessive hypophosphatemia.Objective:Rsl0019009 is a missense variation(A-T). leading to a serine to cysteine change at amino acid position 69. The region including 67-83 amino acids is conserved in DMP1 gene, i.e. glycosaminoglycan region. The region plays an important role in mineralized nucleation. As a result, we believe that rs10019009 may be a functional site leading to AS, so we perform experimental verification for the locus of rs10019009.Method:Subjects:From Shandong Provincial Hospital, Shandong University of Traditional Chinese Medicine, Affiliated hospital, Jining Medical University and Linyi People's Hospital, we collected 350 cases of AS. The patients met the modified New York criteria for AS. Blood of 400 healthy donors was collected from Ji'nan Central Blood Bank.DNA extraction:Extraction of peripheral blood genomic DNA. Using Omega EZ 96 blood genomic DNA extraction kit (EZ 96 Blood DNA Kit) we extracted DNA from 250μl blood, for the detail operations, seeing DNA extraction kit. Via Spectrophotometer oMerinton SMA1000 DNA. we measured the concentration of DNA. The final concentration of DNA was 100ng/μl. The measured A260/A280 value was 1.65-1. 80. The DNA was diluted to 20-50ng/μl. and stored at-40°Genotyping:we used TaqMan-PCR method of genotyping. The probes of rs 10019009 were ordered from Biosystems (Applied B iosystems) synthesis.10μl Reaction included probes Mix, Lightcycler480 Probe Master, PCR Grade H2O and DNA samples. The reaction was performed on the Lightcycler480 PCR instrument. Endpoint was used for Genotyping Analysis of the results. Using online software SHEsis and SPSS 11.5 package for statistical analysis, statistical process exclude individual genotyping failure.Results:Association analysis within case-control:1. The association between rs 10019009 and AS was not statistically significant, p= 0.242, but the risk allele T was linkage disequilibrium with AS.2. To put the first part of the experimental group and the second part of the experimental groups together, via the association analysis within case-control, we found the a strong correlation between rs 10019009 and AS, even after correction for multiple testing, p values were still less than 0.05.Conclusion:Rs 10019009 is a important SNP within DMP1gene, and may be involved in the pathogenesis of AS.PartⅤthe study of effect for the rs10019009 in DMP1 gene on mineralization mechanism of U20S cellsObjective:To investigate the mechanisms involved in mineralization of U2OS cells from SNP rs 10019009, so as to control the ossification of AS to provide the role of target.Method:1. Using protein analysis software, we analyzed the DMP1 tertiary structure, function conserved region, and the active site region. Via SNP function prediction software, we forecasted if rs 10019009 was a functional locus.2. U2OS cell culture. U2OS cell is low phosphatase activity, no mineralization of human osteoblast-like cells. RT-PCR was performed to measure the expression levels of DMP1 in U2OS cells before gene transfection.3. Plasmid construction. We constructed the pEGFP-1-DMP1 expression plasmid. Site-directed mutagenesis of rs 10019009 Rs 10019009 [bases a (Ser)-bp t (cysteine)-base c (Arg) base g (glycine)] were performed. After site-directed mutagenesis the plasmid was transfected into U2OS cells.4. Experimental groups. The experiment was divided into groups with mineralized inducer and groups without mineralized inducer; the DMP1-transfected groups and the non-transfected groups. And in different groups, after cell culture 0 days,48 hours,5 days the phenotype was test.5. Cell phenotype testing. The cell phenotype testing were included Alizarin red staining of mineralized nodules, alkaline phosphatase ALP activity detection and RT-PCR detection of ALP, DMP1, osteocalcin, RUNX2, BMP2, BMP6 expression.Results:1. Rs 10019009 is located in the DMP1 conservative region, and have impact on the function of DMP1.2. Successful site-directed mutagenesis of the rs 10019009 SNP site, and successfully transfected into U2OS cells, transfection efficiency of 75%.3. We didn't find expressing of DMP1 in U2OS without exogenous gene transfection.Conclusion:1. Rs 10019009 SNP is a important DMP1 gene locus, and may affect the function of DMP1.2. U2OS cells were cell model fit for studied the DMP1 gene function. The studies laid the foundation for study the gene function of DMP1.