Association Of The Programmed Cell Death 1 (PD-1), PD-1 Ligands Gene And Asporin Repeat Polymorphism With Ankylosing Spondylitis (AS) And AS Susceptibility Gene Defined By Meta-analysis

ObjectiveTo investigate the association of the PD-1 and PD-1 ligands gene polymorphisms and the haplotypes with ankylosing spondylitis (AS) in a Chinese population sample. To explore the role of the functional polymorphism consisting of an aspartic acid (D) repeat polymorphism located in the ASPN gene in the susceptibility to and clinical outcome of AS.To investigate whether there is any consistent evidence of linkage across multiple studies, and to identify novel AS susceptibility loci by using GSMA method. To explore the association between AS with polymorphism in -308 site of TNF-αpromoter region.Methods1. A total of 196 Chinese patients with ankylosing spondylitis and 180 controls of the same ethnic origin and matched for age and sex were included in the study. The PD-1 and PD-1 ligands polymorphism was genotyped using polymerase chain reaction-restriction fragment length polymorphism and allele-specific PCR and fluorescence melting curves.2. The asporin D repeat polymorphism was genotyped using polymerase chain reaction with a fluorescent primer.3. Genome-search meta-analysis (GSMA) method was applied to genome scans of AS and spondyloarthropathy (SpA) to assess evidence for linkage across studies.4. The literature about AS and polymorphism in -308 site of TNF-αpromoter region were seached and the meta analysis were performed.Results 1. The distribution of genotypes PD-1 rs2227981C/T in AS patients and healthy controls were in agreement with genomic balance (P=0.711; P=0.17668) using HWE detection. No significant difference was found for the distribution of PD-1 rs2227981 between AS patients and healthy controls (P=0.226; P=0.283).2. The distribution of genotypes PD-1 rs2227982C/T in AS patients and healthy controls were in agreement with genomic balance (P=0.1712; P=0.0556) . A significant difference was found for the frequencies of genotypes PD-1 rs2227982 T/T, C/T and C/C between AS and healthy controls (P=0.004). In AS group, the frequencies of genotypes PD-1 rs2227982 T/T, C/T and C/C were 9.69%, 36.22% and 54.08% respectively; in healthy controls, the frequencies were 5%, 24.44% and 70.56% respectively. The frequency of PD-1rs2227982T was higher in AS than healthy controls (28.53%Vs17.22%; P=0.000) .When gender was taken into consideration, the frequencies of alleles and genotypes of PD-1 rs2227982 were significantly high in male AS patients (P=0.004; P=0.019) .While in female AS patients, only PD-1 rs2227982T expression was higher than controls (P=0.004) , no statistic difference was found for other genotypes (P=0.089) .3. Haplotype analysis of PD-1 rs2227981 and rs2227982 showed CT haploid was significant high in AS group than in control group (19.2%Vs11.4%; P=0.003) , and CC haploid was more common in controls (47.1%Vs59.9%; P=0.001) .4. For the study of association between genotypes of PD-1 rs2227982 and clinical manifestation of AS, AS patients with genotype TT showed a higher risk of hip joints and knee joint involvement (P=0.000; P=0.003) , and a severer cacroiliac joint destruction (P=0.000) .5. The distribution of genotypes PD-L1 rs822336 in AS patients and healthy controls were in agreement with genomic balance (P=0.1477; P=0.0794) using HWE detection. A significant difference was found for the frequencies of genotypes PD-L1 rs822336 C/C,C/G和PG/G between AS and healthy controls (P=0.007). In AS group, the frequencies of genotypes PD-L1 rs822336 C/C,C/G和G/G were 9.69%, 48.98% and 40.31% respectively; in healthy controls, the frequencies were 10%, 34.44% and 55.56% respectively. The frequency of PD-L1 rs822336C was higher in AS than healthy controls (34.69%Vs27.22%; P=0.027) . When gender was taken into consideration, the frequencies of alleles and genotypes of PD-L1 rs822336 were significantly high in male AS patients (P=0.005; P=0.002) ; while no statistic difference was found for all the alleles and genotypes in female AS patients (P=0.454; P=0.731) .6. For the study of association between genotypes of PD-L1 rs822336 and clinical manifestation of AS, AS patients with genotype CC showed a higher incidence of low back pain and iritis (P=0.037; P=0.000).AS patients with genotypes CC and CG showed a severer sacroiliitis (P=0.002).7. The distribution of genotypes PD-L2 rs1009759 in AS patients and healthy controls were in agreement with genomic balance (P=0.1740; P=0.1902) using HWE detection. No significant difference was found for the distribution of alleles and genotypes of PD-L2 rsl009759 between AS patients and healthy controls (P=0.371; P=0.695).8. The distribution of genotypes PD-L2 rs6476985 in AS patients and healthy controls were in agreement with genomic balance (P=0.6747; P=0.0517) using HWE detection. No significant difference was found for the distribution of alleles and genotypes of PD-L2 rs6476985 between AS patients and healthy controls (P=0.061; P=0.106) .9. Haploid analysis of PD-L2 rs1009759 and rs6476985 showed the frequencies of CC,CT,TC,TT between AS patients and healthy controls were 26.6% Vs 30.1%, 24.1% Vs 23%, 23.4% Vs 29.1%, 25.9% Vs 1.8% respectively, no statistic difference was found (P >0.05) .10. Seven ASPN-D alleles detected in AS patients and healthy controls.The frequency of each allele was in agreement with genomic balance (P=0.273; P=0.488). ASPN-D polymorphism showed a significant difference between AS patients and healthy controls (P=0.000) . A higher frequencies of D14 and D16 were found in AS patients (P=0.019; P=0.001) . When it was stratified by gender, a significant difference of ASPN-D polymorphism was found between male AS patients and male healthy controls (P=0.000) , while no significant difference was found in females (P=0.595) . Patients with D13/- had a later onset of the disease, and D16 heterozygote showed a earlier, but these did not reach significant differences (P > 0.05) .11. Association analysis of genotypes of ASPN-D and clinical manifestation of AS showed AS patients with genotype D13D16 had a higher risk of hip joints involvement, alagia (P=0.000,P=0.000) and stronger destruction of cacroiliac joint(P=0.001) ; D14D14 patients showed a higher risk of hip joints involvement (P=0.006) and iritis (P=0.000) ; D13D13 patients showed a weaker destruction of cacroiliac joint (P=0.034) .12. Four AS genome scans including 479 families with 1,151 affected individuals were used. Suggesting these bins most likely contain AS-linked loci; bin 6p22.3-p21.1,6pter-p22.3,17pter-p12,2pl2-q22.1 and 5q34-qter.Four AS genome scans and one SpA scan including 544 families with 1,331 affected individuals were used. The GSMA produced genome-wide evidence for linkage on bin 6p22.3-p21 and 16q23.1-qter.13. Eight studies enrolled 987 AS patients and 922 controls in total.The analysis showed that the frequencies of alleles and the genotypes have no statistically difference between AS group and the control group [OR=0.86(0.53,1.38),P=0.53; OR=0.90(0.52,1.55),P=0.69]. But the frequencies of alleles of western origin have statistically difference between AS group and the control group[OR=0.75(0.59,0.96), P=0.02]; The TNF-αgene promoter polymorphism may play a role in the severity of sacroiliitis[OR=0.37(0.15,0.90),P=0.03].Conclusion1. The frequency of PD-1 rs2227982- T was significantly higher in AS patients than in healthy people. Haploid analysis showed PD-1 rs2227981/ rs2227981 CT haploid was significant high in AS patients than in controls. It implies that PD- 1 plays an important role in the immunopathogenesis of AS.2. AS patients with genotype PD-1 rs2227982T/T showed a higher risk for periphery joints involvement and a stronger destruction of cacroiliac joint. PD-1 rs2227982T codes the mutation from Val to Ala. Its effect is not clear whether it is from protein structure or functional change.3. The frequency of PD-L1 rs822336C was significantly high in AS patients than in controls. No statistic differences were found between AS and controls for the frequencies of PD-L2 rs6476985 and rsl009759. It implies that it is PD-1/PD-L1 pathway but not PD—1/PD—L2 pathway which plays an important role in the pathogenesis of AS.4. For the study of association between genotypes of PD-L1 rs822336 and clinical manifestation of AS, AS patients with genotype CC showed a higher incidence of low back pain and iritis. AS patients with genotypes CC and CG showed a severer sacroiliitis. It suggests that PD-L1 might be associated with clinical manifestation of AS.5. The results support a role for asporin D repeat polymorphism in the susceptibility to AS and an influence of this gene on the outcome of the disease.6. This GSMA added the evidence of the HLA loci as the greatest susceptibility factor to AS and showed evidences of chromosome 6,17p,2,5q and 16q as non-HLA susceptibility loci.7. The meta-analysis revealed that the alleles of -308 site of TNF-αpromoter region may be associated with AS in western origin group and play a role in the severity of sacroiliitis.