Study On The Association Of CTLA-4,PDCD1 With Genetic Susceptibility And Phenotypes Of Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE) is a multifactorial inheritance disease, genetic factors play an important role in the development of SLE.In order to explore the association of genetic factors with SLE and its phenotypes,we applied case -control study,case -parental study and case -only study design in Southern Chinese population,with the aid of some molecular biologic techniques,such as polymerase chain reaction(PCR) and restriction fragment length polymorphism(RFLP).The results are as follows:PartⅠThe study on genetic susceptibility to SLEDistributions of genotypes and allelles on polymorphic sites of genes1.For locus CTLA-4-1722T＞C,the results showed that genotypic frequencies of T/T, C/T and C/C in case groups were significantly different from controls(χ~2=7.828, P=0.020).Individuals with genotype T/T had a higher onset risk of SLE compared with those having genotype C/C,with OR=1.98(95%CI:1.21-3.23),and C/T or T/T genotype carriers' OR were 1.65(95%CI:1.06-2.57).For the allelic frequencies of -1722T＞C,there was also significant difference between patients and controls (χ~2=8.070,P=0.005),while there was a higher propotion of allele -1722T in SLE patients than that in controls(63.6%vs 55.3%).2.Both the genotypic and allelic frequencies on polymorphic site of-318C＞T for CTLA-4 gene were not significantly different between SLE patients and controls (P＞0.05).3.For locus PDCD1-PD12A＞G,the results showed that genotypic frequencies of A/A, A/G and G/G in case groups were significantly different from controls(χ~2=37.313, P＜0.001).Individuals with genotype A/G and G/G had a higher onset risk of SLE as compared with those having genotype A/A,and Ors of patients vs controls with respect to genotypes A/G and G/G were 2.70(95%CI:1.90-3.84),3.84(95%CI: 1.94-7.59),respectively.A/G or G/G genotype carriers' OR were 2.84(95%CI: 2.02-3.98).For the allelic frequencies of PD12A＞G,there was also significant difference between patients and controls(χ~2=34.452,P＜0.001),while there was a higher propotion of allele G in SLE patients than that in controls(36.4%vs 20.7%).4.For locus PDCD1-PD15C＞T,the results showed that genotypic frequencies of C/C, C/T and T/T in case groups were significantly different from controls(χ~2=7.699, P=0.021).Individuals with genotype C/T had a higher onset risk of SLE as compared with those having genotype C/C,with the OR=1.60(95%CI:1.14-2.45),C/T or T/T genotype carriers' OR were 1.52(95%CI:1.08-2.12).For the allelic frequencies of PD15C＞T,there was also significant difference between patients and controls (χ~2=4.473,P=0.034),while there was a higher propotion of allele G in SLE patients than that in controls(24.4%vs 19.2%).5.For locus PDCD1-PD16A＞G,the results showed that genotypic frequencies of A/A, A/G and G/G in case groups were significantly different from controls(χ~2=20.842, P＜0.001).Individuals with genotype A/G and G/G had a higher onset risk of SLE as compared with genotype A/A,and ORs were 1.83(95%CI:1.29-2.59),5.93(95%CI: 2.17-16.17),respectively.A/G or G/G genotype carriers' OR were 2.04(95%CI: 1.45-2.85).For the allelic frequencies of PD16A＞G,there was also significant difference between patients and controls(χ~2=20.769,P＜0.001),while there was a higher propotion of allele G in SLE patients than that in controls(28.6%vs 17.0%).Linkage disequilibrium test and analysis of association of haplotype with SLE1.The calculation of the ambi-matched-pairs linkage disquilibrium parameter and the hypothesis tests were conducted using the software of Arlequin31.It was found that there were significant linkage disquilibriums(LD) among two polymorphic sites of CTLA-4 gene as well as between three polymorphic sites of PDCD1 gene.2.The assessment of haplotype structure of genes was done using the software of PHASE2.1 and haplotype association analysis were done by SPSS13.0.It was found that the frequencies of haplotypes in CTLA-4 gene were significantly different between SLE patients and controls.When using individuals with no certain haplotype as reference,the haplotype of T-C and T-T in CTLA-4 gene had a higher onset risk of SLE,while the haplotype of C-C showed a protective effect on SLE.The frequencies of haplotypes in PDCD1 gene were also significantly different between SLE patients and controls.The frequencies of haplotype A-C-G,A-T-A,G-C-A had higher onset risk of SLE,while the haplotypes of A-C-A showed protective effect on SLE.The transmission/disequilibrium test on SLEThe family-based association was carried out by program TDT/sTDT1.1 on PC,it can calculate TDT,STDT and their combined scores.It was found that CTLA-4 gene -1722T allele,PD15T allele were significantly more frequently transmitted to the affected offspring,with Z=2.121 and 2.020 respectively.No excess of transmission of any allele in PD12 locus and PD16 locus was found(respectively Z=0.560,0.695, P＞0.05).The results suggested that CTLA-4 gene -1722T allele,PD15T allele may play a more important role in influencing susceptibility to SLE among the Han nationality population in South China.PartⅡAssociation analysis of genes with SLE phenotype1.The study of gene-phenotype association showed that CTLA-4-1722T＞C locus was associated with anti-SSA.The -1722T/C and T/T genotype frequency in patients with positive anti-SSA were higher than that of patients with negative anti-SSA,with OR=3.12,95%CI:1.16-8.36;OR=2.91,95%CI:1.11-7.65,respectively.The -318C/T and T/T combined group in patients with positive anti-RNP antibody had lower frequency than that of patients with negative anti-RNP antibody(P=0.044,OR=0.41, 95%CI:0.17-0.95).PD16 locus was associated with anti-SSA.The G/G genotype frequency in patients with positive anti-SSA were lower than that of patients with negative anti-SSA(P=0.016,OR=0.23,95%CI:0.07-0.76).PD12 locus was associated with serosa and kindy manifestation,individuals with genotype G/G had a higher onset risk of serosa and kindy,with OR=7.31,95%CI:2.08-25.74;OR=3.91, 95%CI:1.36-11.21,respectively.2.In the association analysis of haplotypes and phenotypes,it was found that when using those individuals with no certain haplotype as reference,the CTLA-4 C-C haplotype showed protective effect on ANA,anti-SSA and C3C4,but had reverse effect on ds-DNA,anti-Sm,anti-RNP,skin,joint,serona,kindy and blood manifestation.T-C haplotype was a risk factor for ds-DNA,anti-SSA,anti-SSB, C3C4,skin and blood manifestation,but had reverse effect on ANA,anti-Sm,joint, serona and kindy.T-C haplotype was a protective factor for ds-DNA,anti-SSA, anti-RNP,C3C4,skin and blood manifestation,but had reverse effect on ANA, anti-Sm,kindy,nerve and blood manifestation.Meanwhile,PDCD1 phenotypes were also found positive association,A-C-A haplotype showed protective effect on anti-Sm, anti-SSB,anti-RNP,C3C4,joint,kindy and nerve manifestation,but had reverse effect on ds-DNA,anti-SSA and blood.A-T-A showed protective effect on ANA,and reverse effect on anti-Sm,anti-SSB and joint.G-C-A showed protective effect on ds-DNA,anti-SSA and joint,while it was a risk factor for anti-Sm,anti-SSB,C3C4, skin,serona,kindy,nerve and blood.G-C-G showed protective effect on anti-RNP, C3C4,nerve and blood manifestation,while it was a risk factor for ANA,ds-DNA, anti-SSA,anti-Sm,anti-SSB,skin and joint.G-T-A showed protective effect on ANA, ds-DNA,anti-SSA,anti-Sm and joint,while it was a risk factor for anti-SSB, anti-RNP,C3C4,skin,serona and blood manifestation.G-T-G haplotype showed protective effect on ANA,anti-Sm,anti-SSB,anti-RNP,C3C4,joint,serona,nerve and blood manifestation,while it was a risk factor for ds-DNA,anti-SSA,skin and kindy manifestation.