| Objective To develop in-house the molecular diagnosis methods for routine detection of 15 single nucleotide polymorphisms (SNPs) from RTKN2、LDLR、APOB、APOC1、ARL15、 HLA-DMA、NFKB2、CRP、ACE、NOS3、ENPP1 and IQSEC1 genes in clinical laboratory, and to explore the relationship of 15 SNPs with the susceptibility of rheumatoid arthritis (RA) and coronary heart disease (CHD) as well as serological indicator in Chinese Han population of Lanzhou area.Methods High resolution melting (HRM) technology was used to establish PCR-HRM assays for detecting eight SNPs (rs3125734 C＞T、rs688 C＞T、rs693 C＞T、 rs4420638 A>G、 rs255758 A>C、rs1063478 C＞T、 rs397514331-/A and rs397514332 C＞T) from seven RA associated genes RTKN2、LDLR、APOB、APOC1、ARL15、HLA-DMA and NFKB2 and seven SNPs (rs1205C＞T、rs4305 A>G、rs4353 G>A、rs7830 C＞A、rs12528076 C＞T、rs939899 A>G and rs1108640 C＞G) from five CHD associated genes CRP, ACE, NOS3、ENPP1 and IQSEC1. Two case-control studies were conducted in 588 RA patients with 200 healthy matched controls and 260 CHD patients with 238 healthy matched controls. The accuracy and clinical applicability of the established assays were verified by directly sequencing on PCR products. Through the case-control analyses, we will screen the SNP loci associated with RA and CHD susceptibility and know the correlation of serum RF and anti-CCP antibodies with the SNPs in Lanzhou Chinese Han population.Result All samples were successfully genotyped using HRM analysis after PCR amplification and the correct rate of assays are found up to 100% by direct sequencing verification. (1) The case-control analysis of RA patients with control subjects showed ①rs3125734C>T and rs688C＞T genotype and allele frequencies were significantly different between RA and control groups (x2=6.144，P=0.046;x2=5.793,P=0.016;x2=8.582,P=0.014;x2=5.409,P=0.020); rs255758 A>C and rs1063478C>T genotype frequencies were significantly different between the two groups (x2=6.877,P=0.032;x2=0.324,P=0.569). ②The result of gender stratification showed that only rs3125734 C＞T genotype and allele frequencies were significantly different between male RA patients and control groups (x2=6.527,P=0.011;x2=5.899,P=0.015); rs688C＞T and rs255758A>C genotype and allele frequencies were significantly different between female RA patients and control groups (x2=8.916,P=0.012;x2=7.180,P=0.007;x2=10.788,P=0.006;x2=4.235, P=0.039). ③The results of serological index stratification showed that rs3125734C＞T allele frequencies were significantly different in CCP positive RA patients with control groups (x2=4.708,P=0.030), and genotype and allele frequencies were significantly different between RF-positive RA and control groups (x2=7.246,P=0.027;x2=6.370,P=0.012); rs4420638A>G genotype frequencies were significantly different between RF-negative RA and control groups (x2=4.105,P=0.043).④The logistic regression model of mutation genotype showed that the heterozygous genotype CT of the rs3125734C＞T increases RA risk (x2=4.011,P=0.045,OR=1.613), the homozygous genotype TT of the rs688C＞T decreases RA risk (x2=6.853,P=0.009,OR=0.273), the heterozygous genotype AC of the rs255758A>C decreases RA risk between RA and control groups (x2=6.641,P=0.010,OR=0.640); The heterozygous genotype CT of the rs1063478C＞T increases RA risk between male RA patients and control groups(x2=6.029,P=0.014,OR=3.204), the genotype TT/AC mutant of the rs688C>T and rs255758A>C decrease RA risk between female RA patients and control groups(x2=5.704,P=0.017,OR=0.244;x2=9.410,P=0.002,OR=0.531); the heterozygous genotype CT of the rs3125734C>T increases RA risk between RF-positive RA and control groups (x2=4.110,P=0.043,OR=1.853); The heterozygous genotype AG of the rs4420638A>G increases RA risk between RF-negative RA and control groups (x2=4.046, P=0.044, OR=1.799). ⑤The analysis of serum lipid levels showed that the rs693C＞T and rs4420638A>G are correlated to RA patients with HDL-C levels (x2=4.762,P=0.029,OR=3.385;x2=8.226,P=0.004,OR=5.416).The statistical differences were founded using mean-variance analysis on rs693C>T and rs4420638A>G mutant and wild-type genotype in HDL-C level (F=7.312,P=0.008;F=12.482,P=0.001). (2) CHD case-control analysis showed ①rs4353G>A allele frequencies were significantly different between CHD and control groups (x2=3.998,P=0.045,OR=1.297). ②rs7830A>C genotype frequencies were significantly different between the two groups (x2=6.369,P=0.041,OR=0.790). ③ rs12528076C＞T allele frequencies were significantly different between the two groups (x2=3.918,P=0.047,OR=1.335). ④The analysis of serum lipid levels showed that the CHD patient groups rs1205 locus is correlated to CHD patients with HDL-C and LDL-C levels (x2=7.371,P=0.007,OR=7.920;x2=5.892,P=0.015,OR=0.320). The statistical significance was founded using mean-variance analysis on rs1205 mutant genotype CT/TT and wild-type genotype CC in HDL-C and LDL-C levels, which is that there was significant difference only with HDL-C level (F=14.067,P=0.001).Conclusion The established PCR-HRM molecular diagnosis methods on 15 SNPs can correctly genotyped for clinical samples to achieve detection performance of clinical routine molecular diagnosis.The genes of RTKN2、LDLR、APOC1、ARL15 and HLA-DMA are associated with susceptibility to RA in Lanzhou Chinese Han population, of which rs693C＞T and rs4420638A>G are associated HDL-C levels may predict the occurrence of RA. The genes of ACE、NOS3 and ENPP1 are associated with susceptibility to CHD in Lanzhou Chinese Han population, of which rs1205 C＞T is associated HDL levels may predict the occurrence of CHD. |
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