Establishment Of Molecular Diagnostic Methods For Genotyping SNPs Associated With Gastric Cancer And Rheumatoid Arthritis Based On HRM Technology

Objective:To establish the polymerase chain reaction-high resolution melting（PCR-HRM）methods for genotyping SNPs（rs13361707（PRKAA1）,rs9841504（ZBTB20）,rs7574865（STAT4）,rs7234029（PTPN2）,rs2233945（PSORS1C1）,rs33980500（TRAF3IP2））associated with gastric cancer and rheumatoid arthritis（RA）and evaluate their detection performance.To investigate the relationship between the gene polymorphisms of rs7574865,rs7234029,rs2233945,rs33980500 and susceptibility to RA in Han population from Lanzhou region.Methods:The primers of rs13361707 and rs9841504 were obtained by consulting literatures,and the specific primers of rs13361707,rs9841504,rs7574865,rs7234029,rs2233945 and rs33980500 were designed by online software Primer-BLAST to establish a PCR-HRM molecular diagnostic method for genotyping SNPs associated with gastric cancer and rheumatoid arthritis.Sanger sequencing was taken as a gold standard to evaluate sensitivity and specificity.Repeat tests were implemented to assess the repeatability and reproducibility of the method.The robustness evaluation mainly investigated the six main factors（the concentration of primers,the amount of reaction system,DNA template concentration,the PCR cycle number,the types of fluorescent dye and the types of instrument）affecting the PCR-HRM method.The clinical applicability was evaluated by genotyping for 280samples.Results:（1）The self-established PCR-HRM methods for 6 genotyping SNPs（rs13361707,rs9841504,rs7574865,rs7234029,rs2233945 and rs33980500）can accurately genotype,and the sequencing results verified that the sensitivity and specificity of these were 100%in this study.The repeatability and reproducibility results of 6 SNPs genotyping,show the range of variation coefficients of Tm were（0.01%-0.07%）and（0.01%-0.05%）respectively.The change of the concentration of primers（0.1μmol/L,0.2μmol/L and 0.3μmol/L）,the amount of reaction system（10μl,15μl,20μl）,DNA template concentration（10 ng/μl,20 ng/μl,50 ng/μl,and 100 ng/μl）,the PCR cycle number（38,40,42 cycles）,the types of fluorescent dye(（LC Green,Syto 13 and Eva Green）,and the types of instrument（Rotor-Gene 6000,Roche LightCycler?480 and Roche LightCycler?96）had no significant effect on the genotyping of the method.（2）Data showed that the genetype distribution of rs2233945 and rs7574865 were significantly different between the cases and controls(x²=13.063,P=1.45×10^-3;x²=31.044,P=1.81×10^-7).Under the dominant model,the A allele carriers of rs2233945（CA hererozygote and AA homozygote）were found to significantly decrease the risk for RA developnt（OR=0.481,95%CI:0.222-0.081,P<0.05）.the T allele carriers of rs7574865（GT hererozygote and TT homozygote）were found to significantly increase the risk for RA development（OR=4.586,95%CI:2.455-8.566,P<0.05）.Conclusion:The self-developed molecular diagnostic methods genotyping for 6SNPs were proved to be highly reliable and suitable for clinical routine testing.Polymorphisms of rs7574865 and rs2233945 are associated with RA risk in Han population from Lanzhou region.