ABSTRACT
Objective To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P4502C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology. Methods Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results. Results The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4％and 10.9％, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results. Conclusion A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.
ABSTRACT
Objective@#To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology.@*Methods@#Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results.@*Results@#The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results.@*Conclusion@#A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.
ABSTRACT
Objective To perform a meta-analysis on the associations of interleukin (IL)-6-174G ＞ C (rs1800795) and-634C ＞ G (rs1800796) polymorphisms with type 2 diabetic nephropathy (DN).Methods The data on the studies about the associations of IL-6-174G ＞ C and-634C ＞ G polymorphisms with type 2 DN were collected from Pubmed,Embace,CNKI,Wan Fang and VIP database during their inception and April 2017.The statistical analysis was performed with STATA 14.0 and Review manager 5.3 softwares.The heterogeneity in the eligible studies was assessed by Q-statistic and I2 statistic.When the significant heterogeneity was found,the random effect model was used for meta-analysis,otherwise,the fixed effect model was used.The publication bias was evaluated with funnel and Begger graphs.The pooled odds ratios (OR) and corresponding 95％ confidence intervals (95％ CI) were calculated for evaluating the associations of IL-6-174G ＞ C and-634C ＞ G polymorphisms with type 2 DN.In addition,the sub-group analysis was performed according to the regions of subjects.Results A total of 11 studies were enrolled,The studies on the association of IL-6-174G ＞ C polymorphism with type 2 DN included 1 688 subjects,while those on the association of IL-6-634C ＞ G with type 2 DN included 2 180 subjects.In the association analysis of IL-6-174G ＞ C polymorphism with type 2 DN of Asian population,the significant relationship was detected in an allelic genetic model (OR =0.461,95％ CI:0.274-0.777,P ＜ 0.01),a homozygote model (OR =0.126,95％ CI:0.022-0.734,P =0.021),a recessive genetic model (OR =0.146,95％ CI:0.026-0.827,P =0.030) and a dominant genetic model (OR =0.504,95 ％ CI:0.273-0.930,P =0.028),but not in a heterozygote model (OR =0.606,95 ％ CI:0.321-1.143,P =0.122).There was no significant relationship between IL-6-174G ＞ C polymorphism and type 2 DN in European population.In the association analysis of IL-6-634C ＞ G polymorphism with type 2 DN of Asian population,the significant relationship was found in an allelic genetic model (OR =1.467,95％ CI:1.238-1.737,P ＜0.01),a homozygote model (OR =2.793,95％ CI:1.844-4.230,P =0.021),a recessive genetic model (OR =2.296,95 ％ CI:1.586-3.323,P ＜ 0.01) and a dominant genetic model (OR =1.377,95％CI:1.109-1.711,P ＜ 0.01),but not in a heterozygote model (OR =1.733,95％ CI:0.932-1.476,P =0.174).There was no significant relationship between IL-6-634C ＞ G polymorphism and type 2 DN in European population.Conclusion In Asian population,IL-6-174CC genotype may prevent the progression of type 2 DN,however,IL-6-634GG genotype may promote the development of type 2 DN.But in European population,there is no relationship between IL-6-174G ＞ C and-634C ＞ G polymorphisms and type 2 DN.