UGT1 A10基因SNP rs10187694对吗替麦考酚酯代谢的影响

Effect of SNP rs10187694 of UGT1A10 gene on metabolism of Mycophenolate mofetil

摘要:

体外研究尿苷二磷酸葡萄糖醛酸转移酶（UGT1A10 ）基因SNP rs10187694 多态位点G/A变异对吗替麦考酚酯（MMF）代谢的影响，明确该位点变异是否与MMF个体代谢差异有关。方法采用基因重组、定点突变技术，构建UGT1A10基因SNP rs10187694位点含有不同等位基因的重组过表达载体，POLO 3000 转染法将重组过表达载体转染入HEK293细胞，采用液相色谱- 质谱联用仪（LC/MS/MS）系统，检测霉酚酸（MPA）代谢产物7-O- 葡醛酸苷（MPAG）的生成量，以评估转染不同等位基因HEK293 细胞中酶的活性。结果成功 构建pIRES2-EGFP-prom（G）和pIRES2-EGFP-prom（A）重组过表达载体，并转染HEK293 细胞。LC/MS/MS系统检测结果显示，携带A 等位基因的突变型1 10代谢MMF 产生MPAG 的能力降低，其24 h MPAG的生成量为（226.00±14.57）nmol/L，而野生型的生成量为（269.00±14.07）nmol/L， UGT1A10突变型MPAG 的生成量是野生型的84.00%，两者比较，差异有统计学意义（ p<0.05）。结论1 10基因SNP rs10187694位点G/A突变可影响MMF代谢产物MPAG的生成，可能是造成不同个体MMF代谢差异的重要原因之一。

Abstract:

To study the effect of G/A mutation in SNP rs10187694 site of uridine diphosphate glucuronosyl transferase (UGT1A10 ) on Mycophenolate mofetil metabolism. Methods The recombinant overex-pression vectors of gene were constructed by gene recombination technique and the strategy of site directed mutagenesis. Then, the recombinant plasmid was transfected into HEK293 cells by POLO3000 method. By detecting the yields of mycophenolic acid (MPA) metabolite 7-o-glucuronide (MPAG), the activity of enzyme in the HEK293 cells transfected with different alleles were evaluated. Results The overexperession vectors pIRES2-EGFP-prom (G) and pIRES2-EGFP-prom (A) were successfully constructed and transfected into HEK293 cells. The results analyzed by LC/MS/MS showed that the cells containing mutant type of gene with A allele produced less MPAG than those with G allele, the concentration of MPAG produced by mutant type in 24 hours was (226.00 ±14.57) nmol/L, however, the concentration in wild type was (269.00 ±14.07) nmol/L. The generation amount of MPAG in mutant type was 84.00% of that in wild type in 24 hours( p< 0.05). Conclusions gene SNP rs10187694 can significantly affect the production of MPAG,which may be one of the reasons causing the difference in Mycophenolate mofetil metabolism.