新发现青少年痛风易感基因PFKP致病机制研究

In recent years, the susceptible population of gout prone to be adolescents. Based on whole genome sequencing, we discovered a novel risk gene, PFKP, in an adolescent cohort (300 gout cases vs. 500 controls) with phase-I replication in another cohort (500 gout cases vs. 500 controls). The 1155-257 G>C SNP was found in the intron 12 on PFKP gene,which is highly related to gout (P=6.44×10^-11). Patients with this SNP have significant elevated serum urate levels compared with those without SNP. Bioinformatics predicates this SNP locates in the transcription factor HIF-1α binding region of the PFKP gene. These preliminary results suggest that the SNP may be involved in the pathogenesis of gout via affecting the binding effects and the following transcription regulation of PFKP in human hepatic cells. The glycolysis and pentose phosphate pathway (PPP) are influenced as well. This study aims to (1) explore the effects of the SNP on the PFKP transcription by dual-luciferase reporter assay system and the specific binding function of HIF-1α with PFKP by ChIP and EMSA in human hepatic cell lines, (2) evaluate the effects of PFKP and the SNP of PFKP on urate synthesis by transfecting cells with high- and low- expression of PFKP and SNP knock-in. Our study will shed light on a novel pathogenesis mechanism for adolescent gout and indicate a new strategy for early-phase diagnosis and treatment of gout.

近年来痛风发病呈年轻化趋势，但对青少年痛风遗传机制鲜有报道。课题组前期完成了300例青少年原发性痛风和500例青少年健康对照全基因组测序。通过独立青少年样本验证（500例痛风vs.500例健康对照）发现了一个全新的青少年痛风易感基因PFKP。该基因第12内含子1155-257G>C SNP与痛风关联密切（P=6.44×10^-11），该SNP携带者血尿酸水平明显高于非携带者。生物信息学预测显示，该SNP位于PFKP基因转录因子HIF-1α结合区，推测该SNP可能通过影响与HIF-1α结合，参与肝细胞PFKP基因转录调控，影响糖酵解和磷酸戊糖途径，参与痛风发病。本课题拟通过EMSA等实验，研究该SNP对基因转录及对HIF-1α结合力的影响；构建过表达、敲降和SNP敲入稳转肝细胞系，观察该基因和该SNP对肝脏尿酸合成的影响。该研究将揭示全新的青少年痛风发病机制，为痛风的早期防治提供新策略。

近年来痛风发病呈年轻化趋势，但对青少年痛风遗传机制鲜有报道。课题组前期完成了青少年原发性痛风和健康对照全基因组测序。通过独立青少年样本验证发现了一个全新的青少年痛风易感基因PFKP。经生物信息学及初步预实验推测该基因通过影响肝脏细胞糖酵解和磷酸戊糖途径，参与痛风发病。对PFKP基因参与糖酵解的可能机制展开深入研究，结果显示：成功构建PFKP基因过表达、敲降的BRL 3A细胞，发现PFKP可影响大鼠肝细胞BRL 3A中糖酵解过程中关键酶的表达，影响尿酸生成以及葡萄糖的摄取，可溶性尿酸刺激可使PFKP蛋白表达水平升高、PFK酶活性水平升高，干扰葡萄糖、果糖及能量代谢过程。通过能量代谢组学分析，我们发现了与炎症相关的基因及通路。该研究揭示了新的青少年痛风发病机制，为制定新的痛风诊疗策略和开发治疗痛风的新药奠定基础。