HRM技术在瘦素基因启动子多态性与肝硬化发生相互关系研究中的应用

Application of PCR-HRM in the study of relationship between leptin gene promoter polymorphisms and liver cirrhosis

目的:探讨PCR-高分辨率熔解曲线分析(HRM)技术筛选和分析肝硬化患者瘦素基因启动子C2549和G2548位点突变的可能性,同时研究突变基因型与肝硬化患者生理生化指标之间的关系。方法:采用PCR-HRM新型技术,同时对比传统方法 PCR-限制性片段长度多态性分析(RFLP),对对照组(n=100)和肝硬化组(n=100)瘦素基因启动子C2549和G2548位点突变进行分析,同时检测生理生化指标。结果:(1)PCR-HRMA技术能够有效、高通量、准确地实现对瘦素基因启动子多态性的检测;(2)肝硬化患者瘦素基因启动子主要突变位点在C2549,而G2548位点没有发现突变;(3)肝硬化组中瘦素、游离瘦素指数(FLI)、空腹胰岛素(FINS)和胰岛素抵抗指数(HOMA-IR)高于对照组,胰岛素敏感指数(ISI)及可溶性瘦素受体(sOB-R)低于对照组,除瘦素外,均有显著差异(P<0.05);FLI与FINS和HOMA-IR呈正相关(r=0.45、r=0.53,均P<0.05),与ISI呈负相关(r=-0.34,P<0.05);(4)肝硬化患者C2549杂合型占优势,肝硬化纯合型和杂合型突变个体中HOMI-IR、瘦素、sOB-R及FLI均高于野生型,除瘦素及sOB-R外均有显著差异(P<0.05)。结论:PCR-HRM能准确及高通量地实现对瘦素基因启动子多态性进行分析,并验证了A位点基因频率的增高可能和肝硬化发生有关;肝硬化患者存在高胰岛素血症及胰岛素抵抗,瘦素可能与肝硬化患者胰岛素抵抗发生有关。

AIM： To analyze the feasibility of PCR- high -resolution melting curve analysis （HRM） for de- tecting the site mutations of C2549 and G2548 in leptin gene promoter from the patients with liver cirrhosis, and to explore the relevance between mutant genotypes and physiological and biochemical indexes in liver cirrhosis patients. METHODS： Compared with the method of PCR - restriction fragment length polymorphism （ RFLP）, the present research used the method of PCR - HRM to analyze the site mutations of C2549 and G2548 in leptin gene promoter in control group （ n = 100 ） and liver cirrhosis group （ n = 100）. The physiological and biochemicaI indexes of the patients were also detected and compared. RESULTS： Leptin gene promoter polymorphism was detected using PCR - HRM with effectiveness, high flux and accuracy. Preliminary results showed that the main mutation of the patients with liver cirrhosis was in C2549 site, but not found in G2548 site. Leptin, free leptin index （FLI）, fasting insulin （FINS） and insulin resistance index estimated by homeostatic model assessment （ HOMA - IR） in liver cirrhosis group were higher than those in control group. Insulin sensi- tivity inde~ （ISI） and soluble leptin receptor （ sOB - R） in liver cirrhosis group were lower than those in control group with significant difference except leptin level. Meanwhile, FLI showed positively correlated with FINS and HOMA - IR （ r = 0.45, r = 0.53, P 〈 0. 05 ）, and negatively with ISI （ r = - 0.34, P 〈 0. 05 ）. In the patients with liver cirrhosis, C2549A heterozygous mutation was predominant. The indexes of HOMI - IR, leptin, sOB - R and FLI of C2549A homozygotes and heterozygotes were higher than those of the wildtypes, which showed significant difference except leptin and sOB - R levels （P 〈0. 05）. CONCLUSION： PCR -HRM can be more accurate for identifying leptin promoter polymorphism. The in- crease in the frequency of C2549A mutation may be closely related with liver cirrhosis. Existence of hyperinsulinemia and insulin resistance may be correlated with leptin level in the patients with liver cirrhosis.