应用高分辨率熔解曲线法分析FTOrs9930506变异与北京居民代谢综合征的关联

The application of a high resolution melting-based genotyping method in studing the association between FTO rs9930506 polymorphism and metabolic syndrome in Beijing population

目的基于高分辨率熔解（HRM）技术，建立敏感、经济、快速、适合临床应用的FrO基因rs9930506分型方法，并评价该基因变异与北京居民代谢综合征（MS）的关系。方法参考GenBank的序列，设计rs9930506特异性的C3-spacer封闭的非标记探针，预混LC-Greenplus染料，经PCR扩增和HRM反应，扫描荧光信号并分析基因型。设立酶切法和测序法为方法学对照，评价准确度和可操作性。在北京地区MS患者（500例）和对照组（500例）中应用该方法，探讨rs9930506变异与MS的关系。结果3种方法均能较好实现rs9930506基因分型，HRM法最为简便快速，应答率为100％，以测序法为金标准，抽样准确度为99．3％。MS组中，AA、AG、GG基因型分别有290例、185例和25例，对照组中分别为344例、138例和18例。对照组与HapMap数据库中北京汉族人各基因型分布无统计学差异（P=0．520）并满足Hardy-Weinberg平衡。AA基因型可能降低MS的发生风险（OR=0．626，95％CI=0．483-0．812）。结论Fro基因s9930506AA基因型的携带，可能是北京居民MS的保护因素；基于HRM技术建立的FTO基因rs9930506分型方法快速、准确、经济。

Objective To set up a new method, which is sensitive, low cost, rapid and suitable for clinical application for FTO gene rs9930506 variant genotyping basing on high resolution melting （ HRM ） platform, and to preliminarily put into practice in susceptibility analysis for metabolic syndrome （ MS ） in Beijing. Methods Unlabelled probe with C3-spacer block specific for rs9930506 variant has been designed according to the Refseq from GenBank. With LC-Green plus dye pre-mixed, we scanned the signal for the genotype analysis after PCR amplification and HRM reaction. Restriction fragment length polymorphism （RFLP） and PCR-sequencing methods were designed as 2 control genotyping methods for the evaluation of accuracy and convenience. Afterwards, the HRM-based method was put into practice in metabolic syndrome patients （n = 500 ） and control groups （n = 500 ） for rs9930506 genotyping, and primarily study the association between rs9930506 and MS. Results All the 3 methods could genotype rs9930506 appropriately, although the 2 control methods seemed to be a little time-inefficient. The call rate of HRM- method was 100% and sampling accuracy reached 99.3% according to sequencing results. In the MS group, AA, AG and GG genotypes were found in 290, 185 and 25 cases, respectively. And in the control group, those were found in 344, 138 and 18 cases. No genotype distribution difference was detected between control group and HapMap-CHB data （P = 0. 520 ） . The genotype distributions were all in Hardy-Weinberg equilibrium in each group. AA genotype of rs9930506 seemed to reduce the risk for MS（ OR = 0. 626, 95% CI = 0. 483-0. 812 ）. Conclusions The AA genotype of rs9930506 variant in FTO might be a protective factor for MS in Beijing population. The susceptibility related genotyping in clinical samples could be more rapid, precise and inexpensive with the development of HRM in genotyping.