DHPLC技术筛查卵巢早衰患者TGFBR-3基因多态性的临床研究

Application of DHPLC screening TGFBR-3 gene in Chinese women with idiopathic premature ovarian failure

【摘要】目的评价变性高效液相色谱（DHPLC）技术筛查卵巢早衰转化生长因子β受体3（TGFBR．3）基因第11、12外显子多态性的临床应用价值。方法选择2009年2月至2011年12月在南方医科大学附属深圳市妇幼保健院确诊的卵巢早衰患者110例为卵巢早衰组，以月经规则、性激素检查在正常范围内的年龄匹配的妇女110例为对照组。采用DHPLC技术筛查TGFBR-3基因第11、12外显子多态性，并以DNA测序结果为金标准，计算DHPLC技术筛查的灵敏度、特异度、假阴性率、假阳性率、Youden指数、阳性预测值和阴性预测值等指标。并随机抽取20％的样本（22例），以相同的实验条件，进行第二次DHPLC技术筛查，与第1次结果进行比较，计算Kappa值。结果TGFBR-3基因第11外显子未检测到多态性，而第12外显子有两个单核苷酸多态性。2022T／C位点多态性：卵巢早衰组CC、TC、TT基因型频率分别为0．9％（1／110）、22．7％（25／110）、76．4％（84／110），C、T等位基因频率分别为12．3％（27／220）、87．7％（193／220）；对照组CC、TC、TT基因型频率分别为0、9．1％（10／110）、90．9％（100／110），C、T等位基因频率分别为4．5％（10／220）、95．5％（210／220）；两组间各基因型频率及等位基因频率分别比较，差异均有统计学意义（P均〈0．05）。2161-75C／T多态性位点基因频率、等位基因频率卵巢早衰组和对照组比较，差异均无统计学意义（P均〉0．05）。以DNA测序结果作为基因多态性判断金标准，DHPLC技术筛查结果的灵敏度为100％、特异度为97．9％、Youden指数97．9％、阳性预测值为96．3％、阴性预测值100％。两次DHPLC技术筛查结果Kappa值为0．888（P〈0．05）。结论DHPLC技术筛查TGFBR．3基因多态性具有较高的真实性、可靠性和实用性。

Objective To evaluate clinical value of denaturing high performance liquid chromatography （DHPLC） used in detecting transforming growth factor beta receptor 3 （ TGFBR-3 ） exons 11 and 12 polymorphism in women with idiopathic premature ovarian failure （ POF）. Methods From Feb. 2009 to Dec. 2011, 110 patients with idiopathic POF undergoing treatment at Shenzhen Maternal ＆ Child Health Institute affiliated to Southern Medical University were enrolled as POF group in this study. In the mean time, 110 women under 40 years old with normal hormonal level and menstrual cycles as control group. The exons 11 and 12 of TGFBR-3 gene polymorphism were screened by using DHPLC, and results of DNA sequencing was as golden standard. Some related indexes were calculated, such as sensitivity, specificity,false negative value, false positive value, Youden index, positive predictive value, and negative predictive value. At the same time, 20% of the tested specimens were chosen randomly and detected by DHPLC again. The value of Kappa index were calculated by comparing the results between the first and second DHPLC analysis. Results The exon 11 of TGFBR-3 were not identified gene polymorphism and two nucleotide polymorphisms were identified in exon 12. For 2022 T/C polymorphism, the frequencies of CC with 0. 9% （1/110）, TC with 22.7% （25/110）, TY with 76. 4% （84/110）, C with 12. 3% （27/220） and T with 87.7% （193/220） in POF group were significantly different from CC with O, TC with 9. 1% （10/110） and TT with 90. 9% （100/110）, C with 4. 5% （10/220） and T with 95.5% （210/220） in control group （all P 〈 O. 05 ）. Allelic and genotypic frequencies of 2161-75 C/T were not differed significantly between the two groups （ all P 〉 0. 05 ）. As DNA sequencing as golden standard, DHPLC showed that the sensitivity was 100% , specificity was 97.9% , Youden index was 97. 9%, positive predictive value was 96. 3%, negative predictive value was 100%, and Kappa index was 0. 888 （ P 〈 0. 05 ）. Conclusion DHPLC analysis is higher validity, reliability and practicability method in detecting TGFBR-3 polymorphism in idiopathic premature ovarian failure.