高分辨熔解曲线技术在筛查21羟化酶缺乏症患者热点基因突变中的临床应用价值

目的探讨利用高分辨熔解曲线技术对21羟化酶缺乏症患者中致病基因热点突变筛查的可行性及临床应用价值。方法针对CYP21A2基因上的4个热点突变c.293-13A/CG(I2G)、c.518TA(p.I173N)、c.955CT(p.Q319X)及c.1069CT(p.R357W)设计相应的特异性引物,应用高分辨熔解曲线技术进行分析,并结合DNA测序技术进行验证。结果高分辨熔解曲线技术能对CYP21A2基因上的该4个热点突变明确分型,且与基因序列测序结果比对准确性良好;对基因突变型标本进行3次重复检测,Tm值变异系数为0.168%~0.620%。结论高分辨熔解曲线检测技术可用于健康人群或高危人群CYP21A2基因热点突变的筛查,是一种高效、准确、低成本的筛查方法,具有一定的临床应用价值。

Clinical application value of high resolution melting curve for screening hot spot mutations in patients with 21 hydroxylase deficiency

Objective?To explore the feasibility and application value of using high resolution melting curve technology to screen hot-spot mutations of pathogenic genes in 21 hydroxylase deficiency.?Method?Aimed at the four hot-spot mutations on?CYP21A2?gene, i.e., c.293-13A/C>G(I2G), c.518T>A(p.I173N), c.955C>T(p.Q319X) and c.1069C>T (p.R357W), the corresponding specific primers were designed. High resolution melting curve technology was used and DNA sequencing was combined to verify the results.?Results?High-resolution melting curve technique could definitely distinguish the four hot-spot mutations in?CYP21A2?gene and the results were consistent with Sanger sequencing showing fine correctness. The repeated analyses of three times showed that the coefficients of variance of Tm value were very small (0.168% to 0.620%).?Conclusion?The high-resolution melting curve technology as an efficient, accurate and low-cost screening method could be used for screening hotspot mutations on?CYP21A2?gene in healthy or high-risk population with certain clinical application value.