新一代测序技术无创产前检测胎儿FGFR3基因突变

Accuracy and feasibility of non-invasive prenatal testing for FGFR3-related skeletal dysplasia by next-generation sequencing

目的探讨新一代测序技术(next-generation sequencing,NGS)无创产前筛查FGFR3相关疾病的可行性与准确率。方法 4例软骨发育不全、2例致死性侏儒Ⅰ型胎儿基因组DNA(g DNA)片段化后,以相应母体产后血浆游离DNA为背景,按照10%、6%、3%、1%、0.5%的浓度进行稀释,形成30例阳性模拟样本,建立孕妇血浆游离DNA模型,同时采集13例阴性对照孕妇血浆游离DNA,运用NGS技术分别检测FGFR3致病突变位点,计算检测灵敏度、特异度、阳性预测值、阴性预测值。结果 30例模拟样本中,胎儿g DNA浓度为10%、6%、3%、1%、0.5%时,突变检出例数分别为6/6、6/6、6/6、3/6、1/6,13例阴性突变检出例数为0/13;胎儿g DNA浓度≥3%时,NGS的灵敏度为100%(95%CI:81.5%~100%),特异度为100%(95%CI:75.3%~100%),阳性预测值为100%(95%CI:81.5%~100%),阴性预测值为100%(95%CI:75.3%~100%)。结论胎儿g DNA浓度≥3%时,NGS可以在孕妇血浆游离DNA模型中准确检出胎儿FGFR3基因突变,提示基于NGS技术的无创产前检测在新发突变或父源遗传疾病的产前诊断领域具有一定的应用前景。

Objective To explore the test accuracy and feasibility of non-invasive prenatal testing for FGFR3-related skeletal dysplasia based on next-generation sequencing（NGS）. Method Fragmented fetal genome DNA（g DNA） of achondroplasia（n=4） or thanatophoric dysplasia typeⅠ（n=2） were diluted by corresponding post-delivery maternal cell-free DNA into different concentrations including 10%, 6%, 3%, 1% and 0.5%（n=30）. Cell-free DNA collected from pregnant women（n=13） whose fetuses were confirmed free of FGFR3 mutation by Sanger sequencing served as negative control. Then NGS was carried out to detect the fetal mutant allele in these samples. Sensitivity, specificity, positive predictive value, negative predictive value were calculated to evaluate the test performance. Results When the fetal g DNA concentrations were 10%, 6%, 3%, 1% and 0.5%,the detection rates were 6/6, 6/6, 6/6, 3/6 and 1/6, respectively. NGS had a sensitivity of 100%（95% CI： 81.5%-99.9%）when the fetal genome DNA concentration was equal to or more than 3%. There was no positive result in the negative controls and the specificity of NGS was 100%（95% CI： 75.3%-100%）. The positive predictive value and negative predictive value were 100%（95% CI： 81.5%-100%） and 100%（95% CI： 75.3%-100%）, respectively. Conclusion NGS has a high accuracy in detecting FGFR3 mutation when the concentration of fetal g DNA is equal to or more than 3%, highlighting its promising value in developing non-invasive prenatal test for monogenic disorders, especially for the denovo and paternal inherited diseases.