基于胎儿有核红细胞建立遗传性耳聋无创产前诊断的新方法

A New Non-invasive Method for Prenatal Diagnosis of Hereditary Hearing Loss Using Fetal Nucleated Red Blood Cells

目的基于原位捕获孕妇外周血中的胎儿有核红细胞(fetal nucleated red blood cells, FNRBCs)建立遗传性耳聋产前无创诊断的新方法。方法将FNRBCs抗体固定在一种功能和结构化医疗导丝上,制备成FNRBCs采集器。利用血液循环模拟装置,对3例男性新生儿脐血进行体外捕获和FISH检测以验证体外捕获体系的可行性。然后,对5例孕妇外周血进行FNRBCs捕获,对捕获细胞全基因组扩增产物和孕妇外周血DNA进行STR分型以分析FNRBCs含量和纯度;对耳聋基因的致病性位点进行Sanger测序,并与已知胎儿羊水穿刺结果对比从而判断捕获细胞来源。结果 FISH结果显示,捕获细胞为有核细胞,即体外捕获体系可行;STR分型表明捕获细胞中存在一定数量的FNRBCs;Sanger测序结果表明1例孕妇外周血的捕获细胞为FNRBCs,其余4例均为母体细胞。结论基于原位捕获孕妇外周血中FNRBCs,有望建立遗传性耳聋产前无创诊断的新方法。但是,原位捕获FNRBCs的特异性、效率和稳定性还有待于进一步提高。

Objective To establish a new method for non-invasive prenatal diagnosis of hereditary hearing loss based on in situ capture of fetal nucleated red blood cells(FNRBCs) in pregnant women’s peripheral blood. Method A FNRBCs collector was prepared by immobilizing a functional and structured medical guidewire with an anti-FNRBCs antibody. In a circulation simulation device, umbilical cord blood from 3 male newborns was used to capture FNRBCs in vitro, which were detected using FISH testing, to verify the feasibility of the extracorporeal capture system. Subsequently, peripheral blood from 5 pregnant women was used to capture FNRBCs. STR typing was performed using the whole genome amplification product of captured cells and the women’s peripheral blood DNA to determine the amount and purity of captured FNRBCs, and Sanger sequencing was used to detect pathogenic sites associated to hearing loss.The results were compared with known fetal amniocentesis results to determine the source of captured cells. Results FISH results showed that the captured cells were nucleated cells, confirming the feasibility of the extracorporeal capture system. STR typing suggested presence of FNRBCs among captured cells. Sanger results confirmed that captured cells from one woman’s peripheral blood were FNRBCs, while those from the other four women were maternal cells. Conclusion In situ capture of FNRBCs from peripheral blood in pregnant women may allow non-invasive prenatal diagnosis of hereditary hearing loss, but its specificity, efficiency and stability need to be further studied and improved.