荧光定量PCR技术用于行遗传性耳聋基因检测胎儿的21三体综合征筛查的可行性

Detection of trisomy 21 by quantitative iluorescent PCR in clinical samples undergoing prenatal diagnosis for hereditary hearing loss

目的探讨荧光定量PCR（QF-PCR）技术用于产前行遗传性耳聋基因检测的胎儿筛查21三体综合征的可行性。方法选择2009年3月至2010年5月在解放军总医院行产前遗传性耳聋基因检测的54例孕妇为研究对象。所有行遗传性耳聋基因检测孕妇的家庭中的先证者均经过解放军总医院耳鼻咽喉研究所聋病分子诊断中心明确了耳聋致病基因。54例孕妇平均年龄31岁，其中9例〉135岁；孕周为18～26周，其中〉23孕周的孕妇有5例。孕18～23周的49例孕妇取羊水15～20ml进行胎儿耳聋基因检测，孕周〉23周的5例，取新生儿脐带血1～2ml进行相关耳聋基因检测。年龄〉135岁的9例孕妇中，5例同时进行了羊水细胞培养和染色体核型分析。采用QF—PCR技术诊断21三体综合征，共选择21号染色体杂合度高的9种短串联重复序列（STR）分子标记，选定6对引物分两种组合进行多重扩增，21三体综合征诊断标准为21号染色体2个以上STR位点出现1：1：1等比例峰及2：1比例峰，或1：2比例峰，即可明确诊断。结果（1）遗传性耳聋基因的检测及其随访结果：54例胎儿均成功进行了相应的耳聋基因（包括GJB2基因、SLC26A4基因等）检测。10例胎儿的耳聋基因与先证者的基因类型相同，选择终止妊娠；其余44例没有重复先证者的基因类型，临床预测不会罹患由先证者基因改变引起的耳聋．选择继续妊娠，现均已分娩，新生儿出生后由专人随访，检查其听力均正常。（2）QF—PCR技术诊断21三体综合征的结果：54例胎儿全部检测成功，两对组合引物同时扩增后，均明确排除21三体综合征的诊断。5例高龄孕妇同时进行了胎儿染色体核型分析也均未见异常。新生儿出生后随访其发育情况，从外观也排除了21三体综合征的诊断。QF—PCR技术检测结果在1～3d即可获得，无漏诊及误诊。结论对于产前行遗传性耳聋基因诊断及其他产前基因诊断的胎儿，同时行QF—PCR技术检测可明确胎儿是否为21三体综合征；QF—PCR技术检测21三体综合征具有快速、准确、价格低廉及高通量等优点，且不增加羊水及脐血标本的抽取量。

Objective To establish the genetic test technique of trisomy 21 concurrently conducts with prenatal diagnosis for hereditary hearing loss. Methods Fifty-four pregnant women who underwent prenatal diagnosis for hearing loss of their fetuses in Chinese People＇s Liberation Army General Hospital from March 2009 to May 2010 were enrolled in thi：s study. All probands from the deaf families have confirmed the causative mutation for hearing loss in Genetic Testing Center in Chinese People＇s Liberation Army General Hospital. The mean age of 54 pregnant women is 31 years at pregnancy of 18 - 26 weeks, 5 cases 〉 pregnancy of 23 weeks, 9 cases ≥ 35 years. All subjects did not conduct the serologic tests for trisomy 21 before. Fifteen to twenty ml amniotic fluid was drawn from 49 cases at pregnancy of 18 - 23 weeks and 5 cases 〉 pregnancy of 23 weeks. One to two ml umbilical blood was drawn from 5 cases 〉 pregnancy of 23 weeks. For 9 cases ≥35 years, amniotic fluid cell culture and karyotyping analysis were conducted concurrently. A multiple quantitative fluorescent （QF） diagnosis trisomy 21. The samples with peaks of 1： 1：1 PCR and six microsatellite markers were applied to or 2：1 at two microsatellite markers can be diagnosed as trisomy 21. Results （ 1 ） Fifty-four fetuses were successfully conducted prenatal genetic diagnosis for hearing loss （ included GJB2 and SLC26A4）. Ten fetuses copied the exactly same genotypes as the probands. The other 44 cases fetuses did not copy the same genotypes as the probands and won＇t develop hearing loss. The hearing test showed normal hearing for the neonates. （2） All the 54 fetuses were excluded of trisomy 21 by QF-PCR and were verified after birth. Five fetuses with advanced maternal age were performed karyotyping analysis and showed normal. The diagnostic results of QF-PCR can be obtained in 1 - 3 days without misdiagnosed. Conclusions QF-PCR is an efficient, rapid and accurate technique for detection of trisomy 21 without increasing sample amount. It can be used for fetuses who were undertaken hearing loss gene test or other prenatal gene test.