Zhu Xiang-Yu, Hu Ya-Li, Wang Ya-Ping, Zhu Hai-Yan, Li Jie, Zhu Rui-Fang, Zhang Ying, Wu Xing, Yang Ying
Department of Obstetrics and Gynecology, Affiliated Drum Tower Hospital of Medical College, Nanjing University, Nanjing 210008, China.
Zhonghua Fu Chan Ke Za Zhi. 2008 Nov;43(11):818-23.
To explore the feasibility of application of multiplex quantitative fluorescent PCR with non-polymorphic loci in prenatal diagnosis of aneuploidies.
From Mar 2006 to Nov 2007, a total of 63 samples were collected from the Department of Obstetrics and Gynecology, Affiliated Drum Tower Hospital of Medical College, Nanjing University, including 54 villous samples obtained for karyotyping because of spontaneous abortion, six amniotic fluid samples of second trimester and three umbilical cord blood samples of third trimester. Blood samples of 60 healthy adults were obtained at the same time as a control group, including 30 males and 30 females. Non-polymorphic QF-PCR was performed on both testing group and control group for the detection of aneuploidies. The Amelogenin gene (AMXY) was selected as an internal control, and dosage quotiety (DQ) of each locus was calculated according to the known formula. If DQ was between 0.7 and 1.3, the sample was considered as normal. If the figure turned out to be > 1.3 or < 0.7, a potential duplication or deletion of the corresponding gene or chromosome was indicated. If the results implied numerical abnormalities in more than one euchromosome, sex chromosome aneuploidies should be considered. Cell culture and karyotyping were carried out for every sample simultaneously. The results of non-polymorphic QF-PCR were checked with karyotypes.
(1) In the control group, all female samples presented only an AMX peak for sex chromosome while all males showed AMX and AMY amplified peaks. The AMY/AMX ratios were between 0.7 - 1.3, and SD was between 0.05 - 0.12. (2) Among 19 QF-PCR abnormal cases, 13 cases were proved by karyotyping. Of the six cases which turned out to be conflicting, one case of trisomy 18 shown by karyotyping was not completely detected by QF-PCR, a locus on chromosome 18 implied trisomy, while another turned out to be normal (DQ = 1.28). Four cases were detected by non-polymorphic QF-PCR as trisomies but showed normal female karyotype because of maternal contamination during cell culture. A karyotypingly '46, XY' case did not present an AMY peak. Thirty-six out of 44 (82%) normal results implied by non-polymorphic QF-PCR were in accordance with cytogenetic analysis. Of the other eight cases, one case which failed cytogenetic analysis was detected by QF-PCR as normal. Four cases showed multiploidy by karyotyping but normal in QF-PCR analysis, including three cases of 69, XXX, one case of 92, XXXX and one case of 45, XX, rob (13;21). The other two cases that showed normal male results turned out to be normal female karyotypes.
Prenatal aneuploidy detection by non-polymorphic QF-PCR is feasible in a clinical diagnostic setting. With the advantages of high throughput, rapidness and low cost, this method shows a good prospect in clinical application.
探讨应用具有非多态性位点的多重定量荧光聚合酶链反应（QF-PCR）进行非整倍体产前诊断的可行性。
2006年3月至2007年11月，从南京大学医学院附属鼓楼医院妇产科收集63份样本，其中包括因自然流产而获取的54份绒毛样本用于核型分析、6份孕中期羊水样本和3份孕晚期脐带血样本。同时采集60名健康成年人的血液样本作为对照组，其中男性30名，女性30名。对检测组和对照组均进行非多态性QF-PCR以检测非整倍体。选择牙釉蛋白基因（AMXY）作为内对照，并根据已知公式计算每个位点的剂量商（DQ）。若DQ在0.7至1.3之间，则样本被视为正常。若结果大于1.3或小于0.7，则提示相应基因或染色体可能存在重复或缺失。若结果提示不止一条常染色体存在数目异常，则应考虑性染色体非整倍体。同时对每个样本进行细胞培养和核型分析。将非多态性QF-PCR结果与核型结果进行核对。
（1）在对照组中，所有女性样本的性染色体仅呈现一个AMX峰，而所有男性样本均显示AMX和AMY扩增峰。AMY/AMX比值在0.7 - 1.3之间，标准差在0.05 - 0.12之间。（2）在19例QF-PCR异常病例中，13例经核型分析证实。在6例结果不一致的病例中，核型分析显示的1例18三体病例未被QF-PCR完全检测到，18号染色体上的一个位点提示三体，但另一个位点结果正常（DQ = 1.28）。4例经非多态性QF-PCR检测为三体，但因细胞培养过程中母血污染而显示女性核型正常。1例核型为“46, XY”的病例未出现AMY峰。非多态性QF-PCR提示的44例正常结果中有36例（82%）与细胞遗传学分析结果一致。在另外8例中，1例细胞遗传学分析失败的病例经QF-PCR检测为正常。4例核型分析显示为多倍体但QF-PCR分析正常，包括3例69, XXX、1例92, XXXX和1例45, XX, rob(13;21)。另外2例显示男性正常结果的病例实际为女性正常核型。
在临床诊断中，应用非多态性QF-PCR进行产前非整倍体检测是可行的。该方法具有高通量、快速和低成本的优点，在临床应用中具有良好的前景。
