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Application of fetal dna in Maternal Plasma in Noninvasive Prenatal Diagnosis

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2004年第1期24卷 59-61页

作者：赵茵邹丽 Department of Obstetrics and Gynecology

To explore the application of fetal dna in maternal plasma for noninvasive prenatal diagnosis, the dna template was extracted by hydroxybenzene chloroform from 44 maternal (7-41 weeks) plasma. The Fetus derived Y sequence DYZ 1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00 %. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88.89 % at early pregnant stage, 100.00 % at medium pregnant stage, and 96.55 % at late stage respectively. The final accuracy of 95.45 % was obtained in all cases. It was concluded that by means of hydroxybenzene chloroform extraction the authors of this article promoted the concentration and purity of the dna template, and diagnosed more accurately. The results showed that free fetal dna in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.

关键词： prenatal diagnosis PCR fetal dna

Detection of fetal Nucleated Erythrocytes and fetal dna from Peripheral Blood of Pregnant Women

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2003年第1期23卷 65-67页

作者：张铭陈汉平 DepartmentofObstetricsandGynecology TongjiHospitalTongjiMedicalCollegeHuazhongUniversityofScienceandTechnology

In order to search for a more reliable method of sorting fetal nucleated red blood cells (NRBCs) and dna from maternal peripheral blood and to identify origin of NRBCs and dna, NRBCs were isolated from peripheral blood of 88 pregnant women by density gradient centrifugation and fluorescence activated cell sorter (FACS) respectively. Nested polymerase chain reaction was used to detect normal male SRY gene from blood plasma dna of 65 pregnant women. The results revealed that fetal NRBCs were found in 14 of 27 maternal samples by density gradient centrifugation. The number of cells was from 1 to 10. Using FACS, CD71 + cells were identified among all 61 samples. The frequency was (0.35±0.25)×10 -2; The detectable rate of the SRY gene of blood plasma dna from 46 women carrying male fetuses was 65.22 % (30/46). Non-detectable rate for 19 women carrying female fetuses was 94.74 % (18/19). It was concluded that the methods of sorting fetal NRBCs and dna have already made great progress. The methods for fetal NRBCs and plasma dna from maternal peripheral blood to diagnose genetic diseases seem to be the best methods of noninvasive prenatal diagnosis.

关键词： nucleated red blood cells fetal dna prenatal diagnosis

Advances on circulating fetal dna in maternal plasma

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Chinese Medical Journal 2007年第14期120卷 1256-1259页

作者： FU Xian-hu CHEN Han-ping Department of Obstetrics and Gynecology Tongji Hospital of Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China

The discovery of cell-free fetal dna in maternal plasma in 1997 has opened up new possibilities for noninvasive diagnosis. By RT-PCR, circulating fetal dna can be detected in the plasma of pregnant women, even in the first trimester of pregnancy, and thus can be used for noninvasive prenatal diagnosis of sex-linked disorders, the RhD status of fetuses, and single gene disorders such as beta-thalassaemia, congenital adrenal hyperplasia, and achondroplasia. In addition, quantitative aberrations of circulating fetal dna may indicate various pregnancy-associated disorders, including preeclampsia, preterm labor and fetal trisomy 21.

关键词： noninvasive prenatal diagnosis fetal dna

Effect of formaldehyde treatment on the recovery of cell-free fetal dna from maternal plasma at different processing times

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Effect of formaldehyde treatment on the recovery of cell-fre...

Non-invasive Prenatal Diagnosis of Trisomy 21 by Dosage Ratio of fetal Chromosome-specific Epigenetic Markers in Maternal Plasma

中国遗传学会第八次代表大会暨学术讨论会

作者：张毅孙树汉 Department of Medical Genetics The Second Military Medical University

Cell-free fetal dna in maternal circulation has huge potential for noninvasive prenatal ***,the low proportion of fetal dna in a high background of maternal dna poses significant challenges for its future clinical applications.A resent study reported that fetal dna in maternal plasma could be dramatically enriched by formaldehyde *** finding was initially thought to be ***,follow-up studies have generated conflicting *** present study is aimed to further evaluate the effect of formaldehyde and investigate the underlying *** results indicated that,in blood portions without formaldehyde addition,the concentration of total and fetal dna in plasma stayed constant within the first 6 h of in vitro storage of blood *** prolonged storage for 24 h or more,the concentration of total dna significantly increased due to the release of maternal dna from lysed blood cells,whereas the concentration of fetal dna decreased due to the degradation by plasma dnase,thus leading to percentage of fetal dna dramatically declined from original baseline of 15.8%(8.0%-21.9%) [median(interquartile range,IQR)]at 6 h to 4.9%(3.4%-7.3%)at 36 *** samples processed at 6 h after blood collection,formaldehyde addition showed no impact on concentrations of both total and fetal dna,therefore on enrichment of fetal dna was achieved by formaldehyde treatment at this time *** samples processed at 36 h,however,formaldehyde addition inhibited the blood cell lysis and nuclease-mediated degradation of cell-free dna,so that the variations in concentrations of both total and fetal dna were prevented,thus the decline in the percentage of fetal dna was impeded by formaldehyde *** median(IQR)percentage of fetal dna increased from 4.6%(3.9%-7.9%)(without formaldehyde addition)to 13.9%(10.1%-16.7%)(with formaldehyde addition)at 36 *** these results,we conclude that the effect of formaldehyde on the percentage of fetal dna in maternal

关键词： Formaldehyde Circulating nucleic acid fetal dna dnase Cell lysis

来源： cnki会议评论

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2011年第5期31卷 687-692页

作者：张铭李涛陈静怡李莉周春王燕刘文惠张元珍 Reproductive Medicine Center Zhongnan Hospital Wuhan University Department of Ophthalmology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology

This study examined the methylation difference in AIRE and RASSF1A between maternal and placental dna, and the implication of this difference in the identification of free fetal dna in maternal plasma and in prenatal diagnosis of trisomy 21. Maternal plasma samples were collected from 388 singleton pregnancies, and placental or chorionic villus tissues from 112 of them. Methylation-specific PCR （MSP） and methylation-sensitive restriction enzyme digestion followed by fluorescent quantitative PCR （MSRE ＋ PCR） were employed to detect the maternal-fetal methylation difference in AIRE and RASSF1A. Diagnosis of trisomy 21 was established according to the ratio of fetal-specific AIRE to RASSF1A in maternal plasma. Both methods confirmed that AIRE and RASSF1A were hypomethylated in maternal blood cells but hypermethylated in placental or chorionic villus tissues. Moreover, the differential methylation for each locus could be seen during the whole pregnant period. The positive rates of fetal AIRE and RASSF1A in maternal plasma were found to be 78.1% and 82.1% by MSP and 94.8% and 96.9% by MSRE ＋ PCR. MSRE ＋ PCR was superior to MSP in the identification of fetal-specific hypermethylated sequences （P〈0.05）. Based on the data from 266 euploidy pregnancies, the 95% reference interval of the fetal AIRE/RASSF1A ratio in maternal plasma was 0.33-1.77, which was taken as the reference value for determining the numbers of fetal chromosome 21 in 102 pregnancies. The accu-racy rate in 98 euploidy pregnancies was 96.9% （95/98）. Three of the four trisomy 21 pregnancies were confirmed with this method. It was concluded that hypermethylated AIRE and RASSF1A may serve as fetal-specific markers for the identification of fetal dna in maternal plasma and may be used for noninvasive prenatal diagnosis of trisomy 21.

关键词： fetal dna differential methylation AIRE RASSF1A non-invasive prenatal diagnosis