[Clinical application of noninvasive prenatal diagnosis using cell free fetal DNA in maternal plasma]

To investigate the clinical value of non-invasive prenatal diagnosis using cell free fetal DNA (cff-DNA) in maternal blood.

From Sep. 2010 to Mar. 2012, 103 pregnant women who came to Henan Province People's Hospital in the first trimester for prenatal diagnosis of sex-linked inherited diseases were included in the first trimester group. From Oct. 2010 to Jan. 2012, 205 pregnant women undergoing amniotic fluid sampling for fetal karyotype analysis in the same hospital were included in the second trimester group. Real time quantitative PCR and fluorescent PCR were used to detect sex determining region of Y chromosome gene (SRY) and amelogenin gene (AML) on cff-DNA of the first trimester group. Moreover, 12 Y chromosome STR loci analysis were performed for 33 male fetuses and their fathers. Massively Parallel Signature Sequencing (MPSS) was used for aneuploidy analysis in cff-DNA of the second trimester group.

(1) In the first trimester group, there were 53 SRY positive and 50 SRY negative. Compared with the results of cff-DNA of chorionic villus samples, there was one SRY false positive and one false negative results, with a sensitivity of 98% and specificity of 98%. For the AML gene test, there were two PCR products of male fetuses:102 bp fragment originating from X chromosome (AML X) and 108 bp fragment from Y chromosome (AML Y); but only AML X was found in products from female fetuses. In the first trimester group, 102 bp and 108 bp fragments were detected in 52 cases, and only 102 bp fragment was found in the other cases. Compared to AML results from chorionic villus samples, there were 2 false negative results, with a sensitivity of 96% and specificity of 100%. (2) For cff-DNA with plasma SRY over 30 copy/ml, Y STR loci were analyzed on cff-DNA of 33 fetuses and their fathers. The Y STR loci less then 200 bp were successfully detected, while Y STR loci with PCR products between 200-300 bp showed low signal or could not be amplicated; and no PCR products more than 300 bp were detected from cff-DNA. Comparing the detected Y STR loci of cff-DNA to the fathers, 32 fetuses were concordant with their fathers'. Exogenous contamination was found in the rest one sample. (3) In the second trimester group, 6 fetuses with abnormal karyotype (two trisomy 21, three trisomy 18 and one 45, XO) were detected by cff-DNA and were proved by karyotype analysis. Moreover, the MPSS results of cff-DNA revealed one 45, Y and one trisomy 16 whose karyotype analysis showed normal results. And in one case, MPSS suggested less chrX or chrY, that was proved to be 47, XYY by karyotype analysis.

(1) Cff-DNA in maternal blood can be used to determine fetal gender in early prenancy with considerable sensitivity and specificity. But the trace cff-DNA and the high maternal DNA background might have impact on the result. (2) Analysis of cff-DNA in maternal blood of the second trimester women showed that MPSS could be used for prenatal screening of trisomy 21 and trisomy 18. However, further research should be done for other chromosomes aneuploidy detection.