[Application of multiplex quantitative fluorescent PCR with non-polymorphic loci in prenatal diagnosis]

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.