Detection Of Fetal–specific C21orf105, PLAC4 MRNA On Chromosome 21 In Maternal Plasma

Trisomy 21 which is also called Down’s syndrome, is the most common chromosome abnormality in live-born infants with an incidence of 1/800~1/600, and associated with intellectual disability, typical facial features and other medical problems. At present, the gold standard for prenatal diagnosis of fetal chromosomal aneuploidies is karyotyping of fetal cells, such as chorionic villus sampling and amniocentesis, and may impose potential risks to the mother or the fetus. Although the maternal serum screening properties are rather good with a detection rate of85–90% with a false-positive rate of 5–9%, the test lacks diagnostic power. As a screening method, the noninvasive prenatal testing(NIPT) based on cell free fetal DNA in maternal plasma is relatively expensive. Pregnant women therefore highly demand for a reliable and practical method with high diagnostic sensitivity and specificities. Recently, the demonstration of the presence of cell-free fetal RNA in maternal plasma has opened up new possibilities for noninvasive prenatal assessment of fetal chromosome aneuploidies.C21orf105 and PLAC4 gene was a chromosome 21-encoded gene located within the Down syndrome critical region(DSCR), and was highly expressed in placental tissues. As well as C21orf105 and PLAC4 m RNA which was fetal-specific could be detectable in maternal plasma during all trimesters of pregnancy. C21orf105 and PLAC4 m RNA in maternal plasma was a promising biological marker for noninvasive prenatal screening for Down’s syndrome. However, for the storage temperature and processing time of blood samples, different results have been reported. In many studies, blood samples were left at room temperature for 3h、6h、24h or at 4°C for 72 h before centrifugation. It is necessary to study the specific C21orf105 and PLAC4 m RNA in the plasma of pregnant women. A number of investigators have shown that the plasma concentration of a chromosome21–transcribed placental C21orf105 and PLAC4 m RNA were increased in trisomy 21 pregnancies, whereas other studies demonstrated that the concentration of C21orf105 and PLAC4 m RNA in maternal plasma was not significantly increased in trisomy 21 pregnancies compared with normal pregnancies. Further studies are required to investigate the relation between the expression level of fetal-specific C21orf105 and PLAC4 m RNA in maternal plasma and trisomy 21 fetuses. In the present study, we aimed to determine effect of storage temperature and treatment time on the stability of fetal–specific C21orf105 and PLAC4 m RNA in maternal plasma and compare the expression level of C21orf105 and PLAC4 m RNA in maternal plasma of pregnancies with euploid and trisomy 21 fetuses using real-time PCR.ObjectiveThe purpose of this research was to detect the stability of fetal-specific C21orf105, PLAC4 m RNA in maternal plasma and to investigate whether the levels of C21orf105, PLAC4 m RNA could be able to discriminate pregnancies whose fetus is trisomy 21.Materials and Methods1. Study participantsPre- and post-delivery peripheral blood samples(n = 10) from the Third Affiliated Hospital of Zhengzhou University were collected and we collected peripheral blood samples from 10 pregnant women and 10 non-pregnant to verify the pregnant specificity and clearance of C21orf105,PLAC4 m RNA.Blood samples were collected randomly from 30 healthy women with singleton uncomplicated pregnancies and detected the expression levels of the fetal-specific C21orf105, PLAC4 m RNA from pregnant women who attended to the Third Affiliated Hospital of Zhengzhou University to detect the stability of C21orf105,PLAC4 m RNA.Singleton pregnancies were recruited for our study, in which 40 blood samples from normal pregnancies and 38 samples from trisomic pregnancies were collected to compare the levels of C21orf105, PLAC4 m RNA in maternal plasma between normal pregnancies and trisomy 21 pregnancies.2. MethodsUsing Real-time PCR we detected the expression expression levels of the fetal-specific C21orf105, PLAC4 m RNA from pregnant womenResults1. The clearance and pregnant specificity of C21orf105 and PLAC4 m RNA in maternal plasmaWe can detected C21orf105,PLAC4 m RNA in pre-delivery plasma, but not in the postpartum samples 24 h after delivery. C21orf105,PLAC4 m RNA was detected in 10 plasma samples obtained from pregnant women, but not in those from non-pregnant women.plasma.2. The Stability of C21orf105 and PLAC4 m RNA in maternalThe ANOVA under the significance level of 0.05 for factorial designed data indicated that the main effect of the storage temperature was not statistically significant(F C21orf105=0.417 P=0.519;F PLAC4=0.408 P=0.524). No significant difference was observed in the levels of C21orf105 and PLAC4 m RNA in samples that had been left for the same time at different temperature(P>0.05). And the main effect of the storage time was also no significantly different(F C21orf105=2.608P=0.052; F PLAC4=1.555 P=0.201). At the same preserved temperature, no significant difference was found for plasma C21orf105 and PLAC4 m RNA levels that had been left for 6,24,72 h(P>0.05). As well as no statistically significant interaction between the two factors was found(F C21orf105=0.244 P=0.866;F PLAC4=0.668P=0.573). There were no obvious effect of temperature and time on the C21orf105 and PLAC4 m RNA levels in maternal plasma(P>0.05).3. The relationship between the expression levels of C21orf105 and PLAC4 m RNA in normal pregnancies and trisomy 21 pregnancies.The expression levels of C21orf105 and PLAC4 m RNA in trisomy 21 pregnancies plasma were 0.166±0.062, 9.374±0.313,and in normal pregnancies the expression levels were 0.068±0.037、6.103±0.206. We detected significant increases in plasma C21orf105 and PLAC4 m RNA expression level in the trisomy 21pregnancies(P<0.01).ConclusionsThe fetal chromosome 21-encoded C21orf105 and PLAC4 m RNA in maternal plasma is stable enough and not affected by the storage temperature and processing time. C21orf105 and PLAC4 m RNA are potential makers for prenatal screening of pregnancies with trisomy 21.