Pregnancy associated plasma protein-A2: a novel biomarker for Down syndrome

Abstract

Introduction: In an effort to improve prenatal screening for Trisomy 21, we evaluated pregnancy associated plasma protein-A2 (PAPP-A2) as a potential novel second trimester biomarker for Trisomy 21.

Methods: Trisomy 21 and normal control mid-trimester placental samples were subjected to quantitative rt PCR analysis of seven genes we had previously found to be differentially expressed in Trisomy 21 placentae. The localization and differential expression of PAPP-A2 in second trimester placentae from normal and Trisomy 21 pregnancies was determined by immunohistochemistry. PAPP-A2 maternal serum protein levels in ten Trisomy 21 and ten diploid pregnancies were compared by Western blotting. Maternal serum PAPP-A2 levels were measured in 30 Down syndrome cases and 142 normal controls, using ELISA. Regression analysis was used to determine the correlation of PAPP-A2 with other existing markers of Trisomy 21.

Results: PAPP-A2 (aka PLAC 3) mRNA and protein expression were both increased in Down syndrome placentae as compared to diploid placentae. PAPP-A2 was also increased in maternal serum from Down syndrome pregnancies as compared to diploid pregnancies. PAPP-A2 expression correlated weakly with established markers.

Discussion: This work takes advantage of our previously performed systematic approach to the discovery of novel maternal serum biomarkers for Trisomy 21, using cDNA microarray analysis. Beginning with the validation of the microarray results, we have tracked PAPP-A2 overexpression in Down syndrome from placental mRNA to maternal serum protein.

Conclusion: PAPP-A2 could serve as an additional maternal serum marker in prenatal screening for Trisomy 21.

Keywords: Biomarker; Down syndrome; Maternal serum; Placenta; Pregnancy associated plasma protein-A2; Second trimester.

Fig. 1

Differential expression of various genes in Trisomy 21 placentae (n=7) . Results are expressed as fold changes in Trisomy 21 placentae compared to normal placentae. mRNA levels are normalized to GAPDH. Error bars refer to standard deviations. All samples were run in triplicate.

Fig. 2

PAPP-A2 immunohistochemical staining in diploid and Trisomy 21 placentae. Representative immunohistochemistcal staining of PAPP-A2 in second trimester normal (n=7, A, C) and diploid placentae (n=7) at 200× (A, B) and 400× (C, D). Prominent staining is apparent in the cytoplasm of syncytiotrophoblasts. Signal intensity is much stronger in Trisomy 21 placentae (B, D) as compared to normal placentae (left). Significantly increased PAPP-A2 expression (p<0.05) was seen in the cytoplasm of syncytiotrophoblasts of Trisomy 21 placentae (n=7) relative to normal placenta (n=7) in the second trimester (E).

Fig. 3

Western blot analysis of PAPP-A2 in maternal sera from second trimester Trisomy 21 (DS, n=10) and normal (NS, n=10) pregnancies (A). The band intensities were measured using image J software from the NIH and normalized to albumin. Serum levels of PAPP-A2 were significantly elevated in Trisomy 21 pregnancies compared to uncomplicated pregnancies (B) (p<0.05).

Fig. 4

Scatter plot of concentration of PAPP-A2 (ng/ml) versus weeks of gestation in normal (NS) and Trisomy 21 (DS) maternal serum (A). Box plot representing concentrations of PAPP-A2 (ng/ml) between 15 and 20 weeks of gestation in normal (NS) and Trisomy 21 (DS) maternal serum (B).

Fig. 5

Correlation of [PAPP-A2] with other maternal serum markers. The Pearson correlation coefficient (R) value was 0.104 for AFP (A), 0.032 for uE₃ (B), 0.327 for hCG (C), 0.289 for inhibin (D) in normal maternal serum samples at 15–20 weeks of gestation.