Expression of transcription factors controlling alpha inhibin gene expression in placental tissues from pregnancies affected by fetal Down syndrome

Maternal serum inhibin A is a clinically accepted marker in assessing risk of fetal Down syndrome during pregnancy. Second trimester levels of inhibin A are, on average, about two times higher in affected than in control pregnancies, although the pathological basis of this alteration is not understood. The genes for inhibin A subunits are not located on chromosome 21, eliminating a simple dosage effect. Nevertheless, previous studies have shown up-regulated inhibin A subunit mRNA levels in placental tissue from pregnancies affected with Down syndrome. Inhibin A is a dimeric protein consisting of an alpha and betaA subunit. It has been previously established, using in vitro studies, that the alpha subunit synthesis plays a key role in production of dimeric inhibin. The alpha inhibin promoter has been characterized in placenta and shown to lack a TATA element, but to contain a repeat TG element, multiple initiation sites and several binding sites for cis-acting elements. We sought to determine the factors regulating alpha inhibin expression in Down syndrome placenta, among candidates known to regulate alpha inhibin gene expression in reproductive tissues (placenta and/or ovary). Among the candidates studied were the alpha inhibin transcription repressors; Wilms’ tumor suppressor (WT1), inducible cAMP early repressor (ICER), and CCAAT/enhancer-binding protein beta (CEBP/beta) as well as the alpha inhibin transcription activators; nuclear receptor 5A2 (NR5A2, also known as LRH-1), GATA factors, cAMP response element binding protein (CREB), and transcription factor activating proteins 2 alpha and 2 gamma (TFAP2A and TFAP2C). The mRNA expression of these regulatory factors and inhibin subunits was compared in Down syndrome and unaffected placental tissues. Residual placental tissue was collected after routine examination with approval of the Institutional Review Board for Human Studies at Women and Infants Hospital. Samples were obtained in the second trimester after spontaneous loss or termination and in the third trimester after delivery. Chorionic villous tissue was sampled by Perinatal Pathology staff and stored in RNA later at 80 C before processing. Total RNA was extracted using Trizol, digested with deoxyribonuclease on Qiagen RNeasy columns, and analyzed for quality using a bioanalyzer. The total RNA was then reverse transcribed with avian myeloblastosis virus-reverse transcriptase. Following reverse transcription, the mRNA levels of select genes were determined by real-time PCR on a 7300 real-time PCR system using SYBR Green PCR master mix, as previously described. Primers were designed according to the complete human cDNA sequences of the above genes. The primer sequences are provided in Table 1. The relative mRNA level for each gene of interest was obtained by normalizing to the mRNA level of a control gene, ribosomal protein L19 (RPL19). Specificity of all the real-time PCR reactions was confirmed by a single peak in the melt curves and a single band of the predicted size after agarose gel electrophoresis of the PCR products (data not shown). Ten samples from Down syndrome pregnancy and eight control samples were collected in the second and third trimesters of pregnancy, at gestational weeks 14 to 39. The mean gestational age at sample collection was 26weeks ( 10) in each group. There was no difference in the distribution of fetal gender with 60% female in each group (three control samples were not assigned a fetal gender). Maternal age was slightly, but not significantly, higher in women having Down