44 single-nucleotide polymorphisms expressed by placental RNA: assessment for use in noninvasive prenatal diagnosis of trisomy 21.

To the Editor: For noninvasive prenatal diagnosis, markers that directly reflect changes in chromosome dosage are preferred over indirect markers that are associated with epiphenomena (1, 2). The RNA:single-nucleotide polymorphism (SNP) allelic ratio strategy was described recently as a means to directly assess fetal chromosome dosage in maternal plasma (2). Quantitive comparison of the allelic expression ratios of a placentally expressed, chromosome 21– encoded gene, placenta-specific 4 (PLAC4), enabled detection in maternal plasma of the differences between 2 (normal) or 3 copies of chromosome 21 (2). The RNA:SNP ratio strategy is currently limited to a subset of the population with hetero-zygosity of the SNP used. Theoretically , an increase in population coverage can be obtained by inclusion of additional SNPs within PLAC4 or other chromosome 21– encoded transcripts with placental expression and detectability in maternal plasma (2). We therefore tested 44 SNPs expressed by 7 chromosome 21– encoded , placentally expressed genes (2), PLAC4, collagen, type VI, alpha 2 (COL6A2), collagen, type VI, alpha 1 (COL6A1), BTG family, member 3 (BTG3), ADAM metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1), chromosome 21 open reading frame 105 (C21orf105), and amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) (APP), for their potential use in noninvasive prenatal diagnosis. All SNP markers were tested for their presence in 1st-trimester plasma and their absence in nonpregnant women. Peripheral blood samples were collected from pregnant women attending the Prenatal Diagnostic Centre of the VU University Medical Center. All participants gave informed consent before study inclusion. The study was approved by the ethics committee at our institution. We collected EDTA blood samples between weeks 9 and 14 of pregnancy , before invasive diagnostic procedures were performed. Samples were processed and RNA extracted as described previously, with automated isolation (BioRobot MDX) (1). RNA extraction from PAX gene tubes was performed using the BioRobot MDX with a standardized protocol (Qiagen). For selected genes, allele frequencies were determined by cycle sequencing with a Big Dye terminator, followed by capillary electrophoresis (ABI 3100XL). Within the transcripts of the 7 genes of interest, 44 SNPs were identified (www.hapmap.org) (Table 1). Primers flanking these SNPs were designed with similar thermody-namic characteristics to permit RT-PCR analysis in single runs. All primers were intron spanning, except for the primers of PLAC4. Using a sensitive, 2-step, 1-tube RT-PCR assay (Superscript II RT-PCR, In-vitrogen) supplemented with 1 mol/L betaine to increase reverse transcrip-tase efficiency and enzyme …