Noncoding RNA and DNA as biomarkers: toward an epigenetic fetal barcode for use in maternal plasma.

The human genome contains a large layer of hidden biological information that is not accessible by proteomic or metabolic methods (1, 2 ). This information does not involve the typical (end)products of gene expression such as proteins. Instead, it involves genes that are transcribed but not translated (noncoding RNA), and DNA sequences that are neither transcribed nor translated (noncoding DNA) (1, 2 ). When this information, despite being noncoding in nature, is unique for the fetus or at least different from that in maternal blood cells (1 ), it can be used as a biomarker during pregnancy for the purpose of noninvasive prenatal diagnostics such as detection of Down syndrome. This application, along with a systematic approach to target this category of mostly unexplored information, is elegantly shown by Chim and coworkers in the current issue of Clinical Chemistry (2 ). Why is the reporting of these findings so timely? According to the Fantom cDNA3 database (2005 release), the percentage of biological information presented by noncoding RNA is at least one-third of the more than 100 000 transcripts expressed in humans (3 ). Noncoding RNAs include the rapidly expanding family of small (21–35 nucleotides in length), noncoding microRNAs (miRNA) (4, 5 ), including small interfering RNA (siRNA), repeat-associated RNA (rasiRNA) (6 ), piwi interacting RNA (piRNA) (7 ), and mirtrons (8 ). The second category consists of noncoding DNA carrying differential DNA markers (2 ). When these signatures involve (chemical) DNA modifications, such as methylation of CpG dinucleotides, in the absence of sequence variations, they are called epigenetic. Epigenetic signatures that differ from those of fetal placenta cells and maternal blood cells can be used for noninvasive prenatal diagnostics (1 ). As for noncoding RNA, the type, number, and nature of noncoding DNA sequences has gained increased interest, with strong reappraisal of their biological significance. The paper by Stephen Chim and coworkers in the current issue of Clinical Chemistry (2 ) is an excellent demonstration of how the hidden and largely unexplored class of biological information provided by noncoding DNA and carried by placental DNA can be retrieved. Retrieval was done systematically and reliably with near completeness for the chromosome region of interest (2 ). By exploring methylated DNA markers, which differ from those of placenta and maternal blood cells, only those biomarkers were targeted that allow reliable discrimination between fetal target DNA and maternal background DNA when used for prenatal diagnostics. By focusing on markers on the long arm of chromosome 21, which are directly related to and therefore informative for the disease of interest (trisomy 21, Down syndrome) (9, 10 ), and analyzing all suitable markers present (114 CpG islands), Chim et al. identified multiple chromosome 21–specific DNA markers. By a combined approach, executed thoroughly, a large and complete set (n 22) of differentially methylated fetaland chromosome 21q–specific DNA markers was identified. Two of these markers, both unmethylated in the placenta, U-PDE9A and U-CGI137, and selected for their greatest difference in median methylation index, were validated in maternal plasma samples. For the latter gene, the method of analysis was successfully adapted to overcome the low number of differentially methylated CpG sites. The results of this investigation clearly demonstrate that markers of this nature, in which noncoding sequences represent and complement the fetal genetic barcode, can be detected reliably and specifically in the maternal plasma (2 ). The consequences of these findings are manifold. Although plasma is usually the primary specimen for clinical testing and contains the largest version of the human proteome (11 ), maternal plasma has not been useful in this way with respect to the completeness of the biological information from the fetus. At least 19.3% of additional data, not accessible by proteomic or metabolic methods, is available by molecular targeting for this class of hidden information (2 ). Epigenetic differences between the placenta and maternal blood cells were found on 22 of 114 CpG islands studied on chromosome 21q (2 ). The same gain in information can be expected for other chromosomes and for noncoding RNA, including microRNA. Recent studies show that of 345 miRNAs analyzed, 275 (80%) are expressed in the human placenta, and 53 (15%) show preferential or exclusive expression in the placenta (12 ). Therefore, the percentage of additional biological information on fetal well-being that becomes available by targeting noncoding RNA and noncoding DNA likely ranges between 20% and 30%. By analogy with the exploration of chromosome 21q (2 ), large-scale searches for epigenetic and related noncoding markers Clinical Chemistry 54:3 456–457 (2008) Editorial