Transrenal nucleic acids: from proof of principle to clinical tests.

In spite of numerous publications on potential diagnostic application of circulating DNA and transrenal nucleic acid (Tr-NA) analysis, few, if any, tests based on this technology are available in clinical labs. This delay in test development and implementation is caused, at least in part, by the deficit in robust methods for isolation of short nucleic acid fragments from bodily fluids, as well as in techniques for analyzing these fragments. We have developed a new anion exchanger-based method for the isolation of cell-free nucleic acid fragments from large volumes of bodily fluids, and analyzed these fragments by PCR techniques specially designed to amplify "ultrashort" templates. The combination of these two techniques not only revealed the presence in urine of 10-150 bases or bp DNA and RNA fragments in addition to previously observed 150-200-bp DNA fragments and high molecular weight DNA, but also significantly increased the sensitivity of Tr-DNA detection. Additionally, we detected in urine a variety of miRNAs, including those excreted transrenally, thereby opening new diagnostic possibilities for Tr-NA analysis.

Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center.

Imprinted genes in mammals are often located in clusters whose imprinting is subject to long range regulation by cis-acting sequences known as imprinting centers (ICs). The mechanisms by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an approximately 2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27 methyltransferase and that maintenance of CpG methylation of the PWS-IC in mouse ES cells requires the function of G9a. We show by RNA fluorescence in situ hybridization (FISH) that expression of Snrpn, an imprinted gene regulated by the PWS-IC, is biallelic in G9a -/- ES cells, indicating loss of imprinting. By contrast, Dnmt1 -/- ES cells lack CpG methylation of the PWS-IC but have normal levels of H3 Lys-9 methylation of the PWS-IC and show normal monoallelic Snrpn expression. Our results demonstrate a role for histone methylation in the maintenance of parent-specific CpG methylation of imprinting regulatory regions and suggest a possible role of histone methylation in establishment of these CpG methylation patterns.

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse.

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.