| Abstract: | The maternal and paternal genomes possess distinct epigenetic marks that distinguish them at imprinted loci. In order to identify imprinted loci, we used a novel method, taking advantage of the fact that uniparental disomy (UPD) provides a system that allows the two parental chromosomes to be studied independently. We profiled the paternal and maternal methylation on chromosome 15 using immunoprecipitation of methylated DNA and hybridization to tiling oligonucleotide arrays. Comparison of six individuals with maternal versus paternal UPD15 revealed 12 differentially methylated regions (DMRs). Putative DMRs were validated by bisulfite sequencing, confirming the presence of parent-of-origin-specific methylation marks. We detected DMRs associated with known imprinted genes within the Prader-Willi/Angelman syndrome region, such as SNRPN and MAGEL2, validating this as a method of detecting imprinted loci. Of the 12 DMRs identified, eight were novel, some of which are associated with genes not previously thought to be imprinted. These include a site within intron 2 of IGF1R at 15q26.3, a gene that plays a fundamental role in growth, and an intergenic site upstream of GABRG3 that lies within a previously defined candidate region conferring an increased maternal risk of psychosis. These data provide a map of parent-of-origin-specific epigenetic modifications on chromosome 15, identifying DNA elements that may play a functional role in the imprinting process. Application of this methodology to other chromosomes for which UPD has been reported will allow the systematic identification of imprinted sites throughout the genome.Imprinting is a phenomenon in which the expression status of a gene is dependent on the sex of the parent from which it is inherited. Imprinted genes generally exhibit monoallelic expression accompanied by parent-of-origin-specific epigenetic marks such as differential DNA methylation and histone modifications that distinguish the maternal and paternal genomes at these loci (Reik and Walter 2001; Dindot et al. 2009). More than 60 imprinted genes have been identified in humans (http://www.geneimprint.com/), and their clustered nature suggests that many are regulated by regional control mechanisms.To date, the discovery of imprinted sites in both mouse and human has largely been driven through the use of phenotype-based approaches. The vast majority of loci subject to parent-of-origin effects were first recognized through the observation that maternal and paternal transmission of the same genetic mutation results in different phenotypes (Nicholls et al. 1989). For example, the identification of imprinted gene clusters in 15q11-q13 associated with Prader-Willi/Angelman syndrome, 11p11.5 associated with Beckwith-Wiedemann syndrome, and imprinted loci at 14q32, 6q24, and 20q13.2 were all catalyzed by the initial observation that genetic disease occurred specifically in patients with either uniparental disomy (UPD) or deletions of these regions of specific parental origin. In combination with chromosomal engineering techniques that can systematically generate defined aneuploidies, this notion has been applied to screen the mouse genome for imprinting with great success, resulting in the identification of more than 130 murine imprinted genes (Williamson et al. 2009). However, because this methodology relies on the recognition of overt phenotypic differences between individuals to detect imprinting, it is likely to miss imprinting that may cause subtle phenotype differences or those that manifest in ways that are not easily recognized by typical methods of phenotypic characterization. Further, imprinted genes will also be missed or masked by phenotypes that are lethal.In order to circumvent this limitation, a variety of genomic techniques have been developed to identify parent-of-origin effects. Several previous studies have attempted to detect imprinting based on the differential expression of parental alleles at imprinted loci. Studies using subtractive cDNA hybridization (Kaneko-Ishino et al. 1995) and high-throughput cDNA sequencing in hybrid mouse strains (Wang et al. 2008) have been used to detect imprinted expression with some success. However, these approaches are limited in that they can only assay the subset of genes expressed in the tissue(s) under investigation, and for some genes, imprinted expression is only observed in specific tissues or at certain developmental stages (Deltour et al. 1995; Rougeulle et al. 1997; Zhou et al. 2006). Furthermore, sequencing-based approaches are only able to assay allelic bias in genes containing transcribed polymorphisms (Daelemans et al. 2010).Alternative approaches to detect imprinting have used the fact that the maternal and paternal genomes have differential epigenetic marks at most imprinted loci. This approach has the advantage over expression-based methods, in that these differential methylation marks are generally conserved, even in tissues that lack imprinted expression (Dockery et al. 2009). The presence of overlapping euchromatin and heterochromatin marks has been used to highlight imprinted domains in human (Wen et al. 2008), and restriction landmark genome scanning (Hayashizaki et al. 1994) and methylation-sensitive representational difference analysis (Kelsey et al. 1999; Smith et al. 2003) have been applied as methods to detect differentially methylated regions in the mouse genome. However, the reliance of these latter techniques on restriction enzyme digestion means that they can only assay a small subset of CpGs that overlap the enzyme recognition site, and if used in outbred genomes, are liable to artifacts generated by the presence of single nucleotide variants that alter restriction patterns.Because one of the key features of imprinted genes is the presence of parent-of-origin-specific methylation, we hypothesized that the systematic comparison of DNA methylation patterns in maternal versus paternal chromosomes should represent an optimal method for the detection of imprinted loci. Based on this hypothesis, we have taken advantage of the fact that uniparental disomy provides a unique system that allows the separate study of chromosomes derived from a single parent and combined this with a methodology in which the methylation of entire chromosomes can be analyzed in an unbiased fashion. By analyzing methylation patterns in cases of maternal UPD15 (matUPD15) and paternal UPD15 (patUPD15) using immunoprecipitation of methylated DNA and high-density tiling arrays with complete coverage of human chromosome 15, we generated separate methylation profiles of the maternally and paternally derived alleles. Comparison of the two parental epigenotypes identifies numerous loci on chromosome 15 that show parent-of-origin-specific methylation differences, defining a set of DNA elements that are likely responsible for the establishment and/or maintenance of imprinting on this chromosome. We identify novel imprinted loci both within and outside of the known PWS/AS imprinted domain, suggesting candidate loci that may exert parent-of-origin effects in several human phenotypes. |
