WGIMPRINT · A Whole-Genome Screen for Novel Imprinted Loci

A Whole-Genome Screen for Novel Imprinted Loci

The initial aims of this project were to construct genome-wide DNA methylation maps of two types of uniparental tissues, Ovarian Teratomas (OT) and Complete Hydatidiform Moles (CHM). By comparing epigenetic maps in these two tissues, it was thought that this should allow the identification of differentially methylated regions (DMRs) corresponding to imprinted loci. DNA methylation maps in two OTs and two CHMs were generated in collaboration with Roche Nimblegen, and a computational pipeline was developed to identify regions that showed different methylation between OT and CHM. Bisulfite PCR validation was performed on ~50 sites that were scored as differentially methylated from the microarray data, confirming that the majority of sites identified from statistical analysis were genuine regions of differential methylation between OT and CHM. However, this also showed that many DMRs identified did not correspond to imprinted loci that showed differential methylation dependent upon parental origin, but instead represented sites that were differentially methylated between different tissue types. This finding poses a considerable problem to the project as originally defined, as it meant that the comparison of methylation in samples from CHM and OT as a means to identify imprinted loci is compromised due to the inherent differences of these two tissue types. Although imprinted sites can be identified, these represent only a subset of the total, with many other DMRs instead corresponding to tissue-specific DMRs. As a result, alternative strategies to identify imprinted loci were considered that could utilise uniparental samples that were derived from the same tissue type that may show better specificity for imprinted regions. It was hypothesised that the same methodology could be applied to study patients with uniparental disomy (UPD) to identify DMRs on the specific chromosome that showed uniparental inheritance. DNA samples from patients with maternal UPD15 and paternal UPD15 were obtained from collaborators, and methylation profiles for chromosome 15 generated by meDIP and hybridisation to chr15 tiling arrays. Results showed that DMRs between matUPD15 and patUPD15 patients occurred specifically at imprinted regions, resulting in the identification of many novel imprinted loci on chromosome 15. This work was recently published in Genome Research, and presentation of this data at the European Society of Human Genetics meeting, Gothenburg, Sweden, resulted in the award of the Young Investigator Award for Outstanding Science to the Marie-Curie Fellow, Dr. Andrew Sharp. The work was subsequently published in Genome Research. We subsequently established a collection of >50 DNA samples from patients with both maternal and paternal UPD for a number of different human chromosomes that could be used to systematically screen for imprinting across large parts of the human genome. This work is currently ongoing. This collection included samples from patients with Turner syndrome (45,X karyotype), who possess a single X chromosome of either maternal or paternal origin. Although analysis of these cases did not detect any imprinted sites on the human X chromosome, relating the methylation data to that obtained in normal females allowed important insights to be made into the process of X chromosome inactivation. A manuscript on this work is currently in revision at Genome Research.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз