Profiling of DNA Methylation in Uniparental and Biparental Tissues Identifies New Imprinted Genes

Imprinted genes are critical for normal human growth and neurodevelopment. Most imprinted genes are clustered and respond to signals in cis from nearby differentially methylated regions (DMRs). Such DMRs, comprised of CpG-rich regions of DNA, demonstrate ∼50% methylation because either the maternal or the paternal chromosomes (alleles) are methylated in a parent of origin-specific manner. We developed a strategy whereby DNA differentially methylated regions were used to identify novel human imprinted genes. We compared genomewide methylation profiles of biparental and uniparental human tissues. We used bisulfite modified genomic DNA from 21 normal biparental tissues (placenta and blood) and 4 uniparental tissues and hybridized it to the Illumina methylation 27 array. This array covers ∼27,600 CpG sites (∼14,500 genes). Candidate DMRs were selected by identifying CpGs with putative allelic differential methylation in normal biparental tissues. In parallel, we looked for parent of origin-specific patterns in DNA methylation in paternally derived human androgenetic complete hydatidiform mole, and maternally derived mature cystic ovarian teratoma. Using this approach, we selected known DMRs associated with imprinted genomic regions. Furthermore, our analysis revealed previously unidentified DMRs for the known imprinted genes, NAP1L5 and ZNF597. Using QIAGEN bisulfite Pyrosequencing, we were able to validate several DMRs identified by the arrays. The DMR for one novel candidate, AXL, is validated by methylation studies in human. In mouse embryos, we showed that Axl is expressed preferentially from the maternal allele. This parent of origin-specific expression pattern is shown to be DNA methylation dependent. This approach permitted the characterization of a new candidate imprinted gene, AXL, in humans. This is one of the few instances of a new imprinted gene being identified first in humans and then in mice.


Sanaa Choufani

Dr. Sanaa Choufani, PhD, is a Research Associate in the Genetics and Genome Biology program at The Hospital for Sick Children in Toronto, Canada. She is working in the research group of Dr. Rosanna Weksberg. Dr. Choufani’s research focuses on the epigenetic mechanisms causing genes to be expressed depending on their parental origin. She works on the identification of new imprinted genes and their role in normal development and. In addition, she studies the epigenetic basis of growth dysregulation in the cancer predisposition disorder, Beckwith-Wiedemann syndrome. An important complementary research focus of Dr. Choufani involves the investigation of the effects of assisted reproductive technologies on genomic imprinting and their impact on human health and development.