Australasian Microarray & Associated Technologies Association, AMATA

Oliver, V*, Cutfield, W**, Miles, H**, Hofman, P** and Morison, I*
*Department of Biochemistry, University of Otago, 710 Cumberland Street, Dunedin North, Dunedin, Otago 9054, New Zealand;**Liggins Institute and the National Research Centre for Growth and Development, University of Auckland, Private Bag 92019, Auckland, New Zealand

In vitro fertilisation (IVF) potentially provides a profoundly abnormal environment for an embryo. Studies with mice, sheep and cattle have indicated that the culture environment of the embryo can affect the imprinting of genes and the phenotype of the animal. Approximately 2% of human births worldwide are conceived using IVF. Recent studies have suggested that IVF causes a small but increased risk of epigenetic imprinting aberrations such as Angelman syndrome and Beckwith-Wiedemann syndrome. Given that mosaicism for the imprinting defect has been observed in Angelman syndrome and Beckwith-Wiedemann syndrome, we hypothesised that low-level, mosaic imprinting defects may be present in phenotypically normal individuals conceived using IVF.

DNA samples from peripheral blood were obtained from 69 IVF-conceived pre-pubertal children and 71 matched controls. DNA methylation of CpG sites within the H19, IGF2, SNRPN and KvDMR1 loci was accurately quantified using methylationsensitive restriction digest followed by real-time quantitative PCR (MSQ-PCR). Global DNA methylation was also examined by using MSQ-PCR on the Satellite 2 repeat region. No differences in the percentage of methylation between the IVF-conceived and control children were observed at the examined CpG sites.

Physiological measurements of these IVF children revealed that they were taller, have higher levels of the growth hormone IGFII and also have higher levels of high-density lipoprotein than their non-IVF counterparts. To discover differentially methylated genes between the IVF and control cohorts immunoprecipitation with an anti-methylcytosine antibody (meDIP) was used to pull down methylated DNA. This was compared to total DNA using the Aviva Systems Biology directed selection and ligation (DSL) technology on 20K promoter arrays.

Currently several differentially methylated genes have been identified using this approach and are undergoing validation using Sequenom.