Analysis of genomic imprinting by quantitative allele-specific expression by pyrosequencing

Genomic imprinting is a phenomenon in which gene expression is restricted to the allele inherited from only one parent. It has been described from flowering plants and eutherian mammals and may have evolved due to parental conflicts over resource allocation: accordingly, imprinted genes are responsible for ensuring correct rates of embryo development, and for preventing parthenogenesis. The molecular basis of imprinting depends upon the presence of differential epigenetic marks on the alleles inherited from each parent although in plants the exact mechanisms that control imprinting are still unclear in many cases. Recent studies have identified large numbers of imprinted genes from Arabidopsis thaliana and other plants (see Claudia Köhler chapter in this volume) providing the tools for more thorough investigation into how the imprinted gene network (IGN) is regulated. Analysis of genomic imprinting in animals has revealed important information on how the IGN is regulated during development, which in many cases is related to the existence of intermediate levels of imprinting. In some instances, small but significant changes in the degree of parental bias in gene expression have been linked to developmental traits, livestock phenotypes and even human disease. As some of the imprinted genes recently reported from plants show partial rather than complete imprinting, there is a clear need for tools that can quantify the degree of allelic bias occurring at a transcribed gene. In this chapter, we will describe the use of Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) as a tool suitable for this challenge. We describe in detail the factors which ensure that a pyrosequencing assay will be suitable for giving robust QUASEP robust and the problems which may be encountered during the study of imprinted genes by pyrosequencing, with particular reference to our work in A. thaliana and in cattle. We also discuss some considerations with respect to the statistical analysis of the resulting data. Finally, we provide a brief overview of the future possibility of adapting pyrosequencing for analysing other aspects of imprinting including the analysis of methylated regions.