Although the monoallelic mechanism of imprinting was first identified in 1984 [8
], quantitative variance in expression of the two different alleles was only first acknowledged in 2002, in a small study where 6 of only 13 genes investigated showed allelic differences [9
]. Since these early studies drew attention to possible cis
-regulatory effects causing ASE, additional individual loci were queried by PCR-based methods, such as real-time quantitative PCR or discrimination of PCR products by differing primer extension [10
]. However, in order to identify and characterize this variation on a more genome-wide level, PCR techniques were coupled with microarray technology. Initially performed by Lo et al
] using an early Affymetrix HuSNP array with approximately 1,000 exonic SNPs in 602 genes, a surprisingly high estimate of >50% of genes showed some ASE pattern and the majority of these were not known to be imprinted.
Thereafter, many further studies have used this approach dependent upon heterozygous SNPs residing within the gene's coding region and, subsequently, compared ratios of copy DNA (cDNA) from RNA to quantify differences [12
]. The direct measurement of both alleles within the same system removes the possible confounding influence of trans
-acting environmental factors. This can identify plausible imprinted genes, which familial studies can verify. However, they may also be developmental-, time- or tissue-specific [13
] or display ASE that will show co-segregation through a pedigree.
Two widely used commercially developed techniques for ASE analysis include the BeadArray platform and the Oligo Pool All (OPA) method (Illumina Inc.). In the BeadArray method, genomic and converted RNA are assessed for the ratio of each allele by primer extension assays with fluorescence-labeled allele-specific primers. The resolution allows a 1.5-fold ASE change to be detected robustly from experimental noise. This method was used by Serre et al
. to estimate that approximately 20% of human genes display ASE [14
]. The OPA method is based on the Golden Gate assay [15
]. By excluding any SNP within within 45 base pairs (bp) of the start or end of exons, in order to ensure that there was an equivalent chance of working between genomic DNA and converted DNA, this method was used to investigate the unrelated 210 individuals within the HapMap population [16
]. By exploring the interaction between non-synonymous SNPs and cis
-regulatory features, this study estimated ASE to be approximately 18%.
The major issues with these aforementioned techniques are threefold, and various adaptations have been developed to overcome them. Firstly, the influence of bias in PCR amplification in these ASE examinations has been acknowledged and a custom ASE array has been developed that removes this possible confounding factor [17
]. Secondly, issues of cross-hybridization were also reduced in the custom ASE array study by the use of longer probes (39 to 49 bp) and a new probe design. The use of shorter probes may have also contributed to a possible overestimation of ASE in earlier studies. Probes were designed for the mismatch base to have a balanced Tm
on either side, thus placing it at the most thermodynamically disruptive location. Thirdly, the necessity of a SNP to reside within the transcript limits the number of genes for which there are informative haplotypes. However, when multiple SNPs occur, robust results can be elucidated with consistent ratio differences, which is considerably aided if these SNPs are in strong linkage disequilibrium (LD) with each other. Verlaan et al
] have modified their method to investigate unspliced primary transcripts, thereby including the intronic regions and thus greatly increasing the number of polymorphisms that can be used to delineate allele calls. These results estimated that >10% of genes expressed in a lymphoblastoid cell line exhibited ASE. An alternative approach, independent of the transcription of coding region SNPs, is to use a marker of transcriptional activity (such as phosphorylated RNA polymerase II (Pol II)), as developed in the haploChIP method [19
]. By immunoprecipitation of phosphorylated Pol II cross-linked to chromatin, the relative DNA fragment amounts of these protein-DNA interactions are differentiated by the use of any SNP within the location of interest.