4 D2S336 i D2S338 3 D2S345 A Calpain-iO

4 D2S336 i D2S338 3 D2S345 A Calpain-iO

Figure 11.10 Allele-specific expression for PKD2and CAPN10. Two CEPH family pedigrees are shown together with relative allelic expression for lymphoblastoid cell lines established from individuals heterozygous for a marker SNP. Allele-specific differences were seen for alleles bearing particular haplotypes of PKD2 (2,2,1,G) and CAPN10 (1,1,1,G). From Yan et al. (2002), reprinted with permission from AAAS.

association studies (Straub et al. 1995; Allen et al. 2008). The specific functional variants remain elusive but it is notable that expression of DTNBP1 is reduced in some areas of the brain in patents with schizophrenia (Talbot et al. 2004; Weickert et al. 2004) and expression of specific haplotypes of DTNBP1 shows some correlation with risk or protective alleles, being reduced or increased, respectively (Bray et al. 2005; Williams et al. 2005a).

Further important insights into allele-specific gene expression were provided by the analysis of inbred mouse strains (Cowles et al. 2002). Here analysis of gene expression for 69 genes using transcribed SNP markers was carried out in F1 hybrid mice resulting from crossing parental strains homozygous for the markers. Allele-specific gene expression in three different tissues was determined using a quantitative assay based on primer extension with a threshold for reliable detection of allelic imbalance set at a 1.5-fold difference in expression between alleles. Six per cent of genes showed consistent allele-specific differences, confirmed by resequencing to identify additional linked transcribed markers for use in allelic discrimination. The analysis demonstrated that allele-specific effects may be tissue-specific, allelic differences in expression for example being found for Ccnf (encoding cyclin F) and Hmgcr (encoding HMG coenzyme A reductase) in liver only while for Uros (encoding uroporphyrinogen III synthase) differences were seen in liver, spleen, and brain tissue (Cowles et al. 2002).

11.5.2 Large scale analysis of allele-specific gene expression

Analysis of allele-specific gene expression provides a complimentary and informative approach to investigate the role of genetic variation in gene expression. Could the approach be applied at a genome-wide level to define the extent of allele-specific expression and specific gene loci, which would be candidates for identification of cis-regulatory variants and compliment the rapidly growing datasets of genome-wide association with disease? A number of studies have addressed this question by using array-based technologies to interrogate allele-specific expression in human samples including lymphoblastoid cell lines (Bjornsson et al. 2008; Serre et al. 2008), fetal tissues (Lo et al. 2003), and peripheral blood cells (Pant et al. 2006). These studies indicate allele-specific expression is common, involving approximately 20% of genes, with both monoallelic expression consistent with genomic imprinting and more modest differences in expression, which in more than half of cases shows variation among individuals informative for a given marker SNP. This is consistent with the need for further analysis to resolve genetic and epigenetic effects, including the specific cis-acting regulatory variant(s) responsible for allele-specific expression, which may be present on only a proportion of chromosomes bearing the marker SNP.

In reviewing these studies in more detail it is worth considering again yeast as a powerful model organism for studying the genetics of gene expression. Kruglyak and colleagues sought to build on and compliment their earlier linkage analysis of haploid segregants by investigating diploid hybrids of the same two parental yeast strains, RM and BY (Section 11.3.3) (Brem et al. 2002; Yvert et al. 2003). In particular they utilized an oligonucleotide array to allow simultaneous genotyping and allele-specific expression, which proved informative but had a high false positive rate for the detection of allele-specific expression. A total of 692 unique open reading frames (ORFs) were interrogated with 1049 polymorphic marker probes and significant evidence of allele-specific expression found at 70 ORFs, of which 24 were likely false positives (Ronald et al. 2005). The RM-derived allele was underrepresented among these, being preferentially expressed in only 11 of 70 ORFs.

Given the power of genetic analysis in yeast it was possible to investigate whether these allele-specific effects in diploid hybrids corresponded to sites of linkage with gene expression among the haploid segregants from earlier studies. There was indeed significant enrichment, with ORFs showing allele-specific expression also showing evidence of local linkage and higher expression of the predicted allele: the allele associated with relatively higher expression on analysis of allele-specific expression in the diploid hybrid was associated with greater expression in the haploid segregant (Ronald et al. 2005).

Early evidence of the extent of allele-specific expression across the human genome was provided by Lo and colleagues who utilized an oligonucleotide genotyping array to investigate relative allelic expression for 1063 transcribed SNPs corresponding to 602 genes (Lo et al. 2003). They analysed gene expression for kidney and liver samples from seven different fetuses and were able to detect with confidence allelic differences in expression greater than two-fold in magnitude. A high proportion of genes analysed appeared to show allele-specific expression, 54% at greater than two-fold difference and 28% at greater than four-fold. For known imprinted genes, monoallelic expression was found in five out of six genes analysed. Variation between individuals was noted together with tissue specificity; genes showing allelic differences included known and novel clusters but were generally scattered across the genome. Overall the authors estimated between 20% and 50% of genes showed allele-specific expression.

Other studies suggest that the proportion of genes showing allele-specific expression may be closer to 20%. Pant and colleagues, for example, investigated gene expression for white blood cells from 12 unrelated individuals using oligonucleotide arrays interrogating 8406 exonic SNPs in 4012 genes (Pant et al. 2006). Of these genes, 1389 were expressed in white blood cells and 731 showed allele-specific expression greater than 1.5-fold in magnitude between alleles in at least one individual. Further analysis of 60 genes where allele-specific expression was found in at least three individuals revealed that 5% had monoallelic expression, confirmed as restricted to the maternal copy from experiments in lymphoblas-toid cell line pedigrees. In 54% of the remaining genes, all heterozygotes showed an allelic difference consistent with the c/s-regulatory variant being present among one of the pair of alleles assayed. Importantly, independent RNA preparations showed high correlation, there was good concordance between multiple informative SNPs at a given gene, and an independent methodology for quantification of allele-specific expression also showed good concordance. Overall the authors estimated that on average 22% of exonic SNPs assayed showed allele-specific expression for a given individual (Pant et al. 2006).

A similar proportion was found in a more recent study by Serre and colleagues who found that approximately 20% of genes showed allele-specific expression in a study of 643 expressed genes among 80 lymphoblas-toid cell lines established from individuals of European ancestry (Serre et al. 2008). The study utilized an allele-specific expression bead array technology based on primer extension together with quantitative sequencing of the products of RT-PCR reactions. The assay platforms were validated and found to be quantitative; allelic differences greater than 1.5-fold being detected with confidence above experimental noise. The study also showed that the allelic differences in expression were consistent when lymphoblastoid cell lines were repeatedly sampled after different numbers of passages of culture. Comparison with earlier data from Stranger and colleagues based on association of SNP markers with gene expression (Stranger et al. 2005) showed six out of 21

genes with strong evidence of c/s-regulatory effects also had significant evidence of allele-specific expression (Serre et al. 2008).

11.5.3 Allele-specific expression based on RNA polymerase loading

Analysis of allele-specific gene expression at a transcript level was dependent on finding a transcribed SNP marker to define allelic origin and hence relative allelic expression. Studying nascent pre-spliced mRNA expanded the number of informative SNPs to include intronic variants (Pastinen et al. 2004) although this approach appears limited to more highly expressed genes given the low proportion of unspliced mRNA to spliced transcripts (Serre et al. 2008). The number of haplotypes that could potentially be interrogated was increased further by approaches based on quantification of the relative allelic abundance of RNA polymerase II (Knight et al. 2003; Maynard et al. 2008).

RNA polymerase II binding had been shown to reflect levels of gene expression, notably when specific phosphor-ylated states of serine residues in the carboxy-terminal domain of the RNA polymerase were assayed (Weeks et al. 1993; O'Brien et al. 1994). The haplotype-specific chromatin immunoprecipitation (haploChIP) methodology was established to quantify allele-specific recruitment of RNA polymerase II (Box 11.7) (Knight et al. 2003). The assay involved immunoprecipitation of RNA polymerase crosslinked to DNA, with the abundance of DNA fragments quantified in an allele-specific manner following reversal of crosslinks. Application of the approach to lymphoblas-toid cell lines allowed resolution of a low producer haplo-type of LTA, encoding lymphotoxin alpha at chromosome 6p21.3. This haplotype was shown to be associated with allele-specific recruitment of the transcriptional repressor protein activated B cell factor 1 (ABF-1) in the presence of the A allele of a specific SNP in the 5' untranslated region (UTR) of the LTA gene (rs2239704) (Knight et al. 2004). Possession of this SNP has subsequently been associated with leprosy and malaria (Section 13.2.6).

The analysis of allele-specific RNA polymerase II loading has subsequently been analysed at a genome-wide level to advance our understanding of the nature and extent of allele-specific gene expression (Maynard et al. 2008). Microarray platforms have proved a powerful approach

Box 11.7 Chromatin immunoprecipitation

The chromatin immunoprecipitation (ChIP) technique allows protein-DNA interactions to be assayed in living cells or tissues. Proteins are crosslinked to DNA, typically by exposure of cells to formaldehyde. Chromatin is then extracted and sonicated, and subjected to immunoprecipitation with specific antibodies, for example to RNA polymerase II or specific histone modifications. The crosslinks between immunoprecipitated protein and DNA are reversed, and relative enrichment of

DNA fragments compared to input chromatin quantified. This is done either for specific genomic loci by quantitative PCR; or by amplification, labelling, and hybridization to microarray platforms to allow genome-wide analysis using the 'ChIP-on-chip' approach (Ren et al. 2000). Next generation, high throughput sequencing technologies (Box 1.24) will further advance application of ChIP for genome-wide mapping of protein-DNA interactions, dubbed 'ChIP-Seq' analysis (Jothi et al. 2008).

to analyse the products of ChIP experiments and enable genome-wide analysis of sites of protein-DNA interactions (Box 11.7). Maynard and colleagues extended this to analyse the products of ChIP for RNA polymerase II by hybridization to a SNP genotyping array and define allele-specific binding for heterozygous SNPs. Human lung fibroblasts were analysed using 317 513 SNPs, of which 119 821 were heterozygous in this cell line, and 11 028 showed enrichment relative to input DNA. Of these, 466 SNPs showed significant allele-specific enrichment corresponding to 239 genes including known imprinted loci such as SNRPN (Maynard et al. 2008). A similar approach based on the analysis of specific histone modifications demonstrated for lymphoblastoid cell lines from CEPH family pedigrees that allele-specific chromatin modifications occur and show familial aggregation (Kadota et al. 2007). The relative roles of genetic and epigenetic variation in determining gene expression is complex and incompletely understood, but further dissection of allele-specific effects at specific loci and genome-wide offers an opportunity to advance our understanding of this fundamentally important area.

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