Allelespecific gene expression

11.5.1 Allele-specific gene expression among autosomal non-imprinted genes

Further important insights into the genetics of gene expression have been provided by the definition of allele-specific gene expression. This is seen in its most extreme form when expression is restricted to a single allele (monoallelic expression), as classically seen among imprinted genes (Box 11.5) where only one of the two copies of an autosomal gene is expressed, or in females where one X chromosome is inactivated (Plath et al. 2002). Among autosomal non-imprinted genes allele-specific expression was found, somewhat surprisingly at the outset of such studies, to occur relatively commonly and show evidence of heritability (Knight 2004). The magnitude of allele-specific expression is variable and often relatively modest, however the definition and analysis of genes showing such differential expression has provided powerful evidence to support the existence of c/s-regulatory variation.

By comparing relative allelic expression within a cellular sample, much of the potential heterogeneity inherent to comparison between individual samples can be avoided. Typically, individuals heterozygous for a given transcribed genetic variant are selected for analysis which allows the allelic origin of the transcript to be defined (Fig. 11.9; Box 11.6). In this case the exonic SNP is being used as a genetic marker to define allelic origin; it may be acting as a regulatory variant but more probably the

Box 11.5 Genomic imprinting

Imprinted genes show monoallelic expression in a parent-of-origin-specific manner. Genomic imprinting arises due to epigenetic marks involving methylation of DNA and modifications of histones. Imprinting is an example of an epigenetic process - heritable changes in gene expression that do not involve changes in the DNA sequence and are transmitted through cell divisions. Up to 1% of human genes are thought to be imprinted with 90 described to date (Luedi et al. 2007; Ideraabdullah et al. 2008). Imprinted genes are found across the genome but with a number of clusters identified, characterized by regulation through parent-specific epigenetic modifications of imprinting control regions. These regions have been shown to act, for example, as insulators and promoters for non-coding RNAs (Ideraabdullah et al. 2008). Imprinted genes have been shown to be important for normal development with the parental imprint set in the germline; often their expression is tissue- and developmental stage-specific. The consequences of dysregulation of imprinting through fine scale or structural genomic variation can be profound, with at least nine human imprinting syndromes described (Amor and Halliday 2008). These include the Prader-Willi and Angelman syndromes, most often arising through deletions of chromosome 5q11-q13. These lead to loss of function of the paternally imprinted gene SNRPN in Prader-Willi syndrome or the maternally imprinted gene UBE3A in Angelman syndrome (Section 5.2.6). A number of databases cataloguing imprinted genes are available online including the Imprinted Gene Catalogue (www.otago.ac.nz/IGC; Morison et al. 2001) and the genomic imprinting website maintained by the Jirtle lab at Duke University (http://www.geneimprint.com).

Figure 11.9 Allele-specific gene expression. Quantification of a transcribed SNP, here an exonic G to A nucleotide substitution, allows the allelic origin of the transcript to be defined with increased expression of allele 1 versus allele 2 shown. This allele-specific expression may be the result of a number of different possible regulatory variants, the exonic SNP is serving as a marker to distinguish allelic origin and is not itself necessarily functional. In this figure several genetic variants are shown including a GA dinucleotide repeat and a T insertion polymorphism. However in this example, the allele-specific expression results from a functional ris-acting regulatory variant, a T to C single nucleotide substitution which in the presence of the T substitution increases transcription from allele 1. The mechanism for this may involve allele-specific recruitment of a specific transcription factor in the presence of the T substitution. Redrawn and reprinted from Trends in Genetics (Knight 2004), copyright 2004, with permission from Elsevier .

Figure 11.9 Allele-specific gene expression. Quantification of a transcribed SNP, here an exonic G to A nucleotide substitution, allows the allelic origin of the transcript to be defined with increased expression of allele 1 versus allele 2 shown. This allele-specific expression may be the result of a number of different possible regulatory variants, the exonic SNP is serving as a marker to distinguish allelic origin and is not itself necessarily functional. In this figure several genetic variants are shown including a GA dinucleotide repeat and a T insertion polymorphism. However in this example, the allele-specific expression results from a functional ris-acting regulatory variant, a T to C single nucleotide substitution which in the presence of the T substitution increases transcription from allele 1. The mechanism for this may involve allele-specific recruitment of a specific transcription factor in the presence of the T substitution. Redrawn and reprinted from Trends in Genetics (Knight 2004), copyright 2004, with permission from Elsevier .

Box 11.6 Quantification of allele-specific gene expression

Singer-Sam and colleagues established the utility of allele-specific transcript quantification using transcribed marker SNPs for which the individual was heterozygous. Their methodology involved PCR amplification of a genomic region followed by a primer extension reaction, the primer being designed such that the 3' end was immediately adjacent to the polymorphic nucleotide. On primer extension, the nucleotide corresponding to the variant nucleotide would be incorporated and detected

(Singer-Sam et al. 1992b). The approach was successfully applied to mouse embryos in the analysis of parental imprinting (Singer-Sam et al. 1992a) and adapted in later studies with fluorescent-labelled dideoxynucleotides used for primer extension (Yan et al. 2002). Quantification of primer extension products by matrix-assisted laser desorption/ion-ization (MALDI) time-of-flight (TOF) mass spectrometry has also been used (Knight et al. 2003; Jurinke et al. 2005).

functional c/s-acting variant lies elsewhere on the same chromosome. A clear definition of the underlying genetic variation and haplotypic structure is therefore critical to dissecting the functional basis of observed allele-spe-cific expression. Further resolution can then be achieved through correlation of specific haplotypes and SNPs with allele-specific expression as demonstrated in lymphoblastoid cell lines using dense genotyping data available from the HapMap Project (Pastinen et al. 2005; Forton et al. 2007).

Given that the transcribed SNP is being used as an allelic marker to analyse individuals heterozygous for that SNP and not necessarily because of a high probability of it being a functional regulatory variant, it is perhaps not surprising that in many cases where allele-specific expression is observed it occurs in only a proportion of individuals analysed for that marker. This was seen in an important early study demonstrating the occurrence of allele-specific expression in human cells by Yan and colleagues (2002). Here lymphoblastoid cell lines established from 96 individuals in the CEPH families were analysed for potential allele-specific expression in 13 different genes, selecting cell lines where individuals were heterozygous for SNPs in those genes.

Allele-specific transcript abundance was quantified using a fluorescence-based dideoxy terminator method (Box 11.6) and found to occur commonly - in six out of 13 genes selected - with between 1.3- and 4.3-fold differences in expression between the alleles resolved. However, allele-specific differences were present in only

3-30% of individuals heterozygous for a given SNP. The availability of family pedigrees allowed the heritability of allele-specific differences in gene expression to be assessed. Clear evidence of coinheritance of allele-spe-cific expression with underlying haplotype as defined by microsatellite markers were seen for PKD2 (encoding polycystic kidney disease 2) and CAPNT0 (encoding calpain 10) (Fig. 11.10). The potential relevance of such data is apparent from studies showing that genetic variation at CAPNW is strongly associated with risk of type 2 diabetes, with allele-specific analysis of gene expression providing a functional approach to defining regulatory variants (Cox et al. 2004).

In primary human cells allele-specific differences in gene expression were also seen. Bray and colleagues analysed gene expression using RNA from post mortem brain tissue of 60 different individuals (Bray et al. 2003). Following PCR amplification, a primer extension-based technique allowed allele-specific quantification sensitive to detection of as low as a 20% difference in relative allelic expression. For the 15 genes analysed, allele-spe-cific expression greater than this level was seen in seven genes for at least one individual (Bray et al. 2003). Like Yan and colleagues, the allelic differences in expression varied between individuals heterozygous for a given SNP marker. Among the genes identified as showing allele-specific expression was DTNBP1 at chromosome 6p22.3 which encodes a neuronal protein, dystrobrevin binding protein 1. Genetic variation at this locus is significantly associated with schizophrenia from linkage analysis and

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