Using measures of relative allelic expression across multiple brain regions, within individual subjects, we have shown that the effects of cis
-variation on gene expression can significantly differ between sampled areas of the adult brain. Only by testing within individual subjects could we be certain that the genetic cis
-regulatory variants were constant between brain regions. For all five assayed genes, we observed significant regional differences in allelic expression in multiple subjects, suggesting this to be a common phenomenon. In the case of ZNF804A
, a robustly supported susceptibility gene for schizophrenia (11
), we observed a general regional pattern of allelic expression at the assayed SNP. Our data therefore extend previous observations of tissue specificity of allelic expression e.g. (4
) in showing that this can also differ between regions of the adult human brain.
All five assayed genes have been reported to show genetic association with neuropsychiatric illness (11
). Previous evidence exists for common genotypic effects on the expression of all of these genes (12
), with the exception of TCF4
[a susceptibility gene for schizophrenia (15
) which is mutated in Pitt–Hopkins syndrome (19
)]. We therefore biased our screen in favor of genes that would contain common (genetic) cis
-regulatory variants. However, for none of these genes was there any prior evidence that effects of cis
-regulatory variation could differ depending on brain region.
We have not attempted to relate our findings to DNA variants for which phenotypic associations have been reported (as exemplified in 21
) because our subject numbers are too small for meaningful statistical comparison between genotypes. The precise mechanisms underlying AEI of these genes, and its variation between brain regions, therefore remain open to speculation. AEI can have a genetic or epigenetic basis, the former explained by heterozygosity for DNA sequence variants affecting cis
-regulatory elements, and the latter by allele-specific modifications of DNA or chromatin. A plausible (genetic) explanation for our findings is that the effects of DNA variation in cis
-regulatory elements (which might include DNA variants associated with disease) vary depending on regional differences in the level of interacting trans
-regulators (e.g. transcription factors, hormones, microRNA). This explanation is consistent with in vitro
findings of cellular differences in the effect of DNA variants in transcription factor binding sites on gene expression (5
). An alternative (epigenetic) explanation is that the assayed genes are subject to an imprinting mechanism occurring in only a proportion of cells, which are then differentially sampled between brain regions. However, imprinting appears to affect only a small proportion of human genes (22
), and there is no previous evidence that any of the five genes assayed in this study are subject to such regulation. Recent evidence suggests that a significant proportion of human genes are subject to random epigenetic allele silencing that can persist in clonal cell lines (23
). However, this also appears an unlikely explanation for our findings, since the number of sampled cells from each region would presumably result in any purely random bias for either allele being canceled out. Within subjects, we observed a general consistency in which allele was over-expressed, again arguing against a purely random mechanism. Recent studies have indicated that common allelic differences in DNA methylation are usually associated with DNA sequence variation (24
). Moreover, there is evidence that such allele-specific methylation can differ between tissues (25
). Differences between brain regions in the extent of methylation at variable DNA sites may therefore provide an alternative explanation for our findings.
In the case of ZNF804A
, we observed a general consistency across subjects in which allele was relatively over-expressed, suggesting either that the assayed SNPs have direct effects on gene expression or (perhaps more likely) that they are in linkage disequilibrium with DNA variants of that effect. General AEI of ZNF804A
in human brain tissue has recently been reported at an exonic SNP with which ours is likely to be in perfect linkage disequilibrium (r2
= 1 in the HapMap CEU sample), and which is itself in strong linkage disequilibrium with the intronic SNP showing strongest association with schizophrenia (12
). It should be noted, however, that in a comparison between heterozygotes and homozygotes for the risk SNP, the authors found no difference in (cortical) allelic expression, leading to the conclusion that the risk SNP was not in itself responsible for AEI. In the present study, we have found that allelic expression of ZNF804A
is generally pronounced in some brain regions and very little in others. In contrast, in our assay of TCF4
, we observed similar levels of general AEI across all assayed brain regions. However, on the individual subject level, we found that, while there was directional AEI of TCF4
in all brain regions, the extent of this imbalance would frequently differ significantly (and reproducibly) between regions. Given that there appears to be little consistency in the region(s) showing pronounced AEI across subjects, it is possible that there are multiple regionally sensitive regulatory variants in TCF4
, leading to individual differences. Alternatively, there may be a single cis
-regulatory variant with differential effects on the many TCF4
transcripts known to exist, which are then differentially expressed/sampled between regions and subjects. Transcript-specific assays of allelic expression are likely to be informative for this and other genes assayed in the present study.
The human brain serves a wide range of functions, from physiological homeostasis to higher cognition. While multiple brain regions are typically recruited for any given function, it is clear that there exists a high degree of regional specialization. The effects of cis-regulatory variation are therefore likely to have distinct phenotypic consequences depending on where in the brain they are manifest. Our data show that such effects are often far more restricted than expression of the regulated gene per se.
Our demonstration of differential allelic expression across brain regions has important implications for studies of cis-regulatory variation in the brain. First, it highlights the caveat that it may not be possible to generalize findings to brain areas that have not been assayed. Second, it suggests that power to detect association between particular gene variants and gene expression may be improved by careful matching of tissue samples. Third, it shows that specific regions of the brain where the effects of cis-regulatory variants are manifest (or pronounced) can be delineated. Using such an approach, it should therefore be possible to define brain areas, and perhaps even cell populations, in which cis-regulatory variants conferring susceptibility to neuropsychiatric disease are active.