DISC1 mRNA expression is not influenced by common Cis-acting regulatory polymorphisms or imprinting.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 147B:1065– 1069 (2008) DISC1 mRNA Expression is Not Influenced by Common Cis-Acting Regulatory Polymorphisms or Imprinting J.B.G. Hayesmoore,1 N.J. Bray,1,2 M.J. Owen,1 and M.C. O’Donovan1* 1 Department of Psychological Medicine, School of Medicine, Cardiff University, Cardiff, UK Centre for the Cellular Basis of Behaviour, Department of Neuroscience, Institute of Psychiatry, King’s College London, London, UK 2 The hypothesis that genetic variation in Disrupted in Schizophrenia 1 (DISC1) influences risk of schizophrenia and other major psychiatric disorders is supported by a growing body of genetic association data and plausible functional biology. Risk of psychiatric disorder is not attributable to non-synonymous changes that alter the protein coding sequence of DISC1, although certain such variants possibly contribute to risk haplotypes. Thus, it is widely hypothesized that the risk variants at DISC1 influence its expression. As a complicating factor, it has also been recently proposed that DISC1 is subject to imprinting, a hypothesis that would profoundly influence the interpretation of current genetic studies. We have tested these two main hypotheses using allelic expression analysis. Of 148 human brain mRNA samples, 65 were informative for analysis. However, only a single sample showed evidence for unequal expression of paternal and maternal transcripts. Analysis of the proximal promoter region in that subject revealed the presence of a previously unknown duplication of the 22 nucleotides 168 to 147 relative to the transcription start site. However, the altered expression in that subject did not appear to be explained by this insertion. Our data robustly demonstrate that DISC1 is not imprinted in the adult human brain, and strongly suggest that reports of genetic association between DISC1 and psychiatric disorder are not explicable by sequence changes that alter mRNA abundance. ß 2008 Wiley-Liss, Inc. KEY WORDS: schizophrenia; bipolar disorder; allelic expression; regulatory polymorphism; imprinting Please cite this article as follows: Hayesmoore JBG, Bray NJ, Owen MJ, O’Donovan MC. 2008. DISC1 mRNA Expression Is Not Influenced by Common cis-Acting Regulatory Polymorphisms or Imprinting. Am J Med Genet Part B 147B:1065–1069. Grant sponsor: MRC (UK). *Correspondence to: M.C. O’Donovan, Department of Psychological Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK. E-mail: firstname.lastname@example.org Received 5 October 2007; Accepted 14 December 2007 DOI 10.1002/ajmg.b.30715 Published online 12 February 2008 in Wiley InterScience (www.interscience.wiley.com) ß 2008 Wiley-Liss, Inc. INTRODUCTION St Clair et al.  described a large Scottish family in which a balanced translocation t(1;11)(q42.1;q14.3) cosegregated with schizophrenia, bipolar disorder and other major mental illnesses. The translocation breakpoint on chromosome 1 was subsequently found to directly interrupt two novel genes, which were named Disrupted in Schizophrenia 1 and -2 (DISC1, DISC2) [Millar et al., 2000a]. Human DISC1 spans 414 kb of chromosome 1q42, includes 13 exons and encodes a full-length protein of 854 amino acids. The translocation interrupts the coding sequence, potentially resulting in a truncated protein (mutDISC1) that lacks the C-terminal 257 amino acids [Millar et al., 2000a; Ozeki et al., 2003]. DISC2 appears to specify a non-coding RNA that is transcribed from the antisense strand, and which may regulate DISC1 expression [Millar et al., 2000a]. The results of genetic linkage and association studies that have followed the Scottish family report suggest that DISC1 may operate more generally as a susceptibility gene for psychiatric illness. Chromosome 1q42 has been reported to show linkage to schizophrenia [Ekelund et al., 2001, 2004; Hwu et al., 2003], schizoaffective disorder [Hamshere et al., 2005], and bipolar disorder [Curtis et al., 2003; Macgregor et al., 2004]. A frameshift variant within DISC1 has been identified in a second family affected by schizophrenia [Sachs et al., 2005]. Initially postulated to be a rare highly penetrant mutation, this variant was subsequently reported in controls [Green et al., 2006] making this hypothesis unlikely. Evidence for genetic association between DISC1 SNPs and haplotypes and schizophrenia, schizoaffective disorder, and bipolar disorder has been reported in several independent populations [Hennah et al., 2003; Hodgkinson et al., 2004; Callicott et al., 2005; Cannon et al., 2005; Thomson et al., 2005; Chen et al., 2007; Qu et al., 2007] as have negative findings [Devon et al., 2001; Kockelkorn et al., 2004; Zhang et al., 2005], the latter unsurprising even for true susceptibility genes given the presumed genetic complexity of these disorders. It should also be noted that, across the diverse populations, not all of the associated haplotypes overlap, indeed when put together, they span the entire gene [Hennah et al., 2006]. This may reflect population differences in linkage disequilibrium structure, or more likely given the distance across which the signals span, the presence of multiple susceptibility variants. If variation in DISC1 confers susceptibility to mental illness in the general population, unless the mRNA influences cellular function directly, DISC1 must contain sequence variation that influences the structure and/or expression of DISC1 protein. A common non-synonymous base substitution, encoding Ser704Cys, has been reported to be associated with schizophrenia and to influence hippocampal structure and function [Callicott et al., 2005]. Two other non-synonymous polymorphisms in DISC1, encoding Arg264Gln and Phe607Leu, form part of haplotypes that have been reported to show altered representation in schizophrenia [Hennah et al., 2003; Cannon et al., 2005]. However, these and other associated haplotypes 1066 Hayesmoore et al. include non-coding variants that do not appear to tag polymorphism with obvious effects on DISC1 protein structure, raising the possibility that associations are mediated, at least in part, by sequence variation affecting DISC1 expression. The potential pathogenic importance of altered DISC1 expression is suggested by the observation that lymphoblastoid cell lines derived from family members carrying the t(1;11) translocation show 50% lower DISC1 expression than cell lines from family members without the translocation [Millar et al., 2005]. No truncated protein was detected in that study leading the authors to suggest that haploinsufficiency of DISC1 underlies increased susceptibility to psychiatric illness in the translocation cases. That DISC1 contains regulatory variation operating in general populations is suggested by a recent study in which a putative risk haplotype for bipolar disorder was associated with reduced DISC1 expression in lymphoblastoid cell lines [Maeda et al., 2006]. However, others have found no correlation between SNPs that showed association with schizophrenia and DISC1 mRNA abundance in human brain [Lipska et al., 2006]. Thus, the mechanism by which variation in DISC1 influences the function of the encoded protein and thereby susceptibility to mental disorders remains open to dispute. Assays based on measures of a gene’s absolute expression level can be influenced by a host of trans-acting factors—both genetic and environmental—that can impair their sensitivity to detect cis-acting genetic influences on expression and also have the potential to produce spurious results. An alternative method for investigating cis-acting regulatory influences involves comparing the relative level of each mRNA transcript in individuals who are heterozygous for an expressed polymorphism [Yan et al., 2002; Bray et al., 2003]. In heterozygous subjects, an expressed single nucleotide polymorphism (SNP) enables discrimination between mRNA molecules transcribed from each parental chromosome. In the absence of cis-acting variation affecting expression of the assayed transcript, each parental transcript will be equally expressed. In contrast, where an individual is also heterozygous for a cis-acting regulatory variant affecting expression of that transcript, the two transcripts will be expressed at different levels. This will occur regardless of where that cis-acting polymorphism is located relative to the gene, or whether that polymorphism is in linkage disequilibrium with the assayed SNP. By assaying a series of individuals, it is thus possible to indirectly screen a gene for the presence of cis-acting variation affecting expression of that gene in the selected tissue. Importantly, this ‘within-subjects’ approach allows detection of genuine cisacting effects, whilst controlling for the trans-acting factors that can confound measures of total expression between samples. This is because within an individual, factors that affect mRNA quality or quantity such as tissue state, use of medication, and population variation in trans-regulatory proteins, are expected to equally influence the mRNA transcribed from each chromosome [Bray et al., 2003]. In the present study, we have applied this method to DISC1 expression using a large number of human brain samples. In addition to looking for the influence of regulatory cis-acting polymorphism, our data also allowed us to investigate whether the gene is imprinted in humans, a possibility suggested by two recent studies of the murine genome [Luedi et al., 2005; Zhao et al., 2006]. This is important because if DISC1 is imprinted in human brain, interpreting the extant association data would have to be re-evaluated in the context of parent of origin effects. MATERIALS AND METHODS Samples Post-mortem brain tissue derived from frontal, parietal or temporal cortex of 148 unrelated individuals was obtain- ed from three reputable tissue sources (The MRC London Neurodegenerative Diseases Brain Bank, UK; The Stanley Medical Research Institute Brain Bank, USA; The Karolinska Institute, Sweden). Genomic DNA was extracted by standard phenol–chloroform procedures. Total RNA was extracted using the RNAwizTM isolation reagent (Ambion, Huntingdon, UK) and then treated with DNase. Reverse transcription was performed using random decamers and the RETROscriptTM kit (Ambion). Genotyping All PCR reactions were carried out by standard procedures using ‘Hot Star’ Taq polymerase (Qiagen, Crawley, UK). PCR primers were designed by use of the Primer3 program (sequences on request). Genotyping was performed by primer extension using the SNaPshot Multiplex Kit (Applied Biosystems, Warrington, UK). Allelic Expression Assay Genomic DNA from all subjects was initially genotyped to identify heterozygotes for expressed SNP rs3738401. cDNA samples from heterozygous subjects were then assayed twice, each time as two separate RT reactions, alongside the corresponding genomic DNA samples. Samples were PCRamplified using primers based on a single exonic sequence, capable of amplifying either cDNA or genomic DNA. RNA samples did not yield detectable levels of product in the absence of an RT step. The same analytic conditions were used for genomic DNA and cDNA so that we could use, for each assay, the average of the ratios observed from genomic DNA (representing a perfect 1:1 ratio of the two alleles) to correct allelic ratios obtained from cDNA for any inequalities in allelic representation specific to that assay [Hoogendoorn et al., 2000]. Allelic representation was measured by primer extension using SNaPshot chemistry (Applied Biosystems), as described previously [Bray et al., 2003]. Peak heights of allelespecific extended primers were determined using Genotyper version 2.5 software (Applied Biosystems). The ratio of cDNA peak heights, corrected using the average genomic ratio, was used to calculate relative expression of the two alleles in each individual sample. To confirm altered allelic expression, individuals showing expression of one allele at a level 20% or more than the other were assayed on four further occasions, again as two separate RT reactions, alongside genomic DNA. In line with previous studies [Yan et al., 2002; Bray et al., 2003], an allelic expression difference >20% for any individual sample was chosen as indicative of the influence of cis-acting polymorphism since measurements surpassing this threshold are generally reproducible and unlikely to reflect measurement error. Finally, where an average difference in allelic expression of >20% was maintained over the subsequent eight reactions, as an additional test, allelic expression was assessed at a second expressed SNP, rs821616, using identical methodology. Sequencing Genomic DNA from selected individuals was sequenced in both directions using BigDye chemistry (Applied Biosystems) according to manufacturer’s protocols. Prior to sequencing, amplicons with and without the duplicated sequence were isolated by agarose gel electrophoresis using standard protocols. RESULTS One hundred forty-eight individuals were genotyped for the expressed SNP rs3738401, which was used for the primary analysis of DISC1 relative allelic expression. This SNP, located Cis-Acting Influences on DISC1 Expression 1067 Fig. 1. The organization of the DISC1 gene and its four major mRNA transcripts, and the position of SNPs assayed in this study. L ¼ Long isoform, Lv ¼ Long variant isoform, S ¼ Short isoform, Es ¼ Extremely short isoform. Transcripts are based on reference sequences NM_018662, NM_001012957, NM_001012959, and NM_001012958, respectively. The Long variant transcript differs from the Long transcript in that it lacks the 30 terminal 66 bp of exon 11 as indicated by the white rectangle with black outline. Protein-coding portions of exons are represented by black rectangles, and 50 and 30 untranslated regions are represented by gray rectangles. Asterisks indicate the position of SNPs rs3738401 in exon 2, and rs821616 in exon 11, which were used for relative allelic expression analysis. in exon 2, and encoding Arg264Gln, is present in all known DISC1 transcripts (see Fig. 1). SNP rs3738401 is not predicted to be transcribed as part of DISC2. However, exon 2 of DISC1 has been found to be contained in low abundance hybrid transcripts formed through intergenic splicing of DISC1 and the adjacent gene translin-associated factor X (TSNAX) [Millar et al., 2000b]. Sixty-five individuals were heterozygous for rs3738401, and therefore informative for allelic expression analysis. Their mean age at death was 57 years (SD ¼ 19.69), 36 were male, and 29 were female. Analyses of cDNA ratios showed good reproducibility, with an average coefficient of variation (SD/mean) of 0.09. Only one of the 65 assayed individuals met the relative allelic expression threshold >20% at SNP rs3738401. The individual was a 57-year-old anonymous male with no known history of neurological or psychiatric disorder. For this individual, the average A/G ratio over eight replicate cDNA reactions was 0.66 (95% CI ¼ 0.60–0.72). Thus, in this individual, expression of the A-allele was reduced by 34% (using the G-allele as an arbitrary reference point), or expression of the G-allele increased by 51% (using the A-allele as an arbitrary reference point). Observed cDNA ratios from this individual were significantly different from ratios derived from genomic DNA (P ¼ 1.89 1013). We assessed relative allelic expression in this individual at a second exonic SNP, rs821616, for which this individual was also heterozygous. SNP rs821616 is located in exon 11 and is predicted to be contained in the long (L) and long-variant (Lv) DISC1 transcripts, but absent from the short (S) and extremely short (Es) DISC1 transcripts (see Fig. 1). SNP rs821616 is not predicted to be contained in the DISC2 transcript as defined by NCBI sequence GI 61806647. When assayed at this SNP, the average A/T ratio in cDNA from this individual was 0.63 (95% CI ¼ 0.52–0.73). This accords well with the ratio observed at rs3738401, and indicates that the identified cis-effect operates on the L and/or Lv DISC1 transcripts, although an effect on the S and Es transcripts cannot be excluded. To explore whether the large difference in allelic expression observed in the one individual can potentially be accounted for by changes in the proximal promoter region, we sequenced approximately 1 kb immediately upstream of the transcription start site for all major DISC1 transcripts in this individual and an additional three individuals that did not show altered allelic expression. A novel insertion polymorphism was identified, a duplication of the 22 nucleotides that are 168 to 147 relative to the DISC1 transcription start site (bases 675–696 in reference sequence AF222982). The duplication was inserted immediately 50 to and in tandem with the original sequence. The individual that showed consistent allelic expression imbalance was heterozygous for this duplication. However, after screening a further eight samples, two additional individuals that did not show allelic imbalance (A/G ratio ¼ 1.06 and 1.03) were also found to be heterozygous for this duplication, suggesting that it does not in itself alter DISC1 expression. The novel duplication includes a TG repeat that has previously been reported to be polymorphic [Devon et al., 2001]. In the present study, all chromosomes carrying the duplicated sequence carried one (TG)8 repeat unit and one (TG)10. In principle, it is possible that the altered allelic expression reflects differences in repeat size between the duplicated and non-duplicated chromosomes. However, this does not seem likely since the individual that showed consistent allelic imbalance carried a (TG)9 on the chromosome without an insertion, a repeat which in size is close to both those on the duplicated chromosome. Moreover, our data do not suggest the repeat is per se functional, since some individuals that showed no allelic expression imbalance were heterozygous for two alleles that differed to a greater extent in size (e.g., 9 and 14 repeats). Thus, our data suggest neither the insertion nor the TG repeat polymorphism influence DISC1 expression in brain. DISCUSSION Alteration of DISC1 expression is a potential mechanism by which DISC1 variants increase risk for psychiatric illness. The present study sought to test this hypothesis by applying a powerful assay of relative allelic expression to a large number of human brain samples. At the same time our data allowed us to seek evidence for the more recent hypothesis that DISC1 is imprinted, a hypothesis that if correct, would have important ramifications for interpreting the existing association findings. Out of 65 informative heterozygous brain samples, only one showed allelic expression differences, this being 50%, whilst the remainder showed approximately equal expression of both parental alleles. These data robustly demonstrate that DISC1 is not imprinted in the adult human brain, and that imprinting is therefore unlikely to be of relevance to the association findings to date. Our data also show that haplotypes that alter DISC1 expression are rare in our population, with a frequency of approximately 0.01. This frequency is much lower than that of most of the haplotypes that have been reported to be associated with psychiatric illness in Caucasian populations. Thus, while our study supports the existence in rare cases of cis-acting variation that influences DISC1 expression in adult brain, our study does not support the hypothesis that the relatively common haplotypes associated with schizophrenia and bipolar disorder tag functional variation that acts through this mechanism. It remains possible that our rare observation 1068 Hayesmoore et al. of allelic expression is relevant to one of the many rare haplotypes that have been reported to be associated with illness [e.g., see Thomson et al., 2005], but this hypothesis is difficult to test using inferred haplotypes from a single subject. Finally, as for all quantitative assays, we cannot exclude the possibility that more common variants exist but that their effects are below the threshold for detection. Most of what is currently known about the function of DISC1 relates to its role in brain development. DISC1 expression peaks during periods of active neurogenesis [Schurov et al., 2004], again at puberty [Schurov et al., 2004], and is expressed in some regions of the developing brain that do not express DISC1 in adulthood [Austin et al., 2004]. It remains possible that risk alleles or haplotypes exert effects on expression exclusively in a developmental context, but testing this hypothesis is currently impractical as it would require large numbers of fetal brain samples, each at different developmental time points. We should also stress that our study does not address quantitative post-transcriptional effects, for example in the abundance of the protein or its sub-cellular distribution. The latter may be of importance, one recent study reporting altered sub-cellular distribution of DISC1 in the orbitofrontal cortex of patients with schizophrenia [Sawamura et al., 2005]. These caveats aside, we conclude that most reports of genetic association between DISC1 and psychiatric disorder are neither explicable by sequence changes that alter protein structure nor, as we have shown, changes that alter mRNA abundance. 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