This study suggests that the HTR2A
gene may be up-regulated in CFS through allele-specific expression. An integrated analysis of genetics, methylation, gene expression and clinical measurements using SEM reveal that this up-regulation may be dependent on the cis-regulatory transcription factors E47, GR and Sp1. We previously showed that the minor allele A of the promoter polymorphism rs6311 (−1438G/A) in HTR2A
was more common in CFS than NF subjects (Smith et al. 2008
). This SNP, which has also been associated with other complex disorders including depression, PTSD and schizophrenia, results in the creation of a binding site for E47 (Smith et al. 2008
) and also results in the loss of CpG methylation site at −1,439. Our current study examined HTR2A
methylation in subjects from a population-based clinical study of CFS and identified two CpG sites, −1,224 and −1,420 that showed differential methylation between CFS and NF subjects and dependence on sequence variation at position −1,438. We recently demonstrated the first experimental evidence for the binding of GR at CpG site −1,420 (Falkenberg and Rajeevan 2010
), whereas binding of Sp1 at CpG site −1,224 and the genotype-dependent binding of E47 at −1,438 were reported earlier (Smith et al. 2008
; Zhu et al. 1995
). Changes at these cis-regulatory elements, two of which are potentially heritable (methylation at −1,439 and −1,420), may have contributed to increased expression of A-allele and to the overall up-regulation of HTR2A
While the specific mechanism for the association of allele-specific expression with CFS is not known, we present qualitative (Fig. ) and quantitative (Fig. ) models that may account for the potential influence of E47, GR and Sp1 on HTR2A
expression. As a result of reduced cortisol production in some CFS subjects (Heim et al. 2000
), GR binding would also be reduced. This state of hypocortisolism is included in the model as leading to inhibition of GR-mediated transcriptional repression (Sorensen et al. 2009
). The qualitative model suggests that in situations of high methylation at −1,420 and low GR activity as in CFS subjects, the A-allele is over-expressed in relation to the G-allele. On the other hand, when methylation at −1,420 is low and GR activity is high as in NF subjects, the G-allele is over-expressed. The joint contributions of transcription factor activation and promoter methylation in the context of sequence variation may explain the lack of consensus between previous investigations that used only isolated functional studies to examine the role of the rs6311 promoter polymorphism in the regulation of HTR2A
(Myers et al. 2007
; Norton and Owen 2005
; Parsons et al. 2004
; Polesskaya et al. 2006
). These joint contributions of several regulatory factors may also explain the lack of simple direct correlations between methylation and expression levels or instances of deviations from simple genetic models as revealed by some results (Fig. a) in this study.
Quantitative analysis by SEM supports two significant aspects of the qualitative model of HTR2A transcriptional regulation. First, the most important contribution of GRE to the transcription of HTR2A is indirect, mediated through competition with E47 (direct pathway coefficient is only −0.054, whereas GRE to the E47 pathway coefficient is 0.49). Second, there is a weaker but suggestive interaction between Sp1 binding and HTR2A expression (although the path coefficient of 0.28 is not significant, this study is underpowered for determining coefficients in this range).
Although the model posits that GR binding to −1,420 would be important for the inhibition of E47 binding, only a small amount of the variance in the 24-h urinary free cortisol contributes to the model (about 10%, vs. the unaccounted for variance of 90%). However, compared to methylation at −1,420, variance in cortisol contributes a relatively substantial amount to the latent variable GRE, (0.32 vs. 0.93, or about 25% of the total). In addition, excluding cortisol from the model drives the model (Chi-squared) P value down to 0.12 and the P value of the RMSEA up to 0.15, both indicating a worse fit. This indirect contribution of GRE to transcriptional regulation also raises the possibility that other GR family members with different kinetics and tissue distributions, such as mineralocorticoid receptor, may be more important for regulating HTR2A expression. As illustrated by the use of SEM in this study to provide a quantitative evaluation of the interaction of different TFBS on HTR2A allele-specific expression, SEM is an additional computational tool for analyzing complex transcriptional regulation paradigms.
The significances of our observed association of methylation levels between CpG sites at −1,439 and −1,420 in CFS but not in NF subjects are presently unknown. It is not clear how commonly an association of methylation between sites occurs, nor is it clear what accounts for a disease-specific association. Correlation of methylation between adjacent CpG sites in the distal region of the serotonin transporter promoter has been reported (Philibert et al. 2008
), so further investigation into this phenomenon is clearly needed. Further investigation is also needed to assess the impact of other SNPs in this region on HTR2A
There are several limitations to this study. Since we enforced strict quality control on CpG site-specific methylation analysis, information on methylation levels of all 17 CpG sites on all 185 subjects was not available. This contributed to small sample sizes in certain stratified analyses of genotype-dependent methylation levels. On the other hand, since methylation changes being close to the action of genome and a quantifiable molecular phenotype, it is likely that polymorphic CpG methylation at −1,439 may exhibit higher effect size than complex disease phenotypes that often involve several biological pathways. As expected, the nearly perfect concordance (r2 = 0.99, P = 0.0261–0.0392) between methylation levels at −1,439 and −1,438 genotypes (Fig. bottom panel) suggests that our conclusions are unlikely to be influenced by sample size. With respect to the allele-specific expression, the data presented included all 22 CFS and NF subjects heterozygous for the −1,438 G/A promoter polymorphisms. As required by the case definition, this analysis (Fig. c) excluded those with exclusionary medical/psychiatric conditions. However, we examined this finding in a larger group of individuals (n = 35) by including subjects who reported exclusionary medical and psychiatric conditions but otherwise met criteria for CFS and NF in this study. The results with the larger group of subjects (data not shown) support the original finding (Fig. c) of allele-specific expression of HTR2A in PBMCs that differ with the fatigue status of the subjects. We recognize that this allele-specific expression finding, although statistically significant, should be considered as suggestive because of the relatively small sample size. Future studies are needed to replicate this finding.
Analytically, the close fit between the SEM model and the data does not mean that the model shown is the one true model, only that it is statistically consistent with the data. The better P values for this model, given what is typical for SEM in the literature, may be reflecting relative paucity of the data and simplicity of the model instead of overfitting of the data. On the other hand, the small sample size likely contributed to some type II errors in path identification, e.g. the study is underpowered to identify the link between Sp1 and HTR2A if indeed the path coefficient is in the range shown. Also, the average 24-h urinary free cortisol used in the SEM is not an ideal measurement for modeling the contribution of cortisol to HTR2A gene expression in PBMCs.
Potential influences of E47, GR and Sp1 binding sites were considered in our qualitative and quantitative models to provide a mechanistic understanding of the allele-specific expression. In silico computational analyses and competitive EMSA provide evidence for direct binding of transcription factors E47, GR and Sp1 in the HTR2A promoter. While EMSA is an accepted method to validate the possibility of functional binding, in vivo binding assays such as chromatin immunoprecipitation (Chip) should be considered in future studies. We believe that evidence for direct binding to specific sites is sufficient for modeling and integrating genetic and epigenetic risk in complex traits.
As depression is a recognized comorbid condition in CFS, the specificity of this model for CFS versus depression will require further exploration as serotonin signaling is recognized to be important in depressive disorders. To address the contribution of depression, we tested the association of methylation at each site by stratifying this study population based on Zung depression scores and did not identify an association (data not shown). However, methylation at one of the CpG sites (−1,224) was negatively correlated (r = −0.4315, P = 0.001) with increasing Zung scores and thus a potential association between HTR2A methylation and depression cannot be totally excluded from this study.
Finally, it should be kept in mind that the expression findings were made in PBMCs. Methylation and gene expression are tissue-dependent, and HTR2A
expression in PBMCs may only be an indirect reflection of CFS pathogenesis. However, the model presented for HTR2A
promoter methylation and expression in PBMCs is relevant for understanding mechanisms that may be playing a role in HTR2A
expression in other tissues like the brain (Le-Niculescu et al. 2009
This study underscores the importance of the coordinated influence of both genetic and epigenetic variations in determining an individual’s susceptibility to disease, and the value of integrated functional and computational genomics for simultaneous identification and quantitative evaluation of multiple cis-regulatory elements in major genes of medical interest. To our knowledge, this is the first study to apply SEM to promoter analysis. Novel findings in this study include disease-specific correlations of methylation at adjacent CpG sites and disease-associated allele-specific expression of HTR2A that is influenced by both CpG methylation and genotype-dependent transcription factor binding. These regulatory mechanisms of HTR2A expression may play significant roles in the pathology of common complex diseases with central nervous system abnormalities such as CFS, depression and PTSD. Confirmation of these findings in an independent study population is required.