Despite significant progress in the quantitative genetic analysis of the risk to develop anxiety and depressive disorders, knowledge of the specific genes, genetic mechanisms and physiological intermediates underlying this risk remains limited. Several studies have reported associations between CRH-related genes and clinical anxiety or depression,2–4,33
as well as association between CRH genes and related phenotypes in macaques.34
Several analyses report gene versus environment interactions in which early childhood trauma interacts with CRHR1
genotypes to predict stress-related psychopathology later in life.3,5–8,35,36
However, these data do not address the intermediate neurobiological pathways that lead from inherited genetic differences to altered behavioral and physiological reactivity and ultimately to psychopathology. Due to fundamental similarities in the brain structure and behavioral reactivity to stress, rhesus monkey models are well suited for analysis of genetic effects that alter the function of neural circuits implicated in human psychopathology.37–39
In this study, we identified several common SNPs in the rhesus macaque CRHR1
gene that are independently associated with AT. Specifically, we find that SNP4805, a splice-site mutation that may affect the inclusion of exon 6 into the CRHR1 protein, and another variant (SNP5043), which alters the amino-acid sequence within exon 6, are associated with individual differences in AT. We also obtained support for association between another exon 6 variant (SNP5094) and AT. To understand how variation in the CRHR1
gene increases the behavioral and physiological reactivity to threat that is characteristic of individuals with high levels of AT, we examined brain metabolic activity in regions previously shown to be predictive of AT.17
The results indicate that multiple polymorphisms in the CRHR1
locus are significantly associated with individual variation in metabolic activity of the anterior hippocampus and central nucleus of the amygdala occurring during the human intruder challenge. We also find significant association between CRHR1
and metabolic activity in the IPS, with suggestive evidence for association with the precuneus.
We note that three SNPs that show significant effects on AT are predicted to impact exon 6 of the CRHR1
gene (). SNP4805 alters a splice site for intron 5, while both SNP5043 and SNP5094 change the amino-acid sequence encoded by exon 6. shows an alignment of partial CRHR1 protein sequences from various species, including several primates and non-primate mammals. The amino-acid sequence of the core of the protein (the segment containing the seven transmembrane domains) is overall highly conserved across all mammals ( and Supplementary Figure S2
). However, the segment of the protein encoded by exon 6 (part of the first intracellular loop) in anthropoid primates (human, chimpanzee, gorilla, macaque and marmoset (Callithrix jacchus
) is much less conserved in the strepsirrhine primates (Microcebus murinus
or mouse lemur and Otolemur garnettii
or bushbaby) and in non-primate mammals. It appears that human exon 6 evolved its current structure in the ancestor of living anthropoid primates (that is, living New World monkeys, Old World monkeys and apes) and is quite different from non-primate mammals.
Previous functional analysis indicates that the presence or absence of exon 6 has a significant influence on CRHR1 function. Markovic et al.40
demonstrated that CRHR1-alpha, the form of the protein that does not incorporate the amino acids encoded by exon 6 and which is the dominant isoform expressed in humans, responds differently to protein kinase-C induced phosphorylation compared with CRHR1-beta, which is identical to CRHR1-alpha except for the inclusion of those residues. As a result, CRHR1-beta exhibits substantially impaired signaling activity. Teli et al.41
identified specific amino-acid residues within the human exon 6 that account for impaired responsivity of this receptor. The relative levels of expression of CRHR1-alpha and -beta have not been documented in specific structures within the human or nonhuman primate brain (for example, amygdala and hippocampus). Nor have levels of CRHR1-beta been reported in patients with anxiety or mood disorders. The data presented here raise the possibility that genetic variation in the exon 6 splice site (SNP4805) may alter the relative proportion of CRHR1-beta as compared with CRHR1-alpha in individuals with high levels of anxiety and hippocampal or amygdalar metabolism. Additionally, amino-acid changes within exon 6 could alter the function of CRHR1-beta in CRHR1-beta-expressing tissues, leading to increased metabolism, and ultimately AT.
Our results indicate that polymorphisms involving exon 6 are associated with metabolism in the anterior hippocampus, amygdala, IPS and precuneus, as well as with AT. The recent demonstration29
that genetically engineered mice that do not express CRHR1 in the forebrain exhibit impaired propagation of neural signals within the hippocampus and amygdala and also display reduced anxious behavior, indicates that CRHR1 has a critical role in the expression of AT in response to challenge. The relationship between metabolism in the IPS and precuneus and correlated expression of AT is less clear. It is plausible that increased freezing (behavioral inhibition), a component of our composite measure of AT that results in reduced motor activity and locomotion, explains the reduced metabolic activity in these brain regions. However, it is also true that the IPS and precuneus are part of the default mode network,42
and alterations in the function of regions involved in the default mode may be relevant to AT.
When we extended our analysis to include other non-coding SNPs in CRHR1
, we found that variation in the 5′ flanking region of CRHR1
is also associated with both anterior hippocampal metabolism and AT. The effects of two putative promoter SNPs (SNP2107 and SNP0879) were statistically significant for both these phenotypes. Neither is in close LD with the exon 6 SNPs. This suggests a possible additional mechanism through which variation in the CRHR1
locus may affect AT, that is, differences in overall levels of gene expression. We note that results from the genetic analysis of complex phenotypes in humans suggest that it is common for a single gene to exhibit several different polymorphisms influencing a single phenotype.43–45
Much of the human CRHR1 genetic data involve gene versus environment interactions, especially interactions with early childhood trauma. Our data were obtained from juvenile monkeys that were normally reared and not exposed to extreme trauma. These findings suggest that children with specific CRHR1 genotypes may exhibit differences in brain activity that precede the expression of clinically significant anxiety and depressive disorders. The interaction between this CRHR1-driven genetic effect on altered early neural circuit activity on the one hand and subsequent child abuse, trauma or other serious adverse environmental events on the other may account for the reported gene-by-environment interaction. More specifically, the early effects of CRHR1 genetic variation that lead to increased activity of the neural circuit, which includes the hippocampus and amygdala, may be the diathesis through which childhood adversity further increases the risk to develop later anxiety and depressive disorders.