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Dysregulation in the stress response of the hypothalamic–pituitary–adrenal axis, involving the corticotrophin-releasing hormone and its main receptor (CRHR1), is considered to play a major role in depression and suicidal behavior. To comprehensively map the genetic variation in CRHR1 in relation to suicidality and depression, as a follow-up to our initial report on SNP rs4792887, we analyzed six new single nucleotide polymorphisms (SNPs), in an extended sample of family trios (n = 672) with suicide attempter offspring, by using family-based association tests. The minor T-allele of exonic SNP rs12936511, not previously studied in the context of psychiatric disorders and suicidal behaviors, was significantly transmitted to suicidal males with increased Beck Depression Inventory (BDI) scores (n = 347; P = 0.0028). We found additional evidence of association and linkage with increased BDI scores among suicidal males with an additional SNP, located proximally to the index SNP rs4792887, as well as with two distal SNPs, which were correlated with index SNP rs4792887. Analysis of haplotypes showed that each of the risk alleles segregated onto three separate haplotypes, whereas a fourth ‘nonrisk’ haplotype (‘CGC’) contained none of the risk alleles and was preferentially transmitted to suicidal males with lowered BDI scores (P = 0.0007). The BDI scores among all suicidal males, who carried a homozygous combination of any of the three risk haplotypes (non-CGC/non-CGC; n = 160), were significantly increased (P = 0.000089) compared with suicidal male CGC carriers (n = 181). Thus, while the characteristics of the suicide female attempters remained undetermined, the male suicidal offspring had increased depression intensity related to main genetic effects by exonic SNP rs12936511 and homozygous non-CGC haplotypes.
Suicide is one of the leading causes of death among young and middle-aged men. According to the World Health Organization (WHO), one million people take their own lives each year in the world and at least 10 times as many attempt suicide (Wasserman 2001). Twin, family and adoption studies have shown the involvement of genetic components in suicidality, with heritability estimates in the range of 17–55% (Brent & Mann 2005; Voracek & Loibl 2007). The complexity of suicidality is summarized in a stress-diathesis model, which describes an accumulation of exposures to environmental risk factors as well as genetic predispositions (Mann 2003; Wasserman 2001). Up to date, a number of genetic variants have been studied and implicated as having roles in suicidality, with main focus on the serotonergic system (Bondy et al. 2006; Rujescu et al. 2007).
Dysfunction of the stress-responsive hypothalamic–pituitary–adrenal (HPA) axis is a common feature in anxiety and mood disorders (reviewed in Bale & Vale 2004; Hauger et al. 2006; Nemeroff & Vale 2005; Swaab et al. 2005) as well as in suicidality (Mann & Currier 2007). Furthermore, dysregulation of the HPA axis is the most potent biological marker presently available for predicting suicide among depressed individuals in combination with markers of serotonergic activity (Coryell & Schlesser 2007; Mann et al. 2006). Activation of the HPA axis is controlled and regulated by hypothalamic corticotrophin-releasing hormone (CRH), which activates CRH receptor 1 (CRHR1) in the anterior pituitary, to mediate the production of adrenocorticotrophic hormone, involving, for example transcription factor TBX19 (Lamolet et al. 2001; Liu et al. 2001), in turn promoting the synthesis and release of cortisol from the adrenal gland. The rise of cortisol levels in blood is normally regulated by multipoint feedback loop, at both HPA and also limbic, brainstem and prefrontal brain areas. Furthermore, extrahypothalamic effects are mediated directly through CRHR1 at the central amygdala, affecting, for example the serotonin system. Dysregulations in this system are highly relevant for a variety of psychopathologies (e.g. depression, anxiety and impulsive aggression) commonly found among suicidal individuals.
We previously identified a single nucleotide polymorphism (SNP) in the CRHR1 gene, rs4782887, which showed linkage and association in a subgroup of suicide attempters exposed to low–medium levels of stressful life events (SLEs), among whom most of the males were depressed (Wasserman et al. 2008). Furthermore, a relationship was also shown between neurotic personality traits, suicidality and the genetic variation in the HPA regulatory TBX19 gene (Wasserman et al. 2006a). In the present study, we continued the investigation of the CRHR1 gene with six new SNPs, covering approximately 80% of the genetic variation, in an expanded sample of 672 nuclear family trios with suicide attempter offspring. The objective was to relate the majority of the known genetic variation in the CRHR1 gene with the depression intensity among suicide attempter offspring, taking into account the previously identified subgroup with low–medium levels of SLE exposure (‘SLE1–3’). As discussed previously (Wasserman et al. 2006b, 2008), individuals in such a low-predisposing subgroup of SLE exposure may display increases in genetic effects of certain risk alleles (in contrast to individuals with high levels of SLE exposure in which case the environment may assume dominant effects over certain risk alleles) similar to observations by others (Hansson et al. 2006; Kendler et al. 2005). Because there are differences in suicidal behavior, as well as in the occurrence of depression, between males and females (Wasserman 2001, 2006), the effects were additionally studied in relation to gender.
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The present study is a comprehensive mapping of the genetic variation in the CRHR1 gene, following our initial finding with index SNP rs4792887 in the SLE1–3 subgroup (Wasserman et al. 2008), now further indicating a main genetic effect on depression intensity among suicide attempter males by a new, exonic SNP rs12936511 and homozygous non-CGC haplotypes. The three risk SNPs (rs4792887, rs110402 and rs12936511) indicated here with depression among suicidal males are located within close physical range in the CRHR1 gene, being evenly distributed within a span of 7.4 kb. The SNPs are not well correlated (low r2; Fig. 1), but all three SNP are in LD by Lewontin’s D′ (complete LD, D′ = 1). This is partly because of the greater sensitivity of the r2 measure to differences in allele frequencies and has been similarly observed elsewhere (Long et al. 2004; Tiret et al. 2002). Different changes in the CRHR1 gene may result in similar phenotypic effects (i.e. genetic heterogeneity, as with the prototypic example of the phenylalanine hydroxylase gene) (Scriver 2007). The segregation of the three risk alleles onto separate haplotypes (and their low, pairwise r2 values), but in relationship to a similar phenotype, suggests that there may be more than one causal loci also in CRHR1. Haplotype analysis proved useful for identifying an overall effect of the absence of any of the three risk alleles, risk alleles that each one had less significant results by themself (covering less affected individuals).
Whereas rs4792887 and rs110402 may not likely be functional determinants per se (but are rather in LD with such loci), we speculate that exonic rs12936511 may affect the CRHR1 gene at, for example the level of alternative splicing regulation. While this exonic SNP does not alter the amino acid sequence, our in silico analysis showed that it is located in a putative exonic splicing enhancer and that the presence of the T-allele destroys potential binding of SR protein SRp55, while also altering the binding efficiency of SC35. This is not without relevance here because alternative splicing of CRHR1-messenger RNA is occurring in this region (Fig. 1). Besides affecting splicing, single synonymous SNPs have capacity to have profound effects on a gene by alternative mechanisms (Parmley & Hurst 2007). It would be interesting to determine any possible functional effects linked to the identified SNPs in an experimental setting. Until the causal variants have been identified in full, the use of SNP haplotypes is likely to be the best alternative. The major C-allele of rs12936511 was indicated in the SLE1–3 subgroup and the opposite, minor T-allele with male suicidal depression. We speculate that this dual result may reflect, for example the complexity of splicing regulation, epistasis with other polymorphisms or some form of selective pressure at this SNP. The newly collected trios (n = 178) had more than fourfold less families with heterozygous parents (i.e. TDT informative families) compared with the initially investigated sample (Wasserman et al. 2008), which was insufficient for confirmatory replication of the previous findings with (index) SNP rs4792887 (yielding a low statistical power). All TDT analyses with SNP rs4792887 in the new subsample were nonsignificant with neutral tendencies (data not shown).
Congruent with these results presented and discussed here is the observation that many depressed individuals who committed suicide were also nonresponsive to the dexamethasone suppression test, a biological measure of reduced HPA axis feedback sensitivity (Coryell & Schlesser 2007; Mann et al. 2006). Whereas we are presently, to our knowledge, alone on focusing the study of genetic variation in CRHR1 in relation to suicidality and depression in suicidal males, others have performed studies in relation to depression alone (Bradley et al. 2008; Licinio et al. 2004; Liu et al. 2006, 2007; Papiol et al. 2007). Bradley et al. reported results with SNPs in the similar 5′ region of the CRHR1 gene, as was implicated here, with overlap with two of the SNPs reported here (rs4792887 and rs110402) (Bradley et al. 2008). Among other results, it was shown that the T- and G-alleles were linked with increased depression (Bradley et al. 2008), whereas our results show depression being linked with the T- and A-alleles, of SNPs rs4792287 and rs110402, respectively. Our results were among suicidal males with mainly low–medium SLE scores, whereas Bradley et al. did not investigate the suicidality parameter, having their main findings among predominantly females, which may in part explain the differences between us and Bradley et al. concerning the results with the A-allele of rs110402. Papiol et al. showed association with the A-allele of rs110402 with age of onset and seasonal pattern of major depression (Papiol et al. 2007). This was the opposite allele compared with Bradley et al., but the same allele as presented here with suicidal males. The finding of Papiol et al. with the A-allele was made among nonsuicidal females (Papiol et al. 2007), which is congruent with our results of no relation of the A-allele with depression among suicidal females. This may suggest that the A-allele of rs110402 is indicative of increased risk of depression in nonsuicidal females and according to our results related with suicidal depression among the males.
By performing a randomized, placebo and double-blind trial, Licinio et al. observed that those depressed and highly anxious Mexican–Americans, who were homozygous carriers of a GAG haplotype, had an increased response to an 8-week period of antidepressant treatment (Licinio et al. 2004). These results were later replicated in a population of Han Chinese patients (Liu et al. 2007). Interestingly, Licinio et al. reported no genetic association with presence of depression per se (Licinio et al. 2004). Nevertheless, the GAG haplotype, containing the major A-allele of rs242939 (underlined), was indicated with better treatment response (i.e. major allele = nonrisk allele) and is correlated with our index SNP rs4792287 (Fig. 1). The opposite minor alleles of these SNPs were linked with risk for depression by us and Bradley et al. (i.e. minor alleles = risk alleles). Thus, the results of Licinio et al. are congruent with ours and Bradley et al. describing the relationship between minor/major and risk/nonrisk alleles. We speculate that males who carry the minor risk alleles may not only be less susceptible to pharmacological treatment (Licinio et al. 2004) but perhaps also be more at risk for suicidality in relation to depression, as has been observed among inpatients with failed treatment (Mann & Currier 2007). Liu et al. investigated the same three SNPs as Licinio et al. and found that SNP rs242939 (Fig. 1) had a doubling in the frequency of the minor allele (from 0.07 to 0.14), among Han Chinese patients with major depression (Liu et al. 2006). Similarly with the results of Licinio et al., the GAG haplotype was not found to be associated with depression per se (Licinio et al. 2004; Liu et al. 2006). In summary, the results with rs4792887 and rs110402 can be viewed as partly congruent across the studies, whereas the exonic SNP rs12936511, shown here to be linked with suicidal male depression, has not been studied previously by others. Because our findings with rs4792887 were made in relation to a BDI cutoff of 9 in the SLE1–3 subgroup, whereas the finding with exonic SNP rs12936511 was resolved in relation to a cutoff of 17 among all males, the latter SNP may be regarded as being more strongly implicated with clinically relevant increases in depression intensity among the suicidal males.
The apparent sexual dimorphism showed in the analyses here may be caused by either genetic/biological or phenotypic heterogeneities or both. This former is not unlikely because the stress response of the HPA axis is known to act in a sexually dimorphic manner on, for example mood and anxiety, particularly during certain times of the reproductive life cycle (Leibenluft 1999; Rhodes & Rubin 1999). The latter may be a reflection of the fact that the phenotype of suicidal behavior also displays a sexual dimorphism (Wasserman 2006), reflecting that the male attempters are more likely to end up as suicide completers, while the females are more likely to remain as suicide attempters. We speculate that the gender differences observed here may indeed be caused by a combination such as genetic and phenotypic differences. The results and conclusions presented here for suicidal males and depression can thus be viewed in light of the likelihood that a higher proportion of the depressed males may die by suicide, compared with depressed females, among the general population (Angst et al. 2002; Wasserman 2006).
We have identified genetic variants in the CRHR1 gene, which may be of importance in the prediction and treatment of depressed males, at risk of suicidal behavior. Identification of genetic factors affecting the HPA axis, such as reported here, may further help identify the fraction of individuals that are suicidal, from the many nonsuicidal and depressed individuals in the general population (Wasserman 2001, 2006). The results motivate continued exploration of the genetics of HPA axis and suicidal behavior, as it may present new possibilities for improving the needed specificity of biological predictors, as well as to identify novel therapeutic approaches (Mann & Currier 2007).