We performed a GWA study, with replication of the top hit and genome-wide significant association in the meta-analysis across a total of 4088 patients and 11001 controls, including one sample from a different ethnic background. Together with gene expression data, neuroimaging correlates and evidence from a mouse model of chronic stress our results point towards SLC6A15, a neuronal amino acid transporter, as a novel candidate gene in the pathophysiology of major depression.
Even though the direction of the association of rs1545843 with depression and depressive symptoms was the same in all samples with non-geriatric depression, the effect sizes were heterogeneous, with a much larger effect in the discovery sample (OR=2.8 for the recessive model) as compared to the other samples with odds ratios ranging from 1.18 to 1.61. The strong association and low p-value in the discovery sample is probably due to the “winners’ curse”, but this phenomenon has also been observed for other, now established, disease loci. For example, the association of a SNP in the FGFR2
gene with breast cancer was much stronger in the rather small discovery sample than in any of the subsequent replication samples. However, the direction of the association was consistent and reached a p-value of 2e-76 in close to 30,000 cases and controls (Easton et al., 2007
). This indicates that heterogeneous effect sizes with overestimation of the effect in a small discovery sample may still be in agreement with a true signal. In addition to the genome-wide significance (Dudbridge and Gusnanto, 2008
) observed in our study, replication of the effect in samples of different ethnicities, European and African American, might be a further indicator for a true effect.
In addition to replication in independent samples, the functional relevance of the associated locus is supported by results of gene expression analyses in pre-mortem human hippocampus and EBV-transformed lymphoblastoid cell lines of the HapMap individuals (Stranger et al., 2005
) and peripheral blood monocytes (Heinzen et al., 2008
). While there is a strong indication of the regulatory relevance of the region associated with MD for SLC6A15
expression, we cannot exclude that these variants might also influence the expression of six unspliced brain ESTs and four spliced ESTs of other than brain tissue which have been mapped to the region of association, since they were not probed by the used Illumina chip ( and supplementary text, tab. S1
). Additional non-annotated transcripts, as described in the ENCODE pilot project in regions of the genome previously thought to be transcriptionally silent (Birney et al., 2007
) might also be functionally relevant for this association.
The imaging genomics results provide evidence that the associated SNPs and related functional effects on SLC6A15 expression might be of relevance for the integrity of brain neurocircuits shown to be important in MD (Frodl et al., 2002
). We found lower total hippocampal volumes, particularly of the cornu ammonis, in risk genotype carriers of the patient- but not the control group, indicating a higher vulnerability to the well-documented effects of recurrent depressive episodes on hippocampal volume (Frodl et al., 2002
; Videbech and Ravnkilde, 2004
). Further support for the detrimental effects of the risk allele on neuronal integrity in this brain region came from 1
H-NMR spectroscopy. We noted that healthy risk allele carriers exhibited lower hippocampal NAA compared to non-risk allele carriers. Reduced hippocampal NAA has been reported for different psychiatric disorders and was also decreased in currently depressed unipolar patients in this study (fig. S4b
). In animal models, hippocampal NAA can be decreased by chronic stress (Czeh et al., 2001
; Li et al., 2008
). Thus, a genetic predisposition towards lower hippocampal NAA, similar to a condition induced by chronic stress experiments, may impair an individual’s resilience to stress which is a risk factor for MD (Wang, 2005
While the genetic association data pointed most strongly to rs1545843, gene expression and imaging data association were strong with both tag-SNPs of the locus, rs1545843 and rs1031681. In healthy subjects, genotype effects on hippocampal neurochemistry were more prominent for rs1031681 compared with rs1545843, both in terms of effects on NAA and Glx and in terms of robustness towards multiple test correction. This is an indication that both SNPs tag the likely underlying functional variants that still remains to be identified. To this aim deep-sequencing analyses are currently underway.
Together with the demonstrated downregulation of SLC6A15
expression in stress-susceptible mice, human gene expression and imaging data support a role for hippocampal SLC6A15
function in stress sensitivity and the pathophysiology of MD. This would be in line with a proposed role of the SLC6A15
transporters in neuronal metabolism and the provision of substrates for neurotransmitters, and specifically glutamate synthesis (Broer et al., 2006
). Thus, decreased hippocampal NAA and by extension glutamate neurotransmission (Benarroch, 2008
), related to genetic factors, may limit excitatory signalling capacity with secondary effects on stress response regulation and hippocampal function in general (Herman et al., 2003
In conclusion, the above presented results from human genetics, gene expression, volumetric imaging, spectroscopy, and a mouse model of chronic stress all support the notion that lower SLC6A15
expression, especially in the hippocampus, could increase an individual’s stress susceptibility by altering neuronal integrity and excitatory neurotransmission in this brain region. Recently, the prokaryotic leucine transporter homologue (LeuTaa
) of SLC6A15
has been crystallized from Aquifex aeolicus and was shown to bind tricyclic antidepressant drugs that can directly block leucine transport by closing the molecular gate for the substrate in a non-competitive manner (Zhou et al., 2007
). Due to the high degree of phylogenetic conservation of the antidepressant binding site, these drugs probably also bind to the human transporter. Because SLC6A15 appears amenable to drug targeting, our results may incite the discovery of a novel class of antidepressant drugs.