In the present study, we analyzed the candidate gene SLC1A1, which encodes the neuronal glutamate transporter, in a large collection of OCD probands and controls in light of increasing interest in glutamatergic neurotransmission in OCD development and its treatment. We identified an SLC1A1 haplotype that was strongly associated with OCD that consists of 3 markers between intron 2 and the 3′ untranslated region of SLC1A1, 2 of which we found to be SNPs that predict gene expression levels (ie, eQTLs) in lymphoblastoid cell lines. In addition, another eQTL polymorphism, located somewhat upstream from SLC1A1, was strongly correlated with an OCD subphenotype (hoarding), as evaluated by 2 validated scales. Our data corroborate the hypothesis of dysfunctional glutamatergic neurotransmission in OCD and hoarding, mediated in part through SLC1A1.
Three recently published, family-based association studies have suggested SLC1A1
to be a candidate gene for OCD.16-18
However, no given SNP within SLC1A1
in these 3 studies was consistently positively associated with OCD across the 3 studies when tested individually, with the exception of rs3780412, which was considered significant in 2 studies: the overall and males-only study by Dickel and coworkers17
and in the males-only sample by Stewart and colleagues.18
However, our results do not support a specific involvement of this marker in OCD, nor did we observe any preferential or stronger association of single variants or haplotypes in males compared with females in our case-control sample ( and ). Speculatively, this observation might be related to the increased power of our case-control design compared with the family-based studies published previously.16-18
Clearly, additional studies are warranted to clarify the discrepancy for preferential association in males observed in prior family-based studies,16-18
though the overall finding of involvement of SLC1A1
in OCD now appears more clearly replicable.
One of the most important common findings in these 3 previous SLC1A1
studies was a haplotypic association in the 3′ region of the gene.16-18
Our results are in line with this observation, as 2 of the 3 SNPs of the associated haplotype we identified are located in exon 10 and the 3′ untranslated region. The third SNP of this haplotype, however, lies somewhat upstream (intron 2) of this region, and although all 3 SNPs (rs7858819, rs301430, and rs3087879) were in considerable linkage disequilibrium with each other (), we speculatively interpret this observation as evidence for more than 1 functional locus within SLC1A1
, as has been suggested previously.17,18
Additional support for this tentative conclusion stems from another SNP, rs3933331, located farther upstream and clearly not in linkage disequilibrium with the previously mentioned haplotype markers that we found to be associated with gene expression functionality and that predicted an OCD hoarding subphenotype in our sample ( and ). The possibility of 2 or more functional (possibly causal) variants of differential effect size provides 1 plausible explanation for the molecular discrepancies between present and published SLC1A1
genotyping studies in OCD, especially if these were found to be interacting from within different haplotype blocks. This possibility might be addressed by genotyping markers at higher density or by resequencing large genomic regions.
We have made extensive use of publicly accessible data with the aim of better understanding 1 facet of SLC1A1
functionality—gene expression. Some of the most extensive gene expression data sets currently available were obtained from lymphoblastoid cell lines, and we used these data to better understand how genetic SLC1A1
variation might affect its gene expression levels. To this end, it was first necessary to determine whether or not SLC1A1
is expressed in lymphoblastoid cell lines at sufficiently high levels for quantitative analyses and then to identify the degree to which (if at all) SLC1A1
gene expression in lymphoblastoid cell lines constitutes a heritable trait. The additive heritability (h2
) value of 0.23 determined herein, while highly significant, is quite modest compared with some other genes expressed in lymphoblastoid cell lines, whose heritabilities ranged between 0.18 and 1, with a median of 0.34.45
In light of the relatively modest additive heritability for SLC1A1
expression now determined in our analysis, it is not surprising that the eQTL markers identified herein also had a small effect size (). Nevertheless, our approach proved valuable, not only because it identified a previously OCD-associated SNP, rs301430, as an eQTL, but also because (1) the 3-marker haplotype that was strongly associated with OCD contained 2 eQTLs, and (2) another eQTL marker farther upstream correlated with an OCD hoarding subphenotype.
Given the modest eQTL significance values and effect sizes from our analyses in lymphoblastoid cell lines that would not withstand multiple testing correction (), we conducted additional studies to further assess functionality. We were able to show that 2 of the 3 initial eQTLs, rs7858819 and rs301430, are also associated with SLC1A1
messenger RNA expression in the brain with similar significance levels and the same effect direction ( and ). These results not only strengthen the relevance of these 2 markers for SLC1A1
genetics research, but also suggest that regulation of gene expression in lymphoblastoid cell lines resembles brain regulation for this gene. This would make this readily obtainable cell line suitable for future functional and population-based analyses of SLC1A1
in a manner that has been used extensively for a related molecule, the serotonin transporter (SLC6A4
It also calls for a systematic comparison of gene expression genetics between brain tissue and lymphoblastoid cell lines now that suitable brain data sets have become available.48
The second complementary study, ie, luciferase reporter gene constructs, was aimed at direct functional assessment of individual SNPs. While association analyses with tissue samples or cell lines have the advantage of more closely resembling in vivo conditions, the eQTLs do not necessarily have to directly confer functionality, as they may merely be in linkage disequilibrium with the actual functional polymorphism. We addressed this uncertainty by examining individual polymorphisms in vitro with reporter gene assays. Our luciferase data for rs301430 suggest that this variant is indeed directly functional. Additional studies are now needed to elucidate the molecular mechanisms through which rs301430 affects gene expression.
The H4 haplotype, as detailed in , which was almost twice as common in those with OCD as in controls (odds ratio, 1.89), represents the strongest haplotypic association of SLC1A1
with OCD published to date.16-18
It illustrates the usefulness of our complementary expression–mining approach, because 2 of its markers were eQTLs, and conditional haplotypic association testing with the remaining markers failed to yield positive results (see the “Methods” section).28,32
The individual alleles in H4, however, are not fully compatible with the notion of increased or decreased SLC1A1
gene expression in OCD, as the C allele at rs7858819 was the lower-expressing allele in the regression analyses (), while the C allele at rs301430 in H4 correlated with increased expression ( and ). On the other hand, the mildly protective haplotype H2 in (odds ratio, 0.78), which narrowly survived correction for multiple testing in individual haplotype analysis, contained the lower-expressing allele at both eQTLs. Here, too, it will be essential to conduct gene expression analyses in larger samples and with more densely spaced markers.
The major limitation of our study is the potential susceptibility of the case-control design to population stratification, especially because our controls originated from 3 different groups that did not closely correspond in their geographic origin to our proband sample. While we addressed this issue by matching self-reported ethnicities of probands and controls, spurious effects in case-control studies (which are regarded by some authors as relatively uncommon49
) can be ruled out by genotyping multiple ancestry-informative markers, which was not feasible for this work. Dense genetic information on SLC1A1
markers from different ethnicities and geographic locations will increasingly become available from genome-wide association studies. Such data will allow a better judgment of how strongly (and if at all) population stratification is relevant to the SLC1A1
markers analyzed herein.
Another aspect of genetic studies such as ours with several investigated phenotypes that requires discussion is the multiple comparison problem. In these studies, the multiple statistical tests are clearly not independent of one another given linkage disequilibrium between variants () and the high correlation between subphenotypes (hoarding in this case) and the general diagnosis of OCD. Therefore, methods that correct by the total number of tests (eg, the Bonferroni procedure) would be more conservative than appropriate in our view. We conducted empirical family-wise error correction for the categorical single-marker and haplotype analyses by permutation and determined that 2 haplotypes remain significant (H4 and H2) (). The significance values for our subphenotype analyses are nominally robust (for association of rs3933331 with Y-BOCS score, P=.005, and with SIR-assessed hoarding, P=.009) (). They would, however, not withstand Bonferroni correction and therefore need to be interpreted with caution and observed in larger samples in future studies.
Two linear regression analyses of rs3933331 for the hoarding subphenotype, both factor analysis–derived (A) and on the basis of the Saving Inventory–Revised (SIR) score (B), which were nominally significant. A, P=.005. B, P=.009.
In summary, we have identified a strong association of OCD with a 3-marker haplotype composed of eQTLs for SLC1A1, which in turn were determined by exploiting expression and genotype data repositories and further validated with brain expression studies and reporter assays. Our complementary expression analysis approach might prove to be of use in future genetic studies for genes expressed at quantifiable levels in lymphoblastoid cell lines and other population-based tissue collections. Our haplotypic association results call for continued appreciation of glutamatergic neurotransmission abnormalities in OCD research.