Functional Coding Variants in <i>SLC6A15</i>, a Possible Risk Gene for Major Depression

<div><p>SLC6A15 is a neuron-specific neutral amino acid transporter that belongs to the solute carrier 6 gene family. This gene family is responsible for presynaptic re-uptake of the majority of neurotransmitters. Convergent data from human studies, animal models and pharmacological investigations suggest a possible role of SLC6A15 in major depressive disorder. In this work, we explored potential functional variants in this gene that could influence the activity of the amino acid transporter and thus downstream neuronal function and possibly the risk for stress-related psychiatric disorders. DNA from 400 depressed patients and 400 controls was screened for genetic variants using a pooled targeted re-sequencing approach. Results were verified by individual re-genotyping and validated non-synonymous coding variants were tested in an independent sample (N = 1934). Nine variants altering the amino acid sequence were then assessed for their functional effects by measuring SLC6A15 transporter activity in a cellular uptake assay. In total, we identified 405 genetic variants, including twelve non-synonymous variants. While none of the non-synonymous coding variants showed significant differences in case-control associations, two rare non-synonymous variants were associated with a significantly increased maximal ³H proline uptake as compared to the wildtype sequence. Our data suggest that genetic variants in the <i>SLC6A15</i> locus change the activity of the amino acid transporter and might thus influence its neuronal function and the risk for stress-related psychiatric disorders. As statistically significant association for rare variants might only be achieved in extremely large samples (N >70,000) functional exploration may shed light on putatively disease-relevant variants.</p></div

Repeated Bmax measurement of mutants that showed large differences in ³H proline uptake compared to WT.

<p>The uptake was measured under four different experimental conditions. Each bar represents the ³H proline uptake (mean ± SD) obtained from triplicates for the buffer solution, six samples for the 3 µM and 12 µM L-proline solution respectively and nine samples for the 780 µM L-proline solution. Corrected p-values given in brackets are based on the difference in mean ³H proline uptake between WT and tested mutant.</p

Inhibition of ³H proline transport by the non-radioactive labeled amino acid L-proline.

<p>Concentration of cold L-proline is plotted on the x-axis, ³H proline uptake as counts per minute (cpm) on the y-axis. Each datapoint represents the mean transport activity of triplicate samples.</p

Overview of the mutant plasmids created by site-directed mutagenesis.

<p>Overview of the mutant plasmids created by site-directed mutagenesis.</p

Maximal ³H proline uptake of wildtype (WT) and all tested mutants.

<p>The maximum in uptake was measured in the presence of 3 µM cold L-proline. Data are expressed as means ± standard deviation (SD) obtained from triplicate samples. Mutants with a circle were tested in a second independent experiment.</p

Cellular localization of the SLC6A15 protein.

<p>Both the wildtype cells (left) and the T49A mutant cells (right) showed a similar expression of SLC6A15 in the cell membrane. The localization of the eGFP-h<i>SLC6A15</i> fusion product is indicated in green. Cell nuclei were stained with DAPI (blue).</p

Significant haplotype blocks, both located in the <i>BDNF</i> gene, which showed association with response after 5 weeks (best phenotype of the single marker analysis).

<p>Note, that for optimal gene coverage, haplotype analysis was performed in the discovery sample only (<i>N</i> = 398, 82 SNPs).</p

Forest plots for the three replicated <i>NTRK2</i> SNPs rs10868223 (A), rs1659412 (B) and rs11140778 (C).

<p>For each sample, odds ratios and <i>P</i> values calculated with the Armitage’s test are indicated separately for the two phenotypes remission at discharge (blue boxes) and response after week 5 (pink boxes). Diamonds were used for the combined sample (<i>N</i> = 894).</p

Association with Treatment Outcome.

a<p>Empirical <i>P</i> values for the associations with treatment outcome (FPM analysis) under an allelic model.</p>b<p><i>P</i> value, permutation-based correction for multiple testing (16 SNPs).</p>c<p>Not polymorphic.</p>d<p>Note that this <i>P</i> value would not fulfill a more conservative threshold of alpha = .025 correcting for the fact that two replication samples have been analyzed.</p

Development of HAM-D values during antidepressive treatment depending on the rs10868223 (A), rs1659412 (B) and rs11140778 (C) genotype (combined sample).

<p>Repeated measurements (Greenhouse-Geisser, age and sex as covariates) revealed significant interaction effects for rs10868223 (<i>P</i> = .007) and rs1659412 (<i>P</i> = .012), but not for rs11140778 (<i>P</i> = .645). Stars indicate significant between-subjects differences at different time points (*, p<.05; **, p<.01; GLM with age and sex as covariates). Error bars are standard errors of the means.</p