Genetic variant in SLC1A2 is associated with elevated anterior cingulate cortex glutamate and lifetime history of rapid cycling

Glutamatergic dysregulation is implicated in the neurobiology of mood disorders. This study investigated the relationship between the anterior cingulate cortex (AC) glutamate, as measured by proton magnetic resonance spectroscopy (1H-MRS), and single-nucleotide polymorphisms (SNPs) from four genes (GLUL, SLC1A3, SLC1A2, and SLC1A7) that regulate the extracellular glutamate in 26 depressed patients with major depressive disorder (MDD; n = 15) and bipolar disorder (BD; n = 11). Two SNPs (rs3812778 and rs3829280), in perfect linkage disequilibrium, in the 3′ untranslated region of the EAAT2 gene SLC1A2, were associated with AC glutamate, with minor allele carriers having significantly higher glutamate levels (p < 0.001) in comparison with common allele homozygotes. In silico analysis revealed an association of minor allele carriers of rs3812778/rs382920 with an upregulation of the astrocytic marker CD44 localized downstream of SLC1A2 on chromosome 11. Finally, we tested the disease relevance of these SNPs in a large group of depressed patients [MDD (n = 458); BD (n = 1473)] and found that minor allele carriers had a significantly higher risk for rapid cycling (p = 0.006). Further work is encouraged to delineate the functional impact of excitatory amino acid transporter genetic variation on CD44 associated physiology and glutamatergic neurotransmission, specifically glutamate–glutamine cycling, and its contribution to subphenotypes of mood disorders.

individuals had full capacity to consent and the informed consent process was both verbal and written during a visit to a special trained psychiatric nurse.
Patients in the American cohort were recruited from the Mayo Clinic. Patients in the American genotyping cohort were recruited from the Individualized Medicine Biobank for Bipolar Disorder, a collaborative project between the Mayo Clinic, the Lindner Center of HOPE/University of Cincinnati, and the University of Minnesota, each with site-specific institutional review board approval. The written informed consent process was followed by a comprehension test questionnaire to ensure key points of study participation were understood. The biobank procedures for participant recruitment, clinical phenotyping, and biological specimen sampling, processing, and storage were published previously (Frye et al., 2015).
Eligible participants were adults (aged 18 years or older at enrollment) with clinical diagnoses of BD-I, BD-II, or schizoaffective disorder who were able to provide valid informed consent. Individuals who were actively psychotic or actively suicidal and needing psychiatric hospitalization were not approached initially for participation; however, patients who were initially excluded for these reasons were approached after acute psychiatric problems resolved. Bipolar Biobank procedures included a detailed evaluation at baseline. BD-I and BD-II diagnoses, age of BD symptom onset, and comorbid psychiatric diagnoses were established using the Structured Clinical Interview for the DSM-IV (SCID). The baseline assessment included structured patient-rated and clinician-administered questionnaires to ascertain demographic variables, clinical variables, and illness characteristics (including lifetime history of rapid cycling).
Lifetime history of rapid cycling (RC) was defined as a self-reported history of having four or more distinct bipolar mood episodes in a 12 months period, with each episode separated by a return to baseline mood state for at least 2 months, or a switch to the opposite mood pole. Manic and hypomanic episodes were counted as being on the same mood pole.
Patient with MDD were derived from the PART study, a longitudinal population-based study in Stockholm County, Sweden (Hällström et al., 2003). PART includes data from 8613 randomly-selected Swedish nationals who have responded to an extensive questionnaire on mental health, work and relations twice (in two waves with a 3-yr interval), including the Major Depression Inventory (MDI).
Individuals characterized as having depression were those diagnosed with major depression, mixed anxiety depression or dysthymia, in at least one of the two PART waves. Depression was defined according to DSM-IV and was identified using the MDI.
The collection of the Swedish samples was approved by the Regional Ethical Review Board in Stockholm and informed consent was obtained from all participants.

MRI -Voxel positioning
For the midline anterior cingulate cortex (MACC) voxel, a reference slice was taken from an axial cut approximately 1 cm above the genu of the corpus callosum, demonstrating a continuous view of the anterior and posterior horns of the lateral ventricles. On this reference image, an 8-cm 3 voxel (2x2x2 cm) of predominantly gray (prefrontal) matter was centered on the frontal interhemispheric fissure.
The posterior margin of the voxel was placed immediately anterior to the genu of the corpus callosum in an area corresponding to the pregenual ACC (Brodmann area 24a, 24b, and 32), as described by Vogt and Vogt (2003). For the left dorsolateral prefrontal cortex voxel (LDLPFC), a reference coronal oblique localizer slice was positioned on the sagittal anatomical images such that it was positioned perpendicular to the average plane of the corpus callosum, and the posterior margin of the slice was located immediately anterior to the anterior-most portion of the genu of the corpus callosum. On this reference image, an 8-cm3 voxel (2x2x2 cm) encompassing the LDLPFC was placed such that: 1) the superolateral corner of the voxel abutted, but did include, the skull, 2) the medial margin of the voxel excluded the medial frontal cortex, and 3) the voxel was placed as superiorly as possible given constraints 1 and 2. This positioning typically includes the superior frontal sulcus and large portions of the superior and middle frontal gyri, containing Brodmann's areas 9 and 46.

Supplementary Figure 1. Chromosomal location of SLC1A2 and CD44 and proposed Mechanism for the Impact of Genetic Variations in SLC1A2 on Glutamatergic Neurotransmission and Rapid Cycling
(A) Single nucleotide polymorphisms (SNPs), rs3829280 and rs3812778, are located in the 3'-untranslated region of the excitatory amino acid transporter 2 (EAAT2) gene (SLC1A2) in chromosome 11. CD44 is situated downstream of SLC1A2.