Differential impact of interleukin-6 promoter gene polymorphism on hippocampal volume in antipsychotic-naïve schizophrenia patients.

BACKGROUND: Differential susceptibility model hypothesizes that a genotype need not be unfavorable all the time as postulated in stress-diathesis model but can be beneficial in a supportive context. Single-nucleotide polymorphism (SNP) (rs18000795) within the promoter region of interleukin-6 (IL-6) gene was earlier noted to have a differential susceptibility on hippocampal volume in schizophrenia (SCZ).
MATERIALS AND METHODS: We examined antipsychotic-naïve/free SCZ patients (n = 35) in comparison with healthy controls (n = 68). Hippocampus volumes were assessed in 3 Tesla magnetic resonance imaging using voxel-based morphometry. Region of interest analysis was done using hippocampus mask. IL-6 SNP (rs1800795) was genotyped using TaqMan allelic discrimination assay.
RESULTS: A significantly deficient right (T = 3.03; K E= 392; P SVC-FWE= 0.04) and left (T = 3.03; K E= 47; P uncorr= 0.03) hippocampal gray matter volumes were noted in SCZ patients after controlling for the potential confounding effects of age, sex, and total brain volume. There was a significant diagnosis x rs1800795 genotype interaction involving both left (T = 2.17, K E= 95, P uncorr= 0.02) and right (T = 1.82, K E= 29, P uncorr= 0.04) hippocampal volumes. Patients with GG (left: F =5.78; P = 0.02; right: F =6.21; P = 0.01) but not GC/CC genotype (left: F =0.89; P = 0.34; right: F <0.01; P = 0.95) had volume depletion.
CONCLUSION: A paradoxical smaller hippocampal volume with GG genotype was noted in SCZ. Further elucidation of its mechanistic basis might have translational implications. Copyright:

INTRODUCTION

Schizophrenia (SCZ) is a complex neuropsychiatric condition with heterogeneous manifestations. It is a disorder with multifactorial inheritance, where multiple gene variants of small effect interact with environmental factors resulting in the condition. The priming of the brain is hypothesized to begin as early as in the fetal life in the context of environmental stressors such as maternal infections, malnutrition, and obstetric complications.[1] However, the onset of symptoms generally coincides with the final stages of neurodevelopment and evidence from genetics, neuropathology, and neuroimaging points at aberrant neurodevelopmental process in the pathogenesis of SCZ.[1] The disruption of neurodevelopmental process occurs during the critical phases of brain development, resulting in aberrant brain regions to generate psychotic symptoms by adolescence or early adulthood. The precise molecular events that mediate the detrimental effects of prenatal adversities on developing brain, though is inadequately known, maternal immune activation and/or developmental neuroinflammation has emerged as a predominant mechanism.[234]

Converging evidence suggests that various maternal immune cells such as Th17 lymphocytes, microglia with their inflammatory mediators like cytokines are crucially involved in the developmental priming of fetal brain. They can alter the neurodevelopmental trajectories subsequently leading to manifestation of SCZ-like symptoms in offspring.[567] Data obtained both from preclinical and clinical studies have reported the involvement of cytokines such as interleukin-6 (IL-6) and IL-8 in mediating a link between maternal immune activation and risk of SCZ in the offspring.[89] Of these, IL-6 has emerged as an important immunoinflammatory candidate of SCZ owing to its involvement in a number of systems or pathways relevant to SCZ pathophysiology.[10] For example, peripheral and brain IL-6 levels have been associated with cognitive function and neuronal circuitry imbalances.[11121314] Besides its expression and functional implications in developing brain, IL-6 was also found to mediate the effect of stress on developing brain.[1516] Perinatal insults contributing to increased plasma IL-6 were shown to affect neuronal plasticity in hippocampal region and impair hippocampus-dependent cognitive functioning.[1516] Further, the genes related to IL-6 were seen to determine the intensity of the effect on hippocampus.[17] Taken together, these findings suggest IL-6 as a potential candidate for gene x environment interaction, which could modulate the neurodevelopment.

The classical “stress-diathesis” model hypothesizes that an individual with vulnerability like carrying a risk gene, when exposed to stress in terms of adverse environment will develop the problematic condition. This model explains the synergistic effect of dual risk or double-hit on the development of abnormality, and an absence of vulnerability leads to resilience.[18] Complementary theories have been put forth on the opposite dimension of the interactive spectrum. Two fairly similar models called “biological sensitivity to context model” and “differential susceptibility models” suggest that the “diathesis” need not be restricted to vulnerability rather an “increased sensitivity” to the environmental influence.[1920] Thus, suggesting the presence of a factor/diathesis deemed risk to certain pathology in a given negative context may actually be enhancing the functioning/adaptability if exposed to a positive supportive environmental context. The level achieved in these subjects would be much superior to that attained by a person not having the “risk” factor if provided a supportive environment.[21]

Evidence for this model are being gathered through gene x environmental interaction on the behavioral outcomes, but the neurobiological susceptibility remains less explored. The differential susceptibility model holds promise not just from sociological and evolutionary perspective but also from the biological and translational perspective. The understanding of the biological bases can potentially pave way for biopsychosocial interventions that are not just protective from “worse” but can yield a “better” phenotype in biologically sensitive group. Given the pleiotropic nature of IL-6, its consistent association with SCZ, relevance as a biomarker of SCZ[22] and its functional relevance at the gene–environment interface, IL-6 gene could be a potential determinant of differential susceptibility. Contextually, a previous Indian study had demonstrated a differential susceptibility effect of rs1800795 single-nucleotide polymorphism (SNP) of IL-6 gene on hippocampal volume in antipsychotic-naïve SCZ.[23] Given the promise the finding held, we aimed at replicating the previous finding in an independent larger sample of antipsychotic-naïve SCZ patients.

MATERIALS AND METHODS

Study participants

Patients with SCZ (n = 35) were recruited from the clinical services of the National Institute of Mental Health and Neurosciences (India), who were antipsychotic-naïve or antipsychotic free (at least 6 weeks of treatment with oral or 3 months with depot antipsychotics). Mini-International Neuropsychiatric Interview (MINI) Plus[24] was used for establishing the diagnosis based on DSM-IV TR and was confirmed independently by two psychiatrists by a comprehensive clinical interview. The details related to treatment status and illness features were ascertained by reliable information obtained from at least one primary care-giver from the family. Psychopathology was rated on the scale for the assessment of positive symptoms[25] and the scale for the assessment of negative symptoms.[26]

Healthy controls (n = 68), who volunteered for the study, were screened to rule out any psychiatric diagnosis using the MINI Plus as well as a comprehensive mental status examination. The study subjects (patients and controls) with history or clinical features suggestive of neurological/medical disorder or comorbid alcohol/cannabis dependence or abnormal movements as assessed by Abnormal Involuntary Movements Scale were excluded from the study. Only right-handed subjects were included in this study. Written informed consent was obtained as per the research protocol approved by the institute ethics committee. Clinical assessments and imaging procedures were performed on the same day before starting antipsychotics.

Genotyping of interleukin-6 single-nucleotide polymorphism

Under aseptic precautions, 5 mL of peripheral blood was drawn from the cubital vein into K2 ethylenediaminetetraacetic acid vacutainer (Becton and Dickinson, U.S.A). The commercial spin column method (Qiagen, Inc., Limburg, Netherlands) was used for the extraction of genomic DNA. The quality of the extracted DNA was checked using ultraviolet spectrophotometer (Thermo Scientific, Waltham, U.S.A). The extracted DNA was stored at − 80°C deep freezer.

The genotyping of IL-6 174 G/C SNP (rs1800795) was carried out using the TaqMan 5' nuclease allelic discrimination assay using predesigned primers and allele-specific MGB probes (FAM and VIC dye) from commercially available TaqMan®SNP Genotyping Assay (Applied Biosystems, Foster City, California). Genotyping was performed in a 10 μL of reaction mix containing TaqMan universal polymerase chain reaction (PCR) master mix with AmpErase®Uracil-DNA glycosylase, assay mix and 10 ng of template genomic-DNA per well. The assays were performed in duplicates per sample, including positive controls and no-template controls, with the StepOnePlus™ Real-Time PCR Systems (Applied Biosystems, Foster City, California) in a 96-well format according to manufacturer's instructions. Standard PCR cycling conditions of 30 s at 60°C and 10 min at 95°C, followed by 50 cycles of 15 s at 92°C and 1.5 min at 60°C was followed.

Magnetic resonance imaging acquisition

Structural T1-weighted magnetic resonance imaging (MRI) of the brain was acquired using a 3 Tesla scanner (Siemens Skyra) (TR = 8.1 ms, TE = 3.7 ms, nutation angle = 8°, FOV = 256 mm, slice thickness = 1 mm without interslice gap, NEX = 1, matrix = 256 × 256). Images were de-identified by assigning codes for further analysis.

Voxel-based morphometry

Voxel-based morphometry (VBM) analysis was performed using MATLAB (R2013a, Math-Works, Natick, MA, USA) based tool SPM8 (http://www.fil.ion.ucl.ac.uk/spm). Using the standard unified segmentation model in SPM8, the images were segmented into gray matter (GM), white matter, and cerebrospinal fluid.[27] GM population templates were then generated from the entire image dataset using the diffeomorphic anatomical registration using exponentiated Lie algebra (DARTEL) technique.[28] Affine registration of the GM DARTEL templates to the tissue probability maps in the Montreal Neurological Institute (MNI) space (http://www.mni.mcgill.ca/) was performed followed by nonlinear warping of GM images to the DARTEL GM template in MNI space. Following the spatial normalization procedure, images were modulated to preserve the GM volume and smoothed with an 8 mm full width at half maximum Gaussian kernel. After spatial preprocessing, the normalized, modulated, and smoothed GM images were used for statistical analysis.

The effect of diagnosis and rs1800795 polymorphism on hippocampus GM volume was tested in SPM8. Age, sex, and total intracranial volume were considered as covariates of no interest in the region of interest analysis. Standard hippocampus mask was defined by automated anatomical labeling[29] system using the Wake Forest University Pick-Atlas tool.[30] Voxels with GM values of < 0.1 (absolute threshold masking) were excluded to avoid edge effects.

Statistical analyses

Data analyses were performed using the SPSS (SPSS Inc., Released 2007. SPSS for Windows, Version 16.0, Chicago, SPSS Inc.) The genotypes were grouped into “GG' and “GC/CC” as per the dominant model[23] due to low prevalence CC allele (one CC of 103 subjects). Comparison was done using independent samples t-test and ANCOVA for continuous variables and Chi-square test for categorical variables.

RESULTS

Demographic and clinical details of the participants are presented in Table 1. A significant difference in the mean ages but not in sex distribution was noted across the diagnostic groups.

Table 1

Demographic and illness characteristics of antipsychotic naïve/free patients with Schizophrenia and healthy controls

Characteristic	Patients (n=35)	Control (n=68)	Statistics	p
Age	32.14±6.62	26.43±4.48	4.6*	<0.01
Sex (male: female)	19:16	38:30	0.02†	0.87
Age at onset (years)	26.7±6.85	-	-	-
Duration of untreated illness (months)	36.8±35.25	-	-	-
SAPS	24.45±12.86	-	-	-
SANS	24.54±29.74	-	-	-

*Independent sample t-test, †Chi-square test. SAPS – Scale for the Assessment of Positive Symptoms; SANS – Scale for the Assessment of Negative Symptoms

The genotypes were consistent with Hardy-Weinberg equilibrium proportions in both the groups (Patient: χ2= 0.76; p = 0.38; Control: χ2= 0.04; p = 0.83). There was a comparable frequency distribution of minor allele at rs1800795 between patients and control groups (minor allele frequency of study subjects: SCZ = 9, HC = 14, χ2= 1.06, P = 0.58). Group differences for hippocampus volume revealed, a significant reduction in both right (x, y, z MNI coordinates: 32-15-12, T = 3.03; KE= 719; P = 0.02) and left (x, y, z MNI coordinates: -32 -40 -0, T = 1.92; KE= 47; Puncorr= 0.03) hippocampal volumes, in SCZ patients compared to controls [Figure 1]. The reduction in right hippocampus volume in patients survived small volume correction (SVC) with family wise error (FWE) correction (PSVC-FWE= 0.04), while left side did not.

Figure 1

Differences in the hippocampal volume of patients with schizophrenia and healthy controls (left) and diagnosis-by-genotype interaction with bilateral hippocampus volumes (right) (Yellow-red blobs in the figures represent the significant effects with T value map overlaid on significant cluster as per the respective color bar)

Full factorial GLM analysis, with age, sex, years of education and total brain volume as nuisance covariates revealed a significant diagnosis X genotype interaction with left hippocampus (X =−27, Y = −37, Z = 4, T = 2.17, KE= 95, Puncorr= 0.02) and right hippocampus (X = 32, Y =-36, Z = 3, T = 1.82, KE= 29, Puncorr= 0.04) [Figure 1].

Average GM volume in this significant cluster was extracted using MarsBaR toolbox for SPM[31] and follow-up analyses were performed to visualize the effect of diagnosis X genotype interaction. ANCOVA analysis with the same nuisance variables as used in VBM analysis depicted that, in the subgroup of subjects who carried “GG” genotype, the volume was significantly deficient in patients in comparison with healthy controls (left: F =5.78; P = 0.02; right: F =6.21; P = 0.01), whereas in “GC/CC” genotype subgroup, the differences were not statistically significant (left: F =0.89; P = 0.34; right: F <0.01; P = 0.95) [Figure 2].

Figure 2

Diagnosis-by-genotype interaction on the left and right hippocampus in Schizophrenia and healthy controls. (*Average gray matter volume [mean signal-intensity values] in the extracted cluster of significance)

Within patients, there was no significant difference between GG homozygotes and GC/CC carriers on the left side (F = 2.62; P = 0.11), but a trend level increase in volume in GC carriers on the right side (F = 3.69; P = 0.06) [Figure 2]. Within healthy controls, GG homozygotes had significantly larger volume compared to GC/CC carriers on the left side (F = 4.06; P = 0.047) and no difference on the right side (F = 0.02; P = 0.87).

DISCUSSION

In the current study, using a an independent and larger sample of antipsychotic-naïve/free SCZ patients, we could replicate the previous finding of smaller hippocampal volume and differential effect of IL-6 rs1800795 polymorphism on hippocampus.[23] IL-6 promoter SNP, especially GG genotype could be a genetic determinant of smaller bilateral hippocampi volume in SCZ patients in comparison to healthy controls with similar genotype. In addition, SCZ with GG genotype had a trend towards lower bilateral hippocampi volumes when compared with those carrying C allele. On the other hand, GG genotype had a beneficial effect in healthy controls with larger left hippocampal GM volume in comparison to “GC/CC” genotype.

IL-6 is known to influence the neurodevelopmental process mediating the inflammatory response to maternal infection or psychosocial stressor.[1332] Hippocampus being one of the brain regions with densest IL-6 receptors is naturally impacted by its levels in blood.[33] IL-6 enhances the oxidative reaction,[34] dopamine turnover, and adversely influences the neuronal and dendritic growth in the hippocampus.[1335] The relationship between IL-6 and neuroplasticity is not linear with both excess and absence of IL-6 resulting in structural and functional impairment of hippocampus.[3637] Thus, an optimal level of IL-6 is required for better resilience and growth of hippocampus.

IL6rs18000795 SNP in the promoter region is one of the comprehensively studied SNPs in health and disease.[17383940] The positive influence of GG genotype on hippocampus in healthy adults was demonstrated even in animal models.[1741] The environmental adversities leading to sympathetic nervous system activation elicits upregulation of GATA1 transcription factor through beta-adrenergic neurons in individuals bearing G allele (but not C-allele as they cannot effectively bind to this polymorphic site). This promotes transcription of IL-6 gene in G allele bearers and subsequent inflammatory effects on neurons.[41] Interestingly, this effect of G allele on nervous system varies across life span. Adolescents with G allele exposed to physiological/psychological stressors developed resilience by getting desensitized to inflammatory effects of the stressors, which was not seen in older individuals.[42] Thus, during developmental age, GG genotype would generate optimal IL-6 in healthy individuals thereby achieving the potential hippocampal volumes.[42]

However, it has been demonstrated that GC genotype confers protective effect in individuals in developmental age encountering very high psychosocial adversities.[42] SCZ individuals are known to have elevated IL-6 levels particularly during early stages of illness.[43] This could either be a result of greater physiological and psychological stressors during the neurodevelopmental period[44] and/or an oversensitive neuro-inflammatory response.[45] The GC genotype could mitigate the IL-6 response in SCZ and protect the hippocampus during developmental age. This is of particular importance as hippocampus plays a major role in pathogenesis of SCZ.[464748] This explains the “differential susceptibility” model of pathogenesis of SCZ with genotype environmental interaction mediated by inflammation.[21]

The differential susceptibility of hippocampus to IL-6-mediated effects could be one of the mechanisms of heterogeneity in SCZ.[21] Extrapolating this further, it can be hypothesized that the manifestation of illness in individuals with SCZ risk will be influenced by their experiences and environments. Proper understanding of these factors and their modifiability would assist in developing interventions not just to prevent deficits but also in helping the individuals at risk to achieve the best of their abilities. This phenomenon would be one of the mechanisms behind an overrepresentation of first degree relatives of SCZ in creative professions.[49]

In the current study, IL-6 levels were not measured as the cross-sectional IL-6 levels in the previous study did not show a significant association with hippocampal volume.[23] Rather, a longitudinal study looking at the developmental trajectory of hippocampus would further validate the mechanistic role of rs18000795 polymorphism of IL-6. Supplementing this with the evaluation of physical and psychosocial adversities during the neurodevelopmental age would be recommended for future studies. The major limitation of the study is that we could implicate only the global hippocampal deficits to the polymorphisms, and the interaction with diagnosis could not survive the SVC. Further inquiry would be needed into the effect on subfields and laterality of hippocampus using advanced neuroimaging tools. Understanding the functional implication of the volume differences in SCZ would have translational implication through the regulation of environmental influences. One of the main strengths of this study is the antipsychotic naïve/free patients which avoided the confounding effects of medications and illness courses on the hippocampal volume.

CONCLUSION

IL-6 seems to play a significant role in the neurodevelopment with IL-6 promoter genotypes influencing the pattern of effects. Our observation further supports the notion that the hippocampi volumes in SCZ are differentially susceptible to IL-6 promoter polymorphism. The GG genotype which is generally protective in healthy subjects may adversely impact the hippocampal growth in SCZ.

Financial support and sponsorship

This work is supported by the CEIB Programme Support (BT/PR5322/COE/34/8/2012) and partly by Swarnajayanti Fellowship Grant, Department of Science and Technology, Government of India (DST/SJF/LSA-02/2014-15) to GV and partly by Wellcome Trust DBT India Alliance Intermediate Fellowship grant (IA/CPHI/16/1/502662) to JCN.

Conflicts of interest

There are no conflicts of interest