Impact Of Variant In The Neuro Hypophysis Hormone Receptor Gene On Structure And Function Of Brain In Normal Subjects

Objective:There is increasing evidence indicating the impact of natural variations in the human neurohypophysis hormone receptor(OXTR rs53576and AVPR1a RS3) on anxiety-related disease and autism spectrum disorders, but the mediating neural mechanisms remain poorly understood. This investigation is divided into the following three parts:(1) we combined voxel-based morphometry and resting-state functional connectivity analyses in a large cohort of young healthy Chinese Han population to test the hypothesis that the OXTR gene polymorphism influences an anxiety-related temperamental trait, as assessed by the harm avoidance (HA) subscale from the Tridimensional Personality Questionnaire (TPQ) via modulating the gray matter volume and resting-state functional connectivity (rsFC) of the brain, especially the limbic system;(2) we combined functional connectivity density (FCD) and rsFC to identify modulatory effects of OXTR rs53576gene variation on the rsFCs of the brain (especially the hypothalamus) in270young healthy subjects;(3) we compared structural alterations in a large sample of young healthy subjects of both sexes with different length of the AVPR1a RS3promoter region repeat polymorphism. We adopted a short/long classification scheme for this repeat region length (grouped into long/long, long/short and short/short repeats) and applied whole-brain voxel-based morphometry technique and structural covariance method to investigate association between AVPR1a and morphological measures of the brain.Subjects and Methods:A total of324healthy, young, right-handed subjects were recruited in this study. The HA scores that quantifies anxiety-related personality trait was assessed using a Chinese version of the TPQ. Resting-state fMRI scan, sagittal structural images were all collected using a GE3.0T Signa HDX scanner. During the scanning, all subjects were explicitly instructed to keep their eyes closed, relax, as motionless as possible and think of nothing.1. Genotyping:Standard protocols were used to extract DNA from white blood cells with the EZgeneTM Blood gDNA Miniprep Kit (Biomiga, Inc. San Diego, California USA). The OXTR SNP (rs53576) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Thus subjects who were homozygous and heterozygous for the G-allele were merged as one group of G-allele carriers to improve statistical power and compared with homozygotes for the A-allele. Amplification of the AVPRla RS3microsatellite (corresponding to the (CT)4-TT-(CT)8-(GT)n marker, which is3625bp upstream of the transcription start site) in the promoter region of AVPR1a was achieved using the following primers:(fluorescent)5’-TTGAAGAGCTGCCTTTGAGC-3’(forward) and5’-AATGCTGACCCGCTTTTTC-3’(reverse). Subjects who have short/short or short/long repeat length of AVPR1a RS3were merged into a group of short carriers (SS and SL) and compared with subjects with long/long repeat length of RS3(LL).2. Data preprocessing:All preprocessing steps for high-resolution structural data were carried out using the statistical parametric mapping (SPM8, http://www.fil.ion.ucl.ac.uk/spm) based on Matlab platform. All of the preprocessing steps for the resting-state fMRI data were performed using Resting-State fMRI Data Analysis Toolkit (REST v1.6, by Song et al., http://www.restfmri.net) implemented in Matlab platform and the FMRIB Software Library (FSL)(www.fmrib.ax.ac.uk/fsl). The preprocessing of high resolution structural data including segmentation of gray and white matter, spatial normalization and smoothing with a kernel of4mm full width at half maximum; while the preprocessing of rs-fMRI data including slice timing, realignment, spatial normalization to the MNI, resampling to to2x2x2mm3cubic voxels, and smoothing with a kernel of4mm full width at half maximum. We calculated the local FCD of each voxel using the in-house script that was written in Linux platform.3. We test the hypothesis that the variation of the OXTR rs53576will impact the individual HA differences because this gene variation affects anxiety-related behaviors. If the association between the genetic variation of the OXTR rs53576and HA scores is present, it is interesting to know which circuitries and networks of the brain are involved in mediating this association. Analysis of variance (ANOVA) with genotype and gender as two factors was used to examine the effect of genetic variation of the OXTR rs53576on the gray matter volume (GMV) and rsFC.4. We combined FCD and rsFC analyses to identify modulatory effects of OXTR rs53576gene variation on the rsFCs of the brain (especially the hypothalamus) in270young healthy subjects. Both the main effect of genotype and the gender-by-genotype interaction were considered in both local FCD and seed-based rsFC analyses.5. We performed VBM and structural covariance analysis throughout the whole brain between individuals with short repeats (short/short and short/long, SS and SL) of RS3and individuals homozygous for long repeats (long/long, LL) to identify brain regions with genotype-mediated GMV differences.6. Multiple comparisons were statistically corrected by False Discovery Rate or Monte Carlo simulation. Finally, the corrected statistical parameter mapping was overlapped onto MNI standard template and the coordinate, size and peak t values of each significant cluster were recorded.Results:1. We revealed that female subjects with the OXTR rs53576AA genotype showed increased harm avoidance scores relative to G-carrier females. We also found that female individuals with the AA genotype exhibited significantly smaller amygdala (especially the centromedial subregion) volumes bilaterally with an allele-load-dependent tendency, compared with female individuals with the GG/GA genotypes. Female subjects with the AA genotype demonstrated reduced resting-state functional couplings between the prefrontal cortex and amygdala bilaterally compared to female individuals with the GG/GA genotypes also with an allele-load-dependent trend. Furthermore, the magnitude of the prefrontal-amygdala coupling in the left hemisphere was positively correlated with harm avoidance scores in female subjects.2. We found a significant gender-by-genotype interaction in rsFC between the hypothalamic region and the left dorsolateral prefrontal cortex, but no significant main effect of genotype was found. Post-hoc testing revealed that this rsFC was significantly weaker in male OXTR rs53576AA homozygotes compared to male G-allele carriers.3. We found that male subjects with relatively shorter alleles (long/short and short/short repeats of the AVPR1a RS3), exhibited significantly smaller gray matter volume in the right face fusiform area and significantly weaker anatomical coupling of right face fusiform area and left parahippocampal gyrus compared to male subjects homozygous for long repeats (long/long repeats).Conclusion:1. Our findings highlight a possible neural pathway by which a naturally occurring gene variation of OXTR may affect anxiety-related temperamental trait in female subjects by modulating prefrontal-amygdala functional connectivity.2. Our findings identify gender-dependent mechanisms of OXTR rs53576gene variation impacting the functional connectivity of the hypothalamus in healthy individuals and suggest that these mechanisms are important for understanding ASDs.3. This is the first report in humans demonstrating variations in repeat length (RS3) of the AVPRla gene promoter region contribute to anatomical variability with a sex-dependent manner, which highlights one neurobiological pathway by which this gene variants may increase risk for ASDs. Further research is needed to identify functional correlates of these alterations in regional brain volume.