Neurogenetic underpinnings of nicotine use severity: Integrating the brain transcriptomes and GWAS variants via network approaches

Abstract

Our study focused on human brain transcriptomes and the genetic risks of cigarettes per day (CPD) to investigate the neurogenetic mechanisms of individual variation in nicotine use severity. We constructed whole-brain and intramodular region-specific coexpression networks using BrainSpan's transcriptomes, and the genomewide association studies identified risk variants of CPD, confirmed the associations between CPD and each gene set in the region-specific subnetworks using an independent dataset, and conducted bioinformatic analyses. Eight brain-region-specific coexpression subnetworks were identified in association with CPD: amygdala, hippocampus, medial prefrontal cortex (MPFC), orbitofrontal cortex (OPFC), dorsolateral prefrontal cortex, striatum, mediodorsal nucleus of the thalamus (MDTHAL), and primary motor cortex (M1C). Each gene set in the eight subnetworks was associated with CPD. We also identified three hub proteins encoded by GRIN2A in the amygdala, PMCA2 in the hippocampus, MPFC, OPFC, striatum, and MDTHAL, and SV2B in M1C. Intriguingly, the pancreatic secretion pathway appeared in all the significant protein interaction subnetworks, suggesting pleiotropic effects between cigarette smoking and pancreatic diseases. The three hub proteins and genes are implicated in stress response, drug memory, calcium homeostasis, and inhibitory control. These findings provide novel evidence of the neurogenetic underpinnings of smoking severity.

Keywords: Brain transcriptome; Cigarettes per day; Coexpression network; GRIN2A; PMCA2; SV2B.

Figure 1.. Workflow of the analytical approaches.

(1) BrainSpan’s transcriptome data from the 15 brain regions were used to construct the gene modules of the whole brain and build the intramodular-region-specific coexpression subnetworks in each of the 15 brain regions using WGCNA. (2) Summary statistics from the U.K. Biobank (UKB) GWAS for CPD were converted to gene-based results using MAGMA. (3) Significant subnetworks enriched for CPD-associated genes in each brain region were identified by integrating the gene-based association strength for CPD from the UKB’s GWAS with the brain-region-specific coexpression subnetworks using PINBPA. (4) Confirmation of gene-set association in each region-specific subnetwork as being collectively associated with CPD using the data from the GWAS of the GSCAN without the UKB samples. (5) Performing protein-protein interaction analyses using STRING and evaluating network properties using CentiScaPe. (6) Evaluation of functional enrichment, whether genes in each subnetwork shared specific functional features, using WebGestalt. Abbreviation: CPD, Cigarettes Per Day. GSCAN, the GWAS and Sequencing Consortium of Alcohol and Nicotine. GWAS, Genome-Wide Association Study. MAGMA, Multi-marker Analysis of GenoMic Annotation. PINBPA, Protein-Interaction-Network-Based Pathway Analysis. STRING, Search Tool for the Retrieval of INteracting Genes/Proteins. WebGestalt, WEB-based GEne SeT AnaLysis Toolkit. WGCNA, Weighted Gene Coexpression Network Analysis.

Figure 2.

(A) Histograms of the random permutation for evaluating the intra-modular subnetwork analysis for the amygdala (p<0.01). The histograms are colored based on different percentiles: 70%, cyan; 90%, pink; and 99%, red. The red dashed line shows the observed subnetwork’s total number of edges and the size of the connected component. (B) Network view of the coexpression subnetwork for the amygdala. Genes (nodes) in the network are sized by their betweenness network centrality score. The blue line indicates the connection between genes. The red node is the hub CNTNAP1, while GRIN2A, the red dashed line circled, is the hub of the protein interaction subnetwork in the amygdala. (C) Network view of the protein interaction subnetwork for the amygdala. The network view delineates the network of predicted associations for the brain subnetwork group of proteins. The hub is GRIN2A, while CNTNAP1, the red dashed line circled, is the hub of the coexpression subnetwork in the amygdala. The network nodes and edges represent proteins and the predicted functional associations, respectively. These colored lines denote the seven types of evidence identified in predicting the associations. A red line implies the presence of fusion evidence. A green line implies neighborhood evidence. A blue line implies co-occurrence evidence. A purple line implies experimental evidence. A yellow line implies text-mining evidence. A light blue line implies database evidence. A black line implies coexpression evidence. (D) The eight brain regions were identified to be the subnetworks associated with cigarettes per day (CPD) (all p<0.01).