Identification of KIAA0513 and Other Hub Genes Associated With Alzheimer Disease Using Weighted Gene Coexpression Network Analysis

Abstract

Alzheimer disease (AD) is the most common cause of dementia and creates a significant burden on society. As a result, the investigation of hub genes for the discovery of potential therapeutic targets and candidate biomarkers is warranted. In this study, we used the ComBat method to merge three gene expression datasets of AD from the Gene Expression Omnibus (GEO). During combined analysis, we identified 850 differentially expressed genes (DEGs) from the temporal cortex of AD and cognitively normal (CN) samples. We performed weighted gene coexpression network analysis to build gene coexpression networks incorporating these DEGs to identify key modules and hub genes. We found one module most strongly correlated with AD onset as the key module and 19 hub genes in the key module that were down-regulated in AD brains. According to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, DEGs were mostly enriched in synapse function, and genes in the key module were mostly related to learning and memory. We selected five little-studied genes, AP3B2, GABRD, GPR158, KIAA0513, and MAL2, to validate their expression in AD mouse model by performing quantitative real-time polymerase chain reaction. We found that all of them were down-regulated in cortices of 8-month 5xFAD mice compared to those of wild-type mice. We then further investigated their correlations with β-secretase activity and Aβ42 levels in AD samples of different Braak stages. We found that all five hub genes had significant negative associations with β-secretase activity and that AP3B2 and KIAA0513 had significant negative associations with Aβ42 levels. We tested the differential expressions of the five hub genes in two AD GEO datasets from the blood and found that KIAA0513 was significantly up-regulated in patients with both mild cognitive impairment (MCI) and AD and was able to differentiate MCI and AD from CN in the two datasets. In conclusion, these five novel vulnerable genes were involved in AD progression, and KIAA0513 was a promising candidate biomarker for early diagnosis of AD.

Keywords: Alzheimer disease; dementia; gene expression; hub genes; weighted gene coexpression network analysis.

FIGURE 1

Study workflow. AD, Alzheimer disease; CN, cognitively normal; FC, fold change; GEO, Gene Expression Omnibus; GO, Gene Ontology; GS, gene significance; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; MM, module membership; WGCNA, weighted gene coexpression network analysis; WT, wild type.

FIGURE 2

Circos plot of expression patterns and chromosomal positions of top 100 differentially expressed genes (DEGs). The outer circle represents chromosomes, and lines coming from each gene point to their specific chromosomal locations. The three Alzheimer disease (AD) microarray datasets from Gene Expression Omnibus (GEO) used for combined analysis are represented in the inner circular heatmaps. GSE122063, GSE36980, and GSE5281 are presented from the outside to the inside. The red lines in the inner layer indicate -log₁₀ (adjusted P-value) of each gene. According to |log₂ fold change|, the top five up-regulated genes (red) and the top five down-regulated genes (blue) are connected with red and blue lines in the center of the Circos plot.

FIGURE 3

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of all differentially expressed genes. Chord plots indicate enrichment analysis of genes. (A) Biological process of GO analysis. (B) Cellular component of GO analysis. (C) Molecular function of GO analysis. (D) KEGG pathways.

FIGURE 4

Key module correlated with Alzheimer disease identified by weighted gene coexpression network analysis (WGCNA). (A) Clustering of samples to detect outliers. (B) Scale-free topology model (left) and mean connectivity (right) for finding the soft-thresholding power. The power selected is 5. (C) Cluster dendrogram of genes. (D) Clustering of all modules. The red line indicates the height cutoff (0.25). (E) Heatmap shows the relationships between different modules and clinical traits. (F) Gene significance in different modules associated with AD. AD, Alzheimer disease; CN, cognitively normal.

FIGURE 5

Functional enrichment of the key module. (A) Scatter plot of module eigengenes in the key module. (B) Interaction network of genes in the key module. Genes in black circles are hub genes in the key module. (C) Bubble plots of biological process of gene ontology (GO) analysis. (D) Bubble plots of cellular component of GO analysis. (E) Bubble plots of molecular function of GO analysis. (F) Bubble plots of KEGG pathways.

FIGURE 6

Expression of five hub genes in the temporal cortex. Only hub genes that have been little characterized to be associated with Alzheimer disease (AD) are listed. The green box indicates the cognitively normal group, and the orange box indicates the AD group. T-testing was performed to compare the means of two groups. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. AD, Alzheimer disease; CN, cognitively normal; ns, no significance.

FIGURE 7

Expression of five hub genes in cortex of 5xFAD mice. The results were presented as mean ± standard deviation (t-testing; n = 4 in each group). *P < 0.05; **P < 0.01. WT, wild type.

FIGURE 8

Correlation of five hub genes with β-secretase activity and Aβ42 levels using GSE106241. (A) The five hub gene expression, β-secretase activity, and Aβ42 levels in different Braak stages. Red asterisks indicate significant vs. Braak 0 groups. (B) Correlation between five hub genes and β-secretase activity. (C) Correlation between five hub genes and Aβ42 levels. For Panels B and C, P values in red are significant (P < 0.05). *P < 0.05; **P < 0.01; ****P < 0.0001. AD, Alzheimer disease; CN, cognitively normal.

FIGURE 9

Disease-predicting ability and Gene Set Enrichment Analysis (GSEA) of KIAA0513. (A) The expression of KIAA0513 in two blood GEO datasets, GSE63060 and GSE63061. (B) Receiver operating characteristic (ROC) curve of KIAA0513 for predicting AD and MCI. (C) The top three GSEA terms, according to normalized enrichment scores in the low-expression group of KIAA0513. *P < 0.05; **P < 0.01; ****P < 0.0001. AD, Alzheimer disease; CN, cognitively normal; MCI, mild cognitive impairment.