Identification of Underlying Hub Genes Associated with Hypertrophic Cardiomyopathy by Integrated Bioinformatics Analysis

Abstract

Background: Considered as one of the major reasons of sudden cardiac death, hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease. However, effective treatment for HCM is still lacking. Identification of hub gene may be a powerful tool for discovering potential therapeutic targets and candidate biomarkers.

Methods: We analysed three gene expression datasets for HCM from the Gene Expression Omnibus. Two of them were merged by "sva" package. The merged dataset was used for analysis while the other dataset was used for validation. Following this, a weighted gene coexpression network analysis (WGCNA) was performed, and the key module most related to HCM was identified. Based on the intramodular connectivity, we identified the potential hub genes. Then, a receiver operating characteristic curve analysis was performed to verify the diagnostic values of hub genes. Finally, we validated changes of hub genes, for genetic transcription and protein expression levels, in datasets of HCM patients and myocardium of transverse aortic constriction (TAC) mice.

Results: In the merged dataset, a total of 455 differentially expressed genes (DEGs) were identified from normal and hypertrophic myocardium. In WGCNA, the blue module was identified as the key module and the genes in this module showed a high positive correlation with HCM. Functional enrichment analysis of DEGs and key module revealed that the extracellular matrix, fibrosis, and neurohormone pathways played important roles in HCM. FRZB, COL14A1, CRISPLD1, LUM, and sFRP4 were identified as hub genes in the key module. These genes showed a good predictive value for HCM and were significantly up-regulated in HCM patients and TAC mice. We also found protein expression of LUM and sFRP4 increased in myocardium of TAC mice.

Conclusion: This study revealed that five hub genes are involved in the occurrence and development of HCM, and they are potentially to be used as therapeutic targets and biomarkers for HCM.

Keywords: HCM; WGCNA; bioinformatics analysis; biomarkers; hub gene; hypertrophic cardiomyopathy; weighted gene coexpression network analysis.

Figure 1

Merging of datasets and differential expression analysis. (A, B) Two-dimensional principal component analysis cluster plot before and after merging GSE133054 and GSE141910. (C) Volcano plot of 455 DEGs. (D) The number of DEGs filtered using limma according to the cutoff criteria [adjusted P value < 0.05, |log2 FC| ≥ 1]. (E) Heatmap of all DEGs.

Figure 2

Enrichment analyses of DEGs. (A) GO enrichment analysis of DEGs. (B) KEGG pathway enrichment analysis of DEGs.

Figure 3

Construction of a coexpression network. (A) Sample clustering to detect outliers; N14, N18, N29, H18, H22, H33, H35, and H36 were excluded. (B) Analysis of the scale-free fit index for various soft-threshold powers; the red line was set at 0.90. (C) Analysis of mean connectivity for various soft-threshold powers. (D) Cluster dendrogram of genes in the coexpression network.

Figure 4

Identification of key modules. (A) Module-trait relationships in the constructed network. (B and C) MM versus GS plot of the key modules. (D and E) Gene expression profiles of the key modules.

Figure 5

Functional enrichment analysis of the blue modules and identification of hub genes. (A) GO enrichment analysis of the blue module. (B) KEGG pathway enrichment analysis of the blue module. (C) Interaction network of the hub genes in the blue module; red nodes represent the hub genes; green nodes represent other related genes in the module.

Figure 6

Analysis of the disease-predicting abilities of hub genes. (A) ROC curve analysis of hub genes in the merged dataset. (B) ROC curve analysis of hub genes in the verification dataset GSE36961.

Figure 7

Validation of the expression of hub genes. (A) Expression of hub genes in the merged dataset. (B) Expression of hub genes in the verification dataset GSE36961. (C) Masson’s trichrome staining of mouse hearts. (D) Expression of hub genes in TAC mice. Error bars indicate mean ± standard deviation. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 8

Western blotting tests representing protein levels of hub genes (FRZB, COL14A1, CRISPLD1, LUM, sFRP4) and remodeling gene RCAN in myocardium of sham and TAC mice.