Key Genes Identified in Nonsyndromic Microtia by the Analysis of Transcriptomics and Proteomics

Abstract

As one of the common birth defects worldwide, nonsyndromic microtia is a complex disease that results from interactions between environmental and genetic factors. However, the underlying causes of nonsyndromic microtia are currently not well understood. The present study determined transcriptomic and proteomic profiles of auricular cartilage tissues in 10 patients with third-degree nonsyndromic microtia and five control subjects by RNA microarray and tandem mass tag-based quantitative proteomics technology. Relative mRNA and protein abundances were compared and evaluated for their function and putative involvement in nonsyndromic microtia. A total of 3971 differentially expressed genes and 256 differentially expressed proteins were identified. Bioinformatics analysis demonstrated that some of these genes and proteins showed potential associations with nonsyndromic microtia. Thirteen proteins with the same trend at the mRNA level obtained by the integrated analysis were validated by parallel reaction monitoring analysis. Several key genes, namely, LAMB2, COMP, APOA2, APOC2, APOC3, and A2M, were found to be dysregulated, which could contribute to nonsyndromic microtia. The present study is the first report on the transcriptomic and proteomic integrated analysis of nonsyndromic microtia using the same auricular cartilage sample. Additional studies are required to clarify the roles of potential key genes in nonsyndromic microtia.

Figure 1

Identification and analysis of DEGs in NSM by microarray. A. Volcano plot of transcriptomics. The right dots show 1776 significantly upregulated genes, and the left dots show 2195 significantly downregulated genes; FC > 1.5, false discovery rate (FDR) < 0.05. B. Heatmap showing the top 20 upregulated and top 20 downregulated DEGs based on the expression of genes (red, upregulated; blue, downregulated). (C, D) KEGG analysis of the upregulated and downregulated DEGs. The dot size represents gene count, and the color represents the P-value. P < 0.05 is considered to be statistically significant.

Figure 2

Determination of hub gene modules associated with NSM through WGCNA. (A) Identification of soft-threshold power by analyzing the scale-free index (left) and the mean connectivity (right) in the WGCNA. (B) Dendrogram of all DEGs clustered based on a dissimilarity measure (1-TOM). Clustering DEGs are shown in colors. (C) Numbers of hub genes in each module. (D) Heatmap showing the correlation between ME and NSM. The CC and P-values of each module in NSM and CS are presented in the center of the panels. Positive and negative associations are separately shown in red and blue, respectively.

Figure 3

Scatter plots of the GS vs MM for the modules. (A) Dark turquoise module (CC = 0.82, P = 1.2 × 10^–16). (B) Brown module (CC = 0.71, P = 3.9 × 10^–104). (C) Dark orange module (CC = 0.67, P = 1.2 × 10^–06). (D) Salmon module (CC = 0.64, P = 1.1 × 10^–09). (E) Blue module (CC = 0.64, P = 1.3 × 10^–89). (F) Dark red module (CC = 0.63, P = 5.7 × 10^–59). (G) Green module (CC = 0.57, P = 2.8 × 10^–31). (H) Black module (CC = 0.56, P = 7 × 10^–21). (I) Magenta module (CC = 0.53, P = 1.1 × 10^–09).

Figure 4

Identification of the gene modules highly correlated with NSM. (A) Venn diagram shows that a total of 71 TFs are predicted to have target genes in the same module, of which 19 are predicted by the two databases. (B–D) KEGG analysis for TFs and their target genes in the brown, green, and black modules. The dot size represents gene count, and the color represents the P-value. P < 0.05 is considered to be statistically significant.

Figure 5

Analysis of DEPs by proteomics. (A) Volcano plot of proteomics. The right dots show 94 significantly upregulated proteins, while the left dots show 210 significantly downregulated proteins. FC > 1.5, P < 0.05. B. Heatmap of the top 20 upregulated and top 20 downregulated DEPs based on the expression of proteins (red, upregulated; blue, downregulated). (C, D) KEGG pathway analysis of the upregulated and downregulated DEPs. The size of dots represents gene count, and the color represents the P-value. P < 0.05 is thought to be statistically significant. (E) Protein–protein interactions (PPI) network composes of downregulated DEPs in extracellular matrix (ECM)–receptor interaction and focal adhesion pathways. Different colored nodes indicate the proteins in the two pathways, and the line color represents the type of interaction evidence.

Figure 6

Combined analysis of transcriptomics and proteomics. (A) Scatter plot of the correlation relationships between mRNA and protein levels of all of the genes overlapped in transcriptomics and proteomics. The genes with significant differential expression at protein levels (FC > 1.5, FDR < 0.1) but with no significant change at mRNA level are indicated in blue. The genes that are only significantly regulated (FC > 1.5, FDR < 0.05) in the transcriptomics are depicted in red. The genes with or without significant changes at both levels are presented in purple or gray color. The R-value shows the correlation between the mRNA and protein levels of genes, and the R-value for all genes overlapped in transcriptomics and proteomics is 0.15 (P = 2.8 × 10^–16), while the R-value for the genes marked by purple is 0.6 (P = 4.2 × 10^–05). (B) Venn diagram showing 40 overlapping genes with significantly differential expression at both mRNA and protein levels in NSM. (C) Heatmap showing the 40 genes (red in the square, upregulated; blue in the square, downregulated; font color: navy blue, genes downregulated at both levels; sky blue, genes upregulated at the mRNA level but downregulated at the protein level; red, genes upregulated at both levels; green, genes downregulated at the mRNA level but upregulated at the protein level). The expression value of each gene corresponds to the proteomic data. (D, E) KEGG pathway analysis of the 30 genes with the same trends at mRNA and protein levels. The size of dots represents gene count, and the color represents the P-value. P < 0.05 is considered statistically significant. (F) PPI network composes of the 40 genes with significantly differential expression at both levels in NSM. The color of lines represents the type of interaction evidence.

Figure 7

PRM verification. One upregulated DEPs (FC > 1.5) and 12 downregulated DEPs (FC < 0.67) are consistent with the TMT-based proteomics results.