Comprehensive Analysis of RNA Modifications Related Genes in the Diagnosis and Subtype Classification of Dilated Cardiomyopathy

Abstract

Background: RNA modifications are associated to various human diseases. However, the functions of RNA modification-related genes have yet to be thoroughly investigated in dilated cardiomyopathy (DCM). This study sought to conduct a comprehensive analysis of RNA modification-associated genes for the diagnosis and subtype classification of DCM.

Methods: We collected DCM and control sample RNA modification-related genes from Gene Expression Omnibus (GEO) microarray datasets. Differential expression analysis was performed on these using the "Limma" package in R. Univariate logistic regression, and the LASSO algorithm were used to identify optimal genes for diagnostic model establishment. Furthermore, ConsensusClusterPlus was used to identify RNA modification-molecular subtypes. Lastly, the expression of the hub RNA modification-related genes and their connection to DCM were confirmed using the clinical samples and mouse models.

Results: Twenty-six RNA modification-related genes were identified as dysregulated in DCM, with strong connections noted among these genes. A diagnostic model based on 13 genes (TRMT61B, MBD2, YTHDC2, NOP2, TRMT10C, WDR4, CPSF2, CSTF3, ZBTB4, UNG, NSUN6, TET1, and DNMT3B) with an AUC of 0.980 predicted DCM well. Infiltrating plasma B cells, eosinophils, CD8 T cells, and regulatory T cells correlated strongly with TRMT61B, MBD2, YTHDC2, and CPSF2. Two RNA modification-molecular subtypes (clusters 1 and 2) were identified. Cluster 1 had greater RNA modification scores, lower immune ratings, and lower HLA-DRB1 and HLA-DPB1 expression than Cluster 2. Cluster 2 engaged metabolism-related pathways, while Cluster 1 activated renin-angiotensin system pathways.We further found a substantial link between lower cardiac function and up-regulation of TET1, DNMT3B, and down-regulation of MBD2, TRMT61B in the 13 hub RNA modification-related genes.

Conclusion: In conclusion, our RNA modification-related diagnostic model predicts DCM well. The discovery of two RNA modification-molecular subgroups and four key pivotal genes may assist stratify DCM patients by risk.

Keywords: 5-methylcytosine; N6-methyladenosine; RNA modification; dilated cardiomyopathy; immune infiltration.

Figure 1

The workflow of this study.

Figure 2

RNA modification-related genes in dilated cardiomyopathy. (A) Boxplot showing the expression levels of differentially expressed RNA modification-related genes in dilated cardiomyopathy (DCM) vs Control. (B) Correlation heatmap showing the correlations among the 26 differentially expressed RNA modification-related genes; The significantly enriched biological processes (C) and KEGG pathways (D) for differentially expressed RNA modification-related genes. Compared with CTRL group, 0.01<*P<0.05, 0.005<**P<0.01, ***P<0.005.

Figure 3

RNA modification-related genes-based diagnostic model. (A) Forest plot showing the 26 differentially expressed RNA modification-related genes significantly associated with dilated cardiomyopathy in univariate Cox regression analysis. (B) LASSO coefficient distribution of RNA modification-related genes and 10-fold cross-validated likelihood deviance of the LASSO coefficient for parameter selection. (C) Heatmap showing the expression pattern of 13 optimal diagnostic genes in GSE141910 dataset. (D) ROC curve showing the diagnostic performance of the diagnostic model. (E) Heatmap showing the expression pattern of 13 optimal diagnostic genes in GSE120895 dataset. (F) ROC curve showing the diagnostic performance of the diagnostic model.

Figure 4

Associations of RNA modification-related genes with immune infiltration. (A) Boxplot showing the difference in infiltration abundance of 22 immune cells between dilated cardiomyopathy (DCM) and control samples; (B) Correlation heatmap showing the correlations between RNA modification-related genes expression and infiltration abundance of differential immune cells. Compared with CTRL group, 0.01<*P<0.05, 0.005<**P<0.01, ***P<0.005.

Figure 5

Consensus clustering analysis. (A) Consensus clustering grouped dilated cardiomyopathy samples into two clusters based on expression of RNA modification-related genes; Boxplots showing the difference in RNA modification score (B), stromal score (C) and immune score (D) between two clusters; (E) Boxplot showing the difference in infiltration abundance of 22 immune cells between two clusters. Compared with Cluster 1 group, 0.01<*P<0.05, ***P<0.005.

Figure 6

Expression of HLA family genes and co-stimulatory molecules. Boxplots showing the difference in expression of HLA family genes (A) and co-stimulatory molecules (B) between two clusters. Compared with Cluster 1 group, 0.01<*P<0.05, 0.005<**P<0.01, ***P<0.005.

Figure 7

Gene expression and pathways between two clusters. Heatmap (A) and volcano plot (B) showing the different expression pattern of genes between two clusters; Bar charts showing the significantly enriched biological process (C) and KEGG pathways (D) for differentially expressed genes; (E) The pathways differentially enriched between two clusters in gene set variation analysis.

Figure 8

Verification of 13 optimal RNA modification-related genes by qRT-PCR in Control and DCM group. Compared with CTRL group, 0.01<*P<0.05, 0.005<**P<0.01.

Figure 9

The relationship between TET1, DNMT3B, MBD2 and TRMT61B mRNA levels, and DCM in mice model. (A) The body weight in the Control and DCM group. (B and D) The echocardiography features in the Control and TAAD groups. (C) TET1, DNMT3B, MBD2 and TRMT61B mRNA levels in the CTRL and DCM group. (E) The correlations between four optimal hub genes TET1, DNMT3B, MBD2 and TRMT61B mRNA levels, and cardiovascular functional parameters in the CTRL and DCM group. Compared with the CTRL group, 0.01<*P<0.05, 0.005<**P<0.01, ***P<0.005.