Multiple effect mechanisms of FLNC in dilated cardiomyopathy based on genetic variants, transcriptomics, and immune infiltration analysis
Authors: Cai ChunYu , He Bin , Yu DianYou , Quan LiPing , Li ChengBan , Liu Yan , Wei QinJiang , Zhou LiuFang , Mo JianJiao , Pan XingShou , Huang JianJun , Liu Li 
Published: April 2023

Abstract
Background: In recent years, the FLNC gene has been shown to participate in dilated cardiomyopathy (DCM) through different mechanisms, and its variants are a common cause of hereditary DCM. This study aimed to systematically evaluate multiple FLNC effect mechanisms in DCM and to expand the spectrum of FLNC gene variations.
Methods:Based on five microarray expression profile datasets downloaded from the Gene Expression Omnibus (GEO) database, comprising DCM tissue and healthy control groups, the difference in FLNC gene expression levels between the two groups was evaluated. Subsequently, differentially expressed genes (DEGs) among 81 DCM tissues were identified based on FLNC grouping, and gene ontology, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, correlation analysis, and protein–protein interaction (PPI) network construction were conducted for DEGs. Based on single-sample Gene Set Enrichment Analysis method, we then evaluated differences in immune infiltration levels between the two groups using ''student 's'' and the correlation between FLNC gene expression.and the immune cells we using '' Spearman's correlation '' methods. Then, we constructed a ce-RNA network of FLNC based on several databases.Finally,100 blood samples from DCM and non-DCM individuals from the Guangxi Zhuang population in China were selected for FLNC gene sequencing, case-specific newly discovered or rare FLNC gene mutation sites were screened, and the clinical information of patients with FLNC gene mutations and their families were collected for Sanger sequencing verification.
Results: FLNC expression was significantly higher in the DCM group than in the control group. After grouping 81 DCM tissues according to median FLNC expression levels, 54 DEGs were identified. The enrichment analysis shows that downregulated DEGs were more associated with inflammation and immunity, while upregulated DEGs involved actin and mitogen-activated protein kinase signaling pathways. The correlation analysis of DEGs and the PPI network identified genes associated with FLNC. According to the immune infiltration analysis, the DCM group was more associated with immunity, and the infiltrating plasma cells had a strong correlation with the FLNC gene; we identified eight miRNAs and 29 lncRNAs that bind to the FLNC gene. The final gene sequencing results show that a total of eight FLNC-specific missense mutations were detected, among which FLNCT407N and FLNCR437L are rare mutations. Additionally, the mutation frequency and minimum allele frequencies determined by sequence comparison were higher than those of databases such as the 1,000Genomes database, and all were predicted to be harmful mutations by SIFT, PolyPhen-2, and Mutation Assessor software. FLNCR437L, FLNCT834M, FLNCG1264S, FLNCR1567Q, and FLNCL2538F mutations are highly conserved among different species and were verified as heterozygous mutations by Sanger sequencing, while FLNCV452M may be the pathogenic site of DCM.
Conclusion: The data analysis of myocardial tissue samples and the mutation analysis of DCM serum samples provides a rich perspective for exploring the biological functions, molecular mechanisms, immune cell correlations, ceRNA networks, and pathogenic gene mutation sites connected to FLNC in DCM.