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Gene Interactions in Cerebral Cavernous Malformations: A Brief Report

Submitted:

12 May 2024

Posted:

13 May 2024

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Abstract
Cerebral cavernous malformation (CCM) is a collection of irregular small blood vessels that may be present in the brain or spinal cord. These vessels contain slow – moving blood that commonly clot. These malformations are frequently caused by mutations in one of the CCM genes. The CCM1 (also known as KRIT1) gene is essential for vascular morphogenesis however, its interactions with transcriptional regulators are unknown. Inhibitor of DNA-Binding/Differentiation-3 (ID3) has been recognized to be involved in different vascular/blood vessel diseases such as peripheral arterial disease, stroke, arteriovenous malformations, and atherosclerosis. We show interactions between ID3 and additional key differential expressed genes (DEGs) in microarray data of overexpressed CCM1 in endothelial cells through bioinformatic and data analytic tools. Improved understanding of how ID3, CCM1, and DEGs interact will play an important role in adding to the increasing knowledge for creating therapeutic targets for cerebral cavernous malformations.
Keywords: 
cerebral cavernous malformations
;  
CCM1
;  
differential expressed genes
;  
gene networks
;  
ID3
Subject: 
Biology and Life Sciences  -   Life Sciences

Introduction

Cerebral cavernous malformations (CCMs) are a type of vascular lesion. CCMs are not a major underlying cause of cerebrovascular or neurovascular disease but their investigation has provided unexpected genetic and molecular insight into vascular disease and development [1,2]. Sporadic CCMs account for 80 – 85% frequently presented as isolated lesions, while familial CCMs account for 15 - 20% followed by autosomal dominant inheritance patterns and present with multiple lesions. Presently, it is widely known that the initial triggers of both sporadic and familial CCM formation have been accredited to genetic mutations [2,3]. The disease is caused by mutations in one of the three known CCM genes: CCM1 (also known as KRIT1), CCM2 (also known as OSM), or CCM3 (also known as PDC10) [3,4]. CCM1 is expressed in endothelial cells and astrocytes. Furthermore, it can be associated with membranes, adheres junctions, and the nucleus [3,4]. Inhibitor of DNA Binding/Differentiation – 3 (ID3), which is part of a group of genes (ID1, ID2, ID3, and ID4), has been known to be expressed in endothelial cells and important for endothelial cell activation and embryonic vasculogesis [5,6,7,8]. While known as a transcriptional regulator that prevents stem cell differentiation, ID3 has been identified to show overlapping functions such as gene knockout dependent on cellular context and can be activated from external environmental exposure [8,9,10,11,12,13,14,15,16]. While ID3 has been associated with vascular malformations such as Hereditary Hemorrhagic Telangiectasia [6,7], little is known about the association between ID3 and cerebral cavernous malformation. This study shows the interaction between ID3 and additional key genes in overexpressed CCM1 samples in cerebral cavernous malformation. Overall, this information can open up an avenue for further investigation toward diagnostic targets for CCM.

Methods

Data from the NCBI Gene Expression Omnibus (GEO) database, a public functional genomic data repository that supports minimum information about a microarray experiment (MIAME) - compliant data submissions, were used to demonstrate how DEGs and gene - network connections play a vital role in the interaction of overexpressed CCM1 in endothelial cells of cerebral cavernous malformations [17]. The results were supported by GSE18014 which was downloaded from GEO and used during our analysis. The original study information consisted of 4 patient samples (2 overexpressed CCM1 and 2 controls). We used the Limma-Voom R package to identify differentially expressed (DE) mRNAs from the featureCounts output. The Log2 fold change (log2FC) values for each mRNA were calculated by comparing the expression levels in patient samples to those in control samples. The threshold for statistical significance was set at p < 0.05 [18,19,20]. We further examined the interaction of the top 10 upregulated DEGs (log2 fold change > 2) and used GeneMania, a publicly available software that helps predict the function and interaction of genes [21].

Results

Microarray data from GSE18014 deposited in NCBI GEO database was used to investigate gene expression patterns. We performed a differential expressed gene analysis as shown in the volcano plot in Figure 1. Each dot signifies a gene with significant alterations based on their position relative to the fold change and p-value levels. The red dots denote upregulated genes and the blue dots denote downregulated genes. Additionally, we have listed the top 10 upregulated genes shown in Table 1 with genes that have a fold change > 2. These genes include: IGFBP3, CH25H, AQP5, CROT, VIPR1, CLEC3B, HIST1H2BD, TMEM100, ID3, and MOBP. The interaction between the top 10 upregulated DEGs is shown in Figure 2.

Discussion

Our research results add to the collective evidence that key DEGs are significant for future diagnostic and therapeutic targets toward cerebral cavernous malformations. By using NCBI GEO, the R package, and GeneMania; we discovered interactions between important genes within overexpressed CCM1 samples. This has provided valuable insight into the complex molecular and mechanistic processes involved in CCM. Previously, it has been demonstrated that ID3 has interactions with various vascular/blood vessel diseases [5,6,7,8,9,10,11,12,13,14,15,22,23] however; this new information can help to build the foundation for additional research within the focus of gene expression networks, bioinformatics, and data analytics. The small sample size in the GEO study for our analysis of DEGs might restrict statistical power, which increases the risk of type II errors and bias. In order to address this for future studies, larger sample sizes are warranted.

Conclusion

Our study demonstrates the interaction between overexpressed CCM1, ID3, and additional differential expressed genes. Through the integrative use of data analytics and bioinformatic tools; we have successfully mapped gene networks that show connections between these important genes including: IGFBP3, CH25H, AQP5, CROT, VIPR1, CLEC3B, HIST1H2BD, TMEM100, ID3, and MOBP. Further investigation should be focused on gene expression analysis and known transcriptional regulators such as ID3 in order to address the unmet need of targeted therapies for cerebral cavernous malformation. Better understanding of this data can lead to improved diagnostic tools for the future.

Author Contributions

V. A. conceptualized, designed, conducted, and wrote the manuscript.

Funding Sources

This study was not supported by any sponsor or funder.

Data Availability Statement

The data used for the analysis is deposited at NCBI GEO (GSE18014). Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The author has no conflict of interest to declare.

Statement of Ethics

An ethics statement was not required because this study is based on publicly deposited and accessible data.

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Figure 1. The volcano plot shows the differentially expressed genes in the overexpressed CCM1 cerebral cavernous malformation samples versus the control samples. Each dot represents a gene with significant changes based on their position comparative to the fold change and p-value levels.
Figure 1. The volcano plot shows the differentially expressed genes in the overexpressed CCM1 cerebral cavernous malformation samples versus the control samples. Each dot represents a gene with significant changes based on their position comparative to the fold change and p-value levels.
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Figure 2. GeneMania was used to show the gene-interaction and co-expression of the top 10 upregulated genes (fold change > 2). The gene networks show 20 related genes and co-expression (shown by the lines in purple), which accounts for 89.14% of the network.
Figure 2. GeneMania was used to show the gene-interaction and co-expression of the top 10 upregulated genes (fold change > 2). The gene networks show 20 related genes and co-expression (shown by the lines in purple), which accounts for 89.14% of the network.
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Table 1. Top 10 upregulated genes (fold change > 2) in overexpressed CCM1 versus control samples.
Table 1. Top 10 upregulated genes (fold change > 2) in overexpressed CCM1 versus control samples.
Gene Symbol Gene Title Log2 (Fold Change)
IGFBP3 insulin like growth factor binding protein 3 3.074
CH25H cholesterol 25-hydroxylase 2.644
AQP5 aquaporin 5 2.615
CROT carnitine O-octanoyltransferase 2.559
VIPR1 vasoactive intestinal peptide receptor 1 2.269
CLEC3B C-type lectin domain family 3 member B 2.221
HIST1H2BD histone cluster 1, H2bd 2.212
TMEM100 transmembrane protein 100 2.051
ID3 inhibitor of differentiation 3 2.043
MOBP myelin-associated oligodendrocyte basic protein 2.021
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