Abstract
Genetic and epigenetic modifications of genes involved in the key regulatory pathways play a significant role in the pathophysiology and progression of multifactorial diseases. The present study is an attempt to identify single nucleotide variations (SNVs) at CpG sites of promoters of ACAT1, APOB, APOE, CYBA, FAS, FLT1, KSR2, LDLR, MMP9, PCSK9, PHOX2A, REST, SH2B3, SORT1 and TIMP1 genes influencing CpG island (CGI) existence and size associated with the pathophysiology of Diabetes mellitus, Coronary artery disease and Cancers. Promoter sequences located between −2000 to + 2000 bp were retrieved from the EPDnew database and predicted the CpG island using MethPrimer. Further, SNVs at CpG sites were accessed from NCBI, Ensembl while transcription factor (TF) binding sites were accessed using AliBaba2.1. CGI existence and size were determined for each SNV at CpG site with respect to wild type and variant allele by MethPrimer. A total of 200 SNVs at CpG sites were analyzed from the promoters of ACAT1, APOB, APOE, CYBA, FAS, FLT1, KSR2, LDLR, MMP9, PCSK9, PHOX2A, REST, SH2B3, SORT1 and TIMP1 genes. Of these, only 17 (8.5%) SNVs were found to influence the loss of CGI while 70 (35%) SNVs were found to reduce the size of CGI. It has also been found that 59% (10) of CGI abolishing SNVs are showing differences in binding of TFs. The findings of the study suggest that the candidate SNVs at CpG sites regulating CGI existence and size might influence the DNA methylation status and expression of genes involved in molecular pathways associated with several diseases. The insights of the present study may pave the way for new experimental studies to undertake challenges in DNA methylation, gene expression and protein assays.
Highlights
Genetic and epigenetic modifications of genes involved in the key regulatory pathways play a significant role in the pathophysiology and progression of multifactorial diseases
Promoter sequence of ACAT1, Apolipoprotein B (APOB), Apolipoprotein E (APOE), CYBA, Factor associated suicide death receptor (FAS), Fms related tyrosine kinase 1 (FLT1), Kinase suppressor of ras 2 (KSR2), Low density lipoprotein receptor (LDLR), Matrix metalloproteinase 9 (MMP9), Proprotein convertase subtilisin/kexin type 9 (PCSK9), Paired like homeobox 2a (PHOX2A), RE1 silencing transcription factor (REST), SH2B adaptor protein 3 (SH2B3), Sortilin 1 (SORT1) and Tissue inhibitor of metalloproteinase 1 (TIMP1) genes were analysed for the prediction of CpG islands and have observed CpG islands for all the genes (Fig. 2A, B)
In TIMP1 gene, 6 single nucleotide variations (SNVs) were analyzed in 2 CpG islands, the results revealed that 4 SNVs in the first CGI have abolished the entire CGI, whereas the remaining 2 SNVs in the second CpG island have shown a 16-20 bp reduction in their CGI size
Summary
CpG islands (CGIs) in promoter sequence of genes under the study were predicted using MethPrimer v1.1 beta. The tools visualize DNA sequence and their respective annotated genetic variations to identify the SNVs at CpG sites in CpG islands[74,75]. AliBaba2.1 tool was used for the prediction of transcription factor binding sites in wild type and variant alleles of SNVs at CpG sites. It is an online tool to identify transcription factors and their respective binding sites for the input DNA sequence by constructing matrices on Scientific Reports | (2022) 12:3574 |. Gene ontology (GO) enrichment analysis of genes (ACAT1, APOB, APOE, CYBA, FAS, FLT1, KSR2, LDLR, MMP9, PCSK9, PHOX2A, REST, SH2B3, SORT1, TIMP1) was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 online tool (https://david.ncifcrf.gov/home.jsp). GO term enrichment analysis was used to annotate the disease class and functional clustering of genes under the study
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