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DNA methylation analysis of human keratoconus corneas using RRBS sequencing

ABSTRACT: Keratoconus (KTCN) is a complex degenerative eye disorder resulting in loss of visual function. It is known, that its development is affected by both genetic and environmental or behavioral components. In order to verify if DNA methylation may also play a role in KTCN development, reduced representation bisulfite sequencing (RRBS) of human corneas obtained from five KTCN and five non–KTCN individuals was performed.

ORGANISM(S): Homo sapiens

PROVIDER: GSE107189 | GEO | 2019/04/05

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study are to identify differentially expressed genes between keratoconus patient and health control. Methods: We conducted a case: control RNA sequencing study of 7 African American, 12 Middle Eastern subjects, and 7 controls. Total RNA was isolated from individual corneas and sequenced at Macrogen Inc. using the TruSeq stranded RNA library kit. The samples were sequenced at 101 bp using the Illumina HiSeq. 2500 platform. Raw images were generated using the HiSeq Control software v2.2.38, base calling using an integrated primary analysis software, Real time Analysisv1.18.61.0, and converted into FASTQ files using the Illumina package bcl2fastq v1.8.4. The alignment of reads against the human reference genome hg19 was performed using the STAR 2.6.0a software. Followed by gene expression quantification using Cufflinks 2.2.1. The differential expression analysis was conducted by Cuffdiff and DESeq2 (version: 1.24.0). The criteria for significantly differential expression is adjusted p ≤ 0.01, absolute fold change ≥2, and FPKM ≥5 in at least one group. Results: We identified an overwhelming decrease in the expression of anti-oxidant genes regulated by NRF2 and those of the acute phase and tissue injury response pathways, in both patient groups. Concordantly, NRF2 immunofluorescence staining was decreased in patient corneas, while KEAP2, which helps to degrade NRF2, was increased. Diminished NRF2 signaling raises the possibility of NRF2 activators as future treatment strategies in keratoconus. Although separated by geography and ancestry, key commonalities in the two patient transcriptomes speak of disease intrinsic gene expression networks. The African American patient group also showed increases in extracellular matrix transcripts that may be due to underlying profibrogenic changes in this group. Transcripts increased across all patient samples include Thrombospondin 2 (THBS2), encoding a matricellular protein, and cellular proteins, GAS1, CASR and OTOP2, and are promising biomarker candidates. Conclusions: Our approach of analyzing transcriptomic data from different populations and patient groups will help to develop signatures and biomarkers for keratoconus subtypes. Further, RNA sequence data on individual patients obtained from multiple studies may lead to a core keratoconus signature of deregulated genes and a better understanding of its pathogenesis.

Project description:Purpose: We compared the levels of miRNA specific for DEGs in isograft corneas with those in normal corneas, as well as the levels of miRNA specific for DEGs in allograft corneas with those in isograft corneas, to gain a better understanding of molecular variables that affect corneal graft rejection pathways. Methods: Illumina Hiseq 2500 deep sequencing was used to screen for differentially expressed genes (DEGs) in matched pairs of isograft corneas and normal corneas, allograft corneas and isograft corneas. Potential target genes among the DEGs were predicted using target prediction software (TargetScan, Miranda, miRDB, and CLIP), and the overlay portion was analyzed using the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG). An analysis of the interactions between DEG proteins (PPI analysis) and a MetaCore software analysis. Results: Our results showed that 22 miRNAs were significantly upregulated and 4 were significantly downregulated in the isograft group when compared with the control group (P < 0.01), while 17 miRNAs were significantly upregulated and 3 were significantly downregulated in the allograft group when compared with the isograft group (P < 0.01). Among the miRNAs with altered expression levels, miR-155-5p, miR-142-3p, miR-142-5p, and miR-223-3p displayed simultaneous changes in the above two comparisons. Potential target genes among the DEGs were predicted using target prediction software, and the overlay portion was analyzed using the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG). GO and KEGG analyses showed that the DEGs were mainly involved in metabolic pathways, cytokine secretion, and tumor immunity functions. An analysis of the interactions between DEG proteins (PPI analysis) and a MetaCore software analysis of 4 key DEGs revealed that the genes regulated by miR-155-5p played important roles in the miRNA-mRNA regulatory network. Furthermore, the MetaCore analysis identified C/EBP beta, p53, and sp1 as key transcription factors in that network. Conclusions: Our study identified transplanted corneas-specific miRNA in matched pairs of isograft corneas and normal corneas, allograft corneas and isograft corneas. Furthermore, bioinformatics analysis of the key miRNA regulatory network revealed the molecular mechanisms, which suggests miRNAs may as new molecular targets for treating corneal injuries and corneal transplant rejection

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DNA methylation analysis of human keratoconus corneas using RRBS sequencing

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