Bioscience Biotechnology Research Communications

An Open Access International Journal

P-ISSN: 0974-6455 E-ISSN: 2321-4007

Bioscience Biotechnology Research Communications

An Open Access International Journal

Alanoud Bakheet Alzahrani, Hani Mohammed Ali and Ahmad Firoz*

Department of Biological Science, Faculty of Science, King Abdulaziz
University, Jeddah, Kingdom of Saudi Arabia.

Corresponding author email: ahmadfirozbin@gmail.com

Article Publishing History

Received: 25/09/2021

Accepted After Revision: 24/12/2021

ABSTRACT:

Proliferative diabetic retinopathy is the widespread type of DM which causes chronic as well as progressive alterations at microvascular level, which particularly effects the eye. The main characteristic of this disease is the development of few new blood vessels around the retina of eye as well as at the posterior region of eye segments. For our computational analysis 155 differentially expressed genes calculated through paired t test statistics analysis using the GenePattern platform, of proliferative diabetic retinopathy in Saudi patients were downloaded. Among the 155 genes, 95 were upregulated, and 60 were downregulated. The Annotation Cluster (FAC) tool in the (DAVID)

(http://david.abcc.ncifcrf.gov/home.jsp) was used to identify biological processes that are abundant in proliferative diabetic retinopathy (PDR). The functions required for response to mRNA splicing, intracellular protein transport, mRNA processing, microtubule cytoskeleton structure, and atrioventricular canal formation are represented by the GO keywords that are abundant in genes. We used the KAAS web server to identify the biological pathways of these DEGs in addition to DAVID functional analysis and found that the majority of the DEGs were associated with important biological processes, with many being classified in metabolic pathways, Spliceosome, Cell cycle, or being involved in the mRNA surveillance pathway. findings are consistent with those of earlier research. To corroborate the predictions stated in this work, which will demonstrate the role enhanced functional processes, experimental validation will be necessary.

KEYWORDS:

Computational Analysis, Diabetes Mellitus, Gene Expression, Proliferative Diabetic Retinopathy

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Alzahrani A. B, Ali H. M, Firoz A. Gene Expression in Proliferative Diabetic Retinopathy using RNA-Seq Data: A Computational Study on Saudi Patients. Biosc.Biotech.Res.Comm. 2021;14(4).


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Alzahrani A.B, Ali H.M, Firoz A. Gene Expression in Proliferative Diabetic Retinopathy using RNA-Seq Data: A Computational Study on Saudi Patients. Biosc.Biotech.Res.Comm. 2021;14(4). Available from: <a href=”https://bit.ly/31bFKFa“>https://bit.ly/31bFKFa</a>


INTRODUCTION

Diabetes mellitus is one of the multifactorial diseases and a leading cause of death in world and especially in Saudi Arabia. Proliferative diabetic retinopathy is the widespread type of DM which causes chronic as well as progressive alterations at microvascular level, which particularly effects the eye, along with other body parts. If the disease is left untreated it will grow gradually and ultimately leading to the blindness. Progression of disease is not rapid, but gradual starting from mild alterations, moving towards moderate and ultimately severe proliferative diabetic retinopathy. The main characteristic of this disease is the development of few new blood vessels around the retina of eye as well as at the posterior region of eye segments i.e., vitreous (El-Bab et al. 2012; Lee et al. 2015; Alharbi and Alhazmi 2020).

The mechanism by which the DM progresses to diabetic retinopathy is not clearly understood and that’s why the disease pathology is thought to be complex and unclear. However, a lot of studies has been carried out to examine the disease progression by considering the disease history along with other aspects. It has been suggested that multiple interactive mechanisms are playing an important role, causing the damage at cellular level and adaptive changes, which cause the devastation in this disease(El-Asrar et al. 1998; Sinclair and Schwartz 2019; Alharbi and Alhazmi 2020).

Earlier it was considered that DM and especially PDR is not a prevalent disease at Saudi Arabia, due to healthy diet and routine. However, recent studies have reported that prevalence of disease is increasing in Saudi Arabia as bell and the possible risk factors for this progression are supposed to be consumption of more westernized diet leading to increased chances of obesity and ultimately complications of diabetes. Earlier the disease was 23.7% prevalent in Saudi Arabia while by the year (2011), it has reached to increase 30% and increasing day by day with men more affected that females (Ali et al. 2008; Al Dawish et al. 2016; Alharbi and Alhazmi 2020).

Different treatment strategies can be used to treat diabetic retinopathy. Photocoagulation is one of them. Studies has shown that photocoagulation approach causes a decrease in chances of loss of vision by up to 50% (Cantrill 1984). It causes the decrease in visual acuity as well as constricts the posterior visual regions. Intravitreal administration of  about 1.25 mg bevacizumab at the time of cataract surgery could be safe as well as protective in preventing the progression of DR and diabetic maculopathy in patients with cataract and diabetic retinopathy (Cheema et al. 2009; Alghamdi et al. 2021).

MATERIAL AND METHODS

155 differentially expressed genes calculated through paired t test statistics analysis using the GenePattern platform, and identified based on the statistical cutoff of proliferative diabetic retinopathy in Saudi patients with type 2 diabetes were downloaded (Pan et al. 2016). Among the 155 genes, 95 were upregulated, and 60 were downregulated, and has been taken for computational analysis shown in Table 1.

For the functional analysis, on the list of differentially expressed genes with a fold change of >1, DAVID (http://david.abcc.ncifcrf.gov/home.jsp) functional annotation cluster analysis was done. For analysis, only terms with a value of 0.05 and a count number of 5 genes were chosen. DAVID was used to classify enriched biological themes in the collection of DEGGs using the gene ontology (GO) term biological process (BP). The KEGG Automatic Annotation Server (KAAS) (http://www.genome.jp/kegg/kaas/) was used to map pathways (Moriya et al. 2007).

The amino acid sequences of these DEGGs were submitted to the KAAS online site as input, and orthologs were assigned using the single-directional best hit (SBH) technique. KAAS uses BLAST similarity searches against a carefully selected set of ortholog groups in the KEGG GENES database to provide functional annotation of genes in a genome. Genes in the data sets that were mapped to one of KEGG’s reference pathways were given a KEGG orthology (KO) number by KAAS (Amoaku et al. 2020).

Table 1. A list of 155 differentially expressed gene selected for analysis (Pan et al. 2016).

ZNF207 UTRN CTR9 TRPS1 ZNF80 CAV2 IL33
SMAD4 TARDBP YTHDC1 USP8 KCNH3 LOC285847 ADRA1D
SEPT2 SFPQ MIA3 MEAF6 LOC100506124 RAMP1 TAAR1
ORC2 CEP350 UBL4A FAM69B RNPC3 LOC100506995 OR4K17
MTCH1 FAM208B GPR18 MGC72080 TUBG1 PRSS27 KDR
SENP1 ECD KCTD4 RBM17 PSAT1 GPRC5D LOC440910
HELZ CORO7 BTBD2 RQCD1 PCDHGC4 NCKAP5
TTC17 EI24 TUG1 MMGT1 DLC1 HLA-DQA2
MAN2A1 STARD3 C11orf30 LATS1 DDX11L9 LINC00346
KLHL11 SYTL1 ZNF597 WEE1 CTHRC1 RCVRN
PTPN11 EIF3A SEC63 HIST1H3I RNU4-2 DYX1C1
SSR1 ASTE1 STX17 SEPN1 DPY19L2 MFAP5
DUSP11 CHMP6 VPS36 CLOCK NTSR1 OVCH1
TMED10 HNRNPA3 PSMD5 LINC00265 IFITM10 PNMT
DYNC1LI2 PSIMCT-1 FAM190B PPP6C ACY3 PPP1R14C
SLC39A3 TMEM39A DTWD2 LOC729852 FP588 CCDC144NL
SLC33A1 CCND3 RBM25 PAICS NXPH3 LOC100505806
ARHGEF6 PACS2 PRIM2 LOC100652890 LOC100506476 GS52
PAPOLG PDE4D ZNF784 HSP90B1 NRXN2 HRH3
HNRNPU KIAA2026 CG030 EYS LOC100506678 PP2672
ASH1L CALU ABRA MOB3B LOC100507144 CPSF4L
LOC729082 AP3M1 PDCD4 PNN PNMAL2 RTL1
USP48 MASP2 CCAR1 HNRNPA2B1 BCL6B LOC100128081
ZDHHC6 ALG2 SOS1-IT1 SDHAP2 TDRD10 CNPY1
UTRN DDX46 LOC283624 RAB40B HCAR1 UGT2A3


RESULTS AND DISCUSSION

We downloaded the precomputed list of 155 differentially expressed genes for our computational analysis shown in Table-1Among the 155 genes, 95 were upregulated, and 60 were downregulated (Pan et al. 2016; Amoaku et al. 2020).  

For the functional annotation analysis, the Annotation Cluster (FAC) tool in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify biological processes that are enriched in proliferative diabetic retinopathy (PDR) (http://david.abcc.ncifcrf.gov/home.jsp). For annotations and GO terms with statistically significant values from the resultant functional analysis, the name “Biological Process” was utilized. The functions required for response to mRNA splicing, intracellular protein transport, mRNA processing, microtubule cytoskeleton structure, and atrioventricular canal formation are represented by the GO keywords that are abundant in genes in this table (Table 2).

Table 2. Significantly enriched gene ontology (GO) terms detected by FAC in differentially expressed genes.
Only those terms which reported a value of ≤0.05 and count number ≥2 genes were selected for the analysis.

For the pathway analysis, we found the biological pathways of DEGs annotated in the current study in addition to DAVID functional analysis. DEG amino acid sequences in FASTA format were put into the KAAS to predict different pathways. There was a total of 154 routes predicted. Table 3 lists the top 20 KEGG pathways, with Supplementary Table S1 providing a comprehensive list of all pathways. The majority of DEGs were discovered to relate to significant biological processes, with many being categorized in metabolic pathways, spliceosomes, or cell cycle, or being engaged in the mRNA monitoring pathway, as seen in these tables (Amoaku et al. 2020).

Table 3. Top 20 KEGG pathways for DEGs, Number of mapped genes shown in bracket

ko01100 Metabolic pathways (10)
ko03040 Spliceosome (5)
ko04110 Cell cycle (4)
ko05164 Influenza A (4)
ko04144 Endocytosis (4)
ko01110 Biosynthesis of secondary metabolites (4)
ko05205 Proteoglycans in cancer (4)
ko04080 Neuroactive ligand-receptor interaction (4)
ko03015 mRNA surveillance pathway (4)
ko05132 Salmonella infection (3)
ko05200 Pathways in cancer (3)
ko04141 Protein processing in endoplasmic reticulum (3)
ko04151 PI3K-Akt signaling pathway (3)
ko05166 Human T-cell leukemia virus 1 infection (3)
ko04020 Calcium signaling pathway (3)
ko05168 Herpes simplex virus 1 infection (3)
ko05207 Chemical carcinogenesis – receptor activation (3)
ko04510 Focal adhesion (3)
ko04390 Hippo signaling pathway (3)
ko05418 Fluid shear stress and atherosclerosis (3)
ko05014 Amyotrophic lateral sclerosis (3)

Recent studies have reported prevalence of Proliferative diabetic retinopathy (PDR) disease is increasing. Proliferative diabetic retinopathy is the widespread type of DM which causes chronic as well as progressive alterations at microvascular level, which particularly effects the eye. The main characteristic of this disease is the abnormal growth of new vessels occurs (Tarr et al. 2013; Safi et al. 2014). Study shows Involvement of angiogenesis, inflammation, and fibrosis in proliferative diabetic retinopathy and Enrichment of genes and pathways related to lymphatic development indicates that targeting lymphatic involvement in PDR progression.

Several pro-angiogenic cytokines have been described as being involved in the pathogenesis of PDR, although VEGF is accepted as the most significant cytokine in PDR (Amoaku et al. 2020). The present finding shows significance of mRNA splicing, intracellular protein transport, mRNA processing, microtubule cytoskeleton organization and atrioventricular canal development, and associated with important biological processes, many being classified in metabolic pathways, Spliceosome, Cell cycle or being involved in mRNA surveillance pathway These are consistent with those of other studies (Korhonen et al. 2021).

CONCLUSION

The findings of the present study have used a Bioinformatics approach to identify the DEGs enrichment indicate the significance of mRNA splicing, intracellular protein transport, mRNA processing, microtubule cytoskeleton organization and atrioventricular canal development, and associated with important biological processes, many being classified in metabolic pathways, Spliceosome, Cell cycle or being involved in mRNA surveillance pathway The present study’s findings are consistent with those of earlier research. To corroborate the predictions stated in this work, which will demonstrate the role enhanced functional processes, experimental validation will be necessary.

ACKNOWLEDGEMENTS

This study was technologically supported by the Bioinformatics and Computational Biology Unit at Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.

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