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Original Research *Corresponding author Anfal Ibrahim Bahereldeen, Department of medical laboratory sciences, Sudan University of Science and In silico analysis of Single Technology, Khartoum, Sudan; Tel: 249916060120; Email: [email protected] Submitted: 14 October 2019 Nucleotide Polymorphisms Accepted: 23 January 2020 Published: 27 January 2020 (SNPs) in Human HFE Gene ISSN: 2576-1102 Copyright coding region © 2020 Bahereldeen AI, et ail. OPEN ACCESS Anfal Ibrahim Bahereldeen1*, Reel Gamal Alfadil2, Hala Mohammed Ali2, Randa Musa Nasser2, Nada Tagelsir Eisa2, Keywords Wesal Hassan Mahmoud2, and Shaymaa Mohammedazeem • In silico • HFE gene, Polyphen-2 2 Osman • I mutant 1Department of Medical laboratory sciences, Sudan University of science and • Project hope Technology, Sudan 2Department of Medical laboratory sciences, Al-Neelain University, Sudan

Abstract

Background: HFE gene is a HLA class1-like molecule, expressed in the different cells and tissues, mutations on this gene reported to cause about 80-90% of Hereditary haemochromatosis (HHC) and it is increasing the risk of different diseases. In this study; we aimed to analysis the SNPs in HFE gene by using different computational methods.

Methodology: We obtained HFE gene nsSNPs from dbSNP/NCBI database, Deleterious nsSNPs predicted by different bioinformatics servers including; SIFT, polyphen-2, I-mutant and SNPs & GO servers. Protein structure analysis done by using Project hope and RaptorX tools then visualized by Chimera software and function, interaction and network of HFE gene analysis done by Gene MANIA program.

Results: SIFT and Polyphen-2 servers predicted 75 deleterious nsSNPs and nine polymorphisms from them predicted as highly damaging and disease associated.

Conclusion: In silico analysis of single nucleotide polymorphisms is better for understanding different genetic disorders, and give helpful information for future candidate studies.

ABBREVIATIONS frequently found in patients suffering from one of the most common types of porphyria known as porphyria cutanea because HFE: Human Factor Engineering, HHC: Hereditary of abnormal deposition of iron in the body may cause iron toxicity Haemochromatosis; nsSNPs: Non-synonymous Single Nucleotide which cause an abnormal building up of porphyrins [11,12], also Polymorphism; dbSNPs: Single Nucleotide Polymorphism HFE gene variants increase the risk of; a coronary heart disease Database; NCBI: National Center of Bioinformatics; Polyphen: (CHD) [13], Alzheimer disease [14] and increase the risk of the Polymorphism Phenotype; HLA: Human Leukocyte Antigen; hepatocellular carcinoma, breast cancer, colorectal cancer and GWAS: Genome-Wide Association Studies; CHD: Coronary Heart others types of cancers [15-19]. Disease; SNP: Single Nucleotide Polymorphism; DDG: Free Energy Change Value; RI: Reliability Index Numbers of databases used to store the SNPs, like dbSNP Database from NCBI, which containing a large number of a INTRODUCTION discovered SNPs in a human genome and other domains [20], Homeostatic iron regulator (HFE) gene on the short arm also tools such as SIFT, Polyphen2 and others used to analysis of the chromosome 6 in position 6p22.2 within the extended of functional and structural effects caused by the genetic HLA class I region, encode for membrane protein, associated polymorphisms and to study the roles of SNPs on various human with beta-2 micro-globulin, this protein located on the surface diseases to help researchers and aid them in analysis and further of intestinal cells, liver cells and other cells, it interacts with studies of different diseases [21-27]. Therefore, this study focuses different proteins to discover the iron levels [1-4]. Mutations on analysis of non-synonymous missense SNPs of HFE gene by in HFE gene associated with more than 80% of Hereditary using computational tools to highlight deleterious mutations and haemochromatosis (HHC) disorder [1], HHC is an autosomal to predict the structural and functional consequences of these recessive disorder characterized by increasing iron absorption polymorphisms. by the intestinal tract and abnormal deposition in parenchymal MATERIALS AND METHODS organs, without treatment, this condition can cause severe diseases [5,6]. Several genome-wide association studies (GWAS) We have obtained HFE gene SNPs from dbSNP database, performed to investigate the effect of HFE genes mutations one of the largest websites canting wide collection of single on body iron concentration [7-10], HFE gene mutations were nucleotide variations, available at: http://www.ncbi.nlm.nih.

Cite this article: Bahereldeen AI, Alfadil RG, Ali HM, Nasser RM, Eisa NT, et al. (2020) In silico analysis of Single Nucleotide Polymorphisms (SNPs) in Human HFE Gene coding region. J Bioinform, Genomics, Proteomics 5(1): 1040. Bahereldeen AI, et ail. (2020)

gov/snp and FASTA format of the HFE proteins has obtained I-Mutant suite [I mutant 3.0] from ensemble database: https://feb2014.archive.ensembl.org Is an online Bioinformatics server available at http://gpcr2. then the analysis of single nucleotide polymorphisms done by biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi, using various Bioinformatics tools (Figure 1). it depends on numbers of data set including; Gibbs frees energy SIFT software [SIFT Predict effects of nonsynonymous/ change, enthalpy change, transition temperature and heat missense variants] capacity change which obtained from thermodynamic databases, to predict the effect of the amino acid changed on protein stability at; http://sift.bii.astar.edu.sg/, used to predict the effect of and predication of their result depending on the reliability index damageOne causedof the firstsby SNPs online in a Bioinformatics particular gene, website, SIFT is available working (RI), which is ranging from (0-10) [28]. We have submitted HFE by taking a query sequence from protein protein sequence and mutations to predict the effects of damaging and making several comparisons at the mutant amino acids nsSNPs on protein stability, all tests done at temperature 25C° to predict if this mutation tolerated or not depending on the and pH 7.0 and the results predicated as decrease or increase in protein stability. dbSNP reference numbers (rs numbers) of HFE gene as batch 3d specific score determined between 0-1 [21]. We have submitted SNPs & GO [SNPs&GO ]: [predictor of Human Deleterious Single Nucleotide Polymorphisms] as deleterious and those with SIFT score > 0.05 predicted as tolerance.into the server, the results with SIFT score of ≤ 0.05 predicated Online database http://snps.biofold.org/snps-and-go/ snps-and-go.html, acts to drive collection of unique framework PolyPhen-2[Polymorphism phenotyping v2] information about protein sequence, evolutionary information Is a free online Bioinformatics website available at http:// and function as encoded in the terms, and genetics.bwh.harvard.edu/pph2/index.shtml, used to predict the automatically do other available tests methods to predicate if effects of amino acids changing on the structural and function the change of amino acid on protein sequence was disease related of the proteins, it depends on several proteomics databases to or neutral [29,30]. We have submitted HFE protein sequence and search for protein 3D structure and makes multiple alignments mutations then selected all Methods to option the server to do between the homologous sequences and newly-formed amino predictions using S3D-PROF, SNPs & GO and PhD-SNPs methods. acid then determines the difference in amino acid properties Gene MANIA (PSIC) for each substitution, which indicate the possibility of Is a cytoscape server available at http://www.genemania.org, and calculates the position-specific independent count scores the damage [22]. We have submitted protein query sequence used for Function, interaction and network of gene analysis; it in FASTA format with each mutations position separately to the acts on numbers of functional association information including server then the results predicated as probably damaging if the physical interactions, genetic interactions, pathways, co- PSIC score equal 2.00 or more, possibly damaging if PSIC score between (1.40–1.90), potentially damaging if PSIC score between obtained from the different biological database [31]. (1.0–1.30) and benign when PSIC score between (0.00–0.90). expression, phylogenetic profiles, protein domain and other data HFE protein 3D structure modeling and analysis programs HFE gene Project hope [Version0.4.1]: Is an online proteomic database developed at the center of molecular and biomolecular nsSNPs from the dbSNP/NCBI informatics (CMBI) at Radboud University in Nijmegen, the Gene MANIA program available at http://www.cmbi.ru.nl/hope/, it works by obtaining structural information from different sites, including SIFT software 3D structure of protein, sequence annotations in UniProt and predictions from DAS-servers, to analysis the impact of the amino polyhen2 server acid change on the protein structure [32]. We have submitted nsSNPs that predicted as highly damaging by both SIFT and Polyphen-2 databases to project hope server as protein sequence I-mutant server SNPs&GO and PhD servers in FASTA format with position of each mutation, native amino acid and the new substituent, than the server provided us with the complete report about the effect of each SNPs on the structure proteins 3D structures modeling obtained by using: of HFE protein. RaptorX [RaptorX Structure Prediction]: Online free server for protein structure and function prediction available Projecthope server RaptorX server at http://raotorx.uchicago.edu/ [33], we have submitted the HFE protein sequence into the RaptorX server, then it provided Chimera-1.12 server us with secondary and tertiary structures in addition to contact map, solvent accessibility, disordered regions, binding sites for Figure 1 Software used in HFE gene SNPs analysis. the given sequence and reliability scores. The tertiary structures

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of HFE gene that was given by RaptorX treated by Chimera-1.12 exon 2, is the third common HFE gene mutation involved in the Bioinformatic server which is the program of high quality used pathogenesis of haemochromatosis [35-38] for visualization and investigation of molecular assemblies and In this study a total of 1443 Homo sapiens HFE gene related data, it designed by University of California, San Francisco SNPs, obtained from dbSNP/NCBI in September 2018 , for (UCSF) available at: http://www.cgl.ucsf.edu/chimera [34]. computational analysis; 163 SNPs of them were missense, 12 RESULTS AND DISCUSSION nonsenses, eight frame shift and 236 in 3’UTR, 31 in 5’UTR and the remaining were other types. We have selected only non- Two missense mutations; C282Y (rs 1800562) and H63D synonymous coding SNPs for our investigation. (rs 1799945) reported as common HFE gene mutations, C282Y Functional analysis of polymorphism in HFE gene is the most common in Northern European populations and coding region by using SIFT and Polyphen-2 servers H63D has a global distribution and case about 40-70% HFE non-C282Y haemochromatosis, a new HFE amino acid variant SIFT software predicted 84 deleterious SNPs, 75 nsSNPs S65C/rs1800730 that reported in position 193 of the HFE gene of them predicated as damaging by Polyphen2 server and nine

Table 1: Highly damaging nsSNPs results obtained by; PolyPhen-2, SIFT, I-Mutant and SNPs&GO servers. Amino PolyPhen-2 SIFT I MUTANT PhD-SNP SNPs&GO acid Change Effect Score Prediction Index DDG RI Predation Predation probability RI Predation Probability RI Probably C194Y 1 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.844 7 damaging Probably C282Y 0.999 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.845 7 damaging Probably C102Y 1 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.87 7 damaging Probably C190Y 1 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.87 7 damaging Probably C176Y 1 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.876 8 damaging Probably C259Y 1 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.845 7 damaging Probably C279Y 1 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.845 7 damaging Probably C180Y 1 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.843 7 damaging Probably C268Y 0.999 Deleterious 0 0.02 1 Increase Disease 0.977 10 Disease 0.425 2 damaging

Table 2: GeneMANIA results of genes co-expression and share domain with HFE gene. Gene symbol Description Co-expression shared domain ATF2 activating transcription factor 2 Yes No TFRC transferrin receptor Yes No B2M beta-2-microglobulin Yes Yes TFR2 transferrin receptor 2 Yes No MR1 major histocompatibility complex, class I-related Yes Yes GALNS galactosamine (N-acetyl)-6-sulfatase Yes No HLA-E major histocompatibility complex, class I, E Yes Yes ID3 inhibitor of DNA binding 3, HLH protein Yes No IGFBP4 insulin like growth factor binding protein 4 Yes No TPP1 tripeptidyl peptidase 1 Yes No HLA-B major histocompatibility complex, class I, B Yes Yes MICA MHC class I polypeptide-related sequence A Yes Yes HLA-C major histocompatibility complex, class I, C Yes Yes TPMT thiopurine S-methyltransferase Yes No IL13RA1 interleukin 13 receptor subunit alpha 1 Yes No DSE dermatan sulfate epimerase Yes No CFI complement factor I Yes No CD44 CD44 molecule (Indian blood group) Yes No PRICKLE3 prickle planar cell polarity protein 3 Yes No HLA-F major histocompatibility complex, class I, F Yes Yes

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Table 3: GeneMANIA results of HFE gene functions and its appearance nine mutations were replacing the Cysteine residue to Tyrosine in network and genome. mutations identified as highly damaging by both servers, all the Genes in different positions(194, 282, 102, 190, 176, 259, 279, 180, Genes in Feature FDR in network 268)/ (rs 1800562) (Table 1 and Appendix Table 1). genome Antigen binding 3.44E-9 7 59 Note: All 84 nsSNPs that predicted as intolerant by SIFT server, used for farther investigation as fallow: Phagocytic vesicle membrane 2.17E-7 5 22 Antigen processing and 2.26E-7 8 216 Prediction of the protein’s stability by I MUATANT presentation Interferon-gamma-mediated server 5.53E-7 3 76 signaling pathway ER to Golgi transport vesicle It results are 69 SNPs as decrease stability of HFE proteins 5.55E-7 5 31 membrane HFE ER to Golgi transport vesicle 7.55E-7 5 35 protein (Table 1 and Appendix Table 2). (Appendix Table 2) and fifteen SNPs increase stability of early Endosome membrane 7.55E-7 5 35

Investigation of disease-mutations association It done by PhD-SNP method that predicated 43 nsSNPs as disease associated and SNP&GO method that predicated only 27 nsSNPs as disease associated, all the highly damaging nine mutations on (rs 1800562) predicated as disease associated by both methods (Table 1 and Appendix Table 2). Function, interaction and network of HFE gene analysis by using GeneMANIA software Gene MANIA server detected that HFE gene was co-expressed with twenty genes and shares the same protein domain with seven of them (Figure 2 and Tables 2 and 3). HFE protein 3D structure modelling The effect of the amino acid changes on 3D structure of HFE protein ware explained by project hope software for all nine GeneMANIA result of functional interaction between HFE Figure 2 highly damaging mutations [Cysteine residue to Tyrosine in gene and its related genes. different positions(194, 282, 102, 190, 176, 259, 279, 180, 268)/ (rs 1800562)], the reports indicated that the mutations in 100% conserved region for all, the mutant residues were bigger than the wild-type residues and the wild-type residues were more hydrophobic than the mutant residues, the wild-type residues annotated in Uniprot to involve in a cysteine bridge, which is important for stability of the protein, the mutations causes’ loss of this interaction and have a severe effect on the 3D-structure of the protein and the variants annotated with severe disease (Figure 3), but C282Y mutation was not given report by project hope software and their protein 3D structure obtained from Raptor X server and treated by Chimera tool (Figure 3,4,5). Figure 3 Project hope model for SNP ID: rs1800562/ C102Y. CONCLUSION HFE gene SNPs analyzed by using translation bioinformatics prediction tools; for functional and structural analysis of mutations in nonsynonymous coding region and to analysis functions and interaction of HFE gene with functional similar genes. Nine SNPs on (rs 1800562) predicted as highly damaging, increase the protein stability, have a severe effect on the 3D-structure of the protein and disease associated. ACKNOWLEDGMENTS Figure 4, 5 Protein 3D structure models by Chimera server for SNP The authors are acknowledgement; Dr. Hisham Noureldayem ID: 1800562/C282Y. Altaib, Faculty of medical laboratories sciences, Sudan University

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Cite this article Bahereldeen AI, Alfadil RG, Ali HM, Nasser RM, Eisa NT, et al. (2020) In silico analysis of Single Nucleotide Polymorphisms (SNPs) in Human HFE Gene coding region. J Bioinform, Genomics, Proteomics 5(1): 1040.

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