Available online 17 March 2018
In silico analysis of the functional non-synonymous single nucleotide polymorphisms in the human CYP27B1 gene
- Department of Biochemistry, Covenant University, Ota, Nigeria
- Received 19 February 2018, Accepted 7 March 2018, Available online 17 March 2018
Abstract
Background
CYP27B1 gene codes for 25-hydroxyvitamin D3 1-α-hydroxylase, an enzyme that catalyses the activation of vitamin D to the 1-α, 25 dihydroxyvitamin D3. The activity of this enzyme is altered by non-synonymous single nucleotide polymorphisms (nsSNPs) located within its gene. Such alterations consequently affect the synthesis of the active form of the hormone, 1-α, 25 dihydroxyvitamin D3, resulting in vitamin D deficiency or insufficiency.
Objective
We aimed to investigate the impact of nsSNPs in the CYP27B1 gene on the structure and/or function of 25-hydroxyvitamin D3 1-α-hydroxylase.
Methods
The pathogenic nsSNPs in the human CYP27B1 obtained from National Centre for Biotechnology Information (NCBI) were analysed for their structural and functional consequence using mutation analysis algorithms like Consurf, I-Mutant, and MutPred. The effects of the mutation on tertiary structure of the human CYP27B1 protein was predicted using SWISS-MODEL while STRING was used to investigate its protein–protein interaction.
Results
Out of 938 SNPs in the human CYP27B1 gene, 455 that are responsible for missense mutations in the protein were subjected to various prediction algorithms to identify the pathogenic variants. Out of 24 consensus pathogenic nsSNPs, our Consurf analysis showed that mutations at conserved positions T321, R389 and G125 will significantly alter the structure of human CYP27B1 protein. These mutations also alter the metal binding and result in intrinsic structural disorder. These consequently, alter the 3D structure of the protein and could impact its ability to interact with other proteins like Cytochrome P450, family 2, subfamily R, polypeptide 1; Cytochrome P450, family 24, subfamily A, polypeptide 1 and Vitamin D receptor, that are involved in vitamin D pathway, as revealed by STRING.
Conclusion
These nsSNPs could contribute to vitamin D deficiency and its associated pathological conditions.
Keywords
- Polymorphisms;
- CYP27B1;
- Vitamin D;
- Mutation;
- Cancer;
- Diabetes
1. Introduction
Single nucleotide polymorphisms (SNPs) are regarded as one of the most common genetic variations in the human genome. They represent a single base change in a DNA sequence, with a common substitute of two possible nucleotides at a given position. The non-synonymous SNPs (nsSNPs) trigger genomic disparities within protein-coding areas resulting in mutations that could alter the structure and/or function of the protein. Nonetheless, such structural and/or functional modifications due to nsSNPs are not damaging or deleterious in most cases [1]. Non-synonymous SNPs could either alter the structural properties or the function of the protein. Such alterations may result in disease phenotypes. However, SNPs in genes involved in regulating key enzymes of nutrient metabolism may have significant phenotypic outcomes.
One of such enzymes is 25-hydroxyvitamin D3 1-α-hydroxylase and its gene, cytochrome p450 27B1 (CYP27B1) in humans is sited on 12q14.1 (Fig. 1). This is the long (q) arm of chromosome 12 at position 14.1 that also encodes enzymes which belong to the cytochrome P450 superfamily. Generally, cytochrome P450 proteins are monooxygenases with catalytic activities including xenobiotic metabolism and synthesis of steroids and other lipids [2]. The protein encoded by human CYP27B1 is called 25-hydroxyvitamin D3 1-α-hydroxylase and it hydroxylates 25-hydroxyvitamin D3 at the 1-α position. It is localized in the inner mitochondrial membrane where it converts 25-hydroxyvitamin D3 to the active form 1-α, 25 dihydroxyvitamin D3[3] ; [4]. The active form of vitamin D3 then binds to the vitamin D receptor to elicit its biological functions [3] ; [4]. Consequently, this gene is very important to the activation and biological activity of the hormone form of vitamin D, which involves calcium metabolism, cell proliferation and differentiation, and immunomodulatory functions, as well as calcidiol 1-monooxygenase activity. However, mutations in the CYP27B1 gene are one of the genomic factors that influence the levels of active vitamin D in circulation. Vitamin D insufficiency and/or deficiency is linked to rickets and a plethora of several chronic disorders, including Addison’s disease, diabetes, cardiac health, and cancer [5] ; [6].
- Fig. 1.
CYP27B1 Gene on chromosome 12: bands according to Ensembl, locations according to GeneLoc [38].
In view of the major effects of circulating vitamin D, there is a consensus that the amount of active vitamin D available to bind vitamin D receptor is reduced by certain SNPs present in the CYP27B1 gene.
1.1. Aim
The aim of this study was to elucidate the impact of nsSNPs on the structural and functional properties of human CYP27B1.
2. Materials and methods
2.1. Sequence recovery
The nucleotide sequence data on human CYP27B1 gene was extracted from the National Centre for Biotechnology Information (NCBI) while the amino acid sequence of human CYP27B1 protein in FASTA format was retrieved from UniProt (http://www.uniprot.org/) [1].
2.2. Retrieval, mining and mapping of SNPs in human CYP27B1
Several databases were used in collecting the polymorphism data for the human CYP27B1 gene. The databases include: UniProt database (http://www.uniprot.org), NCBI dbSNP database (https://www.ncbi.nlm.nih.gov/SNP/), and Ensembl genome browser (http://www.ensembl.org/index.html) [7]. The SNPs recovered from these databases were grouped into several functional classes, as described by Dakal et al. [1].
2.3. Analysis of nsSNP in human CYP27B1
A number of in silico algorithms were used in predicting functional effects of the nsSNPs in human CYP27B1 [8]. These included: PolyPhen-2 (http://genetics.bwh.harvard.edu/), SIFT (http://sift.jcvi.org/) and PROVEAN (Protein Variation Effect Analyzer) (http://provean.jcvi.org). nsSNPAnalyzer (http://snpanalyzer.uthsc.edu) was used to predict nsSNPs that confer pathogenic effects and were considered as high-risk nsSNPs for further studies [9].
2.4. Evolutionary phylogenetic analysis of human CYP27B1 protein
The amino acid evolutionary conservation in CYP27B1 protein was predicted using the ConSurf web server (consurf.tau.ac.il/) [10]. ConSurf server uses an empirical Bayesian method for the determination of evolutionary conservation plus identification of putative structural and functional residues. The conservation score of 1–4 is considered as variable, 5–6 as intermediate, and 7–9 as conserved amino acid position [11] ; [12].
2.5. Extrapolation of amino acid changes and disease phenotypes
The online server used for predicting the molecular basis of the disease linked amino acid replacement in a mutant protein is MutPred (http://mutpred.mutdb.org/)[13]. It uses several attributes associated with protein structure, function, and evolution. Its accuracy of prediction is increased by combining three servers, SIFT [9], PSI-BLAST [14], and Pfam, profiles alongside TMHMM, MARCOIL which are structural disorder prediction algorithms and DisProt.
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