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In Silico Analysis of Single Nucleotide Polymorphism (SNPs) in Human RAG 1&RAG2 Genes of Severe Combined Immunodeficiency from Functional Analysis to Polymorphisms in microRNA

Mona ShamsAldeen S.Ali*, Tomador Siddig M.Z, Rehab A. Elhadi, Muhammad R.Yousof, Siddig Eltyeb Yousif Abdallah, Maiada Mohamed Yousif Ahmed, Nosiba Yahia Mohamed Hassen, Sulum Omer Masoud Mohamed, Marwa Mohamed Osman, Mohamed A. Hassan

Omdurman Teaching Hospital, Sudan

Abstract: Severe combined immunodeficiency (SCID) is an inherited Primary immunodeficiency PID, which is characterized by the absence or dysfunction of T lymphocytes. Defects in RAG1 and RAG2 are known to cause a T⁻B⁻NK⁺ form of SCID. Recombinase activating genes RAG1 and RAG2 (OMIM 179615,179616 respectively) are expressed exclusively in lymphocytes and mediate the creation of double-strand. DNA breaks at the sites of recombination and in signal sequences during T− and B− cell receptor gene rearrangement. This study was focused on the effect of nonsynonymous single nucleotide polymorphisms in the function and structure of RAG1& RAG2 genes using in silico analysis. Only nsSNPs and 3'UTR SNPs were selected for computational analysis. Predictions of deleterious nsSNPs were performed by bioinformatics software. Five damaging nsSNPs (rs112047157, rs61758790, rs4151032, rs61752933, rs75591129) were predicted in RAG1 and two damaging nsSNPs (rs112927992, rs17852002) in RAG2, all of this nsSNPs found on domain that important in binding and mutation effect in its protein function. We hope to provide more information that needed to help researchers to do further study in SCID especially in our country where consanguineous marriage is common.

Keywords: severe combined immunodeficiency (SCID), primary immunodeficiency (PID), T lymphocytes, RAG1&2, nonsynonymous Single Nucleotide Polymorphisms (nsSNP)

Citation: Mona ShamsAldeen S.Ali, Tomador Siddig M.Z, Rehab A. Elhadi, Muhammad R.Yousof, Siddig Eltyeb Yousif Abdallah, Maiada Mohamed Yousif Ahmed, Nosiba Yahia Mohamed Hassen, Sulum Omer Masoud Mohamed, Marwa Mohamed Osman, Mohamed A. Hassan, In Silico Analysis of Single Nucleotide Polymorphism (SNPs) in Human RAG 1&RAG2 Genes of Severe Combined Immunodeficiency from Functional Analysis to Polymorphisms in microRNA. Biomedicine and Biotechnology, 4 (1). 5-11. January 2016. doi:10.12691/bb-4-1-2

1. Introduction

Severe combined immunodeficiency (SCID) is an inherited primary immunodeficiency, which is characterized by the absence or dysfunction of T lymphocytes affecting both cellular and humoral adaptive immunity [1-5]. estimated to be 1 in 75,000-100,000 of live births [8,9,10,11] and are more common in male subjects, reflecting the overrepresentation of X-linked SCID (XL-SCID), the most common worldwide form (~50%) of SCID in human subjects [10]. However, in cultures in which consanguineous marriage is common, the incidence of autosomal recessive (AR) SCID is higher than has been previously reported [10]. It can be classified as T−B+ and T−B− SCID with further subdivision based on the presence or absence of NK cells [5]. Defects in Recombinase activating genes (RAG1 and RAG2) are known to cause a T-B-NK+ form of autosomal recessive SCID (OMIM: 601457) [12,13].

It is now known that SCID can be caused in humans by mutations in at least 13 different genes that result in aberrant development of T cell [7]. Since the first description of RAG1 and RAG2 deficiency in patients with severe combined immunodeficiency by Schwarz et al. in 1996 [14], a pleiotropic spectrum of phenotypes associated with RAG1 and RAG2 deficiency has been described.

The location of recombination activating gene (RAG1& RAG2) is on chromosome 11 p13 [15,16]. RAG1 and RAG2 are expressed exclusively in lymphocytes and mediate the creation of double-strand DNA breaks at the sites of recombination and in signal sequences during T- and B-cell receptor gene rearrangement [10].

V(D)J recombination is the site-specific DNA rearrangement process that assembles the B cell receptor and TCR genes during lymphoid development. Recombination is initiated by the lymphoid-specific RAG1 and RAG2 recombinase, which introduces double-strand DNA breaks at recombination signal sequences (RSSs) flanking variable (V), diversity (D), and junction (J) gene segments spread along the Immunoglobulin and TCR loci [17,18,19,20]. The recombination process is tightly regulated, occurring at specific stages of development and in specific cell types (e.g., Immunoglobulin and TCR genes are rearranged in B and T cells, respectively). This process takes place in a temporal manner, with Immunoglobulin heavy chain rearrangements preceding Immunoglobulin light chain rearrangements and D-to-J rearrangements preceding V-to-DJ rearrangements [17,18,19,20]. Mutations in either Rag1 or Rag2 genes hamper initiation of V(D)J recombination,hence causing an early block of B and T cell maturation similar to the situation of Rag1 and Rag2 knockout (KO) mice [21].

In this computational study, we focused on the effect of nsSNPs in the function and structure of RAG 1 and RAG 2 Protein using in silico analysis. Hence it is the first study type of RAG1 and RAG2 analysis, we hope to provide more information that needed to help researchers to do further study in SCID especially in our country where consanguineous marriage is common.

2. Materials and Methods

The SNPs sequence of RAG1 and RAG 2 genes were collected in August 2015 from NCBI database (http://www.ncbi.nlm.nih.gov/projects/SNP). They contained a total of 5413 SNPs in RAG1 and 2804 SNPs in RAG2 at the time of the study, out of which 1048 in RAG1 and 508 in RAG2 were coding SNPs, 147 in RAG1 and 28 in RAG2 occurred in the miRNA 3′ UTR, 8 in RAG1 and 10 in RAG2 occurred in 5′ UTR region and 234 in RAG1 and 178 in RAG2 occurred in intronic regions. We selected missense & nonsense nsSNPs and 3′ UTR SNPs for our investigation, Figure 1. The nsSNPs (rs SNPs) of RAG 1&2 were submitted as batch to SIFT server, then the resultant damaging nsSNPs were submitted to Polyphen as query sequences in FASTA Format. Prediction of change in stability due to mutation was performed by I-Mutant 2.0. The protein sequences used were obtained from the ExPASy Database (www.expasy.org/). Project hope software was used to highlight the changes occurred as a result of the deleterious SNPs at the molecular level of the protein 3D structure. The SNPS at the 3′ UTR region were analyzed by Polymirt software. Prediction of the function of genes and their interactions were obtained from GENEMANIA database.

GeneMANIA (http://www.genemania.org/): is an online database that helps you predict the function of your favorite genes and gene sets. GeneMANIA finds other genes that are related to a set of input genes, using a very large set of functional association data. Association data include protein and genetic interactions, pathways, co-expression, co-localization and protein domain similarity. You can use GeneMANIA to find new members of a pathway or complex, find additional genes you may have missed in your screen or find new genes with a specific function, such as protein kinases. Your question is defined by the set of genes you input [22].

SIFT-software In order to detect deleterious nsSNPs SIFT program was used , which is a novel bioinformatics tool to predict whether an amino acid substitution affects protein function , this program generates alignments with a large number of homologous sequences and assigns score for each residue ranging from zero to one . Scores closer to zero indicates evolutionary conservation of the genes and intolerance to substitution, while scores closer to one indicate tolerance to substitution only [23]. (http://sift.jcvi.org/).

PolyPhen-2 Also is an online bioinformatics soft-ware produced by Harvard university it searches for 3D protein structures, then calculates position-specific independent count scores (PSIC) for each of two variant and the computes the PSIC scores difference between two variants. PolyPhen results were assigned probably damaging (2.00 or more) possibly damaging (1.40-1-90), potentially damaging (1.0-1.5), benign (0.00-0.90), [24]. (http://genetics.bwh.harvard.edu/pph2/index.shtml). We used this software to confirm SIFT result and we took only double positive results for further workup.

I-Mutant v2.0c Predictor the stability changes upon mutation from the protein sequence or structure. It shows the amino acid in Wild-Type Protein (WT), New Amino acid after Mutation (NAW), reliability Index (RI), Temperature in Celsius degrees (T) and the PH [25].

Figure 1. distribution of nonsynonymous, 5′ UTR, 3′ UTR and intronic SNPs for RAG1&2

I-Mutant available at: ( http:/www.I-Mutant2.0.cgi).

Chimera is software produced by University of California, San Francisco is used in this step to generate the mutated models of ATP7B protein 3D model. The outcome is then a graphic model depicting mutation [26]. (http://www.cgl.ucsf.edu/chimera/).

Project Hope software (http://www.cmbi.ru.nl/hope/input) is an online web service where the user can submit a sequence and mutation. This software collects structural information from a series of sources, including calculations on the 3D protein structure, sequence annotations in UniProt and predictions from DAS-servers. It combines this information to give analyze the effect of a certain mutation on the protein structure and will show the effect of that mutation in such a way that even those without a bioinformatics background can understand it [27].

PolymiRTS is the database server designed specifically for the analysis of the 3UTR region, we used this server to determine SNPs that may alter miRNA target site [28]. All SNPs located within the 3UTR region were selected separately and submitted to the program. (Available at: http://compbio.uthsc.edu/miRSNP/).

3. Results

Recombination activating proteins 1&2 have many vital functions. Also these genes co-expressed with, share similar protein domain, or participate to achieve similar function are illustrated by using GENEMANIA and shown in Figure 2 below.

3.1. Prediction of Protein Structural Stability

Seven nsSNPs of RAG1&2 genes have been selected on the basis of prediction scores of Sift and polyphen; these SNPs were given to I-Mutant web server to predict the DDG stability and reliability index (RI) upon mutation, in RAG1, three SNPs (rs112047157, rs4151032 and rs75591129) shown decrease in protein stability while other two SNPs (rs61758790 and rs61752933) shown increase in protein stability. Both SNPs in RAG2 predicted to decrease protein stability, Table 1.

3.2. Modeling of Mutant Structure

Protein sequences of the nsSNP were presented to Project Hope revealed the 3D structure for the truncated proteins with its new candidates; in addition, it described the reaction and physiochemical properties of these candidates. Here we present the results upon each candidate and discus the conformational variations and interactions with the neighboring amino acids; All native and mutant structure of RAG1 &2 proteins showed in the Figure 3 and the wild type is displayed by green color while mutant type is displayed by red one.

3.3. SNPs at the 3’UTR Region

SNPs in 3′UTR of RAG1, 2 genes were submitted as batch to PolymiRTS server. The output showed result as following; in RAG1, 19 SNPs were predicted while only three SNPs were predicted in RAG2 gene. The functional classes of both genes are described in Table 2 below.

Figure 2. functions and interaction of RAG1 and RAG2 with functional similar genes was predicted using Gene MANIA software

Table 1. Prediction of nsSNPs in RAG 1& RAG 2 by SIFT, Polyphen-2 and I-Mutant software

Figure 3. 3D model by Chimera and Project HOPE for RAG1&2 proteins

Table 2.A. 3′UTR SNPs of RAG1 as detected by PolymiRTS

Table 2B. 3′UTR SNPs of RAG2 as detected by PolymiRTS

4. Discussion & Conclusion

In RAG1 we found five nsSNPs predicted by SIFT & polyphen and five nsSNP (rs415107, rs34841221, rs4151029, rs2227973, rs4151034) are damaging by SIFT only. In RAG2 we found two nsSNPs predicted damaging by both SIFT & polyphen, however one nsSNP (rs117899975) is damaging by SIFT only.

A/G mutation (rs112047157): methionine 487 valine, This residue is part of an interpro domain named Recombination-Activation Protein 1 (IPR024627).This domain is annotated with the Gene-Ontology (GO)that indicate the domain has a function in DNA Binding (GO:0043565), Nucleic Acid Binding (GO:0003676). The mutant residue is smaller than wild residue; this might lead to loss of interactions, so it might be too small to make multimer contacts. The wild-type residue is located in an α-helix. The mutation converts the wild-type residue in a residue that does not prefer α-helices as secondary structure. The mutated residue is located in a domain that is important for binding of other molecules. Mutation of the residue might disturb the function. (Figure 3-1)

G/T mutation (rs61758790): phenylalanine 520 leucine, the wild-type residue is located in its preferred secondary structure, a β-strand. This residue is part of an interpro domain named Recombination-Activation Protein 1 (IPR024627). This domain is annotated with the Gene-Ontology (GO) that indicate the domain has a function in DNA Binding (GO:0043565), Nucleic Acid Binding (GO:0003676). The mutant residue is smaller than wild residue, this might lead to loss of interactions. The mutant residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized. The mutated residue is located in a domain that is important for binding of other molecules. Mutation of the residue might disturb this function. (Figure 3-2).

C/T mutation (rs4151032): proline 525 serine, prolines are known to have a very rigid structure, sometimes forcing the backbone in a specific conformation, changes a proline with such a function into another residue disturbing the local structure. This variant is annotated with severity: POLYMORPHISM (VAR_029263). This residue is part of an interpro domain named Recombination-Activation Protein 1 (IPR024627). This domain is annotated with the Gene-Ontology (GO)that indicate the domain has a function in DNA Binding (GO:0043565), Nucleic Acid Binding (GO:0003676). The mutant residue is smaller than wild residue; this might lead to loss of interactions. The wild-type residue is more hydrophobic than the mutant residue. Hydrophobic interactions, either in the core of the protein or on the surface, will be lost. The mutated residue is located in a domain that is important for binding of other molecules. Mutation of the residue might disturb this function, (Figure 3-3)

A/G mutation (rs61752933): isoleucine 810 valine, this residue is part of an interpro domain named Recombination-Activation Protein 1 (IPR024627). This domain is annotated with the Gene-Ontology (GO) that indicate the domain has a function in DNA Binding (GO:0043565), Nucleic Acid Binding (GO:0003676). The mutant residue is smaller than wild residue; this might lead to loss of interactions. The mutated residue is located in a domain that is important for binding of other molecules. Mutation of the residue might disturb this function, (Figure 3-4)

A\C mutation (rs75591129): tyrosine 931 serine, the wild-type residue is preferred secondary structure, a β-strand. The mutant residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized. This residue is part of an interpro domain named Recombination-Activation Protein1 (IPR024627). This domain is annotated with the Gene-Ontology (GO) that indicate the domain has a function in DNA Binding (GO:0043565), Nucleic Acid Binding (GO:0003676).The mutated residue is located in a domain that is important for binding of other molecules. Mutation of the residue might disturb this function, (Figure 3-5).

C/T mutation (rs112927992): serine 291 phenylalanine, this residue is part of an interpro domain named V-D-J Recombination Activating Protein 2 (IPR004321). This domain is annotated with Gene-Ontology (GO) these GO annotations indicate the domain has a function in DNA binding (GO:0003677), Nucleic Acid Binding (GO:0003676). This residue is part of an interpro domain named Galactose Oxidase/kelch, Beta-Propeller (IPR011043). The mutant residue is bigger than the wild-type residue, this might lead to bumps. The mutant residue is more hydrophobic than the wild-type residue; this can result in loss of hydrogen bonds and/or disturb correct folding. The mutated residue is located in a domain that is important for binding of other molecules. Mutation of the residue might disturb this function, (Figure 3-6).

T/C mutation (rs17852002): valine 154 alanine, the wild-type residue is located in its preferred secondary structure, a β-strand. The mutant residue is smaller than the wild residue; this might lead to loss of interactions. The mutant residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized. The residue is part of an interpro domain named V-D-J Recombination Activating Protein 2(IPR004321). This domain is annotated with Gene-Ontology (GO) these indicate the domain has a function in DNA binding (GO:0003677), Nucleic Acid Binding (GO:0003676), also is part of an interpro domain named Galactose Oxidase/kelch, Beta-Propeller (IPR011043), is part of an interpro domain named Kelch-Type Beta Propeller (IPR015915), the domain is also important in Protein Binding (GO:0005515), (Figure 3-7).

Both nsSNPs of RAG2 were found in the same domain that important in function of protein leading to disturb of function. Mutations in RAG1 or RAG2 result in the blocking of T- and B-cell development secondary to the inability to initiate recombination of the DNAs variable, diversity, and joining regions, and thereby do not form functional T-cell or B-cell receptors [29].

According to Table 2, we found some SNPs in 3UTR of both RAG 1&2 related to cancer development although those patients with SCID don’t survive [8,9,10] till they develop cancer, but some patients with delayed onset of RAG1 deficiency develop cancer [30].

Our results from this study suggest that the application of computational tools including SIFT, PolyPhen-2, I Mutant 2v, polymRTS, Chimera and Project Hope might provide an alternative approach to select target SNPs in association studies.

Acknowledgements

The authors thank the Africa City of Technology (Sudan) for providing the facilities to carry out this work.

Competing Interests

The authors declare that they have no competing interests.

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