- Email: [email protected]
Association of rs1738074 polymorphism of TAGAP gene with susceptibility to multiple sclerosis in the Iranian population
Accepted Manuscript Title: Association of rs1738074 polymorphism of TAGAP gene with susceptibility to multiple sclerosis in the Iranian population Authors: Ali Jazaeri, Sadeq Vallian PII: DOI: Reference:
S0304-3940(17)30271-9 http://dx.doi.org/doi:10.1016/j.neulet.2017.03.041 NSL 32729
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
18-9-2016 20-3-2017 23-3-2017
Please cite this article as: Ali Jazaeri, Sadeq Vallian, Association of rs1738074 polymorphism of TAGAP gene with susceptibility to multiple sclerosis in the Iranian population, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.03.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Association of rs1738074 polymorphism of TAGAP gene with susceptibility to multiple sclerosis in the Iranian population Ali Jazaeri and Sadeq Vallian* Division of Genetics, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, IR Iran. Running Title: TAGAP polymorphism and MS *Corresponding author: Sadeq Vallian, MSc, PhD Prof. of human molecular genetics Division of Genetics, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran. Tel/Fax: +98 31 37932456 [email protected]
Research Highlights
TAGAP rs1738074 single nucleotide polymorphism is associated with the prevalence of multiple sclerosis in the Iranian population The T/T genotype of rs1738074 shows a suppressor effect on the prevalence of multiple sclerosis in the Iranian population, and therefore could be further studied as a possible risk factor
Abstract Multiple sclerosis (MS) is one of the most common diseases of the central nervous system (CNS) in the Iranian population. To date, association of many genes with the prevalence and progression of the disease have been investigated. In the present study, the impact of rs1738074 single nucleotide polymorphism (SNP) in the TAGAP gene (TAGAP rs1738074) on the risk of MS was evaluated in a sample of the Iranian population. In a case control study, genotyping was performed on 300 patients and normal individuals. The data were analyzed using Pearson's chi-square test. The results showed a significant difference in the SNP frequency between case and control groups (p-value= 0.049). The genotype frequencies of TT, TC and CC in patients were 10.67%, 51.33% and 38%, respectively, and in normal individuals were 20.66%, 42.67% and 36.67%, respectively. The results showed a significant difference in the genotype frequency of T/T between the patient and control groups (p<0.05). Interestingly, individuals with T/T genotype were estimated to be less susceptible to MS ((p-value= 0.025, Fisher’s exact test), odd ratio was 2.18 (controls versus MS patients) with 95% CI: 1.137-4.187. The results suggested that TAGAP rs1738074 polymorphism could be considered as a risk factor in the prevalence of MS in the Iranian population. Keywords: Multiple sclerosis; polymorphic markers; TAGAP gene; Iranian population
Introduction: Multiple Sclerosis (MS) is a severe inflammatory disease of the central nervous system (CNS). The disease attacks on myelin sheath surrounding axons of the neuron cells of the CNS, leading to deterioration of them (Goldenberg 2012). Several reports have shown a notable variation in the prevalence of MS depending on the variations in ethnically, diagnostic criteria, definitions of prevalence, incidence, etc. (Etemadifar, Janghorbani et al. 2006). According to Kurtzke map, proposed for MS prevalence and incidence around the world, Asian and African countries classified as low risk countries. However, recent studies have shown that this classification needs to be changed as the prevalence, incidence, and distribution of MS seem to be very complex. Therefore, the distribution of MS varied even within the countries themselves (Etemadifar, Janghorbani et al. 2006). Iran is a country with diverse racial groups and to date, few studies have been conducted to reveal the epidemiological distribution of MS in the Iranian population (Sharafaddinzadeh, Moghtaderi et al. 2013). Recent studies have shown a sharp increasing in the prevalence and incidence of MS in Iran, especially in the central province of Isfahan (Elhami, Mohammad et al. 2011, Etemadifar, Sajjadi et al. 2013). Young adults are at higher risk for the disease compare to other age groups (Etemadifar, Nasr-Esfahani et al. 2007). MS is a multifactorial disease that seems to arise from interaction of genetic and environmental factors. In recent years genetic studies on MS principally have concentrated on the search for prone genes (Ronaghi, Vallian et al. 2009). To date, no specific gene or genetic factor has been identified to directly resulting in MS. However, several gene loci have been reported to be associated with the prevalence and progression of the disease, and therefore, considered as possible risk factor for the disease (Ronaghi, Vallian et al. 2009, Gourraud, Harbo et al. 2012). The most important genetic loci that seems to be associated with MS is the human leukocyte antigen (HLA) region located on chromosome 6p. In addition to HLA loci related to MS, several non-HLA loci have been reported to be associated with MS. According to some studies, T-cell activation Rho-GTPase activating
protein (TAGAP) is one of these non-HLA genes that has been reported as a susceptible gene in MS (Consortium I.M.S.G. and W.T.C.C.C. 2 2011, Muñoz-Culla, Irizar et al. 2013). TAGAP gene is located on chromosome 6q25.3 and encodes a member of the Rho GTPase-activator protein superfamily. The encoded protein may function as a Rho GTPase-activating protein. According to previous studies, alterations in this gene have shown to be associated with several autoimmune diseases, namely coeliac disease, type 1 diabetes, rheumatoid arthritis and multiple sclerosis in Caucasians (Anaya 2010, Festen, Goyette et al. 2011, Chatzikyriakidou, Voulgari et al. 2013). As the gene name indicates, the TAGAP protein plays an important role in the activation of T cells, which leads to cytoskeleton arrangement and finally their migration (Connelly, Berg et al. 2014). The product of TAGAP gene phosphorylates the GTPases, increasing their innate activity and consequently leads to several changes in T cells and their activity (Mohamed N. S., et al., 2016).. The aim of this study was to investigate the possible relationship between rs1738074 single nucleotide polymorphism (SNP) located in the 5′-UTR region of the TAGAP gene with the risk and susceptibility to MS in the Iranian population.
Material and methods: Human subjects and blood sampling: In the present study 300 individuals (150 patients and 150 controls) were involved in the study. All the patients were considered as “definite MS” according to the McDonald criteria (McDonald, Compston et al. 2001). Before blood sampling, participants in the study were informed and consented for blood donation and subsequent molecular experiments. Blood samples were collected in EDTA containing tubes and kept frozen until DNA extraction and genotyping examinations.
Genomic DNA extraction and genotyping: Total genomic DNA was extracted using standard salting out precipitation method (Miller, Dykes et al. 1988). Allele specific PCR (ASP) was used as the standard method for determining genotype frequencies in patient and control groups (Gaudet, Fara et al. 2009). The Primers were designed using Oligo V7 software and web-based primer1 software (primer1.soton.ac.uk/primer1.html) (Collins and Ke 2012). The rs1738074 SNP results in substitution of T and C alleles with each other in TAGAP gene. Four primers were designed for genotyping of rs1738074 SNP. The sequence of the primers are as follows: Reverse T (allele T): 5′-GCTGTAAAGAATGGGAGAAACAGaAt-3′ Reverse C (allele C): 5′-GCTGTAAAGAATGGGAGAAACAGaAc-3′ Forward outer: 5′-GCCCTAAAAGGAATGAGGAAGC-3′ Reverse outer: 5′-GAGCATATTCCACGGGATAGC-3′. The PCR amplification was carried out in 25 μL total volume in Eppendorf tubes containing 2.5 µL 10X PCR buffer, 1 µL dNTP (10mM), 1 µL MgCl2 (50mM), 1 µL DNA (100 ng/uL), 1µL reverse T (10pM) (vial 1), 1µL reverse C (10pM) (vial 2), 1 µL forward primer (10pM), .25 µL reverse outer primer (10pM), .25 µL Taq DNA Polymerase (5unit/ µL) and deionized H2O up to 25 µL. The PCR conditions were as follows: 5 minutes denaturing at 94ºC, 40 seconds denaturing at 94ºC, 45 seconds annealing at 60ºC and extension at 72ºC for 50 seconds, for a total of 31 cycles and eventually 10 minutes at 72ºC. The PCR products were separated and analyzed on 2% agarose gel electrophoresis (Figure 1). Statistical analysis: Patients and normal controls were examined for any significant differences in their genotype distribution at the population level. Allele frequency was determined, using GENEPOP software (genepop.curtin.edu.au) (Rousset 2008). Statistical analysis was performed using IBM SPSS21 software.
Pearson’s chi-square test was applied to perform homogeneity assessments for distribution of the genotypes between the patients and the control population. The association between genotypes and MS were examined as the odds ratio (OR) with a 95% confidence interval (95% CI). Both patient and control groups were examined for deviation from Hardy-Weinberg equilibrium.
Results Genotyping of TAGAP rs1738074 SNP The DNA samples of 150 cases, and 150 control individuals were genotyped. As shown in Figure 1, for each individual two PCR reactions were performed, one with reverse T primer and the other with reverse C primer. As a consequence, three different genotypes could be detected, which were T/T, T/C, and C/C. For heterozygous individuals two allele bands (T and C) and for homozygous individuals, one allele band (T or C) were expected. After performing PCR amplifications, two product bands were expected on the agarose gels. The forward outer and reverse C/T primers resulted to a 224 bp product. For ensuring of PCR accomplishment in the PCR tubes, the reverse outer primer was designed and used in the PCR amplifications. Amplification with this primer and the forward outer primer resulted in a 372 bp product. As presented in Table 1, the genotype frequencies of TT, TC and CC in patients were 10.67%, 51.33% and 38%, respectively, and in normal individuals were 20.66%, 42.67% and 36.67%, respectively. The allele frequency of rs1738074 was shown in Table 2. Association of the TAGAP rs1738074 TT genotype with the prevalence of MS Statistical analysis using SPSS and Genepop software were performed to investigate the correlation of the SNP genotypes with the prevalence of MS. Both patient and control groups were in accordance with Hardy-Weinberg equilibrium (p-value= 0.22 and p-value=0.14, respectively).
The results showed an association between the TAGAP rs1738074 and the prevalence of MS (p-value= 0.049). Among the genotypes of the TAGAP rs1738074, the frequency of TT genotype was significantly lower in MS patients in comparison with the controls (p-value< 0.05, odds ratio was 2.18, 95% CI: 1.1374.187) (see Table 1). According to these results, it could be suggested that the TAGAP rs1738074 was associated with the prevalence of MS in the Iranian population and that the TT genotype could have a significant role in reducing the prevalence of the disease.
Discussion In recent years, the prevalence of MS has increased significantly in Iran and from this prospect, Iran is now considered among the medium to high risk countries (Ronaghi, Vallian et al. 2009, Izadi, Nikseresht et al. 2014). Many genes have been investigated for association with the prevalence and progression of the disease. However, no specific gene has been reported to directly resulting in MS. To date, a few studies have investigated the molecular pathogenesis of MS in the population of Iran, and most of them have principally explored epidemiological and immunological factors related to MS, such as HLA antigenic polymorphisms (Kalanie, Hosseinzadeh et al. 2011, Reza 2015, Mardasi, Galehdari et al. 2016). TAGAP gene has been reported as one of the genes associated with susceptibility to MS (Muñoz-Culla, Irizar et al. 2013). In the present study association of rs1738074 polymorphism in TAGAP gene with MS was investigated in the Iranian population. This SNP results in the substitution of nucleotide cytosine (C) with thymine (T). The comparison of genotype frequencies between patient and control groups revealed a significant difference in the TAGAP T/T genotype, but not in the T/C and C/C genotypes. Therefore, according to these results, it could be suggested that T/T genotype of rs1738074 could have a preventive effects on the MS prevalence and could be further studied as a possible risk factor for MS. Therefore, individuals
with this genotype could be at a lower risk for MS. These data and also the result of previous studies confirm the effect of TAGAP rs1738074 SNP on prevalence of MS. Moreover, the result of this study are in line with the previous reports on the possible role of the TAGAP rs1738074 polymorphism with prevalence of MS (Consortium I.M.S.G. and W.T.C.C.C. 2 2011). In addition to TAGAP rs1738074, we also examined the allele frequencies of RAGE rs1800624 gene in the same group population. It has been reported that RAGE rs1800624 could play a role in the prevalence of MS (Tiszlavicz, Gyulai et al. 2009, Li, Zhao et al. 2011). However, our data showed no significant association for RAGErs1800624 and MS (Jazaeri, Karimi Moghadam et al. 2016). The clinical and demographic characteristics of the patients were evaluated and presented in Tables 1 and 3. The data indicated that the prevalence of MS is much higher in the Iranian females compare to males. Moreover, analysis of the association of primary progressive, progressive relapsing; relapsing remitting; and secondary progressive of the disease with different genotypes of TAGAP rs1738074 were analyzed. The data showed that the genotype TT compare to other genotypes is more effective in progression of the disease. In summary, the data from the present study showed that patients with the TAGAP T/T genotype were less prone to MS. Therefore, the patients carrying this genotype may require less thorough treatment compared to patients with the TAGAP T/C and TAGAP C/C genotypes. Identification of more associated genes with MS would help completing the genetic map of this disease and even could help to improve related medical treatments.
Acknowledgments: We would like to thank the patients and their family for participation in this study. This study was supported by department of research of University of Isfahan, Isfahan, IR Iran. The study was approved by University of Isfahan institutional review board (IRB) for research and ethics approval (Approval Ref No. 790205).
References: Anaya, J.-M. (2010). "The autoimmune tautology." Arthritis Research and Therapy 12(6): 147. Chatzikyriakidou, A., et al. (2013). "Validation of the TAGAP rs212389 polymorphism in rheumatoid arthritis susceptibility." Joint Bone Spine 5(80): 543-544. Collins, A. and X. Ke (2012). "Primer1: primer design web service for tetra-primer ARMS-PCR." The Open Bioinformatics Journal 6: 55-58. Connelly, T. M., et al. (2014). "T-cell activation Rho GTPase–activating protein expression varies with inflammation location and severity in Crohn's disease." Journal of Surgical Research 190(2): 457-464. Consortium, I. M. S. G. and W. T. C. C. C. 2 (2011). "Genetic risk and a primary role for cellmediated immune mechanisms in multiple sclerosis." Nature 476(7359): 214-219. Elhami, S.-R., et al. (2011). "A 20-year incidence trend (1989–2008) and point prevalence (March 20, 2009) of multiple sclerosis in Tehran, Iran: a population-based study." Neuroepidemiology 36(3): 141-147. Etemadifar, M., et al. (2006). "Prevalence of multiple sclerosis in Isfahan, Iran." Neuroepidemiology 27(1): 39-44. Etemadifar, M., et al. (2007). "Childhood-onset multiple sclerosis: report of 82 patients from Isfahan, Iran." Arch. Iran Med 10(2): 152-156.
Etemadifar, M., et al. (2013). "Epidemiology of multiple sclerosis in Iran: a systematic review." European Neurology 70(5-6): 356-363. Festen, E., et al. (2011). "A meta-analysis of genome-wide association scans identifies IL18RAP, PTPN2, TAGAP, and PUS10 as shared risk loci for Crohn's disease and celiac disease." PLoS Genet 7(1): e1001283. Gaudet, M., et al. (2009). Allele-specific PCR in SNP genotyping. Single Nucleotide Polymorphisms, Springer: 415-424. Goldenberg, M. M. (2012). "Multiple sclerosis review." Pharmacy and Therapeutics 37(3): 175. Gourraud, P. A., et al. (2012). "The genetics of multiple sclerosis: an up‐to‐date review." Immunological Reviews 248(1): 87-103. Izadi, S., et al. (2014). "Significant Increase in the Prevalence of Multiple Sclerosis in Iran in 2011." Iranian Journal of Medical Sciences 39(2): 152. Jazaeri, A., et al. (2016). "Evaluating the Association between Rs1800624 in RAGE Gene and Multiple Sclerosis in Isfahan Population." SSU_Journals 23(10): 923-931. Kalanie, H., et al. (2011). "HLA Profile and Clinical Presentation of Multiple Sclerosis in IRAN." Genetics in the 3rd millennium 8(4): 2235-2239. Li, K., et al. (2011). "A functional p. 82G> S polymorphism in the RAGE gene is associated with multiple sclerosis in the Chinese population." Multiple Sclerosis Journal 17(8): 914-921. Mohamed N. S., et al., (2016) Identification of rheumatoid arthritis biomarkers based on singlenucleotide polymorphisms and haplotype blocks: A systematic review and meta-analysis. Journal of Advanced Research, 7(1): 1–16. Mardasi, F. G., et al. (2016). "Association Study between Multiple Sclerosis Disease and HLA DQA0102* 1 in South-West Iran." Focus on Sciences 2(2). McDonald, W. I., et al. (2001). "Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis." Annals of neurology 50(1): 121-127.
Miller, S., et al. (1988). "A simple salting out procedure for extracting DNA from human nucleated cells." Nucleic Acids Research 16(3): 1215. Muñoz-Culla, M., et al. (2013). "The genetics of multiple sclerosis: review of current and emerging candidates." Appl Clin Genet 6: 63-73. Reza, S. M. (2015). "Analysis of HLA-DQB1* 0602 in Multiple Sclerosis Patients in Khuzestan Province, Iran." Archives of Iranian Medicine 18(10): 698. Ronaghi, M., et al. (2009). "CD24 gene polymorphism is associated with the disease progression and susceptibility to multiple sclerosis in the Iranian population." Psychiatry Research 170(2): 271-272. Rousset, F. (2008). "genepop’007: a complete re‐implementation of the genepop software for Windows and Linux." Molecular Ecology Resources 8(1): 103-106. Sharafaddinzadeh, N., et al. (2013). "The influence of ethnicity on the characteristics of multiple sclerosis: a local population study between Persians and Arabs." Clinical Neurology and Neurosurgery 115(8): 1271-1275. Tiszlavicz, Z., et al. (2009). "RAGE gene polymorphisms in patients with multiple sclerosis." Journal of Molecular Neuroscience 39(3): 360-365.
Legend to Figure 1 Figure 1: Genotyping of TAGAP rs1738074 in the Iranian population. The PCR products were separated on 2% agarose gel. As presented, the 372 bp band shows the internal PCR control confirming the occurrence of PCR in the PCR tubes. 224 bp band shows the specific band. Lanes 2 and 3 show an individual homozygous for TT genotype. Lanes 4 and 5 show a homozygous CC individual, and lanes 6 and 7 show a heterozygous TC individual. The DNA size marker is in lane 1. Figure 1
Table 1: The clinical characteristics and genotype frequency of TAGAP rs1738074 in the Iranian population Genotypes CC
TC
TT
MS patients
57/150 (38%)
77/150 (51.33%)
16/150 (10.67%)
Normal controls
55/150 (36.67%)
64/150 (42.67%)
P-value
NS
NS
Significant**
RR
35%
45%
20%
PP
18%
52%
30%
SP
12%
58%
30%
PR
21%
39%
40%
P-value= 0.049*
31/150 (20.66%)
NS: Not significant; **p-value= 0.025, fisher’s exact test, odds ratio (controls/MS patients) = 2.18, 95% CI: 1.137- 4.187, PP, primary progressive; PR, progressive relapsing; RR, relapsing remitting; and SP, secondary progressive.
Table 2: The allele frequency of TAGAP rs1738074 Allele frequency MS patients Normal controls
C 63.7% 58%
T 36.3% 42%
Table 3: Demographic characteristics of Multiple Sclerosis (MS) Patients and control group. Gender
Age (year)
Disease
Marital status
Occupation
Duration Male
Female
Male
Female
Male/Female
Married
Single
Employed
Unemployed
(year) MS
26%
74%
patients Control group
25-
23-36
2-5
66%
34%
36%
64%
25-36
-
60%
40%
45%
55%
45 25%
25%
2545
S0304-3940(17)30271-9 http://dx.doi.org/doi:10.1016/j.neulet.2017.03.041 NSL 32729
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
18-9-2016 20-3-2017 23-3-2017
Please cite this article as: Ali Jazaeri, Sadeq Vallian, Association of rs1738074 polymorphism of TAGAP gene with susceptibility to multiple sclerosis in the Iranian population, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.03.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Association of rs1738074 polymorphism of TAGAP gene with susceptibility to multiple sclerosis in the Iranian population Ali Jazaeri and Sadeq Vallian* Division of Genetics, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, IR Iran. Running Title: TAGAP polymorphism and MS *Corresponding author: Sadeq Vallian, MSc, PhD Prof. of human molecular genetics Division of Genetics, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran. Tel/Fax: +98 31 37932456 [email protected]
Research Highlights
TAGAP rs1738074 single nucleotide polymorphism is associated with the prevalence of multiple sclerosis in the Iranian population The T/T genotype of rs1738074 shows a suppressor effect on the prevalence of multiple sclerosis in the Iranian population, and therefore could be further studied as a possible risk factor
Abstract Multiple sclerosis (MS) is one of the most common diseases of the central nervous system (CNS) in the Iranian population. To date, association of many genes with the prevalence and progression of the disease have been investigated. In the present study, the impact of rs1738074 single nucleotide polymorphism (SNP) in the TAGAP gene (TAGAP rs1738074) on the risk of MS was evaluated in a sample of the Iranian population. In a case control study, genotyping was performed on 300 patients and normal individuals. The data were analyzed using Pearson's chi-square test. The results showed a significant difference in the SNP frequency between case and control groups (p-value= 0.049). The genotype frequencies of TT, TC and CC in patients were 10.67%, 51.33% and 38%, respectively, and in normal individuals were 20.66%, 42.67% and 36.67%, respectively. The results showed a significant difference in the genotype frequency of T/T between the patient and control groups (p<0.05). Interestingly, individuals with T/T genotype were estimated to be less susceptible to MS ((p-value= 0.025, Fisher’s exact test), odd ratio was 2.18 (controls versus MS patients) with 95% CI: 1.137-4.187. The results suggested that TAGAP rs1738074 polymorphism could be considered as a risk factor in the prevalence of MS in the Iranian population. Keywords: Multiple sclerosis; polymorphic markers; TAGAP gene; Iranian population
Introduction: Multiple Sclerosis (MS) is a severe inflammatory disease of the central nervous system (CNS). The disease attacks on myelin sheath surrounding axons of the neuron cells of the CNS, leading to deterioration of them (Goldenberg 2012). Several reports have shown a notable variation in the prevalence of MS depending on the variations in ethnically, diagnostic criteria, definitions of prevalence, incidence, etc. (Etemadifar, Janghorbani et al. 2006). According to Kurtzke map, proposed for MS prevalence and incidence around the world, Asian and African countries classified as low risk countries. However, recent studies have shown that this classification needs to be changed as the prevalence, incidence, and distribution of MS seem to be very complex. Therefore, the distribution of MS varied even within the countries themselves (Etemadifar, Janghorbani et al. 2006). Iran is a country with diverse racial groups and to date, few studies have been conducted to reveal the epidemiological distribution of MS in the Iranian population (Sharafaddinzadeh, Moghtaderi et al. 2013). Recent studies have shown a sharp increasing in the prevalence and incidence of MS in Iran, especially in the central province of Isfahan (Elhami, Mohammad et al. 2011, Etemadifar, Sajjadi et al. 2013). Young adults are at higher risk for the disease compare to other age groups (Etemadifar, Nasr-Esfahani et al. 2007). MS is a multifactorial disease that seems to arise from interaction of genetic and environmental factors. In recent years genetic studies on MS principally have concentrated on the search for prone genes (Ronaghi, Vallian et al. 2009). To date, no specific gene or genetic factor has been identified to directly resulting in MS. However, several gene loci have been reported to be associated with the prevalence and progression of the disease, and therefore, considered as possible risk factor for the disease (Ronaghi, Vallian et al. 2009, Gourraud, Harbo et al. 2012). The most important genetic loci that seems to be associated with MS is the human leukocyte antigen (HLA) region located on chromosome 6p. In addition to HLA loci related to MS, several non-HLA loci have been reported to be associated with MS. According to some studies, T-cell activation Rho-GTPase activating
protein (TAGAP) is one of these non-HLA genes that has been reported as a susceptible gene in MS (Consortium I.M.S.G. and W.T.C.C.C. 2 2011, Muñoz-Culla, Irizar et al. 2013). TAGAP gene is located on chromosome 6q25.3 and encodes a member of the Rho GTPase-activator protein superfamily. The encoded protein may function as a Rho GTPase-activating protein. According to previous studies, alterations in this gene have shown to be associated with several autoimmune diseases, namely coeliac disease, type 1 diabetes, rheumatoid arthritis and multiple sclerosis in Caucasians (Anaya 2010, Festen, Goyette et al. 2011, Chatzikyriakidou, Voulgari et al. 2013). As the gene name indicates, the TAGAP protein plays an important role in the activation of T cells, which leads to cytoskeleton arrangement and finally their migration (Connelly, Berg et al. 2014). The product of TAGAP gene phosphorylates the GTPases, increasing their innate activity and consequently leads to several changes in T cells and their activity (Mohamed N. S., et al., 2016).. The aim of this study was to investigate the possible relationship between rs1738074 single nucleotide polymorphism (SNP) located in the 5′-UTR region of the TAGAP gene with the risk and susceptibility to MS in the Iranian population.
Material and methods: Human subjects and blood sampling: In the present study 300 individuals (150 patients and 150 controls) were involved in the study. All the patients were considered as “definite MS” according to the McDonald criteria (McDonald, Compston et al. 2001). Before blood sampling, participants in the study were informed and consented for blood donation and subsequent molecular experiments. Blood samples were collected in EDTA containing tubes and kept frozen until DNA extraction and genotyping examinations.
Genomic DNA extraction and genotyping: Total genomic DNA was extracted using standard salting out precipitation method (Miller, Dykes et al. 1988). Allele specific PCR (ASP) was used as the standard method for determining genotype frequencies in patient and control groups (Gaudet, Fara et al. 2009). The Primers were designed using Oligo V7 software and web-based primer1 software (primer1.soton.ac.uk/primer1.html) (Collins and Ke 2012). The rs1738074 SNP results in substitution of T and C alleles with each other in TAGAP gene. Four primers were designed for genotyping of rs1738074 SNP. The sequence of the primers are as follows: Reverse T (allele T): 5′-GCTGTAAAGAATGGGAGAAACAGaAt-3′ Reverse C (allele C): 5′-GCTGTAAAGAATGGGAGAAACAGaAc-3′ Forward outer: 5′-GCCCTAAAAGGAATGAGGAAGC-3′ Reverse outer: 5′-GAGCATATTCCACGGGATAGC-3′. The PCR amplification was carried out in 25 μL total volume in Eppendorf tubes containing 2.5 µL 10X PCR buffer, 1 µL dNTP (10mM), 1 µL MgCl2 (50mM), 1 µL DNA (100 ng/uL), 1µL reverse T (10pM) (vial 1), 1µL reverse C (10pM) (vial 2), 1 µL forward primer (10pM), .25 µL reverse outer primer (10pM), .25 µL Taq DNA Polymerase (5unit/ µL) and deionized H2O up to 25 µL. The PCR conditions were as follows: 5 minutes denaturing at 94ºC, 40 seconds denaturing at 94ºC, 45 seconds annealing at 60ºC and extension at 72ºC for 50 seconds, for a total of 31 cycles and eventually 10 minutes at 72ºC. The PCR products were separated and analyzed on 2% agarose gel electrophoresis (Figure 1). Statistical analysis: Patients and normal controls were examined for any significant differences in their genotype distribution at the population level. Allele frequency was determined, using GENEPOP software (genepop.curtin.edu.au) (Rousset 2008). Statistical analysis was performed using IBM SPSS21 software.
Pearson’s chi-square test was applied to perform homogeneity assessments for distribution of the genotypes between the patients and the control population. The association between genotypes and MS were examined as the odds ratio (OR) with a 95% confidence interval (95% CI). Both patient and control groups were examined for deviation from Hardy-Weinberg equilibrium.
Results Genotyping of TAGAP rs1738074 SNP The DNA samples of 150 cases, and 150 control individuals were genotyped. As shown in Figure 1, for each individual two PCR reactions were performed, one with reverse T primer and the other with reverse C primer. As a consequence, three different genotypes could be detected, which were T/T, T/C, and C/C. For heterozygous individuals two allele bands (T and C) and for homozygous individuals, one allele band (T or C) were expected. After performing PCR amplifications, two product bands were expected on the agarose gels. The forward outer and reverse C/T primers resulted to a 224 bp product. For ensuring of PCR accomplishment in the PCR tubes, the reverse outer primer was designed and used in the PCR amplifications. Amplification with this primer and the forward outer primer resulted in a 372 bp product. As presented in Table 1, the genotype frequencies of TT, TC and CC in patients were 10.67%, 51.33% and 38%, respectively, and in normal individuals were 20.66%, 42.67% and 36.67%, respectively. The allele frequency of rs1738074 was shown in Table 2. Association of the TAGAP rs1738074 TT genotype with the prevalence of MS Statistical analysis using SPSS and Genepop software were performed to investigate the correlation of the SNP genotypes with the prevalence of MS. Both patient and control groups were in accordance with Hardy-Weinberg equilibrium (p-value= 0.22 and p-value=0.14, respectively).
The results showed an association between the TAGAP rs1738074 and the prevalence of MS (p-value= 0.049). Among the genotypes of the TAGAP rs1738074, the frequency of TT genotype was significantly lower in MS patients in comparison with the controls (p-value< 0.05, odds ratio was 2.18, 95% CI: 1.1374.187) (see Table 1). According to these results, it could be suggested that the TAGAP rs1738074 was associated with the prevalence of MS in the Iranian population and that the TT genotype could have a significant role in reducing the prevalence of the disease.
Discussion In recent years, the prevalence of MS has increased significantly in Iran and from this prospect, Iran is now considered among the medium to high risk countries (Ronaghi, Vallian et al. 2009, Izadi, Nikseresht et al. 2014). Many genes have been investigated for association with the prevalence and progression of the disease. However, no specific gene has been reported to directly resulting in MS. To date, a few studies have investigated the molecular pathogenesis of MS in the population of Iran, and most of them have principally explored epidemiological and immunological factors related to MS, such as HLA antigenic polymorphisms (Kalanie, Hosseinzadeh et al. 2011, Reza 2015, Mardasi, Galehdari et al. 2016). TAGAP gene has been reported as one of the genes associated with susceptibility to MS (Muñoz-Culla, Irizar et al. 2013). In the present study association of rs1738074 polymorphism in TAGAP gene with MS was investigated in the Iranian population. This SNP results in the substitution of nucleotide cytosine (C) with thymine (T). The comparison of genotype frequencies between patient and control groups revealed a significant difference in the TAGAP T/T genotype, but not in the T/C and C/C genotypes. Therefore, according to these results, it could be suggested that T/T genotype of rs1738074 could have a preventive effects on the MS prevalence and could be further studied as a possible risk factor for MS. Therefore, individuals
with this genotype could be at a lower risk for MS. These data and also the result of previous studies confirm the effect of TAGAP rs1738074 SNP on prevalence of MS. Moreover, the result of this study are in line with the previous reports on the possible role of the TAGAP rs1738074 polymorphism with prevalence of MS (Consortium I.M.S.G. and W.T.C.C.C. 2 2011). In addition to TAGAP rs1738074, we also examined the allele frequencies of RAGE rs1800624 gene in the same group population. It has been reported that RAGE rs1800624 could play a role in the prevalence of MS (Tiszlavicz, Gyulai et al. 2009, Li, Zhao et al. 2011). However, our data showed no significant association for RAGErs1800624 and MS (Jazaeri, Karimi Moghadam et al. 2016). The clinical and demographic characteristics of the patients were evaluated and presented in Tables 1 and 3. The data indicated that the prevalence of MS is much higher in the Iranian females compare to males. Moreover, analysis of the association of primary progressive, progressive relapsing; relapsing remitting; and secondary progressive of the disease with different genotypes of TAGAP rs1738074 were analyzed. The data showed that the genotype TT compare to other genotypes is more effective in progression of the disease. In summary, the data from the present study showed that patients with the TAGAP T/T genotype were less prone to MS. Therefore, the patients carrying this genotype may require less thorough treatment compared to patients with the TAGAP T/C and TAGAP C/C genotypes. Identification of more associated genes with MS would help completing the genetic map of this disease and even could help to improve related medical treatments.
Acknowledgments: We would like to thank the patients and their family for participation in this study. This study was supported by department of research of University of Isfahan, Isfahan, IR Iran. The study was approved by University of Isfahan institutional review board (IRB) for research and ethics approval (Approval Ref No. 790205).
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Legend to Figure 1 Figure 1: Genotyping of TAGAP rs1738074 in the Iranian population. The PCR products were separated on 2% agarose gel. As presented, the 372 bp band shows the internal PCR control confirming the occurrence of PCR in the PCR tubes. 224 bp band shows the specific band. Lanes 2 and 3 show an individual homozygous for TT genotype. Lanes 4 and 5 show a homozygous CC individual, and lanes 6 and 7 show a heterozygous TC individual. The DNA size marker is in lane 1. Figure 1
Table 1: The clinical characteristics and genotype frequency of TAGAP rs1738074 in the Iranian population Genotypes CC
TC
TT
MS patients
57/150 (38%)
77/150 (51.33%)
16/150 (10.67%)
Normal controls
55/150 (36.67%)
64/150 (42.67%)
P-value
NS
NS
Significant**
RR
35%
45%
20%
PP
18%
52%
30%
SP
12%
58%
30%
PR
21%
39%
40%
P-value= 0.049*
31/150 (20.66%)
NS: Not significant; **p-value= 0.025, fisher’s exact test, odds ratio (controls/MS patients) = 2.18, 95% CI: 1.137- 4.187, PP, primary progressive; PR, progressive relapsing; RR, relapsing remitting; and SP, secondary progressive.
Table 2: The allele frequency of TAGAP rs1738074 Allele frequency MS patients Normal controls
C 63.7% 58%
T 36.3% 42%
Table 3: Demographic characteristics of Multiple Sclerosis (MS) Patients and control group. Gender
Age (year)
Disease
Marital status
Occupation
Duration Male
Female
Male
Female
Male/Female
Married
Single
Employed
Unemployed
(year) MS
26%
74%
patients Control group
25-
23-36
2-5
66%
34%
36%
64%
25-36
-
60%
40%
45%
55%
45 25%
25%
2545