doi: 10.1111/iji.12152
Interleukin-4 single nucleotide polymorphisms in juvenile systemic lupus erythematosus M. Mahmoudi*, F. Tahghighi†,‡, V. Ziaee†,‡, S. Harsini§, A. Rezaei§, S. Soltani¶,**, M. Sadr¶,**, M. H. Moradinejad‡, Y. Aghighi†† & N. Rezaei‡,§,¶,**
Summary Juvenile systemic lupus erythematosus (JSLE) is a chronic, recurrent multisystem inflammatory disease, caused by a combination of environmental events and genetic risk factors. As cytokines, including interleukin-4 (IL-4), seem to have a role in the pathogenesis of JSLE, the investigation was performed to evaluate the associations of specific single nucleotide polymorphisms (SNPs) of IL-4 and IL-4RA genes in a case– control study. Fifty-nine patients with JSLE were recruited in this study as patients’ group and compared with 140 healthy volunteers. Genotyping was performed for IL-4 gene at positions 1098, 590 and 33, as well as IL-4 receptor a (IL-4RA) gene at position +1902, using polymerase chain reaction with sequence-specific primers method. Following alleles were found to be more common among patients with JSLE: C at 590 and 33 and T at 1098 of IL-4 gene (P value < 0.001; OR = 4.6, P value < 0.001; OR = 2.7 and P value < 0.001; OR = 2.1, respectively). Additionally, significant positive associations for the following genotypes were recognized in JSLE cases, compared with controls: C/C at 33, C/C at 590 and T/T at 1098 of IL-4 gene (P value < 0.001; OR = 5.3, P value < 0.001; OR = 29.5
* School of Nutrition and Dietetics, Tehran University of Medical Sciences, Tehran, Iran, † Pediatric Rheumatology Research Group, Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran, ‡ Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran, § Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran, ¶ Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran, ** Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran and †† Department of Pediatrics, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran Received 21 May 2014; revised 28 August 2014; accepted 7 September 2014 Correspondence: Nima Rezaei, MD, PhD, Research Center for Immunodeficiencies, Children’s Medical Center Hospital, Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran. Tel: +9821 6692 9234; Fax: +9821 6692 9235; E-mail: [email protected]
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and P value < 0.001; OR = 3.3, respectively), while following genotypes were less frequent among patients with JSLE: T/C at 33 and 590 and T/G at 1098 of IL-4 gene (P value < 0.001; OR = 0.1, P value < 0.001; OR = 0.03 and P value < 0.001; OR = 0.3, respectively). Furthermore, we noticed an astonishing negative haplotypic association for JSLE for IL-4 (positions 1098, 509 and 33) TTC, GCC and TTT haplotypes (P value < 0.001). There was also a significant relationship between TCC haplotype (IL4 gene at positions 1098, 590 and 33) and having JSLE (P value < 0.001). On the other hand, we found no significant associations between IL-4R polymorphisms and the susceptibility to JSLE. Cytokine gene polymorphisms may influence susceptibility to JSLE. Particular IL-4 gene variants are associated with JSLE and might have a role in the pathophysiology of disease.
Introduction Systemic lupus erythematosus (SLE) is a chronic, recurrent multisystem inflammatory disease, caused by a combination of environmental events and genetic risk factors (Midgley et al., 2009; Sule et al., 2011). Roughly 10–20% of all SLE cases prevail in the first two decades of life (Tucker et al., 1995; Klein-Gitelman et al., 2002; Stichweh et al., 2004). It has been reported that juvenile SLE (JSLE) imposes a greater burden arising from long-term morbidity, on society, as well as having a more aggressive clinical course in comparison with adult-onset SLE (Mok et al., 2005; Perfumo & Martini, 2005; Lee et al., 2007). At the immunopathogenesis level, the involvement of both B cells and T cells in the expression of SLE immunopathology is indubitable (Tsokos et al., 1997). A considerable body of evidence points to a crucial role of cytokines in the pathogenesis of SLE, as an autoimmune disorder (Tsao, 2004). Cytokine genetic variants seem to elucidate, at least to some degree, the variations in serum levels of cytokine among individuals (Grainger et al., 1999; Miyake et al., 2002). Some of these SNPs have been reported to have associations
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 512–517
IL-4 SNPs in juvenile SLE
with SLE, either as disease modifiers (Ohtsuka et al., 1999; Miyake et al., 2002) or as susceptible candidate genes (Rood et al., 2000; Ahmad & Bruce, 2001; Gibson et al., 2001). Interleukin-4 (IL-4), which is a pleiotropic type I cytokine, is produced by T cells, basophils, mast cells and natural killer cells (Midgley et al., 2009; Sule et al., 2011). IL-4 has been identified as an important actor in regulating the differentiation of antigen-stimulated naive T cells to develop into T helper 2 (Th2), which is capable of secreting cytokines such as IL-4, IL-5, IL-6 and IL-10, and contributes towards humoral immune responses (Mosmann & Coffman, 1989; Nelms et al., 1999). Moreover, IL-4 inhibits Th1-mediated immune inflammation through antagonizing the macrophage activating effect of IFN-c produced by Th1 (Mudd et al., 2006). It is well understood that IL-4 mediates its biological activity by binding to IL-4 receptor. It has also been speculated that certain polymorphisms in IL-4 RA could be associated with the risk of SLE development (Xu et al., 2007). Despite identification of several polymorphisms in the cytokine genes to influence both initiation and progression of inflammatory responses in different immunological disorders (Amirzargar et al., 2005, 2006, 2010; Mahdaviani et al., 2009; Rezaei et al., 2009; Barkhordari et al., 2010), there is a paucity of data concerning the possible roles of IL-4 gene SNPs in the immunopathology of juvenile SLE, and to the best of our knowledge, no association study on IL-4 gene polymorphisms has ever been performed involving Iranian paediatric patients with SLE. In this study, we appraised the association of the SNPs in IL-4RA gene at position +1902 and IL-4 gene at positions 1098, 590 and 33 reported to modify the production of IL-4 in a sample of Iranian paediatric patients with SLEs and healthy controls.
Patients and methods Study population
Fifty-nine Iranian children with SLE (10 male, 49 female), with mean age of male 10.10 4.38 and female 11 3.47, who had been referred to the Children’s Medical Center Hospital, the Pediatrics Center of Excellence in Iran, participated in this study. They were among all the patients with SLE who were under follow-up in this centre and were agreed to enrol in this study. The revised criteria of the American College of Rheumatology (ACR) for SLE were used for the diagnosis (Hochberg, 1997). One hundred and forty healthy unrelated volunteers with the same ethnicity of the patients were enrolled into the study as the control group (Amirzargar et al., 2008). The local ethical committee of Tehran University of Medical Sciences approved the study. Written informed consents were taken from all participants or their parents prior to the study procedures.
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 512–517
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Sampling and genotyping
Five millilitres of peripheral blood from all the participants were collected with 5% EDTA as anticoagulant. Genomic DNA was isolated from nucleated cells by modified ‘salting-out’ method (Miller et al., 1988). We have determined the allele and genotype frequencies of IL-4 (G/T at 1098 as well as C/T at 590 and 33) and IL-4RA (G/A at +1902) genes, using polymerase chain reaction with sequence-specific primers (PCRSSP assay kit; Heidelberg University, Heidelberg, Germany) (Amirzargar et al., 2008). Briefly, amplification of the extracted DNA was carried out using a thermal cycler Techne Flexigene apparatus (Rosche, Cambridge, UK), as explained before. The presence or absence of polymerase chain reaction products was observed by 2% agarose gel electrophoresis. Subsequently, the gel was placed on an ultraviolet transilluminator, and a picture was taken for interpretation and documentation. Statistical analysis
Allele, genotype and haplotype frequencies were estimated by direct gene counting and were compared with the controls using the chi-square test. We calculated the odds ratio (OR) and 95% confidence interval (CI) for each allele, genotype and haplotype in both case and control groups, and considered the P value of
Interleukin-4 single nucleotide polymorphisms in juvenile systemic lupus erythematosus M. Mahmoudi*, F. Tahghighi†,‡, V. Ziaee†,‡, S. Harsini§, A. Rezaei§, S. Soltani¶,**, M. Sadr¶,**, M. H. Moradinejad‡, Y. Aghighi†† & N. Rezaei‡,§,¶,**
Summary Juvenile systemic lupus erythematosus (JSLE) is a chronic, recurrent multisystem inflammatory disease, caused by a combination of environmental events and genetic risk factors. As cytokines, including interleukin-4 (IL-4), seem to have a role in the pathogenesis of JSLE, the investigation was performed to evaluate the associations of specific single nucleotide polymorphisms (SNPs) of IL-4 and IL-4RA genes in a case– control study. Fifty-nine patients with JSLE were recruited in this study as patients’ group and compared with 140 healthy volunteers. Genotyping was performed for IL-4 gene at positions 1098, 590 and 33, as well as IL-4 receptor a (IL-4RA) gene at position +1902, using polymerase chain reaction with sequence-specific primers method. Following alleles were found to be more common among patients with JSLE: C at 590 and 33 and T at 1098 of IL-4 gene (P value < 0.001; OR = 4.6, P value < 0.001; OR = 2.7 and P value < 0.001; OR = 2.1, respectively). Additionally, significant positive associations for the following genotypes were recognized in JSLE cases, compared with controls: C/C at 33, C/C at 590 and T/T at 1098 of IL-4 gene (P value < 0.001; OR = 5.3, P value < 0.001; OR = 29.5
* School of Nutrition and Dietetics, Tehran University of Medical Sciences, Tehran, Iran, † Pediatric Rheumatology Research Group, Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran, ‡ Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran, § Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran, ¶ Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran, ** Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran and †† Department of Pediatrics, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran Received 21 May 2014; revised 28 August 2014; accepted 7 September 2014 Correspondence: Nima Rezaei, MD, PhD, Research Center for Immunodeficiencies, Children’s Medical Center Hospital, Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran. Tel: +9821 6692 9234; Fax: +9821 6692 9235; E-mail: [email protected]
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and P value < 0.001; OR = 3.3, respectively), while following genotypes were less frequent among patients with JSLE: T/C at 33 and 590 and T/G at 1098 of IL-4 gene (P value < 0.001; OR = 0.1, P value < 0.001; OR = 0.03 and P value < 0.001; OR = 0.3, respectively). Furthermore, we noticed an astonishing negative haplotypic association for JSLE for IL-4 (positions 1098, 509 and 33) TTC, GCC and TTT haplotypes (P value < 0.001). There was also a significant relationship between TCC haplotype (IL4 gene at positions 1098, 590 and 33) and having JSLE (P value < 0.001). On the other hand, we found no significant associations between IL-4R polymorphisms and the susceptibility to JSLE. Cytokine gene polymorphisms may influence susceptibility to JSLE. Particular IL-4 gene variants are associated with JSLE and might have a role in the pathophysiology of disease.
Introduction Systemic lupus erythematosus (SLE) is a chronic, recurrent multisystem inflammatory disease, caused by a combination of environmental events and genetic risk factors (Midgley et al., 2009; Sule et al., 2011). Roughly 10–20% of all SLE cases prevail in the first two decades of life (Tucker et al., 1995; Klein-Gitelman et al., 2002; Stichweh et al., 2004). It has been reported that juvenile SLE (JSLE) imposes a greater burden arising from long-term morbidity, on society, as well as having a more aggressive clinical course in comparison with adult-onset SLE (Mok et al., 2005; Perfumo & Martini, 2005; Lee et al., 2007). At the immunopathogenesis level, the involvement of both B cells and T cells in the expression of SLE immunopathology is indubitable (Tsokos et al., 1997). A considerable body of evidence points to a crucial role of cytokines in the pathogenesis of SLE, as an autoimmune disorder (Tsao, 2004). Cytokine genetic variants seem to elucidate, at least to some degree, the variations in serum levels of cytokine among individuals (Grainger et al., 1999; Miyake et al., 2002). Some of these SNPs have been reported to have associations
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 512–517
IL-4 SNPs in juvenile SLE
with SLE, either as disease modifiers (Ohtsuka et al., 1999; Miyake et al., 2002) or as susceptible candidate genes (Rood et al., 2000; Ahmad & Bruce, 2001; Gibson et al., 2001). Interleukin-4 (IL-4), which is a pleiotropic type I cytokine, is produced by T cells, basophils, mast cells and natural killer cells (Midgley et al., 2009; Sule et al., 2011). IL-4 has been identified as an important actor in regulating the differentiation of antigen-stimulated naive T cells to develop into T helper 2 (Th2), which is capable of secreting cytokines such as IL-4, IL-5, IL-6 and IL-10, and contributes towards humoral immune responses (Mosmann & Coffman, 1989; Nelms et al., 1999). Moreover, IL-4 inhibits Th1-mediated immune inflammation through antagonizing the macrophage activating effect of IFN-c produced by Th1 (Mudd et al., 2006). It is well understood that IL-4 mediates its biological activity by binding to IL-4 receptor. It has also been speculated that certain polymorphisms in IL-4 RA could be associated with the risk of SLE development (Xu et al., 2007). Despite identification of several polymorphisms in the cytokine genes to influence both initiation and progression of inflammatory responses in different immunological disorders (Amirzargar et al., 2005, 2006, 2010; Mahdaviani et al., 2009; Rezaei et al., 2009; Barkhordari et al., 2010), there is a paucity of data concerning the possible roles of IL-4 gene SNPs in the immunopathology of juvenile SLE, and to the best of our knowledge, no association study on IL-4 gene polymorphisms has ever been performed involving Iranian paediatric patients with SLE. In this study, we appraised the association of the SNPs in IL-4RA gene at position +1902 and IL-4 gene at positions 1098, 590 and 33 reported to modify the production of IL-4 in a sample of Iranian paediatric patients with SLEs and healthy controls.
Patients and methods Study population
Fifty-nine Iranian children with SLE (10 male, 49 female), with mean age of male 10.10 4.38 and female 11 3.47, who had been referred to the Children’s Medical Center Hospital, the Pediatrics Center of Excellence in Iran, participated in this study. They were among all the patients with SLE who were under follow-up in this centre and were agreed to enrol in this study. The revised criteria of the American College of Rheumatology (ACR) for SLE were used for the diagnosis (Hochberg, 1997). One hundred and forty healthy unrelated volunteers with the same ethnicity of the patients were enrolled into the study as the control group (Amirzargar et al., 2008). The local ethical committee of Tehran University of Medical Sciences approved the study. Written informed consents were taken from all participants or their parents prior to the study procedures.
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 512–517
513
Sampling and genotyping
Five millilitres of peripheral blood from all the participants were collected with 5% EDTA as anticoagulant. Genomic DNA was isolated from nucleated cells by modified ‘salting-out’ method (Miller et al., 1988). We have determined the allele and genotype frequencies of IL-4 (G/T at 1098 as well as C/T at 590 and 33) and IL-4RA (G/A at +1902) genes, using polymerase chain reaction with sequence-specific primers (PCRSSP assay kit; Heidelberg University, Heidelberg, Germany) (Amirzargar et al., 2008). Briefly, amplification of the extracted DNA was carried out using a thermal cycler Techne Flexigene apparatus (Rosche, Cambridge, UK), as explained before. The presence or absence of polymerase chain reaction products was observed by 2% agarose gel electrophoresis. Subsequently, the gel was placed on an ultraviolet transilluminator, and a picture was taken for interpretation and documentation. Statistical analysis
Allele, genotype and haplotype frequencies were estimated by direct gene counting and were compared with the controls using the chi-square test. We calculated the odds ratio (OR) and 95% confidence interval (CI) for each allele, genotype and haplotype in both case and control groups, and considered the P value of
