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. 2017 Sep;12(3):164–168.

Preliminary Study Regarding the Association between Tumor Necrosis Factor Alpha Gene Polymorphisms and Childhood Idiopathic Nephrotic Syndrome in Romanian Pediatric Patients

Ioana TIERANU 1,2, Monica I DUTESCU 3, Constantin BARA 4, Cristian G TIERANU 5, Mihaela BALGRADEAN 6,7, Olivia M POPA 8
PMCID: PMC5706754  PMID: 29218062

Abstract

Background:

Childhood idiopathic nephrotic syndrome (INS) is one of the most common glomerular diseases, characterized by heavy proteinuria, hypoalbuminemia, dyslipidemia and generalized edema. Although some progresses were made regarding the pathogenesis of this disease, there are a lot of questions still left unanswered. Some of them involve the implications of several cytokines, including tumor necrosis factor alpha (TNF-alpha), in the development and clinical course of INS.

Objective:

Our objective was to analyze the role of two single nucleotide polymorphisms of TNF-alpha gene in the development of pediatric INS and their implication in the response to corticosteroid therapy.

Material and methods:

Seventy patients with INS and 159 healthy controls were included in this study. They were analyzed for TNF-alpha gene polymorphisms by using polymerase chain reaction. The two SNPs (rs1799724/-857C/T and rs1800629/-308G/A) were genotyped by TaqMan Genotyping Assays, association tests were performed and p values <0.05 were considered significant.

Results:

Minor alleles frequencies were 15.72% in INS patients versus 18.55% in controls for 857*T allele and 11.43% in INS versus 13.2% in controls for 308*A allele. Although the minor alleles were more frequent in controls than in patients, the difference was not statistically significant (p=0.46, OR=0.818 and p=0.59, OR=0.848).

Analyzing the response to corticosteroid therapy, we found a low frequency of 857*T allele in steroid resistant patients (9.09%) compared to steroid sensitive patients (16.95%) and controls (18.55%). Regarding 308*A allele, the frequencies were 18.18% in the corticoresistant group and 10.17% in the corticosensitive one. None of them was statistically significant (p>0.05).

Conclusions:

We conclude that neither -857C/T, nor-308G/A polymorphisms of TNF-alpha gene are associated with the susceptibility and response to steroid treatment of INS in our population. Given the small sample size used, future studies are necessary to clarify the results observed in the present study.

Keywords:nephrotic syndrome, TNF-alpha polymorphisms, steroid response

INTRODUCTION

Idiopathic nephrotic syndrome (INS) is the most frequent glomerular disease in childhood. The alterations of the selective glomerular permeability barrier – caused by damage to podocytes and foot process effacement – lead to intense proteinuria (1), which is the hallmark of this disease. In the USA and Europe, the annual incidence of pediatric INS has been estimated to be between 1 and 3 per 100,000 children below the age of 16 (2-4), varying with age, race and geography.

Children with NS have a decreased quality of life (5), are at risk of a wide range of complications associated with significant morbidity (infection, thromboembolism and dyslipidaemia) and experience mortality rates of up to 2.7% (6).

The pathogenesis of INS is yet unknown, but evidence such as efficacy of immunosuppressive treatment (especially prednisone), indicate that the immune system may play a crucial pathogenic role in non-genetic forms of this disease. However, despite extensive investigation, the mechanism by which immune dysregulation leads to disruption of the glomerular filtration barrier and consequently to proteinuria is poorly understood (7).

Cytokines play a critical role as mediators of inflamation and as progressive factors in INS, some of them being considered prime candidates for mediating INS progression (8-10). Among them, tumor necrosis factor alpha (TNF-alpha), a potent immunomediator and proinflammatory cytokine, has been implicated in the pathogenesis of a large number of diseases (11). Several studies reported the association of TNF-alpha gene variations with childhood INS (12-14).

First line treatment – corticotherapy – is effective in most of the patients, meaning they attain remission following this treatment (steroid sensitive) (15). The ones who fail to respond to treatment are called steroid resistent. However, some of the patients who respond to therapy will undergo relapse at least once, with 50% of them being frequent relapsers or developing steroid dependence (15). The mechanisms underlying the difference in response to steroid the rapy are not well understood, genetic factors may be involved (16).

The present study was conducted to investigate the possible association between TNF-alpha gene polymorphisms and INS in a group of Romanian children.

MATERIALS AND METHODS

Patients and controls

A total number of 229 unrelated Caucasian individuals of Romanian origin was included in the study. Idiopatic nephrotic syndrome patients (N=70 patients, 50 M/20 F, mean age nine years) were recruited from the Nephrology Department of ”M. S. Curie” Emergency Hospital (Bucharest, Romania). All patients with INS were diagnosed according to the Kidney Disease Improving Global Outcomes (KDIGO) guideline criteria (17) and were categorized according to their response to corticotherapy: steroid sensitive – attainment of complete remission within initial four weeks of corticosteroid therapy (N=59, 42 M/7F, mean age eight years) and steroid resistant – failure to achieve complete remission after eight weeks of corticosteroid therapy) (N=11 patients, 8 M/3 F, mean age 9 years 11 months) (17).

We used a total number of 159 controls (103 M/56F, mean age 39 years), with no history of proteinuria and edemas.

Assessment of HP infection was performed using the stool antigen test, Quest DiagnosticsTM, an enzyme immunoassay with a sensitivity and specificity of 96% for detecting HP infection.

The study was approved by the Ethic Committee of “Marie Curie” Hospital, Bucharest, Romania. The details were explained to all patients and controls and consent for genetic screening was obtained.

DNA extraction and genotyping

Genomic DNA was obtained from EDTA treated peripheral blood samples using QIAamp DNA Blood Mini Kit(Qiagen, Hilden, Germany) according to manufacturer protocol. Two single nucleotide polymorphisms of TNF-alpha gene were selected for this study: rs1799724 (-857C/T) and rs1800629 (-308G/A). Single nucleotid polymorphisms (SNPs) genotyping was performed by real-time polymerase chain reaction (PCR) with TaqMan Allelic Discrimination AssaysC_11918223_10 and C_7514879_10, respectively (Real time PCR System, Applied Biosystems, Foster City, CA).

Statistics

The Hardy-Weinberg equilibrium (HWE) was tested using the Chi-square test. The association tests and HWE tests were performed with DeFinetti program (http://ihg2.helmholtz muenchen. de/cgi-bin/hw/hwa1.pl) and p values ≤0.05 were considered statistically significant.

Alleles and genotypes frequencies of the studied SNPs were compared between INS patients and controls and between the steroid sensitive group and the steroid resistant group.

RESULTS

We have performed a case control association study of TNF-alpha gene single nucleotide polymorphisms in a group of Romanian idiopatic nephrotic syndrome patients versus ethnically matched controls.

The control and patients groups showed no departure from HWE for all studied SNPs. The frequencies of TNF-alpha gene SNPs genotypes and alleles in controls and INS patients are presented in Table 1. For rs 1799724, the frequence for minor allele T was 15.72% in the INS group versus 18.55% in the control group. Although the minor allele T was more frequent in controls than in patients, the difference was not statistically significant (p=0.46, OR=0.818).

The analysis of the two groups (INS patients and controls) revealed similar distribution of the investigated polymorphisms.

When the INS patients were subdivided according to steroid responsiveness, no significant statistical difference was found in allele and genotypes frequencies between the steroid-sensitive and steroid-resistant subgroups when compared to each other, nor when compared to controls (Table 2).

DISCUSSION

In the present paper we studied the association between two TNF-alpha gene polymorphisms (G308A and C857T) and the susceptibility for developing INS in Romanian population. We investigated as well the implication of those polymorphisms in the response to corticosteroid therapy.

Tumor necrosis factor alpha (TNF-alpha) is a potent immunomodulator and pro-inflamma tory Th1 cytokine (19). Polymorphism at position -308 of the TNF-alpha gene promoter (G to A base transition) has been linked to increased TNF transcription (20, 21), earlier studies showing an increase of TNF-alpha synthesis and gene expression in patients with INS (22, 23).

Several studies have investigated the association between cytokines gene polymorphisms and INS in worldwide populations, but not in Romanian population (12-14, 18). The results of our study showed no significant difference between the patient group and controls regarding the TNF-alpha gene SNPs genotypes or alleles distribution. This is in agreement with Kim Sung-Do et al., who genotyped the G to A exchange at po si tion -308 of the TNF-alpha gene, and found no difference in allele frequency between the two groups (18). In contrast, Madani et al. concluded in their study that AA genotype and A allele were significantly higher in the INS group than controls (12). The same results were obtained by Jafar T et al. in a study conducted in 2011 (14).

Given the fact that corticosteroids are the first line agents used to treat pediatric INS, but their long term use is marked by a lot of serious adverse effects, it is very important to see if we can find some early markers that would allow us to determine the effectiveness of these agents in remission induction.

We could not find any association between the studied polymorphisms and the response to corticosteroid therapy. In 2014, Madani et al. investigated the role of cytokine genes polymorphisms (TNF-alpha-G308A, IL6-G174C and IL4-C590T) and the response to corticosteroid therapy in children with INS in Egypt. When comparing steroid-sensitive patients with steroid- resistant ones, the results showed that AA genotype is higher in the first group (though not reaching statistical significant difference), but A allele distribution is significantly higher in the steroid-sensitive group compared to the steroidresistant one (12). More than that, in the study of Jafar T et al., when the steroid-resistant group was compared with steroid-sensitive group, significant association was found at both the genotypic level, and allelic level, in the steroid-resistant group (14). In 2008, Tripathy G et al. investigated the association between some cytokine gene polymorphisms (IL-4, IL-6, TNF-alpha) and the response to corticosteroid therapy, in 150 children with INS. They observed that TNF-alpha showed a strong association at both the genotypic and allelic levels in steroid-sensitive and steroid-resistent groups, de mons trating that it may be considered one of the genetic risk factors affecting the steroid response among the North Indian INS patients (13).

Regarding -857C/T TNF alpha gene polymorphism, to our knowledge, this is the first paper adressing this issue. Our data did not show any significant difference between INS population and controls in terms of genotype and allele frequencies; also, we found no role of this polymorphism in predicting response to steroid treatment in Romanian pediatric INS patients.

DNA data should be interpreted carefully, as different populations present different geneticbackground, making results difficult to apply in practice outside the studied population. Given the small sample size used, future studies with a larger scale are necessary to clarify the results observed in the present study and to determine whether TNF-alpha polymorphisms play a role in INS development and course of treatment.

CONCLUSION

Our results show that, in our population, neither -857C/T, nor -308G/A polymorphisms of TNF-alpha gene are associated with the susceptibility and the response to steroid treatment of INS.

Conflict of interests: none declared.

Financial support: none declared.

TABLE 1.

TABLE 1.

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Genotypes and allele frequencies of TNF-alpha gene polymorphisms among INS and control groups

TABLE 2.

TABLE 2.

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Genotypes and allele frequencies of TNF-alpha gene polymorphisms among SSNS and SRNS groups

Contributor Information

Ioana TIERANU, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania; "MS Curie" Children Emergency Hospital, Bucharest, Romania.

Monica I. DUTESCU, "Prof. Dr. C. T. Nicolau" National Institute of Blood Transfusion, Bucharest, Romania

Constantin BARA, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania.

Cristian G. TIERANU, "Elias" Emergency University Hospital, Bucharest, Romania

Mihaela BALGRADEAN, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania; "MS Curie" Children Emergency Hospital, Bucharest, Romania.

Olivia M. POPA, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

REFERENCES

  1. Eddy AA, Symons JM. - Nephrotic syndrome in childhood. Lancet. 2003;9384:629–639. doi: 10.1016/S0140-6736(03)14184-0. [DOI] [PubMed] [Google Scholar]
  2. International Study of Kidney Disease in Children. - Nephrotic syndrome in children: prediction of histopathology from clinical and laboratory characteristics at time of diagnosis. A report of the International Study of Kidney Disease in Children. Kidney Int. 1978;2:159–165. doi: 10.1038/ki.1978.23. [DOI] [PubMed] [Google Scholar]
  3. McKinney PA, Feltbower RG, Brocklebank JT, Fitz patrick MM. - Timetrends and ethnic patt erns of childhood nephrotic syndrome in Yorkshire, UK. Pediatr Nephrol. 2001;12:1040–1044. doi: 10.1007/s004670100021. [DOI] [PubMed] [Google Scholar]
  4. Schlesinger ER, Sultz HA, Mosher WE, Feldman JG. - The nephrotic syndrome. Its incidence and implications for the community. Am J Dis Child. 1968;6:623–632. [PubMed] [Google Scholar]
  5. [No authors listed]. - The primary nephrotic syndrome in children. Identifi cation of patients with minimal change nephrotic syndrome from initial response to prednisone. A report of the International Study of Kidney Disease in Children. J Pediatr. 1981;98:561–564. doi: 10.1016/s0022-3476(81)80760-3. [DOI] [PubMed] [Google Scholar]
  6. [No authors listed]. - [No authors listed]. Minimal change nephrotic syndrome in children: deaths during the fi rst 5 to 15 years’ observation. Report of the International Study of Kidney Disease in Children. Pediatrics. 1984;73:497–501. [PubMed] [Google Scholar]
  7. Colucci M, Corpett i G, Emma F, Vivarelli M. - Immunology of idiopathic nephrotic syndrome. Pediatr Nephrol. 2017.;  doi: 10.1007/s00467-017-3677-5. [DOI] [PubMed] [Google Scholar]
  8. Cho BS, Yoon SR, Jang JY, et al. - Upregulation of interleukin-4 and CD23/ Fcepsilon RII in minimal change nephrotic syndrome. Pediatr Nephrol. 1999;13:199–204. doi: 10.1007/s004670050592. [DOI] [PubMed] [Google Scholar]
  9. Noronha IL, Niemir Z, Stein H, Waldherr R. - Citokines and growth factors in renal disease. Nephrol Dial Transplant. 1995;10:775–786. [PubMed] [Google Scholar]
  10. Tenbrock K, Schubert A, Stapenhorst L, et al. - Type I IgE receptor, interleukin 4 receptor and interleukin 13 polymorphisms in children with nephrotic syndrome. Clin Sci. 2002;102:507–512. [PubMed] [Google Scholar]
  11. Elahi MM, Asotra K, Matata BM, et al. - Tumor necrosis factor alpha-308 gene locus promoter polymorphism: An analysis of association with health and disease. Biochemia et Biophysia Acta. 2009;1792:163–172. doi: 10.1016/j.bbadis.2009.01.007. [DOI] [PubMed] [Google Scholar]
  12. Madani HA, Bazaraa HM, Rady H. - Association of cytokine genes polymorphisms and the response to corticosteroid therapy in children with idiopathic nephrotic syndrome: A pilot study in Egypt. International Research Journal of Medicine and Medical Sciences. 2014;4:84–90. [Google Scholar]
  13. Gaurav Tripathi, TabrezJafar, Kausik Mandal, et al. - Does cytokine gene polymorphism aff ect steroid responses in idiopatic nephrotic syndrome? Indian Journal of Medical Sciences. 2008;10:383–391. [PubMed] [Google Scholar]
  14. Tabrez Jafar, Suraksha Agrawal, Abbas Ali Mahdi, Raj Kumar Sharma, et al. - Cytokine Gene Polymorphism in Idiopathic Nephrotic Syndrome Children. Ind J ClinBiochem. 2011;3:296–302. doi: 10.1007/s12291-011-0126-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Reidy K, Kaskei FJ. - Pathophysiology of focal segmental glomerulosclerosis. Pediatr Nephrology. 2007;22:350–354. doi: 10.1007/s00467-006-0357-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. TripathiG, Jafar T, Mandal K, et al. - Does citokine gene polymorphism aff ect steroid response in idiopathic nephrotic syndrome? Ind J Med Sci. 2008;62:383–391. [PubMed] [Google Scholar]
  17. Kim Do-Sung, Park Jae-Man, Kim Il-Soo, et al. - Association of IL-1β, IL-1ra, and TNF-α gene polymorphisms in childhood nephrotic syndrome. Pediatr Nephrol. 2004;19:295–299. doi: 10.1007/s00467-003-1403-y. [DOI] [PubMed] [Google Scholar]
  18. Mosmann TR, Cherwinski H, Bond MW, et al. - Two types of murine helper T cell clone. 1. Defi nition according to profi les of lymphokine activities and secreted proteins. J Immunol. 1986;136:2348–2357. [PubMed] [Google Scholar]
  19. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. - Single base polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene detectable by NcoI restriction of PCR product. Hum Mol Genet. 1992;1:353. doi: 10.1093/hmg/1.5.353. [DOI] [PubMed] [Google Scholar]
  20. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. - Eff ects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl AcadSci USA. 1997;1:3195–3199. doi: 10.1073/pnas.94.7.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Bustos C, González E, Muley R, Alonso JL, Egido J. - Increase of tumor necrosis factor á synthesis and gene expression in peripheral blood mononuclear cells of children with idiopathic nephrotic syndrome. Eur J Clin Invest. 1994;24:799–805. doi: 10.1111/j.1365-2362.1994.tb02022.x. [DOI] [PubMed] [Google Scholar]
  22. Matsumoto K. - Spontaneous and LPS-stimulated release of tumor necrosis factor-alpha by peripheral blood monocytes in patients with focal glomerular sclerosis. Nephron. 1995;70:118–119. doi: 10.1159/000188559. [DOI] [PubMed] [Google Scholar]

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