Effect of pro-inflammatory cytokine (IFN-γ +874, IL-18–137 G/C,–607 C/A) genes in relation to risk of vesico-ureteral reflux

Abstract

Aim: The aim this work is to estimate whether genetic polymorphisms of +874 of IFN-γ and −137 G/C,−607 C/A of IL-18 genes are implicated in the development of VUR, because a vast literature indicates that genetic variations play a significant role in the pathogenesis of VUR. Materials and methods: The PCR single specific primer (SSP) and amplification refractory mutation system (ARMS) were applied for analyzing the polymorphic sites of −137 G/C,−607 C/A of IL-18 and +874 of IFN-γ genes in 110 healthy controls and 124 VUR children. Results: A significant relationship was found between AT and combined AT + TT genotypes of IFN-γ and highly increased risk of VUR (OR = 4.2, 95% CI, 2.00–9.24; p < 0.0001: OR = 4.00, 95% CI, 1.90–8.70, p < 0.0001, respectively). On the other hand, the genotype frequency of IL18-137 G/C indicated a significant assessment of the decrease risk of VUR for GC and GC + CC genotypes (OR = 0.53, 95% CI, 0.3–0.9; p = 0.02: OR = 0.53, 95% CI, 0.3–0.92 p = 0.01, respectively). No significant association was found between −607 C/A polymorphism of IL-18 and UVR. Conclusion: To the author's best knowledge, this is the first data regarding polymorphism of IFN-γ (+874) cytokine genes that highly increased the risk of VUR. To confirm the presented data, further studies should be done in different populations with a larger sample size.

• IFN-γ
• IL-18
• polymorphism
• VUR

Introduction

Vesicoureteral reflux (VUR) is a frequent finding in children affecting to urinary tract infection (UTI) that ultimately, increase renal damage with consequent infections.1 VUR facilitates the entrance of both infection and transmission of bladder pressure to the kidney, though, the development from VUR and UTI to renal parenchymal damage, renal scarring and reflux nephropathy has not been comprehensively elucidated.2 It often occurs during fetal development with the incidence of more than 10% of the population;3 so, most of them are congenital (primary) and also, high pressure in the bladder can cause VUR (secondary). On the basis of international reflux study in children, there are five grades of severity in reflux.4

Various studies have suggested that more than one genetic change may account for VUR in some families.5 So, there are strong data for supporting the heritability of VUR, which are inherited in an autosomal dominant manner with variable penetrance.6 Genetic factors may also be changed by environmental exposures to express various phenotypes despite the identical gene changes.7 Maybe the genetic difference between the VUR and healthy children reflect a greater risk for inflammatory renal damage in children with VUR. The infection is accompanied by inflammation which is mediated by several soluble factors known as cytokine such as IL-18 and IFN-γ, which are well known to regulate the inflammatory response in UTI and renal damage, but less is acknowledged on their function after resolution of acute infection. IL-18 is a pro-inflammatory cytokine that belongs to the IL-1 super family and is produced by macrophages and other cells.8 It is now documented as a vital regulator of innate and acquired immune responses and plays multiple roles in chronic inflammation.9 IFN-γ is one of the key cytokines of T helper cells. Several studies have indicated that alterable expression of the IFN-γ level has significant effects on the susceptibility of various inflammatory and autoimmune diseases.10,11 The IL-18 gene locus is sited in 11q22.2-q23.3 and some polymorphisms have been detected in its promoter region. Any changes in the regulatory region of IL-18 affect its transcriptional activity, which include substitution of G > C at position −137 which changes the binding site of a histone 4 transcription factor-1 (H4TF-1) and a transform of C > A at position −607 disrupts a cyclic adenosine monophosphate (cAMP).12 Certain studies have confirmed that genetic polymorphisms of the IL-18 gene have been involved in various immune and inflammatory diseases.13,14 A single nucleotide polymorphism (+874T/A) located on chromosome 12q24.1 in the first intron of the human IFN-γ gene can putatively influence the secretion of IFN-γ.15

The main aim of the present study was to highlight the impact of single nucleotide polymorphism +874 of IFN-γ and −137 G/C,−607 C/A of IL-18 genes with the risk of VUR in a sample of children in southeastern of Iran.

Materials and methods

Study subjects

The study was confirmed by the Ethical Committee of Zahedan University of Medical Sciences in southeast of Iran in 2011. It was conducted on 124 VUR patients (including 73 females and 51 males) who had different grades of development in their illness (I = 5, II = 19, III = 40, IV = 14 and V = 6) with the mean age of 2.51 yr ( ± 2.89) and weight of 11.51 kg (±6) and a healthy group without VUR including 110 (44 males and 66 females) that had the mean age of 2.79 yr (±5.9) and weight of 11.23 kg (±5.9). The exclusion criterion was reflux secondary to another condition (e.g., neurogenic bladder, non-urological disorders/syndromes or chromosomal disorders).

According to the International Reflux Classification,17 diagnosis of primary VUR cases referring to the pediatric unit at Aliasghar Hospital (Zahedan, Iran) was done by voiding cystourethrogram (VCUG) and categorized as grades I–V. The control samples were obtained from individuals who had no inflammation disturbance or renal disease.

Sampling and polymerase chain reaction

Genotypic analysis was done on the DNA extracted from the collected blood by venipuncture in EDTA-coated tubes at the time of clinical examination. Polymorphisms of SNPs located in IL-18 (−607 C/A, −137 G/C) and IFN-γ (+874 T/A) genes were estimated by polymerase chain reaction (PCR) using PCR-SSP and ARMS for discovering various alleles and genotype, respectively. The primers sequence and PCR condition were used for detecting variations in IL-18 (−607 C/A, −137 G/C) and IFN-γ (+874 T/A) genes, as already reported.16–18

Statistical analysis

SPSS version 10.0 (SPSS, Chicago, IL) and Epical Info version 3.2 were used for all the statistical analysis. An association of polymorphisms in IL-18 (−607 C/A, −137 G/C) and IFN-γ (+874 T/A) genes with the risk of VUR was recognized by computing odds ratios (OR) and 95% confidence intervals (95% CI) using Epical Info version 3.2.

Results

As shown in Table 1, the allele frequency of IL18-137 G/C was for C 25% in the cases and 66.36% in the controls which was significantly different (p = 0.023) in comparison with wild-type allele (the most frequent). The allele frequency of IFN-γ gene in (+874 T/A) locus was determined as T (48.79%) for VUR individuals and (38.64%) for healthy group, which was statistically different (p = 0.003) in comparison with wild type A.

Table 1. Allele frequency (%) of IFN-γ (+874A/T) and IL-18 (−137G/C, −607C/A) genes among individual with VUR patients and healthy controls.

Genes	Cases (%)	Controls (%)	p Value
IL-18 (−137G/C)
G	186 (75)	74 (33.64)
C	62 (25)	148 (66.36)	0.023
IL-18 (−607C/A)
C	138 (55.65)	287 (53.18)
A	110 (44.35)	103 (46.82)	0.28
IFN-γ (+874A/T)
A	127 (51.21)	135 (61.36)
T	121 (48.79)	85 (38.64)	0.003

Note: The most frequent alleles at each site is chosen as the reference.

The genotype frequencies of IL18-137 G/C for GC were 41.093% in the cases and 56.3% in the controls and the combined genotype of GC + CC was 45.97% and 61.82% for the cases and controls, respectively. In comparison with the wild genotype of GG, the risk of disease (OR = 0.53, 95% CI, 0.3–0.9; p = 0.02: OR = 0.53, 95% CI, 0.3–0.92; p = 0.01) was cleared for GC and GC + CC genotypes, respectively, as shown in Table 2.

Table 2. Number and frequency of IFN-γ (+874A/T) and Il-18 (−137G/C, −607C/A) genotypes in VUR individuals and healthy controls.

SNPs	Cases N = 124	Controls N = 110	OR	CI (95%)	p Value
IL-18 (−137G/C)
GG	67 (54.03%)	42 (38.18%)	Ref	–	–
GC	52 (41.93%)	62 (56.36%)	0.53	0.30–0.93	0.022
CC	5 (4.03%)	6 (5.45%)	0.52	0.12–2.21	0.34
GC + CC	57 (45.97%)	68 (61.82%)	0.53	0.30–0.92	0.018
IL-18 (−607C/A)
CC	17 (13.71%)	9 (8.18%)	Ref
CA	104 (83.87%)	99 (%90)	0.56	0.21–1.40	0.21
AA	3 (2.42%)	2 (1.82%)	0.80	0.08–11.24	1.00
CA + AA	107 (86.29%)	101 (91.82%)	0.56	0.21–1.41	0.21
IFN-γ (+874A/T)
AA	13 (10.48%)	35 (31.82%)	Ref
AT	101 (81.45%)	65 (59.09%)	4.20	2.00–9.24	<0.0001
TT	10 (8.06%)	10 (9.09%)	2.70	0.80–9.10	0.093
AT + TT	111 (89.52%)	75 (68.18%)	4.00	1.90–8.70	<0.0001

With regard to IFN-γ, the frequency of AT genotype was 81.45% for the cases and 59.09% for the controls and the combined AT + TT genotype was specified as 89.52% and 68.18% for the cases and controls, respectively, which highly increased the risk of VUR (OR = 4.2, 95% CI, 2.00–9.24; p < 0.0001: OR = 4.00, 95% CI, 1.90–8.70; p < 0.0001) in comparison with AA reference genotype. As given in Table 3, the analysis of gene–gene interaction between IL-18 (−607 C/A, −137 G/C) and IFN-γ did not find any significant data.

Table 3. Association of different genotypic combination with VUR for the three IFN-γ (+874) and IL-18 (−137G/C, −607C/A) SNPs.

	Cases	Controls	OR	CI	p Value
IFN-γ(+874A/T)/IL-18(−137G/C)
AA/GG	7	11	Ref 0.86
AA/CC	1	2	0.48	0.16–4.70	1.0
AA/CG	5	22		0.18–1.27	0.17
AT/CC	4	4	1.29	0.52–3.17	0.6
AT/CG	46	35	2.07	0.65–6.66	0.26
AT/GG	51	26	3.08	0.46–10.14	0.06
TT/CG	1	5	0.43	0.07–2.81	0.62
TT/GG	9	5	2.83	0.54–15.77	1.14
IFN-γ(+874A/T)/IL-18(−607C/A)
AA/CC	4	4	Ref
AA/CA	9	30	0.30	0.05–2.01	0.19
AT/CC	13	4	1.53	0.73–3.21	0.35
AT/CA	85	60	1.17	0.58–2.38	0.7
AT/AA	3	1	1.50	0.61–3.67	0.57
TT/CC	10	1	1.82	0.89–3.73	0.1
TT/CA	10	9	1.05	0.47–2.38	1.0

Discussion

Cytokines are the yields of host response to inflammation and organize the defense against any infection.1 Various changes in the sequence of cytokine genes may affect their function. One of these variations is SNP in the regulatory region of cytokine genes, as the main cause of interindividual difference of cytokine level.19 Pehlivan et al. found that cytokine gene polymorphisms affected cytokine production and was infectious in various stages of disease and treatment.20 As explained earlier, VUR is characterized by the reflux of urine from the bladder into ureter and kidneys and cause damage of kidney cortex which provokes the secretion of pro-inflammatory cytokine. Okamoto et al.’s results suggested that, there may be an immunological basis for VUR development.21 Thus, any defeat in the inflammation process may be a susceptible host for the specific bacterial agents, microbial products such as lipopolysaccharide (LPS), increase secretion of IL-18 and IL-12 from macrophages and affect natural killer cell and certain T cells, which leads to the release of IFN-γ that plays a significant role in activating macrophages and other cells to increase cell-mediated immune response.22

IL-18 is a pro-inflammatory cytokine that exhibits broad functional effects in innate and acquired immune responses, which has been found in high levels in several chronic inflammatory responses which stimulate the production of IL-6, TNF-α and C-Reactive Protein (CRP).23 The present study tries to highlight the association between some SNP in the regulatory region of IFN-γ and IL-18 with the risk of VUR due to the spars' information on this participant. By the way, AT genotype of IFN-γ showed a statistically significant increased risk of VUR. Zibar et al. demonstrated that IFN-γ gene polymorphisms could be a significant risk factor for acute kidney transplant rejection. This study indicated that the urine levels of IFN-γ receptor in the individuals with VUR were significantly higher than those in the healthy controls.24 Parikh et al.’s results showed that urine IL-18 represent early, predictive biomarkers of delayed graft function, following kidney transplantation.25 VanderBrink et al. concluded that the expression of IL-18 predominantly increased during renal obstruction in tubular epithelial cells.26

In conclusion, the results of the present study provide the first evidence regarding IL-18(−607 C/A, −137 G/C) and IFN-γ (+874 T/A) genes' polymorphism with the risk of VUR in that variation in IFN-γ gene in the site of (+874 T/A) highly increased the risk of VUR in children; therefore, further studies with a larger sample size are recommended to clarify precise effects of these cytokine genes in the development of VUR.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

We give our sincere thanks to the Department of Biology, University of Sistan and Baluchestan, Zahedan, Iran, and the Department of Pediatric Nephrology, Children and Adolescents Health Center, Zahedan University of Medical Sciences, for supporting this project financially.