|Year : 2021 | Volume
| Issue : 1 | Page : 192-196
Interleukin-6 gene -572G>C polymorphism in Egyptian children with idiopathic nephrotic syndrome
Ghada M El-Mashad1, Essam Sh Khattab2, Muhammad S El-Mekkawy1, Nagwan Y Saleh1, Wafaa N Abou El Yazed3
1 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Biochemistry and Molecular Biology, Chemistry, Biochemistry Division, Faculty of Science, Al-Azhar University, Cairo, Egypt
3 Department of Pediatric, Ministry of Health, Gharbiya Governorate, Cairo, Egypt
|Date of Submission||07-Apr-2019|
|Date of Decision||26-Apr-2019|
|Date of Acceptance||01-Jun-2019|
|Date of Web Publication||27-Mar-2021|
Wafaa N Abou El Yazed
Hassan Radwan, Tanta
Source of Support: None, Conflict of Interest: None
To evaluate the association of -572G > C polymorphism with idiopathic nephrotic syndrome (INS) in children.
Interleukin 6 (IL-6) gene promoter polymorphisms, including -572G > C polymorphism, have been implicated in INS.
Patients and methods
A total of 55 Egyptian children with INS were recruited along with 51 healthy controls. Genotyping for IL-6 gene -572G > C polymorphism was performed by PCR followed by restriction fragment length polymorphism.
GC genotype was more prevalent among patients compared with controls [40 vs. 7.8%, odds ratio (OR)=5.5, P = 0.002], but there was no significant difference between patients and controls regarding CC genotype (9.1 vs. 2%, OR = 5, P = 0.14). Under the dominant model, the pooled GC + CC was more prevalent among patients compared with controls (OR = 5.4, P < 0.001). The distribution of C allele was significantly higher among patients compared with controls (29.1 vs. 5.9%, OR = 5.3, P < 0.001). No significant difference was found between patients and controls regarding the prevalence of any of the alleles or genotypes under codominant, recessive, or dominant models.
IL-6 gene -572G > C polymorphism confers susceptibility to INS among children, but it has no influence on the response to steroids.
Keywords: idiopathic nephrotic syndrome, interleukin 6 -572G > C polymorphism, interleukin 6, pediatric
|How to cite this article:|
El-Mashad GM, Khattab ES, El-Mekkawy MS, Saleh NY, Abou El Yazed WN. Interleukin-6 gene -572G>C polymorphism in Egyptian children with idiopathic nephrotic syndrome. Menoufia Med J 2021;34:192-6
|How to cite this URL:|
El-Mashad GM, Khattab ES, El-Mekkawy MS, Saleh NY, Abou El Yazed WN. Interleukin-6 gene -572G>C polymorphism in Egyptian children with idiopathic nephrotic syndrome. Menoufia Med J [serial online] 2021 [cited 2021 May 8];34:192-6. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/192/311992
| Introduction|| |
Nephrotic syndrome is a well-known childhood disease with an incidence of approximately two to seven cases/100 000 children/year. It is characterized by edema, massive proteinuria, marked hypoalbuminemia, and hyperlipidemia . Nephrotic syndrome may be primary or secondary to drugs, infections, autoimmune diseases, or malignancy. Primary nephrotic syndrome may be associated with certain genetic and metabolic disorders. However, the cause is unknown in most cases [idiopathic nephrotic syndrome (INS)] . Overall, 70% of children with INS respond to steroids, whereas 30% are steroid resistant. Minimal change disease is typically steroid responsive. Overall, 26% of patients with steroid-resistant nephrotic syndrome have an underlying single gene defect, of which 53 are currently known . Conversely, the genetic factors underlying steroid-sensitive nephrotic syndrome are largely unknown. T-cell dysfunction and plasma factors, like hemopexin, interleukin 13 (IL-13), and IL-18, causing protein leak from the glomeruli, have been postulated . IL-6 is one of the cytokines that have been implicated in INS pathogenesis. It is produced by phagocytes, lymphocytes, endothelial cells, and fibroblasts. IL-6 possesses both pro-inflammatory and anti-inflammatory properties, and it plays important roles in immune response, inflammation, acute-phase response, hematopoiesis, and organ development . In the kidney, podocyte is a very important source of IL-6. Mesangial and tubular epithelial cells also secrete IL-6, which might be involved in the pathogenesis of various renal diseases . IL-6 gene is located at 7p21–14. Its promoter contains four polymorphisms, which are supposed to influence level of IL-6 gene transcription and consequently IL-6 production . One of these polymorphisms, namely, -572G > C, has been implicated by prior studies in the pathogenesis of several diseases, including hypertension, breast cancer, and tuberculosis . Studies of IL-6 polymorphisms in nephrotic syndrome are rare, and the role of -572G > C polymorphism was not addressed before in INS in children. Here, we aimed to study the role of IL-6 -572G > C polymorphism in susceptibility to INS and in the response to treatment .
| Patients and methods|| |
The study was approved by the ethical committee of the hospital and patients gave an informed consent. In this study, 55 children with INS were recruited from the Nephrology Unit and Outpatient Clinic of Menoufia University Hospital, Menoufia, Egypt, from June to November 2018. Any child from the age of 3 months to 18 years old was eligible for inclusion in the study after parental informed consent. The exclusion criteria included (a) age under 3 month (congenital nephrotic syndrome) or over 18 years; (b) secondary nephrotic syndrome such as owing to drugs, infections, and systemic lupus erythematosus; and (c) nephrotic syndrome associated with genetic syndromes like Cockayne's syndrome and Laurence-Moon-Biedl-Bardet syndrome. A diagnosis of nephrotic syndrome was made in the presence hypoalbuminemia (<2.5 g/dl), urinary protein excretion more than 40 mg/m2/h, or spot urine protein/creatinine ratio more than 2. To determine whether nephrotic syndrome was idiopathic or secondary, a thorough clinical evaluation was performed, including history of drug intake, infections, and autoimmune diseases like systemic lupus erythematosus. Furthermore, several laboratory tests were performed, including complete blood count, viral testing (hepatitis B and C and HIV), anti-nuclear antibody, and complement C3 and C4. Specific tests like renal biopsy, renal ultrasonography, chest radiograph, tuberculin skin testing, and genetic studies were ordered when clinically indicated. Patients were subdivided into steroid-responsive and steroid-resistant patients. Steroid resistance was defined as failure to achieve remission despite 4 weeks of daily prednisone 60 mg/m2/day with or without three methylprednisolone pulses. Remission was diagnosed when urine protein : creatinine ratio was less than 0.2 or if urinary protein was less than 1 + by dipstick for 3 consecutive days. In addition to the patient group, another group of 51 healthy children served as a control group. DNA extraction and genotyping was done as follows: genomic DNA was extracted from peripheral blood leukocytes for all patients and controls samples using a DNA extraction kit (GeneJET Whole Blood Genomic DNA Purification Mini Kit; Thermo Scientific, V. A. Graičiūno g. 8, Vilnius 02241, Lithuania) and then stored at –80°C. The IL-6 -572 polymorphism was analyzed by PCR followed by restriction fragment length polymorphism. In PCR, the following primers were used to amplify a 163-bp fragment: 5′-GAGACGCCTTGAAGTAACTGC-3′ and 5′ GAGTTTCCTCTGACTCCATCGCAG-3′. Thirty-five cycles of amplification were performed: 30 s at 95°C, 45 s at 59°C, and 60 s at 72°C. The PCR product was then digested with BsrBI (Mbil) restriction enzyme (Thermo Scientific) at 37°C for 3 h. Digested products were then electrophoresed on a 2% agarose gel and visualized with ethidium bromide 1%. In the presence of C allele, no digestion occurs, giving a fragment of 163 bp, whereas the presence of G allele yields 2 fragments of 102 and 61 bp. All the genetic studies for all patient and control samples were performed at the Genetic and Endocrinology Unit, Pediatric Department, Faculty of Medicine, Menoufia University.
For quantitative data, the Shapiro–Wilk test for normality was performed. All data followed normal distribution, so values were expressed as mean ± SD. Independent samples t test was performed for comparison between two quantitative variables. Qualitative data were summarized as number and percentage. Pearson's χ2 test of independence was used to assess the presence of association between two qualitative variables. When assumptions of χ2 test were not fulfilled, Fisher's exact test or Fisher-Freeman-Halton exact test was used as appropriate. Binary logistic regression was carried out to assess the association between genotypes and the disease and response to treatment. Significance was adopted at P value less than 0.05 for interpretation of results of the tests. All statistical analyses were performed using IBM SPSS (Statistical Package for Social Science) program, version 20 (SPSS Inc., Chicago, Illinois, USA).
| Results|| |
This study included 55 INS Egyptian children representing the patients' group. There were 34 (61.8%) males and 21 (38.2%) females, and their mean age was 8.3 ± 3.7 years. Moreover, the study included 51 normal participants (control group) matched in age and sex (P > 0.05). There were 24 (43.6%) males and 31 (56.4%) females, with a mean age of 8.2 ± 2.9 years [Table 1]. The patients' mean weight was 37.4 ± 13.3 kg, the mean height was 1.2 ± 0.2 m, and the mean BMI was 26 ± 10.6, whereas the weight of the control group was 33.9 ± 9.8 kg, height was 1.3 ± 0.2 m, and BMI was 19.7 ± 2.4. Statistical differences between patients and controls regarding weight, height, and BMI were as follows: P values more than 0.05, less than 0.05 and less than 0.001, respectively. Twelve (21.8%) patients showed consanguinity and six (10.9%) patients showed a positive family history, with statistically significant difference with controls (P < 0.001 and < 0.05, respectively). Moreover, hypertension was presented in 30 (54.5%) patients in the case group and had a very highly statistically significant difference compared with controls (P < 0.001), as shown in [Table 1]. Laboratory findings showed the mean blood urea nitrogen was 11.3 ± 3.1 and 12.4 ± 2.3 mg/dl in patient and control groups, respectively, and the difference was statistically significant (P < 0.05), whereas the mean creatinine was 0.47 ± 0.12 and 0.5 ± 0.12 mg/dl in patient and control groups, respectively, and the difference was statistically insignificant (P > 0.05), as shown in [Table 1]. The mean duration of the disease was 40 ± 30 months, and the onset of the disease was less than 3 years in 25 (45.5%) patients, from 3 to 6 years in 23 (41.8%) patients, and more than 6 years in seven (12.7%) patients. There were 45 (81.8%) patients with relapse of the disease, whereas 10 (18.2%) patients had no relapse [Table 1]. The study shows that 32 (58.2%) patients responded to steroids and 18 (32.7%) patients did not respond to steroids, whereas only five (9.1%) patients had unknown steroid response [Table 1]. All patients (100%) had chronic dialysis, and renal biopsy showed minimal changes in 11 (20%) patients, focal segmental glomerulosclerosis in seven (12.7%) patients, and was not performed in 37 (67.3%) patients [Table 1]. On studying genotypes and alleles among patients and controls, it was found that the codominant models GG, GC, and CC were seen in 46 (90.2%) patients, four (7.8%) patients, and one (2%) patient, respectively, in the case group and 28 (50.9%), 22 (40%), and five (9.1%), respectively, in the control group. There was a statistically significant difference (P < 0.01) between GG and GC and statistically insignificant difference between GG and CC (P > 0.05). It was found that the dominant models GG and CC + GC were seen in 46 (90.2%) patients and five (9.8%) patients, respectively, in the case group and in 28 (50.9%) patients and 27 (49.1%) patients, respectively, in the control group. There was a statistically significant difference (P < 0.01) between patients and control regarding GG and CC + GC. It was found that the recessive model CC was found in one (2%) patient in the case group and five (9.8%) patients in the control group, and GC + GG was seen in 50 (98%) patients in the case group and 50 (90.9%) patients in the control group. There was a statistically insignificant difference (P > 0.05) between patients and control regarding CC and GC + GG. Regarding the allele G, it was found in 96 (94.1%) patients in the case group and six (5.9%) patients in the control group. There was a statistically highly significant difference between patients and control (P < 0.001) regarding G and C [Table 2] and [Table 3]. On studying genotypes and alleles among steroid-responsive and steroid-resistant patients, it was found that the codominant models GG, GC, and CC were seen in 21 (65.6%) patients, 11 (34.4%) patients, and 0 (0%), respectively, in the case group and two (11.1%), 11 (61.1%), and five (27.8%) individuals, respectively, in the control group. There was a statistically insignificant difference (P > 0.05) between GG and GC and between GG and CC. It was found that the dominant models GG and CC + GC were seen in 21 (65.6%) patients and 11 (34.4%) patients, respectively, in the case group and in two (11.1%) patients and 16 (88.9%) patients, respectively, in the control group. There was a statistically insignificant difference (P > 0.05) between patients and control regarding GG and CC + GC. It was found that the recessive model CC was found in 0 patient and five (27.8%) patients control and CC + GC was seen in 32 (100%) patients and 13 (72.2%) patients controls. There was a statistically insignificant difference (P > 0.05) between patients and control as regard CC and GC + GG. Regarding the allele G, it was found in 53 (82.8%) patients and 15 (41.7%) patients controls, and the allele C was found in 11 (17.2%) patients in the case group and 21 (58.3%) patients in the control group. There was a statistically insignificant difference between patients and control (P > 0.05) regarding G and C [Table 2].
|Table 2: Distribution of genotypes and alleles among patients and controls|
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|Table 3: Distribution of genotypes and alleles among steroid-responsive and steroid-resistant patients|
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| Discussion|| |
IL-6 is one of a long list of cytokines that have been implicated in the pathogenesis of INS along with IL-13 , IL-17 , IL-18 , IL-1, and tumor necrosis factor α. Su et al.  stated that IL-6 has been implicated in the pathogenesis of INS as well as in various other renal diseases, including acute kidney injury, chronic kidney disease, diabetic nephropathy, lupus nephritis, and IgA nephropathy. Here, we hypothesized that IL-6 -572 promoter polymorphism could influence gene expression, with consequent excessive inflammation, which could play a pivotal role in INS pathogenesis. We found that the C allele was more frequent among patients with INS compared with controls. C allele increased the risk of development of nephrotic syndrome, with an odds ratio of 5.3. Moreover, the pooled GC + CC genotypes were more frequent among patients compared with controls under the dominant model. As far as we know, the current study is the first to investigate the role of IL-6 -572 G/C polymorphism in pediatric patients with INS. Chen et al.  stated that IL-6 -572 G/C polymorphism was previously studied in idiopathic membranous nephropathy where both C allele and CC genotype were found to be associated with the disease among a Han Chinese population (OR = 2.42 and 2.71, respectively). However, it should be taken into consideration that membranous glomerulonephritis is the prime cause of nephrotic syndrome in adults but is rare in children. Jafar et al.  mentioned that the polymorphism that was studied in pediatric INS was the IL-6 -174 polymorphism. In the latter study, the prevalence of C allele was significantly higher among patients compared with controls. In addition, genotypes CC and CG were also significantly increased among patients. Furthermore, CC genotype was significantly more prevalent among steroid-resistant compared with steroid-responsive children. Likewise, in a cohort by Tripathi et al.  of adult patients with INS, IL-6 -178 GG genotype was found to be significantly increased in steroid-resistant compared with steroid-responsive patients. Another important aspect of the relationship between nephrotic syndrome and IL-6 is the influence on the response to steroids. The mechanism of action of steroids includes turning off proinflammatory genes; therefore, the presence of a polymorphism that increases IL-6 transcription is expected to be associated with decreased response of patients to steroid treatment. Unfortunately, we failed to find an association of IL-6 -572 G/C polymorphism with the response to steroids. Maybe the small sample size is an explanation. Most likely, the genetic determinants of the response to steroids are complex and involve several genes with different polymorphisms in each gene (haplotypes), and the separate effect of each needs a large patient sample to be elicited. It is also possible that ethnic factors are responsible. Regarding steroid response, it was shown that after 12 months of immunosuppressive therapy, a complete response occurred more frequently among carriers of C allele of IL-6 -174 gene (P = 0.045). Kamyshova et al.  added that renal dysfunction was observed more frequently among carriers of C allele compared with homo-zygotes (P = 0.014). In addition to these studies, a nongenetic study by Al-Eisa et al.  revealed important indications, suggesting a role of IL-6 in nephrotic syndrome. In a study of 37 children with INS, urinary IL-6 level was significantly increased during relapse compared with controls. Similarly, Rizk et al.  stated that serum IL-6 was significantly higher among children with INS compared with controls, and the level was highest in patients with first presentation and during relapse. Likewise, in a study by Wang et al.  on a rat model of experimental minimal change nephrotic syndrome induced by Doxorubicin, IL-6 level was significantly increased in urine and renal tissue, suggesting a role of IL-6 in this condition. Limitations of the present study include the small sample size, which necessitates a larger patient sample for establishing the role of IL-6 polymorphism in INS. In addition, we did not assess serum or urinary IL-6 level to study the correlation between their levels and the promoter polymorphism. Finally, the ultimate IL-6 level may be a function of all the polymorphisms in the promoter and not only -572 polymorphism, which makes it imperative to analyze these polymorphisms to capture a more global view.
| Conclusion|| |
In conclusion, IL-6 gene -572G > C polymorphism confers susceptibility to INS in children, but it has no influence on patients' response to steroids. Further studies in other nations with a larger patient sample are unquestionably required to establish a role of IL-6 -572G > C promoter polymorphism in INS.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]