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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 2  |  Page : 588-594

Interleukin-1 receptor antagonist gene polymorphisms in children with febrile convulsions


1 Department of Pediatric, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pediatric, Al Shohadaa Central Hospital, Menoufia, Egypt

Date of Submission19-Feb-2021
Date of Decision16-Mar-2021
Date of Acceptance21-Mar-2021
Date of Web Publication27-Jul-2022

Correspondence Address:
Mehad I.M. Abd AlKarim
Al Shohadaa, Meet Shehala, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_46_21

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  Abstract 


Objectives
To study the possible contribution of interleukin-1 receptor antagonist (IL-1Ra) gene polymorphisms to the susceptibility of febrile convulsions.
Background
Febrile seizures (FSs) are considered the most common seizure disorder in childhood. They are considered a major cause of emergency department visits and a source of family anxiety. FSs of children involve an interaction between the immune-inflammatory process, cytokine activation, and genetic factors.
Patients and methods
This was a case–control study and included 55 children diagnosed to have febrile convulsions and 28 healthy children with no history of any type of convulsions or any neurological disorders. IL-1Ra gene polymorphisms were genotyped by PCR and compared in both groups.
Results
The most common genotype for the IL-1Ra gene in both groups was I/I. The IL-1Ra I/I homozygote was significantly more frequent in patients with FSs than in healthy controls (76.4 vs. 46.3%, P = 0.01). In addition, IL-1Ra I/II genotype was significantly associated with resistance to FSs (P = 0.001). There were no significant differences between the studied groups regarding the distribution of other genotypes of IL-Ra gene. IL-1Ra allele I is associated with higher susceptibility to FSs among members of the case group (P = 0.03). However, IL-1Ra allele II is associated with resistance to FSs among the healthy control group (P = 0.01).
Conclusion
IL-1Ra I/I homozygous genotype is significantly associated with febrile convulsions.

Keywords: childhood seizures, cytokines, febrile convulsions, gene polymorphisms, interleukin-1 receptor antagonist


How to cite this article:
Mahmoud AT, Tawfik MA, Said NM, AlKarim MI. Interleukin-1 receptor antagonist gene polymorphisms in children with febrile convulsions. Menoufia Med J 2022;35:588-94

How to cite this URL:
Mahmoud AT, Tawfik MA, Said NM, AlKarim MI. Interleukin-1 receptor antagonist gene polymorphisms in children with febrile convulsions. Menoufia Med J [serial online] 2022 [cited 2024 Mar 29];35:588-94. Available from: http://www.mmj.eg.net/text.asp?2022/35/2/588/352203




  Introduction Top


Febrile convulsion is defined by the International League against Epilepsy as convulsions occurring during febrile disease in children aged over 6 months with no previous afebrile convulsion, with no infections involving the central nervous system, and with no identified causes such as electrolyte imbalance, metabolic disorders, intoxication, or trauma. Onset after the age of 7 years is rare[1].

Febrile seizures (FSs) are the most common seizure disorder in childhood, affecting 2–5% of children. They are a major cause of emergency department visits and a source of family distress and anxiety. While they recur in approximately one-third of the children during early childhood, they are an otherwise benign phenomenon and may be associated with a risk of future epilepsy that is slightly higher than the general population[2].

However, a FS that lasts more than 15 min, which has focal signs, or that recurs within 24 h is defined as a 'complex' FS. A prolonged FS is therefore a particular type of complex FS. In case the duration exceeds 30 min, the more appropriate definition is febrile status epilepticus[3].

FSs represent an example of the interplay between genetic susceptibility and environmental factors. Most likely, all children have some increased susceptibility to seizures from fever at a specific age window. The degree of this susceptibility is influenced by several factors, including genetic ones. A febrile illness during the susceptibility period is required for a FS to occur. Suffering from an illness with a high enough fever, many 18-month-old infants will have a FS. Genetic influences are therefore likely to account for some, but not all, of the cases[4].

The genetic contribution to the incidence of FSs is manifested by positive family history of FSs in some patients. In some families, the disorder is usually inherited as an autosomal dominant trait, and multiple single genes that cause this disorder have been identified in these families. However, in most cases, the disorder seems to be polygenic, and many genes predisposing to it remain to be identified. A dysregulation between the proinflammatory interleukin (IL)-1β, IL-6, and IL-8 cytokines and anti-inflammatory interleukin-1 receptor antagonist (IL-1Ra) cytokines has been associated with febrile status epilepticus[5]. This study aimed to detect the possible contribution of IL-1Ra gene polymorphisms to the susceptibility of febrile convulsions.


  Patients and methods Top


This is a case–control study that was conducted on children diagnosed as having febrile convulsions (according to the International League Against Epilepsy criteria of febrile convulsions) in the Neurology Unit or Outpatient Clinic of the Pediatric Department of Menoufia University Hospitals and Shebin El-kom Fever Hospital from March 2019 to March 2020.

This study was approved by the Ethics Committee of the Faculty of Medicine, Menoufia University, Egypt. Informed written consent was obtained from all parents of the children enrolled in the study.

Fifty-five (28 males and 27 females) children with a mean age of 30.7 ± 7.2 months (mean ± SD) diagnosed to have febrile convulsions were assigned to the case group and have the following inclusion and exclusion criteria.

Inclusion criteria

Children aged from 6 months to 6 years. Cases collected from both sexes.

Children who suffered from simple febrile convulsion (fever with generalized tonic–clonic fits lasting <15 min and did not recur in the following 24 h) and those with normal neurological development.

Exclusion criteria

Children with intracranial infections (encephalitis, brain abscess, meningitis), cerebrovascular accident, any neurodevelopmental disorders, or metabolic disturbances. Children with previous attacks of non-FSs, and those who had febrile convulsions beyond 6 years, which is known as FSs plus. Children with a positive family history of epilepsy.

Twenty-eight (11 males and 17 females) children with a mean age of 116.3 ± 20.2 months (mean ± SD), apparently healthy children with no history of any type of convulsions or any neurological disorders were assigned to the control group. All those children were older than 7 years to exclude the occurrence of FSs.

All patients were subjected to thorough history taking focusing on the characteristics of febrile convulsions as regards the type, age of onset of convulsions, duration, frequency per year, and family history of epilepsy or febrile convulsions. The patients were subdivided according to the median value of the duration of attack (6 min) and frequency per year (four attacks per year). Full neurological examination and physical examination of all body systems (cardiovascular, gastrointestinal, respiratory, genitourinary, etc.) were done. Laboratory investigations such as complete blood count, liver function tests (ALT, AST), kidney functions tests (urea, creatinine), and serum electrolytes (sodium, magnesium, and calcium) were done to exclude other causes of seizures.

Genetic study

All children underwent peripheral blood sampling for genotype analyses in the laboratory of genetic studies in the Pediatric Department, Menoufia Faculty of Medicine. A measure of 2 ml of blood was collected in a tube containing EDTA as an anticoagulant for DNA extraction and was stored at −20°C.

Genomic DNA was isolated from peripheral blood leukocytes by means of a genomic DNA purification kit according to the manufacturer's instructions (Thermo Fisher Scientific Inc., Bartlesville, Oklahoma, USA).

Genotyping of IL-1Ra gene polymorphism: PCR reaction was carried out with 50 ng of genomic DNA, 20 pmol of primer, and 12.5 μl Master Mix (Dream TaqTM, Green PCR Master Mix) (Fermentas Life Sciences, Vilnius, Lithuania) in a total volume of 25 μl.

IL-1Ra genotyping was performed using the primer pair (forward, 5′- CTC AGC AAC ACT CCT AT-3′ and reverse, 5′-TCC TGG TCT GCA GGT AA-3′) (Bioneer, Daejeon, Korea) with initial denaturation at 94°C for 4 min followed by 32 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min with a final extension at 72°C for 10 min using a PCR Thermal Cycler (Thermo Hybaid, Birmingham, UK).

PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Alleles 1–5 (IL-1Ra 1–IL-1Ra 5) were detected according to their sizes relative to a 100-bp DNA ladder: allele 1 (four repeats), 410 bp; allele 2 (two repeats), 240 bp; allele 3 (five repeats), 500 bp; allele 4 (three repeats), 325 bp; and allele 5 (six repeats), 595 bp [Figure 1].
Figure 1: Agarose gel electrophoresis of IL-1Ra genotypes. Lane 1100 bp DNA marker. Lane 2, 4, 6, 7, 8: IL-1Ra I/I (410 bp). Lane 3 IL-1Ra I/II (410 and 240 bp). Lane 5 IL-1Ra I/III (410 and 500 bp). IL-1Ra, interleukin-1 receptor antagonist.

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Statistical analysis

Data were collected, tabulated, and statistically analyzed using an IBM personal computer with Statistical Package for the Social Sciences (SPSS), version 23 (SPSS Inc. Released 2015. IBM SPSS statistics for Windows, version 23.0; IBM Corp., Armonk, New York, USA). Two types of statistical analysis were performed: (a) descriptive statistics, for example, qualitative data were expressed in number (n) and percentage (%), while quantitative data were expressed as mean, SD, and range (minimum–maximum). (b) Analytic statistics: (i) Student's t test is a test of significance used for comparison of quantitative variables between two groups of normally distributed data. (ii) χ2 test was used to study the association between qualitative variables. (iii) Z test was used to study the association between two proportions. The significant test results were quoted as two-tailed probabilities. Significance of the obtained results was judged at the 5% level (P ≤ 0.05)[6].


  Results Top


This study included 55 patients with FS, their age ranged from 18 to 44 months (mean age: 30.7 ± 7.2 months) (27 were males and 28 were females) and 28 healthy control children whose characteristics are listed in [Table 1]. Twenty-two patients in the case group had a family history of FS constituting about 40%.
Table 1: Sociodemographic characteristics of the studied groups

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The mean age at the onset of FS was 15.4 ± 3.6 months (range, 8–22 months). The average duration of the seizures was 7.2 ± 3.3 min (range, 3–15 min). The average frequency per year was 3.9 ± 1.1 (range, 1–7 attacks) [Table 2].
Table 2: Present history and examination of cases

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Temperature and causes of fever are shown in [Table 2]. Our data revealed that tonsillitis was the most common cause of fever precipitating febrile convulsions.

IL-1Ra polymorphisms in patients with FSs and healthy control children are summarized in [Table 3]. Our data shows that the most common genotype for the IL-1Ra gene in both groups was I/I. The IL-1Ra I/I homozygote was more frequent in patients with FSs than in healthy controls (76.4 vs. 46.3%, P = 0.01). In addition, the IL-1Ra I/II genotype was significantly associated with less liability to FSs (P = 0.001). There were no significant differences between the studied groups regarding the distribution of other genotypes of the IL-Ra gene.
Table 3: Genetic study of the studied groups

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However, our results did not find any significant association between studied IL-1Ra gene polymorphisms and seizure duration [Table 4], or family history of FS [Table 5] among our patients (all P > 0.05).
Table 4: Comparison between genotypes results regarding the duration of attack (minutes)

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Table 5: Comparison between genotype results regarding family history of febrile convulsions

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  Discussion Top


In this study, we investigated the possible contribution of IL-1Ra gene polymorphisms to the susceptibility of febrile convulsions among the studied children.

In our thesis, the mean age of the studied children in the case group was 30.7 ± 7.2 months, which was significantly different (P < 0.001) from the control group (mean age = 116.3 ± 20.2 months) to exclude the occurrence of febrile convulsions in the control group.

Concerning sex distribution, we found no significant difference between male and female patients regarding the occurrence of febrile convulsions (P = 0.32). This coincides with studies conducted by Gourabi et al.[7], Canpolat et al.[1], and El-Radhi[8], who found no association between sex and increased risk of FSs.

The mean age of first seizure was 15.4 ± 3.6 months (range, 6–22 months) in the case group, which is similar to the mean age reported for first simple FSs (13 months) in a previous study carried out by Al Morshedy et al.[9] and also quite similar to a study done by Serdaroğlu et al.[10], who found that the mean age of first seizure was 19.7 ± 11.2 months.

Young age at first FS is associated with increased risk of at least one recurrence. The risk of a recurrence in a child whose initial FS occurred at less than 1 year of age is about 40% and falls to about 20% if the initial FS occurred after 18 months of age[8].

The number of FSs per year in the case group ranged from 1 to 7 with mean ± SD 3.9 ± 1.1. The average duration of the seizures was 7.2 ± 3.3 min and ranged from 3 to 15 min.

Kumar et al.[11] in their study on Indian children revealed that younger age at onset of first seizure, lower temperature during the seizure, brief duration between the onset of fever and the initial seizure, and family history of FSs were risk factors associated with recurrence of FSs. In their study, recurrence was seen more commonly in children less than 18 months (41.3%) as compared with children more than or equal to 18 months (24.1%).

A positive family history of FSs was found in 22 patients who comprised about 40% of the FS children (55 children), which agrees with the study by Matsuo et al.[12], who reported that positive family history for FSs can be elicited in 25–40% of patients with FSs. Ali and Abdolmejed[13] reported that positive family history for FSs was found in 38 patients comprising 22.2% of their studied patients with FSs (171 children). Also, a positive family history of consanguinity was found in 18 cases, which comprised 32.7% of cases.

Regarding the causes of fever in our study precipitating febrile convulsion, 23 (41.8%) children had tonsillitis. Fifteen (27%) children had gastroenteritis, 12 (21.8%) children had bronchitis, and five (9.1%) children had pneumonia.

Gourabi et al.[7] conducted a cross-sectional study on 214 children having FSs. They concluded that the most common causative factor was upper respiratory infection. In their survey upper respiratory tract infection was the main reason for FS in 74.29% of the cases and gastroenteritis (11.68%) was the second cause of fever.

Regarding laboratory investigations done in our study, serum electrolytes, kidney, and liver functions were all in normal values. Leukocytosis was found in 35 patients (63.6% of the cases group). Anemia was found in 30 children comprising about 54.6% of the cases group.

One of the most noticeable points in our study is that all cases present only with simple FSs unlike other studies in the literature that reported both types of FSs. In almost all of them, there was agreement that simple FSs were more common than complex ones. Simple FSs account for about 80–85% of all FSs[1],[14].

In this study, the mean age of the studied patients and also the mean age of the first FS were much higher compared with those reporting both types of FSs. This supports the previous reports, which concluded that complex FSs are associated with younger age of presentation of the first FSs.

In our study, we compared the distribution of IL-Ra genotypes and allele frequencies in the patient group and the control group. Genotype proportions and allele frequencies for the IL-1Ra in both groups were significantly different between FS cases and the healthy control group.

The most common genotype for IL-1Ra gene in both groups was I/I. The IL-1Ra I/I homozygote was more frequent in patients with FSs than in healthy controls (76.4 vs. 46.3%, P = 0.01). In addition, the IL-1Ra I/II genotype was significantly associated with resistance to FSs (P = 0.001). There were no significant differences between the studied groups regarding the distribution of other genotypes of IL-Ra gene.

The results of the present study show that the IL-1Ra allele I is associated with a significant higher susceptibility to FSs among members of the cases group (P = 0.03). However, IL-1Ra allele II is associated with resistance to FSs among the healthy control group (P = 0.01).

Our results confirm and extend the previous findings of Abdel Rasol et al.[15] who reported similar results in Egyptian children with FS and Chou et al.[16] who found similar results in Taiwanese population.

On the other hand, Serdaroğlu et al.[10] and Ozen et al.[17] observed a significant negative association between IL-1Ra allele I and FS suggesting that allele I was in some manner protective against FSs in Turkish children. However, Haspolat et al.[18] found no significant effects of this polymorphism on FSs in Turkish children.

The discrepancy between results from our Egyptian study and Taiwanese study[16], on one hand, and results from Turkish studies[10],[17], on the other hand, may be explained by the differences in age group, study design or geographic and ethnic variations, or by gene–gene or gene–environmental interaction. Genetic predisposition to FSs seems to be polygenic, with many variants in multiple genetic loci, playing important roles.

The IL-1Ra cytokines are structurally related to IL-1a and IL-1β and competes with these molecules for the occupation of IL-1 cell surface receptors. IL-1Ra can block fever by competitive inhibition at the IL-1 receptor type I that blocks IL-1β signaling, and thus fever. The IL-1β bioactivity is under the genetic control of IL-1Ra[16].

The balance between proinflammatory and anti-inflammatory cytokines may be more critical than the concentration of a single cytokine in the regulation of inflammation. Cytokines are modified, modulated, or substituted by other cytokines. Proinflammatory cytokines such as IL-1b, TNF, and IL-6 participate in inducing acute-phase reactions including fever. Anti-inflammatory cytokines such as IL-1Ra have a negative feedback effect during febrile response[19].

Cells from children prone to seizures may produce higher levels of proinflammatory cytokines that may induce seizures or, alternatively, higher levels of anti-inflammatory cytokines as a defense mechanism against seizures. This may occur by direct action on ionic currents or indirectly by enhancing extracellular glutamate concentrations or reducing GABAA receptor function[20].

The results of this study show that the IL-1Ra allele I is associated with higher susceptibility to FSs among members of the case group (P = 0.03). However, IL-1Ra allele II is associated with resistance to FSs among the healthy control group (P = 0.01).

We could not find any significant association between studied IL-1Ra polymorphisms and seizure duration (all P > 0.05). This is in agreement with the results of Serdaroğlu et al.[10] and Abdel Rasol et al.[15]. However, allele frequencies showed a significant effect on seizure duration. We observed that patients with allele I commonly have seizure attacks with prolonged duration.

There was no significant association between studied IL-1Ra polymorphisms and seizure frequency per year (all P > 0.05). In contrast, Abdel Rasol et al.[15] found a significant relationship between IL-1Ra I/I genotype and the frequency of seizures per year for those with more than five attacks per year.


  Conclusion Top


Our present study suggests that the IL-1Ra I/I genotype is positively associated with febrile convulsions. Therefore, it can be used as useful markers for predicting susceptibility to febrile convulsions. On the other hand, febrile convulsions are not associated with the IL-1Ra I/II genotype. Our finding suggests that cytokine genes may increase or decrease susceptibility to febrile convulsions. Furthermore, the impact of other cytokine polymorphisms on the development of febrile convulsions deserves further study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Canpolat M, Per H, Gumus H, Elmali F, Kumandas S. Investigating the prevalence of febrile convulsion in Kayseri, Turkey: an assessment of the risk factors for recurrence of febrile convulsion and for development of epilepsy. Seizure 2018; 55:36–47.  Back to cited text no. 1
    
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Kumar N, Midha T, Rao YK. Risk factors of recurrence of febrile seizures in children in a tertiary care hospital in Kanpur: a one year follow up study. Ann Indian Acad Neurol 2019; 22:31.  Back to cited text no. 11
    
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Matsuo M, Sasaki K, Ichimaru T, Nakazato S, Hamasaki Y. Increased IL-1β production from dsRNA-stimulated leukocytes in febrile seizures. Pediatr Neurol 2006; 35:102–106.  Back to cited text no. 12
    
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Abdel Rasol HA, Issac MSM, Ghaffar HA, El-Mously S. Interleukin-1 receptor antagonist and interleukin-1β-511 gene polymorphisms among Egyptian children with febrile seizures. Comp Clin Pathol 2014; 23:419–425.  Back to cited text no. 15
    
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Chou IC, Lin WD, Wang CH, Tsai CH, Li TC, Tsai FJ. Interleukin (IL)-1β, IL-1 receptor antagonist, IL-6, IL-8, IL-10, and tumor necrosis factor α gene polymorphisms in patients with febrile seizures. J Clin Lab Anal 2010; 24:154–159.  Back to cited text no. 16
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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