From the Departments of Pediatrics and Medical Genetics (Drs F.-J. Tsai and C.-H. Tsai), Obstetrics and Gynecology (Drs Hsieh and Chang), and Family Medicine (Dr Lin), China Medical College Hospital, Taichung, Taiwan.
To investigate whether interleukin 1β (IL-1β) exon 5 and IL-1 receptor antagonist (IL-1Ra) gene polymorphisms can be used as markers of susceptibility to febrile convulsions in children.
Children were divided into 2 groups: those with febrile convulsions (group 1; n = 51) and normal control subjects (group 2; n = 83). Polymorphisms for IL-1β exon 5 and IL-1Ra gene polymorphisms were detected by polymerase chain reaction. Genotypes and allelic frequencies for IL-1β exon 5 and IL-1Ra gene polymorphisms in both groups were compared.
Genotype and allele frequencies for IL-1β exon 5 in both groups were not significantly different. Proportions of E1 homozygotes and E1/E2 heterozygotes for IL-1β exon 5 were 50 (98.1%) and 1 (1.9%), respectively, in group 1 and 82 (98.8%) and 1 (1.2%), respectively, in group 2. Frequencies of alleles E1 and E2 for IL-1β exon 5 were 101 (99.0%) and 1 (1.0%), respectively, in group 1 and 165 (99.4%) and 1 (0.6%), respectively, in group 2. Genotype proportions and allele frequencies for IL-1Ra between groups were significantly different. Proportions of genotypes I/I and I/II for IL-1Ra were 49 (96.1%) and 2 (3.9%) in group 1 and 69 (83.1%) and 14 (16.9%) in group 2. Frequencies of alleles I and II for IL-1Ra were 100 (98.0%) and 2 (2.0%) in group 1 and 152 (91.6%) and 14 (8.4%) in group 2.
The IL-1Ra allele I is associated with a higher susceptibility to febrile convulsion, which may become a useful marker for predicting the development of febrile convulsions. The IL-1β exon 5 gene polymorphisms are not a useful marker for predicting the susceptibility to febrile convulsions.
FEBRILE CONVULSIONS, the most common form of childhood seizures, occur in 2% to 5% of children before the age of 5 years.1 Febrile convulsions are marked by high or rapidly rising fever and short duration; uncomplicated convulsions do not predispose to epilepsy and are not associated with neurologic abnormalities.2 The pathogenesis of febrile convulsions remains obscure. Possible causes include viral infection of the central nervous system and lowered threshold for convulsions in the presence of fever.3 Recently, febrile convulsions have been suggested to be a gene-related disease.4 In fact, febrile convulsions of children involve a complex interaction between the immunoinflammatory process, cytokine activation, and genetic factors.5
Cytokine is a key factor of the host response to infection, and its effects include induction of fever, leukocytosis, and acute-phase protein synthesis.6 Cells from children prone to seizures may produce more proinflammatory cytokines that may induce convulsions or, alternatively, higher levels of anti-inflammatory cytokines as a defense mechanism against seizure.7 Proinflammatory cytokines, including interleukin 1β (IL-1β), are known to modulate effects of neurotoxic neurotransmitters discharged during excitation or inflammation in the central nervous system.8 Some cytokine polymorphisms might be related to febrile convulsions, including IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1Ra) gene polymorphisms.8 Interleukin 1 belongs to a cytokine family modulating cellular proliferation and has the capacity to induce other cytokines. It is a primary mediator of the inflammatory response and has been shown to induce prostaglandin synthesis.9
The IL-1 genes are associated with several immunoinflammatory diseases.10 The IL-1β polymorphisms are associated with enhanced production of IL-1β and the increased risk of both hypochlorhydria induced by Helicobacter pylori and gastric cancer.11 The IL-1β polymorphisms were related to the regulation of cytokine and growth factor expression in articular chondrocytes and the final development of osteoarthritis.12 Allele E2 in IL-1β exon 5 was related to an increased risk of erosive arthritis.13 The action of IL-1 is complex and regulated in part by its naturally occurring inhibitor, the IL-1Ra.
Genetic studies of multifactorial diseases such as febrile convulsions are difficult to approach because of the uncertainty of polygenic traits. In our laboratory, we have observed that the IL-1β-511*T allele could be used as a genetic marker of susceptibility to Kawasaki disease (Yi-Ru Shi, MS, F.-J.T., and C.-H.T., unpublished data, 2001). In contrast, we noted that the IL-1β (IL-1β-511 promotor, IL-1β exon 5) and IL-1Ra were not useful markers to predict susceptibility to endometriosis and rheumatoid arthritis.14 We also noted that the IL-1β-511 promotor polymorphism is not a useful marker for prediction of the susceptibility to febrile convulsions and epilepsy (F.-J.T., Y.-Y.H., C.-C.C., and C.-H.T., unpublished data, 2001). On the basis of these experiences, we further tried to evaluate whether these polymorphisms are useful markers for predicting susceptibility to febrile convulsions in children.
The study included Taiwanese children with febrile convulsions (group 1; n = 51) and normal control subjects (group 2; n = 83). This study was approved by the Ethical Committee of the China Medical College Hospital, Taichung, Taiwan. Informed consent was signed by all parents of the patients who donated their blood. There were nonsignificant differences between the groups in age, weight, and height. The diagnosis of febrile convulsions was made after the exclusion of other causative agents, including bacterial or viral infection.
All children underwent peripheral blood sampling for genotype analyses. Genomic DNA was isolated from peripheral blood by means of a DNA extractor kit (Genomaker DNA extraction kit; Blossom, Taiwan). A total of 50 ng of genomic DNA was mixed with 20 pmol of each polymerase chain reaction (PCR) primer in a total volume of 25 µL containing 10mM Tris hydrochloride, pH 8.3; 50mM potassium chloride; 2.0mM magnesium chloride; 0.2mM each deoxyribonucleotide triphosphate; and 1 U of DNA polymerase (Amplitaq; Perkin-Elmer, Foster City, Calif). Four PCR primers were used to amplify the correlated gene. The sequences of these primers were as following (from 5′ to 3′ end): IL-1β exon 5: upstream, GTTGTCATCAGACTTTGACC; downstream, TTCAGTTCATATGGACCAGA; and IL-1Ra: upstream, CTCAGCAACACTCCTAT; downstream, TCCTGGTCTGCAGGTAA. The PCR conditions were as follows: 35 cycles at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes, then stand at 72° for 30 minutes and hold at 4°. Biallelic base polymorphisms in exon 5 at position +3953 (IL-1β+3953) for IL-1β gene were detected. The IL-1β+3953 exon 5 polymorphism was analyzed by PCR amplification followed by Taq I restriction analysis.15 The PCR products were directly analyzed for IL-1Ra by electrophoresis on agarose gel, and each allele was recognized according to its size. Allelic frequencies are expressed as a percentage of the total number of alleles. Genotypes and allelic frequencies for IL-1β and IL-1Ra polymorphisms in both groups were compared.
The SAS system with χ2 and Fisher exact test were used for statistical analyses. P<.05 was considered statistically significant.
Genotype proportions and allele frequencies for IL-1β exon 5 in both groups were not significantly different (Table 1). The most common genotype for IL-1 gene in both groups was E1 homozygote. Proportions of E1 homozygote and E1/E2 heterozygote for IL-1β exon 5 were as follows: group 1, 98.0% and 1.9%, respectively; group 2, 98.8% and 1.2%, respectively. There were no E2 homozygotes. Allele E1 and E2 frequencies for IL-1β exon 5 were as follows: group 1, 99.0% and 1.0%, respectively; group 2, 99.4% and 0.6%, respectively (Table 1).
In contrast, the genotype proportions and allele frequencies for IL-1Ra between the groups were significantly different. The most common genotype for IL-1 gene in both groups was I/I. Proportions of I/I and I/II for IL-1Ra were as follows: group 1, 96.1% and 3.9%, respectively; group 2, 83.1% and 16.9%, respectively (Table 2). Allele I for IL-1Ra was associated with febrile convulsions. Allele I/II for IL-1Ra was found in the following proportions: group 1, 98.0% and 2.0%, respectively; group 2, 91.6% and 8.4%, respectively (Table 2).
Cytokines are proteins that play a role in the communication link between the immunologic system and brain tissue. Cytokines are related to leukocyte function and migration, angiogenesis, hematopoiesis, antitumoral effects, and atherosclerosis.16 They are produced by peripheral monocytes and also by astrocytes and glial cells within the central nervous system.17 Cytokines from peripheral-blood mononuclear cells may cross the blood-brain barrier and may thus be involved in the pathogenesis of fever.18
The IL-1 response from sensitized mononuclear cells may have a role in the development of febrile convulsions.3 During the acute phase of febrile convulsions, patients have significantly increased plasma IL-1β levels, which may be responsible for the pathogenesis of febrile convulsions.18 Differences in the distribution of the biallelic polymorphism in the promotor region of the IL-1β gene were found among patients exhibiting temporal lobe epilepsy.8 Interleukin 1 also regulates the development of glial scars at sites of central nervous system injury.8 In contrast, Ichiyama et al19 found no correlation between the cytokines tumor necrosis factor α, IL-1β, and IL-6 in the cerebrospinal fluid and the presence of febrile seizures.
Genetic factors play a major role in the etiology of febrile convulsions. Cytokine genes may be related to cytokine expression and regulation of the immune-mediated pathogenetic process. Cytokine gene polymorphisms have recently attracted considerable interest because distinct alleles of cytokine genes have been discovered to be associated with different immunoinflammatory diseases.7 Single nucleotide polymorphisms are the most abundant types of DNA sequence variation in the human genome.20 Single nucleotide polymorphism markers provide a new way to identify complex gene-associated diseases such as febrile convulsions in children.
Interleukin 1 exists in 2 forms, IL-1α and IL-1β, which are encoded by distinct genes but share the same receptors and biological properties.21 The loci for IL-1α and IL-1β are located on the proximal region of the long arm of chromosome 2.22 The IL-1β polymorphism has been correlated with IL-1β expression.23 These results indicate that the genotype of the IL-1β polymorphisms may affect IL-1β production in an intricate and complicated manner. Different polymorphisms have been described in the IL-1β gene, and at least 2 of them could influence protein production: one located in the promotor region at position -511 (IL-1β-511)12 and the other in exon 5.15 In our previous research, we noted the lack of association between febrile convulsions and IL-1β-511 promotor polymorphism (F.-J.T., Y.-Y.H., C.-C.C., and C.-H.T., unpublished data, 2001). These genes code several proteins that may be key components in the pathogenesis of febrile convulsions.
The IL-1 or IL-1Ra polymorphisms have been found to be related to susceptibility or disease activities for individual diseases, including Alzheimer disease,24 Parkinson disease,25 temporal lobe epilepsy,8 schizophrenia,26 erosive arthritis,13 polymyositis and dermatomyositis,27 multiple sclerosis,28 lymphocytic leukemia,29 atherosclerosis,30 coronary artery disease,31 alcoholic liver disease,32 idiopathic pancreatitis,33 inflammatory bowel disease,34 and IgA nephropathy.35 The regulation of IL-1β and IL-1Ra may be coordinated during inflammation.36 The level of IL-1β messenger RNA showed a positive correlation with that of IL-1β and a negative correlation with the level of IL-1Ra messenger RNA.37
In contrast, some investigators have found no correlation between genetic polymorphisms of IL-1β and susceptibility to rheumatoid arthritis,13 chronic obstructive pulmonary disease,38 multiple myeloma,39 diabetes mellitus,40 postmenopausal osteoporosis,41 and ischemic heart disease.42 Furthermore, IL-1 genes may have a role in the severity of the disease rather than in susceptibility to the disease itself.10 In our previous study, we also observed an association between disease activity of rheumatoid arthritis and a polymorphic IL-1β–511*C gene sequence (Chun-Ming Huang, MD, F.-J.T., and C.-H.T., unpublished data, 2001).
The IL-1Ra is structurally related to IL-1α and IL-1β and competes with these molecules for occupation of IL-1 cell surface receptors. The presence of the IL-1Ra allele II was associated with enhanced IL-1β production in vitro.43 The IL-1Ra allele II is associated with a variety of epithelial-related chronic inflammatory diseases including systemic lupus erythematosus,44 psoriasis,45 alopecia areata,46 lichen sclerosus,47 and ulcerative colitis.48 In this study, we observed that the IL-1Ra allele I is associated with higher susceptibility to febrile convulsions. The results further suggest that the IL-1Ra allele II as well as the increased production of IL-1β might play a role in preventing febrile convulsions. Genotype distributions and allelic frequencies for IL-1Ra gene polymorphism may be the candidate genetic markers in the susceptibility to febrile convulsion. In contrast, the IL-1β exon 5 gene polymorphism is not useful in predicting the susceptibility to febrile convulsion. This discrepancy may be due to different illness classifications and racial and disease variation.
In conclusion, IL-1Ra is a useful marker for predicting susceptibility to febrile convulsions. In contrast, febrile convulsions are not associated with IL-1β exon 5 gene polymorphisms. This could provide the database for further survey of the IL-1 and IL-1Ra polymorphisms. However, the real roles of the IL-1 polymorphisms in febrile convulsions remain to be clarified. Furthermore, the impact of other cytokine polymorphisms on development of febrile convulsions merits further study.
Accepted for publication February 7, 2002.
Interleukin 1β exon 5 and IL-1Ra may be related to the pathogenesis of febrile convulsions. This study examines the potential usefulness of these markers in predicting susceptibility to febrile convulsions in children. We found that interleukin 1 receptor antagonist is a useful marker for predicting susceptibility to febrile convulsions.
Corresponding author and reprints: Fuu-Jen Tsai, MD, PhD, Department of Pediatrics and Medical Genetics, China Medical College Hospital, No. 2 Yuh-Der Rd, Taichung, Taiwan (e-mail: email@example.com).
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Figure e18.1-4. Common (Wild-Type) Allele and 4 Types of Genetic Polymorphisms
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