Kaohsiung J Med Sci November 2007 • Vol 23 • No 11 560
The most common seizure disorder in children, febrile seizures (FS), occurs in 1–10% of children [1]. Although the pathogenesis of FS remains unclear, many studies have revealed that genetic components play an impor-tant role. In 1986, neuropeptide Y (NPY) was found to be widely distributed throughout the central nervous system, including the hippocampus, and was shown to prevent the occurrence of seizures [2]. Woldbye et al observed that NPY administered through the lateral ventricle of rats is a powerful inhibitor of motor
seizures as well as electroencephalographic (EEG) seizures induced by kainic acid [3]. Dube et al also found that endogenous neuropeptide Y prevents the recurrence of experimental febrile seizures by increas-ing the seizure threshold [4]. NPY receptor knockout mice were found to be more sensitive to kainic acid-induced seizures [5], suggesting that NPY is critical for the modulation of the excessive synaptic excita-tion associated with an epileptic seizure. In addiexcita-tion, NPY-deficient mice are susceptible to limbic seizures [6,7]. In a study of patients with alcohol withdrawal syndrome, Okubo and Harada found that seizures occurred more frequently in patients with the single nucleotide polymorphism (SNP) of NPY (nucleotide 5671 C/T) (GenBank Accession no. NW 923240.1) [8]. Based on the above findings, it is reasonable to Received: February 15, 2007 Accepted: April 25, 2007
Address correspondence and reprint requests to: Dr Rei-Cheng Yang, Department of Pediatrics, Kaohsiung Medical University Hospital, 100 Tzyou 1stRoad, Kaohsiung 807, Taiwan. E-mail: [email protected]
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EUROPEPTIDE
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Lung-Chang Lin,1Hung-Shun Lin,2and Rei-Cheng Yang3,4
1Department of Pediatrics and 2Clinical Laboratory, Kaohsiung Municipal Hsiao-Kang Hospital, 3Department of Pediatrics, Kaohsiung Medical University Hospital, and 4Department of
Physiology, Graduate Institute of Physiology and Molecular Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Neuropeptide Y (NPY) has been shown to depress the hyperexcitability of neurons. In the present study, we investigated the association between the nucleotide (nt) 5671 C/T polymorphism of the NPY gene and the plasma NPY level in patients with febrile seizures (FS). Fifty-six patients with FS and 55 control subjects were enrolled. Genotype and allele frequencies were compared. The frequencies of genotypes TT, TC and CC for the NPY gene nt 5671 C/T polymorphism were 21.4%, 28.6% and 50.0%, respectively, in patients with FS, and 14.6%, 40.0% and 45.4%, respectively, in control subjects. The frequencies of alleles T and C were 35.7% and 64.3%, respectively, in patients with FS, while those in the control group were 34.5% and 65.5%, respectively. We found no signifi-cant relationship between the NPY gene nt 5671 C/T polymorphism and FS. The plasma NPY concentrations of the FS group, the age-matched non-FS group, and subjects aged > 6 years in the non-FS group were 48.23± 32.49, 55.36 ± 23.12, and 70.10 ± 60.31 pg/mL, respectively. These results indicate no statistical difference in plasma NPY concentration between FS patients and the non-FS group. However, plasma NPY concentration was found to increase significantly with age.
Key Words:febrile seizures, neuropeptide Y, polymorphism
hypothesize that NPY is responsible for the patho-genesis of FS. To determine whether NPY is associ-ated with FS pathogenesis, the frequency of this NPY genetic polymorphism and the plasma concentrations of NPY were determined in children with FS, and the possible role of NPY was evaluated.
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ATERIALS ANDM
ETHODSThis study enrolled 56 Taiwanese patients who had been clinically diagnosed with FS. All patients met the stringent criteria for FS described by Freeman [9]. FS were further classified as typical or atypical. Atypical FS were defined as seizures that persist for more than 15 minutes, are repeated within the same febrile illness, or are focal. Each participant (or a responsible adult in the case of minor children) signed the informed consent form as approved by the institutional review board of Kaohsiung Municipal Hsiao-Kang Hospital. Fifty-five healthy, unrelated, randomly selected indi-viduals above 6 years of age, without any history of FS, were recruited as controls for the genetic poly-morphism study.
Genomic DNA was extracted from peripheral blood leukocytes using a DNA extraction kit (Gentra Systems Inc., Minneapolis, MN, USA), and part of exon 3 of the NPY gene was amplified and sequenced. DNA amplification was performed in a 50-µL poly-merase chain reaction (PCR) solution containing 50 ng of genomic DNA, 200µM of each dNTP, 2.0 units of Taq DNA polymerase, 0.3µM of each primer and 10% DMSO, using a thermal cycler (Hybaid PxE2.0, Midland, ON, Canada). The sequences of the primers used to amplify the NPY exon 3 were: forward primer, 5⬘-ATGCTTCATACACCTAGCTTGC-3⬘; re-verse primer, 5⬘-TCCTCTGCCTGCTTCTTCA-3⬘. The PCR conditions were as follows: 94°C for 4 minutes followed by 35 cycles at 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds, then 72°C for 10 minutes, and storage at 4°C. Amplification prod-ucts were purified using a Multiscreen filter plate
(Millipore, Billerica, MA, USA) and sequenced on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
The concentration of NPY was determined in some individuals with FS and in the age-matched con-trol group using an enzyme immunoassay (EIA) kit (Phoenix Pharmaceuticals Inc., Belmont, CA, USA). In brief, 50µL of standard or sample was mixed with 25µL of primary antiserum and 25 µL of biotinylated peptide in each well, and then incubated at room tem-perature for 2 hours. Streptavidin-horseradish perox-idase (SA-HRP) solution (100µL/well) was added to each well after the immunoplate had been washed five times with 300µL of assay buffer per well. The immu-noplate was incubated at room temperature for 1 hour and then washed six times with 300µL of assay buffer per well. TMB substrate solution (100µL) was added to each well, and incubation was continued at room temperature for 1 hour. Reactions were terminated by adding 100µL of 2N HCl to each well. Absorbance (optical density) at 450 nm was determined and these data were used for statistical analysis.
The significance of the differences in genotype and allele frequencies between the FS patients and control subjects was calculated using the χ2test and logistic regression. The concentrations of NPY are shown as mean±standard deviation. All statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). Two-sample t tests were used to compare the means of the two groups. A value of p< 0.05 was considered to be statistically significant.
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ESULTSFifty of the 56 FS patients were placed in the typical FS group, and the remaining six (10.7%) in the atypi-cal FS group. Eighteen patients (32.1%) had a positive family history of FS. The female-to-male ratio was 1 to 1.33 (Table 1). Seventeen of the 56 FS patients (30.3%) had had two or more FS attacks by the time of our study. The genotype and allele frequencies of the NPY
Table 1.Demographic data of febrile seizure (FS) and control groups
Typical FS (n= 50) Atypical FS (n= 6) Control (n= 55) Age (mo) 23.96± 11.28 22.67± 12.54 174.62± 116.29
Sex (M/F) 28/22 4/2 24/31
gene nt 5671 C/T polymorphism in each group are shown in Table 2. The frequencies of TT, TC and CC genotypes were 21.4%, 28.6% and 50.0%, respectively, in patients with FS, and 14.6%, 40.0% and 45.4%, re-spectively, in control subjects. There was no significant difference between the two groups. The frequencies of alleles T and C were also not significantly different between the two groups (35.7% and 64.3%, respec-tively, in patients with FS vs. 34.5% and 65.5%, re-spectively, in control subjects). We further divided the FS patients into two groups (one with a positive and one with a negative family history of FS) and then compared the genotypes and allele frequencies of the two groups. The between-group differences in
genotype or allele frequencies were still insignificant (Table 3).
The NPY concentrations in the FS, atypical FS, age-matched non-FS, and non-FS (> 6 years) groups were 48.23± 32.49, 33.24 ± 0.47, 55.36 ± 23.12, and 70.10 ± 60.31 pg/mL, respectively (Table 4). The concentration of NPY was slightly (but not significantly) higher in the age-matched non-FS group than the FS group. How-ever, the difference in NPY concentration between the age-matched non-FS group and the age > 6 years non-FS group was significant, suggesting a possible age-dependence pattern (Figure). In addition, the plasma NPY concentration was lower in patients with atypi-cal FS than in those with typiatypi-cal FS, highlighting the
Table 2.Genotype and allele frequencies at the nt 5671 C/T site of the NPY gene in febrile seizure (FS) and control groups FS patients Control subjects p OR 95% CI TT genotype (%) 12 (21.4) 8 (14.6) 0.583 1.34 0.48, 3.92 TC genotype (%) 16 (28.6) 22 (40.0) 0.313 0.65 0.28, 1.50 CC genotype (%) 28 (50.0) 25 (45.4) 1.00 TT genotype (%) 12 (21.4) 8 (14.6) 0.348 1.60 0.61, 4.44 CC/TC genotype (%) 44 (78.8) 47 (85.4) 1.00 CC genotype (%) 28 (50.0) 25 (45.4) 0.632 1.20 0.56, 2.54 TC/TT genotype (%) 28 (50.0) 30 (54.6) 1.00 T allele (%) 40 (35.7) 38 (34.5) 0.855 1.05 0.61, 1.83 C allele (%) 72 (64.3) 72 (65.6) 1.00
OR= odds ratio; CI = confidence interval.
Table 3.Genotype and allele frequencies at the nt 5671 C/T site of the NPY gene in febrile seizure (FS) patients with and without family history
Family history Yes No p OR 95% CI TT genotype (%) 3 (16.7) 9 (23.7) 0.652 0.70 0.13, 3.06 TC genotype (%) 6 (33.3) 10 (26.3) 0.719 1.27 0.34, 4.59 CC genotype (%) 9 (50.0) 19 (50.0) 1.00 T allele (%) 12 (33.3) 28 (36.8) 0.718 0.86 0.36, 1.95 C allele (%) 24 (66.7) 48 (63.2) 1.00
OR= odds ratio; CI = confidence interval.
Table 4.Concentration of neuropeptide Y in different groups
FS (n= 10) Non-FS Atypical FS (n= 2)
Age≤ 6 yr (n = 9) Age> 6 yr (n = 19)
Age (mo) 29.60± 18.83 29.56± 15.55 232.21± 131.86 32.50± 6.36 NPY (pg/mL) 48.23± 32.49 55.36± 23.12 70.10± 60.31* 33.24± 0.47 *Plasma concentration of NPY was significantly higher in individuals aged > 6 years than in individuals aged ≤ 6 years in the non-FS group, p= 0.036. FS = febrile seizures.
possible role of NPY in atypical FS. Positive family history did not significantly influence plasma NPY concentration in this study (data not shown).
D
ISCUSSIONMany polymorphisms have been studied in Taiwanese patients with FS, including polymorphisms in genes for voltage-gated sodium channel subunits (SCN1A), brain-derived neurotrophic factor (BDNF), interleukin-4,α4-subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4), nicotinic receptor β2 subunit (CHRNB2), and voltage-gated potassium channel KCNQ2 [10–15]. However, only the CHRNA4 gene has been associated with FS. As we already noted, the relationship between the NPY gene and FS has not been investigated.
Several biochemical and electrophysiologic find-ings in experimental models and in tissue from human epilepsy sufferers have suggested that NPY plays an important role in suppressing seizures. Lack of inhibi-tory control by NPY could, therefore, play a role in epileptogenesis [16,17]. In an animal study, agonists of the Y1, Y2and Y5receptors for NPY were observed to reduce seizure-like activity in hippocampal cultures [18]. In contrast, the hyperexcitability of NPY knockout mice was markedly increased when challenged with a convulsing agent such as pentetrazole, and the rate of developing motor convulsions was increased from 30% to 80% [19]. A study of specimens from patients with mesial temporal lobe epilepsy found that NPY may mediate inhibition of glutamate release through upregulation of NPY Y2 receptors [20]. It was also
reported that the expression level of the NPY gene has a tendency to increase with age [21]. Accordingly, it is reasonable to expect that the NPY gene might play a role in the pathogenesis of FS. In our study of Taiwanese subjects with FS, we found no significant difference in the genotype and the allele frequencies of the NPY nt 5671 C/T polymorphism between our FS and non-FS groups. This finding indicates that FS has no apparent association with the nt 5671 site of the NPY gene.
In this study, the plasma NPY concentrations in the FS group and non-FS age-matched control group were not significantly different. However, the plasma NPY concentration was significantly higher in the non-FS age > 6 years group than the age-matched non-FS group. Our results suggest the importance of an age-related increase in plasma NPY concentration in the physiologic regulation of some neuronal functions, and the need for further study of its preventive role in recurrent FS. According to our case-limited data, the plasma NPY concentration is lower in patients with atypical FS than in those with typical FS. Whether this lower concentration indicates that patients lacking NPY are more susceptible to long-lasting seizures or recurrent FS would be worthwhile investigating.
In conclusion, we found that plasma concentration of NPY in patients with no previous FS was signifi-cantly higher in individuals older than 6 years than in their age-matched counterparts. In addition, NPY concentration was found to depend on age. However, further investigation is needed to determine more precisely the relationship between this gene and FS.
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Age (mo) 0 50 100 150 200 250 300 0 100 200 300 400 500 NPY (pg/mL)
Figure.Relationship between plasma NPY and age in non-FS individuals (n= 28).
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