Failure to detect association between polymorphisms of the sodium channel gene SCN1A and febrile seizures in Chinese patients with epilepsy

Summary

A recent study in Caucasians found an association between the single nucleotide polymorphism (SNP) of SCN1A, IVS5N +5 G>A (rs3812718), and febrile seizures (FS). We examined whether this and other tagging SNPs of SCN1A were associated with an increased risk of FS in Han Chinese. A total of 728 Han Chinese patients with focal epilepsy were recruited: 97 had a history of FS (58% male, mean age 35 ± 12 years) and 631 did not (50% male, mean age 40 ± 15 years). Genotyping was performed for IVS5N +5 G>A and seven other tagging SNPs selected from the HapMap database. Genotyping was also performed in 848 ethnically matched population controls (50% male, mean age 37 ± 17 years). There was no statistically significant difference in either allele or genotype frequency of any of the SNPs studied between epilepsy patients with and without FS, and between epilepsy patients with FS and controls. The results do not suggest that SCN1A SNPs are susceptibility factors for FS in Han Chinese.

Febrile seizures (FS) affect approximately 2–9% of young children (Nakayama, 2009). Because SCN1A mutations are recognized as the most common cause of the rare mendelian syndrome of generalized epilepsy with FS plus (GEFS+) (Mulley et al., 2005), the sodium channel gene is considered a potential candidate for the more common, presumably polygenic, forms of FS. Specifically, the single nucleotide polymorphism (SNP) of SCN1A, IVS5N +5 G>A (rs3812718), is believed to be a candidate functional variant because it alters the proportions of the neonatal and adult transcripts of the gene (Heinzen et al., 2007) and has been reported to be associated with the maximal doses of carbamazepine and phenytoin, both sodium channel–blocking antiepileptic drugs, in patients with epilepsy (Tate et al., 2005). Recently, Schlachter et al. (2009) found higher proportions of the A allele and AA genotype of the SNP in a cohort of Caucasian adult epilepsy patients with a history of FS (n = 90) and in children with FS (n = 144), but a similar proportion in epilepsy patients without FS (n = 486), compared to population controls (n = 701).

We undertook a gene-wide tagging approach to examine whether this and other tagging polymorphisms of SCN1A were associated with increased risk of FS in Han Chinese.

Methods

Patients and genotyping

The study included 728 Han Chinese patients with focal epilepsy recruited from four major regional hospitals in Hong Kong. Ninety-seven patients had a history of FS (58% male, mean age 35 ± 12 years) and 631 did not (50% male, mean age 40 ± 15 years). Ninety-four patients had temporal lobe epilepsy with hippocampal sclerosis, 315 other lesional epilepsies, and 319 had cryptogenic focal epilepsy. DNA was extracted from blood samples. Genotyping was performed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MassArray, Sequenom, San Diego, CA, U.S.A.) for IVS5N +5 G>A and seven other tagging SNPs selected from the HapMap database (CHB [Chinese Beijing] SNP genotype data Rel#21/phaseII Jul06 in HapMap.org) as described previously (Kwan et al., 2008), capturing 89 of 99 alleles at r² > 0.8. Genotyping was also performed in 848 ethnically matched population controls (50% male, mean age 37 ± 17 years). The study was approved by the ethics committees of the participating hospitals, and all subjects or their guardians gave written informed consent.

Statistical analysis

Variants in SCN1A were analyzed for any association with febrile seizures between the group of patients with FS and groups without FS (control group or non-FS epilepsy group). As an exploratory analysis we also compared allele and genotype frequencies between all epilepsy patients (combining those with and without FS) and controls. Pearson’s chi-square test and odds ratios [OR; with 95% confidence interval (CI)] were performed to assess allelic differences (SPSS 16.0 for Windows, SPSS Inc., Chicago, IL, U.S.A.). Armitage trend test was used to identify genotype differences (PLINK, http://pngu.mgh.harvard.edu/~purcell/plink/). Hardy-Weinberg equilibrium was analyzed using Haploview 4.0 (http://www.broad.mit.edu/mpg/haploview). Logistic regression was used to adjust for sex, age, and presence of hippocampal sclerosis. P-value ≤0.05 (two-sided) was considered statistically significant.

Power calculations were made using CaTS (http://www.sph.umich.edu/csg/abecasis/CaTS/index.html) software. Assuming a disease prevalence of 3%, the sample size had a statistical power of 80% to detect the A allele of IVS5N +5 G>A as a susceptibility allele for FS with genotypic relative risk (GRR) of 1.83 under an additive model and 1.55 under a multiplicative model for comparing epilepsy patients with FS versus epilepsy patients without FS, and 1.80 and 1.54, respectively, for comparing epilepsy patients with FS versus controls. Based on a reported OR of 2.01 for association of FS with the A allele of IVS5N +5 G>A for comparing epilepsy patients with FS versus epilepsy patients without FS, and an OR of 2.12 for epilepsy patients with FS versus controls (Schlachter et al., 2009), the present study had a power of 98.3% and 99.4%, respectively, to detect the same associations at p < 0.05.

Results

All genotype distributions were consistent with Hardy-Weinberg equilibrium (p > 0.05) except that of rs4667866 in the patient group with FS (p = 0.004). There was no significant difference in either allele or genotype frequency of IVS5N +5 G>A between epilepsy patients with and without FS, and between epilepsy patients with FS and controls (Table 1). No difference was found after adjusting for age, sex, and presence of hippocampal sclerosis by logistic regression. Similarly, there was no significant difference in either allele or genotype frequency of the other seven SNPs between the patient groups and controls. Exploratory analysis found significant differences in allele (p = 0.038, OR 1.16, 95% CI 1.01–1.34) and genotype (p = 0.038) frequencies of IVS5N+5 G>A between all epilepsy patients and controls, with the A allele overrepresented among patients. Likewise, there were significant differences in allele (p = 0.011, OR 1.27, 95% CI 1.06–1.53) and genotype (p = 0.011) frequencies of rs10188577 between all epilepsy patients and controls, with the C allele overrepresented among patients. There was no significant difference in allele or genotype frequencies between epilepsy patients and controls for the other SNPs tested (data not shown).

Table 1.   Frequencies of genotypes and alleles of eight tagging single nucleotide polymorphisms of SCN1A in tested groups

SNP rs#	Group	Allele, no. (%)		p-value^a, odds ratio (95% confidence interval)	Genotype, no. (%)	p-value^b
F, epilepsy patients with febrile seizures; E, epilepsy patients without febrile seizures; C, controls. ^aPearson’s χ^2 test. ^bArmitage trend test.
rs3812718 (IVS5N+5 G>A)	F	A 117 (60)	G 77 (40)	–	AA 32 (33), AG 53 (55), GG 12 (12)	–
	E	A 770 (62)	G 480 (38)	F versus E: 0.73, 0.95 (0.70–1.29)	AA 242 (39), AG 286 (46), GG 97 (16)	F versus E: 0.73
	C	A 967 (58)	G 707 (42)	F versus C: 0.50, 1.11 (0.82–1.51)	AA 279 (33), AG 409 (50), GG 149 (18)	F versus C: 0.49
rs1020853	F	G 53 (35)	T 99 (65)	–	GG 7 (9), GT 39 (51), TT 30 (40)	–
	E	G 493 (40)	T 739 (60)	F versus E: 0.22, 0.80 (0.56–1.14)	GG 103 (17), GT 287 (47), TT 226 (37)	F versus E: 0.22
	C	G 619 (37)	T 1051 (63)	F versus C: 0.59, 0.91 (0.64–1.29)	GG 115 (14), GT 389 (47), TT 331 (40)	F versus C: 0.59
rs2298771	F	A 138 (91)	G 14 (9)	–	AA 62 (82), AG 14 (18), GG 0 (0)	–
	E	A 1119 (91)	G 117 (9)	F versus E: 0.92, 1.03 (0.58–1.84)	AA 507 (82), AG 105 (17), GG 6 (1.0)	F versus E: 0.92
	C	A 1490 (90)	G 178 (10)	F versus C: 0.57, 1.18 (0.67–2.09)	AA 661 (80), AG 168 (20), GG 5 (0.6)	F versus C: 0.56
rs10188577	F	C 29 (19)	T 123 (81)	–	CC 2 (3), CT 25 (33), TT 49 (65)	–
	E	C 251 (20)	T 985 (80)	F versus E: 0.72, 0.93 (0.60–1.44)	CC 24 (4), CT 203 (33), TT 391 (64)	F versus E: 0.72
	C	C 278 (17)	T 1396 (83)	F versus C: 0.44, 1.18 (0.77–1.81)	CC 25 (3), CT 228 (27), TT 584 (70)	F versus C: 0.44
rs4667866	F	C 108 (71)	G 44 (29)	–	CC 33 (43), CG 42 (55), GG 1 (1)	–
	E	C 887 (72)	G 351 (28)	F versus E: 0.88, 0.97 (0.67–1.41)	CC 327 (53), CG 233 (38), GG 59 (10)	F versus E: 0.88
	C	C 1157(69)	G 525 (31)	F versus C: 0.56, 1.11 (0.77–1.61)	CC 393 (47), CG 371 (44), GG 77 (9)	F versus C: 0.55
rs13405797	F	A 64 (42)	G 88 (58)	–	AA 11 (14), AG 42 (55), GG 23 (30)	–
	E	A 514 (42)	G 722 (58)	F versus E: 0.90, 1.02 (0.73–1.44)	AA 105 (17), AG 304 (49), GG 209 (34)	F versus E: 0.90
	C	A 678 (40)	G 1002 (60)	F versus C: 0.67, 1.08 (0.77–1.51)	AA 131 (16), AG 416 (50), GG 293 (35)	F versus C: 0.67
rs1461197	F	A 112 (74)	G 38 (26)	–	AA 40 (53), AG 32 (43), GG 3 (4)	–
	E	A 896 (72)	G 340 (28)	F versus E: 0.57, 1.12 (0.76–1.65)	AA 322 (52), AG 252 (41), GG 44 (7)	F versus E: 0.57
	C	A 1241 (74)	G 425 (26)	F versus C: 0.96, 1.01 (0.69–1.48)	AA 453 (54), AG 335 (40), GG 45 (5)	F versus C: 0.96
rs2169312	F	C 53 (35)	T 99 (65)	–	CC 9 (12), CT 35 (46), TT 32 (42)	–
	E	C 379 (31)	T 835 (69)	F versus E: 0.36, 1.12 (0.83–1.68)	CC 63 (10), CT 253 (42), TT 291 (48)	F versus E: 0.37
	C	C 498 (30)	T 1172 (70)	F versus C: 0.19, 1.26 (0.89–1.79)	CC 74 (9), CT 350 (42), TT 411 (49)	F versus C: 0.19

Discussion

IVS5N+5 G>A was initially reported to be associated with the maximal doses of the sodium channel blocking antiepileptic drugs, carbamazepine and phenytoin, in patients with epilepsy (Tate et al., 2005). Despite failure to replicate the association in subsequent studies (Zimprich et al., 2008), given its effect on splicing and possible resultant alteration of channel properties (Heinzen et al., 2007), the polymorphism has been hypothesized to be a candidate SNP for FS (Schlachter et al., 2009). The hypothesis was supported by findings of higher proportions of the A allele and AA genotype in a cohort of Caucasian adult epilepsy patients with a history of FS and in children with FS, but similar proportion in epilepsy patients without FS, compared to population controls (Schlachter et al., 2009). We examined similar, if not slightly larger, numbers of Han Chinese epilepsy patients (97 with FS and 631 without FS) and controls (n = 848) and did not find associations between FS and this and seven other tagging SNPs of the SCN1A gene.

There may be several potential explanations for the discrepant findings. First, IVS5N+5 G>A may not be causative but in linkage disequilibrium (LD) with the causal SNP(s), which might exhibit different LD patterns between Caucasian and Chinese populations. Indeed, it appears that greater proportions of Chinese carry the AA genotype and A allele compared with Caucasians (33% and 58% vs. 26% and 52% (Schlachter et al., 2009), respectively). Second, although both studies included adults with focal epilepsy, the patients differ in important phenotypic characteristics. In particular, greater proportions of patients had hippocampal sclerosis (34%) in the study of Schlachter et al. (2009), compared with the present study (12.9%). As would be expected, hippocampal sclerosis was overrepresented in patients with FS compared to those without in the former study (73% vs. 27%), which did not adjust for the presence of hippocampal sclerosis in the analysis. Whether the difference in the distribution of underlying pathologies exerted a confounding effect is unknown.

The present study did not include children with FS but without concomitant epilepsy, although a previous study conducted among Chinese children (without epilepsy) in Taiwan (Chou et al., 2003) did not find any association between FS and the SCN1A SNP rs2298771, which was also examined in the present study. Genetic association study of FS in epilepsy patients is subject to the risk of phenotyping error because obtaining from an adult patient a history of childhood FS often relies upon subjective recall, usually by the patient’s parents, who may not be able, or even available, to accurately recollect the event. Both the present study and the study by Schlachter et al. (2009) are further limited by the relatively small numbers of epilepsy patients with FS. Exploratory analysis of the present study hinted that IVS5N+5 G>A and rs10188577 were nominally associated with increased risk of epilepsy in general. Given the uncertainties of assessing FS in retrospect, there is the possibility of having undetected cases of FS among the group of epilepsy patients. Alternatively the risk phenotype of SCN1A variations, if it exists, could be a broader one than manifesting FS solely. Further large-scale, preferably prospective studies are needed to clarify the role of SCN1A variants in the genetic susceptibility of FS as well as that of epilepsy in general.

Acknowledgment

Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. CUHK4466/06M). We would like to thank Drs. Brian Tomlinson and Gary Wong, both of the Chinese University of Hong Kong, for providing some of the DNA samples of the population controls. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. None of the authors has any confl ict of interest to disclose.

Disclosure

The authors declare no conflicts of interest.