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Positive association between benign familial infantile convulsions and LGI4
Brain & Development 32 (2010) 538–543 www.elsevier.com/locate/braindev
Original article
Positive association between benign familial infantile convulsions and LGI4 Atsushi Ishii a, Bo Zhang b, Sunao Kaneko c, Shinichi Hirose a,* a
Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan Department of Cardiology, School of Medicine, Fukuoka University, Fukuoka, Japan c Department of Neuropsychiatry, School of Medicine, Hirosaki University, Hirosaki, Japan b
Received 26 May 2009; received in revised form 4 September 2009; accepted 7 September 2009
Abstract Purpose: LGI4 is located in 19q13.11, where the locus of benign familial infantile convulsions (BFIC) has been mapped. LGI4 belongs to a family of proteins with the epilepsy-associated repeat (EAR) domain and is associated with various epilepsies. We investigated whether LGI4 is a candidate gene for BFIC. Methods: Fifteen patients with BFIC were examined for mutations and/or polymorphisms of LGI4 by using a direct sequencing method. Results: Several frequent polymorphisms were identified. The genotype frequency distribution of c.1722G/A polymorphism was significantly different between patients with BFIC and control subjects (p < 0.05). Logistic regression analysis showed that the G allele of c.1722G/A polymorphism had significant recessive effects on the increased relative risk for BFIC (p < 0.05). There was no association between c.1722G/A polymorphism and benign familial neonatal convulsion, an epilepsy phenotype similar to BFIC but genetically distinguished from BFIC. Discussion: The positive genotypic association between BFIC and c.1722G/A polymorphism suggests that LGI4 might contribute to the susceptibility to BFIC. Ó 2009 Elsevier B.V. All rights reserved. Keywords: Idiopathic generalized epilepsy; LGI4 polymorphisms; Benign familial infantile convulsions (BFIC); Benign familial neonatal convulsions (BFNC)
1. Introduction Benign familial infantile convulsions (BFIC) represent an autosomal dominant hereditary idiopathic epilepsy syndrome characterized by frequent seizures in infancy with subsequent spontaneous remission [1]. Four susceptible loci of BFIC on 1p36.12-p35.1, 2q24, 16p12-q12, and 19q12-q13.1 have been reported previously [2–5]. An association with idiopathic epilepsies for several channel genes on these loci has also been reported. Seven mutations of SCN2A at 2q24 *
Corresponding author. Address: Department of Pediatrics, School of Medicine, Fukuoka University, 45-1, 7-chome, Nanakuma, Jonanku, Fukuoka 814-0180, Japan. Tel.: +81 92 801 1011; fax: +81 92 863 1970. E-mail address: [email protected] (S. Hirose). 0387-7604/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2009.09.006
encoding the a2 subunit of the sodium channel were detected in BFIC and benign familial neonatal-infantile seizure (BFNIS). In addition, SCN1B, which encodes the b1 subunit of the sodium channel, is located at 19q13.1. It is known that mutations of SCN1B are the cause of an idiopathic familial epilepsy syndrome called generalized epilepsy with febrile seizures plus (GEFS+; OMIM 604233) [6]. Furthermore, mutations of the genes encoding the neuronal sodium channel may produce diverse epilepsy phenotypes and thus mutations of SCN1B could be the cause of BFIC. However, a previous study did not find any association between SCN1B and BFIC [7]. Another candidate is the LGI4 gene at 19q13.11. The LGI4 molecule belongs to a family of proteins with epilepsy-associated repeat (EAR) domain [8]. This family includes LGI1 and MASS1, of which mutations were
A. Ishii et al. / Brain & Development 32 (2010) 538–543
identified in autosomal dominant lateral temporal epilepsy (ADLTE) and febrile seizures, respectively [9– 11]. The association between LGI4 and BFIC has not been examined. The present study was designed to determine the association between LGI4 and BFIC. For this purpose, we checked for polymorphisms and mutations in individuals with BFIC. Furthermore, the distribution of the identified genetic variations was compared with that of healthy volunteers and individuals with benign familial neonatal convulsions (BFNC). The epilepsy phenotype in BFNC resembles that of BFIC except that convulsions predominantly afflict neonates [12–20]. 2. Materials and methods 2.1. Subjects and families A total of 15 and 25 Japanese individuals with BFIC and BFNC, respectively, were recruited for the study. The diagnoses of BFIC and BFNC were made based on the following clinical manifestations: for BFIC, seizures occurring from the age of 3–24 months and spontaneously remitted in infancy; and for BFNC, seizures occurring from the age of 1–7 days and spontaneously remitted in the neonatal period. For both syndromes, the possibility of any other etiology was excluded using blood tests, cerebrospinal fluid examination, electroencephalography, and neuroimaging studies. In addition, all patients had a family history of convulsions either in infancy or the neonatal period. Linkage analysis was not performed because of the small number of the population sample. A total of 96 unrelated healthy Japanese volunteers who did not have seizures or any history of epilepsy were included as the control group. The parent or guardian of each participant signed an informed consent form and this study was approved by the Ethics Review Committee of Fukuoka University and similar committees of the participating institutions.
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2.2. Polymerase chain reaction (PCR) and analysis of the products Genetic analyses for LGI4 were performed as described previously [16,20–22]. In brief, genomic DNA was prepared from blood samples containing EDTA-Na2 using QIAamp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) according to the method described by the manufacturer. The complete LGI4 coding sequence (accession number; RefSeq: NM_139284), including adjacent short intronic sequences of the gene (Accession No. NT011196), was amplified by PCR with genomic DNA obtained from one affected individual in each of the family. Primer sequences and corresponding PCR protocols are available on request. After amplification and purification on a 96-well PCR Clean-up Kit (Whatman, Piscataway, NJ), PCR products were sequenced directly by a dye terminator sequencing method using the amplification primers on an ABI PRISMÒ 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). One individual with BFNC in whom exon 8 of LGI4 could not be sequenced was excluded from the statistical analysis of the corresponding polymorphism, including exon 8 by the dye terminator sequencing method. 2.3. Statistical analyses Deviation of the genotype distribution from the Hardy–Weinberg equilibrium and differences between groups were examined by the Fisher’s exact test. The additive, dominant, and recessive effects of LGI4 allele were assessed by multiple logistic regression analysis. The odds ratio, 95% confidence interval (95% CI), the Wald v2 value, and two-tailed p values were computed. A p value < 5% was considered significant. All statistical analyses were performed using the Statistical Analysis System software (version 9.1, SAS Institute Inc., Cary, NC).
c.1069G>A IVS2+47G>A
c.1915C>T c.1914A>G c.2143C>T IVS9-53C>G IVS8+52T>C c.2144G>A c.1722G>A c.1353C>G
c.2214C>T
IVS6-33G>A
10.69 kb Fig. 1. Structural organization of LGI4 and genetic variations. Vertical bars: LGI4 exons, open bars: untranslated regions, closed bars: coding regions. Arrow indicates the positions of the polymorphisms identified.
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Table 1 Genotype and allele frequencies of LGI4 polymorphisms in controls and subjects with BFIC and BFNC. Polymorphism
Allele frequencies
p valuea
A/A 44 (0.4583) 8 (0.5333) 12 (0.4800)
G allele 0.2969 0.3333 0.3000
A allele 0.7031 0.6667 0.7000
– 0.0734 0.7610
G/A 42 (0.4375) 4 (0.2667) 8 (0.3200)
A/A 19 (0.1979) 3 (0.2000) 4 (0.1600)
f(G) 0.5833 0.6667 0.6800
f(A) 0.4167 0.3333 0.3200
– 0.4220 0.4095
G/G 92 (0.9583) 15 (1.0000) 24 (0.9600)
G/A 3 (0.0313) 0 (0.0000) 1 (0.0400)
A/A 1 (0.0104) 0 (0.0000) 0 (0.0000)
f(G) 0.9740 1.0000 0.9800
f(A) 0.0260 0.0000 0.0200
– 1.0000 1.0000
96 15 25
C/C 2 (0.0208) 2 (0.1333) 1 (0.0400)
C/G 33 (0.3438) 4 (0.2667) 9 (0.3600)
G/G 61 (0.6354) 9 (0.6000) 15 (0.6000)
f(C) 0.1927 0.2667 0.2200
f(G) 0.8073 0.7333 0.7800
– 0.1352 0.6702
Controls BFIC BFNC
96 15 24
G/G 3 (0.0313) 3 (0.2000) 2 (0.0833)
G/A 35 (0.3646) 3 (0.2000) 9 (0.3750)
A/A 58 (0.6042) 9 (0.6000) 13 (0.5417)
f(G) 0.2135 0.3000 0.2708
f(A) 0.7865 0.7000 0.7292
– 0.0415 0.4321
Controls BFIC BFNC
96 15 25
T/T 61 (0.6354) 10 (0.6667) 15 (0.6000)
T/C 33 (0.3438) 3 (0.2000) 9 (0.3600)
C/C 2 (0.0208) 2 (0.1333) 1 (0.0400)
f(T) 0.8073 0.7667 0.7800
f(C) 0.1927 0.2333 0.2200
– 0.0943 0.6702
Controls BFIC BFNC
96 15 25
C/C 60 (0.6250) 10 (0.6667) 15 (0.6000)
C/G 32 (0.3333) 4 (0.2667) 9 (0.3600)
G/G 4 (0.0417) 1 (0.0667) 1 (0.0400)
f(C) 0.7917 0.8000 0.7800
f(G) 0.2083 0.2000 0.2200
– 0.6129 0.9224
Controls BFIC BFNC
96 15 25
A/A 5 (0.0521) 2 (0.1333) 3 (0.1200)
A/G 33 (0.3438) 4 (0.2667) 7 (0.2800)
G/G 58 (0.6042) 9 (0.6000) 15 (0.6000)
f(A) 0.2240 0.2667 0.2600
f(G) 0.7760 0.7333 0.7400
– 0.3357 0.4469
Controls BFIC BFNC
96 15 25
C/C 59 (0.6146) 9 (0.6000) 15 (0.6000)
C/T 33 (0.3438) 4 (0.2667) 7 (0.2800
T/T 4 (0.0417) 2 (0.1333) 3 (0.1200)
f(C) 0.7865 0.7333 0.7400
f(T) 0.2135 0.2667 0.2600
– 0.2984 0.3139
Controls BFIC BFNC
96 15 25
C/C 35 (0.3646) 7 (0.4667) 9 (0.3600)
C/T 25 (0.2604) 5 (0.3333) 6 (0.2400)
T/T 36 (0.3750) 3 (0.2000) 10 (0.4000)
f(C) 0.4948 0.6333 0.4800
f(T) 0.5052 0.3667 0.5200
– 0.4164 1.0000
Controls BFIC BFNC
96 15 25
G/G 92 (0.9583) 13 (0.8667) 24 (0.9600)
G/A 0 (0.0000) 0 (0.0000) 0 (0.0000)
A/A 4 (0.0417) 2 (0.1333) 1 (0.0400)
f(G) 0.9583 0.8667 0.9600
f(A) 0.0417 0.1333 0.0400
– 0.1856 1.0000
Controls BFIC BFNC
96 15 25
C/C 92 (0.9583) 13 (0.8667) 24 (0.9600)
C/T 1 (0.0104) 0 (0.0000) 0 (0.0000)
T/T 3 (0.0313) 2 (0.1333) 1 (0.0400)
f(C) 0.9635 0.8667 0.9600
f(T) 0.0365 0.1333 0.0400
– 0.2531 1.0000
Group
n
Genotype frequencies
Controls BFIC BFNC
96 15 25
G/G 5 (0.0521) 3 (0.2000) 2 (0.0800)
G/A 47 (0.4896) 4 (0.2667) 11 (0.4400)
Controls BFIC BFNC
96 15 25
G/G 35 (0.3646) 8 (0.5333) 13 (0.5200)
Controls BFIC BFNC
96 15 25
Controls BFIC BFNC
IVS2 + 47G > A
IVS6–33G > A
c.1069G > A
c.1353C > G
c.1722G > A
c.IVS8 + 52T > C
c.IVS9–53C > G
c.1914A > G
c.1915C > T
c.2143C > T
c.2144G > A
c.2214C > T
a
p value vs. controls, by Fisher’s exact test.
3. Results LGI4 spans 10.69 kb and consists of nine exons ranging in size from 72 bp to 995 bp, and 12 genetic variations were identified in this study (Fig. 1). Eleven were silent polymorphisms and one was a single nucleotide
polymorphism (SNP) c.1069G > A, resulting in an amino acid exchange (Ala184Thr) identified in a patient with BFNC. No other variations that could affect mRNA splicing were detected. The genotype and allele frequencies of 12 SNPs are shown in Table 1. The frequencies of the observed geno-
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Table 2 Multiple logistic regression analysis of the association between LGI4 polymorphism and BFIC. Polymorphism
Model studied
Effect of allele
r
SE
Odds ratio
95% CI
Wald v2
p value
IVS2 + 47G > A c.1353C > G c.1722G > A c.IVS8 + 52T > C
G/G vs. G/A and A/A C/C vs. C/G and G/G G/G vs. G/A and A/A T/T and T/C vs. C/C
Recessive Recessive Recessive Dominant
1.515 1.978 2.048 1.978
0.792 1.043 0.872 1.043
4.551 7.231 7.750 0.138
0.850–21.104 0.813–64.647 1.310–46.281 0.015–1.231
3.658 3.599 5.512 3.599
0.056 0.058 0.019 0.058
r: regression coefficient, SE: standard error.
types did not significantly deviate from those predicted by the Hardy–Weinberg equilibrium. The genotype frequency distribution of c.1722G/A polymorphism was significantly different between patients with BFIC [GG/GA/AA: 3 (20.0%)/3 (20.0%)/9 (60.0%)] and control subjects [GG/GA/AA: 3 (3.1%)/35 (36.5%)/58 (60.4%)], respectively (p = 0.042). The genotype frequency distributions of the other two SNPs in patients with BFIC tended to be different from those of the control group [IVS2 + 47G/A: GG/ GA/AA:3 (20.0%)/4 (26.7%)/8 (53.3%) vs. 5 (5.2%)/47 (49.0%)/44 (45.8%), respectively, p = 0.073, IVS8 + 52T/C: TT/TC/CC:10 (66.7%)/3 (20.0%)/2 (13.3%) vs. 61 (63.5%)/33 (34.4%)/2 (2.08%), respectively, p = 0.094]. The association between LGI4 polymorphisms and BFIC was examined by multiple logistic regression analysis (Table 2). The recessive effects of the G and C alleles in IVS2 + 47G > A and c.1353C > G polymorphism, respectively, and the dominant effects of the T allele of IVS8 + 52T > C polymorphism tended to be significant. The results of the analysis showed that the G allele of c.1722G/A polymorphism had significant recessive effects on the increased relative risk for BFIC (GG vs. GA and AA, odds ratio (95% CI): 7.8 (1.3–46.3), p = 0.019). However, there was no significant association between LGI4 polymorphisms and BFNC (data not shown) and no pathogenic mutation was detected in subjects with BFNC. 4. Discussion The present study provided the first evidence for an association between LGI4 and BFIC. The c.1722G/A polymorphism was associated significantly with BFIC: the G allele of c.1722G/A polymorphism had significant recessive effects on the increased relative risk for BFIC. However, the results showed no significant association between LGI4 and BFNC. LGI4 is located on chromosome 19q13.11 where the genetic locus of BFIC was assigned in a previous linkage analysis of Italian families [4]. LGI4 belongs to a family of proteins with the signature repeat at the C-terminus. The repeat is referred to as EAR domain or Epitempin (ETPT) repeat since two molecules of the family, LGI1 and MASS1, were found to be closely associated with
epilepsy [11,23–26]. LGI1 is similar to LGI4 in sharing a leucine-rich repeat along with EAR and its mutations were identified in ADLTE, a familial idiopathic epilepsy [8,27]. A mutation of MASS1 was also reported in febrile seizures and a mutation of Mass1, the mice homologous gene, was found in the Frings mouse, a model of audiogenic epilepsy [11,25,26]. The functions of LGI or LGI4 proteins are not clear at present. However, LGI1 has been reported to be a subunit of Kv1.1-associated protein complexes and acts in inactivation gating of presynaptic A-type channels [28]. Hence, defects of LGI1 may contribute to the epileptic activity via channel dysfunction. Furthermore, a recent study has shown that both LGI1 and LGI4 are associated with ADAM22 in mouse brain [29]. ADAM22 is a cell-surface receptor predominantly expressed in neuronal tissues [30] and ADAM22deficient mice have hypomyelination in the peripheral nerves and develop ataxia and seizure [31], suggesting LGI1 and LGI4 can support ADAM22 to maintain proper neuronal function in mammals. Thus, LGI1 or LGI4 mutations might affect neuronal cell migration, axon guidance, or synaptogenesis. However, no causative mutation in the LGI4 coding region was found in the present study. This finding implies several possibilities. One possibility is that the positive association of LG14 with BFIC suggests that LGI4 could be perhaps a suitable marker of BFIC in local linkage disequilibrium. In such case, causative mutations for BFIC may be found in one of the adjacent genes. SCN1B is one such candidate since it is located in direct proximity to LGI4 within an interval of 60 K [8]. However, a previous study could not identify mutation of SCN1B in patients with BFIC [7]. It is also possible that the causative mutations of LGI4 that may underlie BFIC were overlooked in the present study. The methods used in the present study might have failed to identify such mutations (e.g., mutations in the promoter regions or microdeletions of the gene). In fact, the etiologies of various epilepsies include microdeletions of several genes [32]. It is also possible that LGI4 could contribute to susceptibility to BFIC with other putative genetic factors. A previous study described the genetic association between LGI4 and childhood absence epilepsy, an idiopathic epilepsy associated with a genetic etiology [33].
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Our findings of a positive association between LGI4 and BFIC might enhance our understanding of the pathomechanisms of epilepsy. Disclosure of conflicts of interest None of the authors has any conflict of interest to disclose. 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.
Acknowledgments We are indebted to all members of the family for their helpful cooperation in this study. We thank Ms. Takako Umemoto and Hideko Takeda for formatting and typing the manuscript and Ms. Minako Yonetani and Akiyo Hamachi for the technical assistance. This study was supported in part by Grants-in-Aid for Scientific Research (S) 16109006, (A) 18209035 and 21249062, Exploratory Research 1659272, and “High-Tech Research Center” Project for Private Universities-matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology, 2006–2010 “The Research Center for the Molecular Pathomechanisms of Epilepsy, Fukuoka University”, Research Grants (19A-6 and 21B-5) for Nervous and Mental Disorders and Health and Labor Science Research Grant 21210301 from the Ministry of Health, Labor and Welfare and the Central Research Institute of Fukuoka University. 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. References [1] Echenne B, Humbertclaude V, Rivier F, Malafosse A, Cheminal R. Benign infantile epilepsy with autosomal dominant inheritance. Brain Dev 1994;16:108–11. [2] Baulac S, Gourfinkel-An I, Picard F, Rosenberg-Bourgin M, Prudhomme JF, Baulac M, et al. A second locus for familial generalized epilepsy with febrile seizures plus maps to chromosome 2q21–q33. Am J Hum Genet 1999;65:1078–85. [3] Caraballo R, Pavek S, Lemainque A, Gastaldi M, Echenne B, Motte J, et al. Linkage of benign familial infantile convulsions to chromosome 16p12–q12 suggests allelism to the infantile convulsions and choreoathetosis syndrome. Am J Hum Genet 2001;68:788–94. [4] Guipponi M, Rivier F, Vigevano F, Beck C, Crespel A, Echenne B, et al. Linkage mapping of benign familial infantile convulsions (BFIC) to chromosome 19q. Hum Mol Genet 1997;6:473–7. [5] Li HY, Li N, Jiang H, Shen L, Guo JF, Zhang RX, et al. A novel genetic locus for benign familial infantile seizures maps to chromosome 1p36.12–p35.1. Clin Genet 2008;74:490–2. [6] Wallace RH, Wang DW, Singh R, Scheffer IE, George Jr AL, Phillips HA, et al. Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B. Nat Genet 1998;19:366–70.
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Original article
Positive association between benign familial infantile convulsions and LGI4 Atsushi Ishii a, Bo Zhang b, Sunao Kaneko c, Shinichi Hirose a,* a
Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan Department of Cardiology, School of Medicine, Fukuoka University, Fukuoka, Japan c Department of Neuropsychiatry, School of Medicine, Hirosaki University, Hirosaki, Japan b
Received 26 May 2009; received in revised form 4 September 2009; accepted 7 September 2009
Abstract Purpose: LGI4 is located in 19q13.11, where the locus of benign familial infantile convulsions (BFIC) has been mapped. LGI4 belongs to a family of proteins with the epilepsy-associated repeat (EAR) domain and is associated with various epilepsies. We investigated whether LGI4 is a candidate gene for BFIC. Methods: Fifteen patients with BFIC were examined for mutations and/or polymorphisms of LGI4 by using a direct sequencing method. Results: Several frequent polymorphisms were identified. The genotype frequency distribution of c.1722G/A polymorphism was significantly different between patients with BFIC and control subjects (p < 0.05). Logistic regression analysis showed that the G allele of c.1722G/A polymorphism had significant recessive effects on the increased relative risk for BFIC (p < 0.05). There was no association between c.1722G/A polymorphism and benign familial neonatal convulsion, an epilepsy phenotype similar to BFIC but genetically distinguished from BFIC. Discussion: The positive genotypic association between BFIC and c.1722G/A polymorphism suggests that LGI4 might contribute to the susceptibility to BFIC. Ó 2009 Elsevier B.V. All rights reserved. Keywords: Idiopathic generalized epilepsy; LGI4 polymorphisms; Benign familial infantile convulsions (BFIC); Benign familial neonatal convulsions (BFNC)
1. Introduction Benign familial infantile convulsions (BFIC) represent an autosomal dominant hereditary idiopathic epilepsy syndrome characterized by frequent seizures in infancy with subsequent spontaneous remission [1]. Four susceptible loci of BFIC on 1p36.12-p35.1, 2q24, 16p12-q12, and 19q12-q13.1 have been reported previously [2–5]. An association with idiopathic epilepsies for several channel genes on these loci has also been reported. Seven mutations of SCN2A at 2q24 *
Corresponding author. Address: Department of Pediatrics, School of Medicine, Fukuoka University, 45-1, 7-chome, Nanakuma, Jonanku, Fukuoka 814-0180, Japan. Tel.: +81 92 801 1011; fax: +81 92 863 1970. E-mail address: [email protected] (S. Hirose). 0387-7604/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2009.09.006
encoding the a2 subunit of the sodium channel were detected in BFIC and benign familial neonatal-infantile seizure (BFNIS). In addition, SCN1B, which encodes the b1 subunit of the sodium channel, is located at 19q13.1. It is known that mutations of SCN1B are the cause of an idiopathic familial epilepsy syndrome called generalized epilepsy with febrile seizures plus (GEFS+; OMIM 604233) [6]. Furthermore, mutations of the genes encoding the neuronal sodium channel may produce diverse epilepsy phenotypes and thus mutations of SCN1B could be the cause of BFIC. However, a previous study did not find any association between SCN1B and BFIC [7]. Another candidate is the LGI4 gene at 19q13.11. The LGI4 molecule belongs to a family of proteins with epilepsy-associated repeat (EAR) domain [8]. This family includes LGI1 and MASS1, of which mutations were
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identified in autosomal dominant lateral temporal epilepsy (ADLTE) and febrile seizures, respectively [9– 11]. The association between LGI4 and BFIC has not been examined. The present study was designed to determine the association between LGI4 and BFIC. For this purpose, we checked for polymorphisms and mutations in individuals with BFIC. Furthermore, the distribution of the identified genetic variations was compared with that of healthy volunteers and individuals with benign familial neonatal convulsions (BFNC). The epilepsy phenotype in BFNC resembles that of BFIC except that convulsions predominantly afflict neonates [12–20]. 2. Materials and methods 2.1. Subjects and families A total of 15 and 25 Japanese individuals with BFIC and BFNC, respectively, were recruited for the study. The diagnoses of BFIC and BFNC were made based on the following clinical manifestations: for BFIC, seizures occurring from the age of 3–24 months and spontaneously remitted in infancy; and for BFNC, seizures occurring from the age of 1–7 days and spontaneously remitted in the neonatal period. For both syndromes, the possibility of any other etiology was excluded using blood tests, cerebrospinal fluid examination, electroencephalography, and neuroimaging studies. In addition, all patients had a family history of convulsions either in infancy or the neonatal period. Linkage analysis was not performed because of the small number of the population sample. A total of 96 unrelated healthy Japanese volunteers who did not have seizures or any history of epilepsy were included as the control group. The parent or guardian of each participant signed an informed consent form and this study was approved by the Ethics Review Committee of Fukuoka University and similar committees of the participating institutions.
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2.2. Polymerase chain reaction (PCR) and analysis of the products Genetic analyses for LGI4 were performed as described previously [16,20–22]. In brief, genomic DNA was prepared from blood samples containing EDTA-Na2 using QIAamp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) according to the method described by the manufacturer. The complete LGI4 coding sequence (accession number; RefSeq: NM_139284), including adjacent short intronic sequences of the gene (Accession No. NT011196), was amplified by PCR with genomic DNA obtained from one affected individual in each of the family. Primer sequences and corresponding PCR protocols are available on request. After amplification and purification on a 96-well PCR Clean-up Kit (Whatman, Piscataway, NJ), PCR products were sequenced directly by a dye terminator sequencing method using the amplification primers on an ABI PRISMÒ 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). One individual with BFNC in whom exon 8 of LGI4 could not be sequenced was excluded from the statistical analysis of the corresponding polymorphism, including exon 8 by the dye terminator sequencing method. 2.3. Statistical analyses Deviation of the genotype distribution from the Hardy–Weinberg equilibrium and differences between groups were examined by the Fisher’s exact test. The additive, dominant, and recessive effects of LGI4 allele were assessed by multiple logistic regression analysis. The odds ratio, 95% confidence interval (95% CI), the Wald v2 value, and two-tailed p values were computed. A p value < 5% was considered significant. All statistical analyses were performed using the Statistical Analysis System software (version 9.1, SAS Institute Inc., Cary, NC).
c.1069G>A IVS2+47G>A
c.1915C>T c.1914A>G c.2143C>T IVS9-53C>G IVS8+52T>C c.2144G>A c.1722G>A c.1353C>G
c.2214C>T
IVS6-33G>A
10.69 kb Fig. 1. Structural organization of LGI4 and genetic variations. Vertical bars: LGI4 exons, open bars: untranslated regions, closed bars: coding regions. Arrow indicates the positions of the polymorphisms identified.
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Table 1 Genotype and allele frequencies of LGI4 polymorphisms in controls and subjects with BFIC and BFNC. Polymorphism
Allele frequencies
p valuea
A/A 44 (0.4583) 8 (0.5333) 12 (0.4800)
G allele 0.2969 0.3333 0.3000
A allele 0.7031 0.6667 0.7000
– 0.0734 0.7610
G/A 42 (0.4375) 4 (0.2667) 8 (0.3200)
A/A 19 (0.1979) 3 (0.2000) 4 (0.1600)
f(G) 0.5833 0.6667 0.6800
f(A) 0.4167 0.3333 0.3200
– 0.4220 0.4095
G/G 92 (0.9583) 15 (1.0000) 24 (0.9600)
G/A 3 (0.0313) 0 (0.0000) 1 (0.0400)
A/A 1 (0.0104) 0 (0.0000) 0 (0.0000)
f(G) 0.9740 1.0000 0.9800
f(A) 0.0260 0.0000 0.0200
– 1.0000 1.0000
96 15 25
C/C 2 (0.0208) 2 (0.1333) 1 (0.0400)
C/G 33 (0.3438) 4 (0.2667) 9 (0.3600)
G/G 61 (0.6354) 9 (0.6000) 15 (0.6000)
f(C) 0.1927 0.2667 0.2200
f(G) 0.8073 0.7333 0.7800
– 0.1352 0.6702
Controls BFIC BFNC
96 15 24
G/G 3 (0.0313) 3 (0.2000) 2 (0.0833)
G/A 35 (0.3646) 3 (0.2000) 9 (0.3750)
A/A 58 (0.6042) 9 (0.6000) 13 (0.5417)
f(G) 0.2135 0.3000 0.2708
f(A) 0.7865 0.7000 0.7292
– 0.0415 0.4321
Controls BFIC BFNC
96 15 25
T/T 61 (0.6354) 10 (0.6667) 15 (0.6000)
T/C 33 (0.3438) 3 (0.2000) 9 (0.3600)
C/C 2 (0.0208) 2 (0.1333) 1 (0.0400)
f(T) 0.8073 0.7667 0.7800
f(C) 0.1927 0.2333 0.2200
– 0.0943 0.6702
Controls BFIC BFNC
96 15 25
C/C 60 (0.6250) 10 (0.6667) 15 (0.6000)
C/G 32 (0.3333) 4 (0.2667) 9 (0.3600)
G/G 4 (0.0417) 1 (0.0667) 1 (0.0400)
f(C) 0.7917 0.8000 0.7800
f(G) 0.2083 0.2000 0.2200
– 0.6129 0.9224
Controls BFIC BFNC
96 15 25
A/A 5 (0.0521) 2 (0.1333) 3 (0.1200)
A/G 33 (0.3438) 4 (0.2667) 7 (0.2800)
G/G 58 (0.6042) 9 (0.6000) 15 (0.6000)
f(A) 0.2240 0.2667 0.2600
f(G) 0.7760 0.7333 0.7400
– 0.3357 0.4469
Controls BFIC BFNC
96 15 25
C/C 59 (0.6146) 9 (0.6000) 15 (0.6000)
C/T 33 (0.3438) 4 (0.2667) 7 (0.2800
T/T 4 (0.0417) 2 (0.1333) 3 (0.1200)
f(C) 0.7865 0.7333 0.7400
f(T) 0.2135 0.2667 0.2600
– 0.2984 0.3139
Controls BFIC BFNC
96 15 25
C/C 35 (0.3646) 7 (0.4667) 9 (0.3600)
C/T 25 (0.2604) 5 (0.3333) 6 (0.2400)
T/T 36 (0.3750) 3 (0.2000) 10 (0.4000)
f(C) 0.4948 0.6333 0.4800
f(T) 0.5052 0.3667 0.5200
– 0.4164 1.0000
Controls BFIC BFNC
96 15 25
G/G 92 (0.9583) 13 (0.8667) 24 (0.9600)
G/A 0 (0.0000) 0 (0.0000) 0 (0.0000)
A/A 4 (0.0417) 2 (0.1333) 1 (0.0400)
f(G) 0.9583 0.8667 0.9600
f(A) 0.0417 0.1333 0.0400
– 0.1856 1.0000
Controls BFIC BFNC
96 15 25
C/C 92 (0.9583) 13 (0.8667) 24 (0.9600)
C/T 1 (0.0104) 0 (0.0000) 0 (0.0000)
T/T 3 (0.0313) 2 (0.1333) 1 (0.0400)
f(C) 0.9635 0.8667 0.9600
f(T) 0.0365 0.1333 0.0400
– 0.2531 1.0000
Group
n
Genotype frequencies
Controls BFIC BFNC
96 15 25
G/G 5 (0.0521) 3 (0.2000) 2 (0.0800)
G/A 47 (0.4896) 4 (0.2667) 11 (0.4400)
Controls BFIC BFNC
96 15 25
G/G 35 (0.3646) 8 (0.5333) 13 (0.5200)
Controls BFIC BFNC
96 15 25
Controls BFIC BFNC
IVS2 + 47G > A
IVS6–33G > A
c.1069G > A
c.1353C > G
c.1722G > A
c.IVS8 + 52T > C
c.IVS9–53C > G
c.1914A > G
c.1915C > T
c.2143C > T
c.2144G > A
c.2214C > T
a
p value vs. controls, by Fisher’s exact test.
3. Results LGI4 spans 10.69 kb and consists of nine exons ranging in size from 72 bp to 995 bp, and 12 genetic variations were identified in this study (Fig. 1). Eleven were silent polymorphisms and one was a single nucleotide
polymorphism (SNP) c.1069G > A, resulting in an amino acid exchange (Ala184Thr) identified in a patient with BFNC. No other variations that could affect mRNA splicing were detected. The genotype and allele frequencies of 12 SNPs are shown in Table 1. The frequencies of the observed geno-
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Table 2 Multiple logistic regression analysis of the association between LGI4 polymorphism and BFIC. Polymorphism
Model studied
Effect of allele
r
SE
Odds ratio
95% CI
Wald v2
p value
IVS2 + 47G > A c.1353C > G c.1722G > A c.IVS8 + 52T > C
G/G vs. G/A and A/A C/C vs. C/G and G/G G/G vs. G/A and A/A T/T and T/C vs. C/C
Recessive Recessive Recessive Dominant
1.515 1.978 2.048 1.978
0.792 1.043 0.872 1.043
4.551 7.231 7.750 0.138
0.850–21.104 0.813–64.647 1.310–46.281 0.015–1.231
3.658 3.599 5.512 3.599
0.056 0.058 0.019 0.058
r: regression coefficient, SE: standard error.
types did not significantly deviate from those predicted by the Hardy–Weinberg equilibrium. The genotype frequency distribution of c.1722G/A polymorphism was significantly different between patients with BFIC [GG/GA/AA: 3 (20.0%)/3 (20.0%)/9 (60.0%)] and control subjects [GG/GA/AA: 3 (3.1%)/35 (36.5%)/58 (60.4%)], respectively (p = 0.042). The genotype frequency distributions of the other two SNPs in patients with BFIC tended to be different from those of the control group [IVS2 + 47G/A: GG/ GA/AA:3 (20.0%)/4 (26.7%)/8 (53.3%) vs. 5 (5.2%)/47 (49.0%)/44 (45.8%), respectively, p = 0.073, IVS8 + 52T/C: TT/TC/CC:10 (66.7%)/3 (20.0%)/2 (13.3%) vs. 61 (63.5%)/33 (34.4%)/2 (2.08%), respectively, p = 0.094]. The association between LGI4 polymorphisms and BFIC was examined by multiple logistic regression analysis (Table 2). The recessive effects of the G and C alleles in IVS2 + 47G > A and c.1353C > G polymorphism, respectively, and the dominant effects of the T allele of IVS8 + 52T > C polymorphism tended to be significant. The results of the analysis showed that the G allele of c.1722G/A polymorphism had significant recessive effects on the increased relative risk for BFIC (GG vs. GA and AA, odds ratio (95% CI): 7.8 (1.3–46.3), p = 0.019). However, there was no significant association between LGI4 polymorphisms and BFNC (data not shown) and no pathogenic mutation was detected in subjects with BFNC. 4. Discussion The present study provided the first evidence for an association between LGI4 and BFIC. The c.1722G/A polymorphism was associated significantly with BFIC: the G allele of c.1722G/A polymorphism had significant recessive effects on the increased relative risk for BFIC. However, the results showed no significant association between LGI4 and BFNC. LGI4 is located on chromosome 19q13.11 where the genetic locus of BFIC was assigned in a previous linkage analysis of Italian families [4]. LGI4 belongs to a family of proteins with the signature repeat at the C-terminus. The repeat is referred to as EAR domain or Epitempin (ETPT) repeat since two molecules of the family, LGI1 and MASS1, were found to be closely associated with
epilepsy [11,23–26]. LGI1 is similar to LGI4 in sharing a leucine-rich repeat along with EAR and its mutations were identified in ADLTE, a familial idiopathic epilepsy [8,27]. A mutation of MASS1 was also reported in febrile seizures and a mutation of Mass1, the mice homologous gene, was found in the Frings mouse, a model of audiogenic epilepsy [11,25,26]. The functions of LGI or LGI4 proteins are not clear at present. However, LGI1 has been reported to be a subunit of Kv1.1-associated protein complexes and acts in inactivation gating of presynaptic A-type channels [28]. Hence, defects of LGI1 may contribute to the epileptic activity via channel dysfunction. Furthermore, a recent study has shown that both LGI1 and LGI4 are associated with ADAM22 in mouse brain [29]. ADAM22 is a cell-surface receptor predominantly expressed in neuronal tissues [30] and ADAM22deficient mice have hypomyelination in the peripheral nerves and develop ataxia and seizure [31], suggesting LGI1 and LGI4 can support ADAM22 to maintain proper neuronal function in mammals. Thus, LGI1 or LGI4 mutations might affect neuronal cell migration, axon guidance, or synaptogenesis. However, no causative mutation in the LGI4 coding region was found in the present study. This finding implies several possibilities. One possibility is that the positive association of LG14 with BFIC suggests that LGI4 could be perhaps a suitable marker of BFIC in local linkage disequilibrium. In such case, causative mutations for BFIC may be found in one of the adjacent genes. SCN1B is one such candidate since it is located in direct proximity to LGI4 within an interval of 60 K [8]. However, a previous study could not identify mutation of SCN1B in patients with BFIC [7]. It is also possible that the causative mutations of LGI4 that may underlie BFIC were overlooked in the present study. The methods used in the present study might have failed to identify such mutations (e.g., mutations in the promoter regions or microdeletions of the gene). In fact, the etiologies of various epilepsies include microdeletions of several genes [32]. It is also possible that LGI4 could contribute to susceptibility to BFIC with other putative genetic factors. A previous study described the genetic association between LGI4 and childhood absence epilepsy, an idiopathic epilepsy associated with a genetic etiology [33].
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Our findings of a positive association between LGI4 and BFIC might enhance our understanding of the pathomechanisms of epilepsy. Disclosure of conflicts of interest None of the authors has any conflict of interest to disclose. 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.
Acknowledgments We are indebted to all members of the family for their helpful cooperation in this study. We thank Ms. Takako Umemoto and Hideko Takeda for formatting and typing the manuscript and Ms. Minako Yonetani and Akiyo Hamachi for the technical assistance. This study was supported in part by Grants-in-Aid for Scientific Research (S) 16109006, (A) 18209035 and 21249062, Exploratory Research 1659272, and “High-Tech Research Center” Project for Private Universities-matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology, 2006–2010 “The Research Center for the Molecular Pathomechanisms of Epilepsy, Fukuoka University”, Research Grants (19A-6 and 21B-5) for Nervous and Mental Disorders and Health and Labor Science Research Grant 21210301 from the Ministry of Health, Labor and Welfare and the Central Research Institute of Fukuoka University. 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. References [1] Echenne B, Humbertclaude V, Rivier F, Malafosse A, Cheminal R. Benign infantile epilepsy with autosomal dominant inheritance. Brain Dev 1994;16:108–11. [2] Baulac S, Gourfinkel-An I, Picard F, Rosenberg-Bourgin M, Prudhomme JF, Baulac M, et al. A second locus for familial generalized epilepsy with febrile seizures plus maps to chromosome 2q21–q33. Am J Hum Genet 1999;65:1078–85. [3] Caraballo R, Pavek S, Lemainque A, Gastaldi M, Echenne B, Motte J, et al. Linkage of benign familial infantile convulsions to chromosome 16p12–q12 suggests allelism to the infantile convulsions and choreoathetosis syndrome. Am J Hum Genet 2001;68:788–94. [4] Guipponi M, Rivier F, Vigevano F, Beck C, Crespel A, Echenne B, et al. Linkage mapping of benign familial infantile convulsions (BFIC) to chromosome 19q. Hum Mol Genet 1997;6:473–7. [5] Li HY, Li N, Jiang H, Shen L, Guo JF, Zhang RX, et al. A novel genetic locus for benign familial infantile seizures maps to chromosome 1p36.12–p35.1. Clin Genet 2008;74:490–2. [6] Wallace RH, Wang DW, Singh R, Scheffer IE, George Jr AL, Phillips HA, et al. Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B. Nat Genet 1998;19:366–70.
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