BRIEF COMMUNICATION
Mutation of CHRNA2 in a family with benign familial infantile seizures: Potential role of nicotinic acetylcholine receptor in various phenotypes of epilepsy *1Marina Trivisano, †1Alessandra Terracciano, ‡Teresa Milano, §Simona Cappelletti, *¶Nicola Pietrafusa, †Enrico Silvio Bertini, *Federico Vigevano, and *Nicola Specchio Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
SUMMARY
Marina Trivisano is researcher at the Neuroscience Department of Bambino Gesu Children’s Hospital, Rome.
Nicotinic acetylcholine receptor genes are involved mainly in nocturnal frontal epilepsy. Despite extensive studies, to date, the a2 subunit did not show a strong association with this peculiar epileptic phenotype. We report CHRNA2 missense mutation in a family with benign familial infantile seizures (BFIS). TrueSeq Custom Amplicon (TSCA) sequencing approach was used to screen 10 ion channel genes in patients with idiopathic epilepsies. TSCA revealed a heterozygous single-nucleotide substitution in CHRNA2 gene (c.1126 C>T; p. Arg376Trp) that segregated in a family with BFIS; based on bio-informatics inspection, the change was predicted to be pathogenic. The investigated family includes parents and their three daughters. In affected individuals, seizures started between 6 and 24 months of age. Seizures were mainly in cluster and well-controlled. Outcome was good in all subjects. Even if nicotinic acetylcholine receptor genes are traditionally associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), this single-family description can open new possibilities in the genetic diagnosis, molecular characterization, and management of CHRNA2-related epilepsy. The pathogenic conversion of arginine 376 to tryptophan alters all of these interactions in the cytoplasmic domain, never reported to be involved in epileptogenic mechanism. Further functional tests will be necessary to strongly relate CHRNA2 mutation with BFIS phenotype. KEY WORDS: Benign familial infantile epilepsies, CHRNA2 gene, Acetylcholine receptor, Benign epilepsies, Autosomal dominant nocturnal frontal lobe epilepsy.
Mutations in genes encoding the neuronal acetylcholine receptor (nAChRs) are typically associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), Accepted February 11, 2015; Early View publication April 3, 2015. *Division of Neurology, Bambino Gesu Children’s Hospital, IRCCS, Rome, Italy; †Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, Bambino Gesu Children’s Hospital, IRCCS, Rome, Italy; ‡Department of Biochemical Science “Rossi Fanelli,” Sapienza University of Rome, Rome, Italy; §Unit of Clinical Psychology, Bambino Ges u Children’s Hospital, IRCCS, Rome, Italy; and ¶Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari, Bari, Italy 1 Both authors equally contributed to this paper. Address correspondence to Nicola Specchio, Division of Neurology, Bambino Ges u Children’s Hospital, IRCCS, P.zza S. Onofrio 4, 00165 Rome, Italy. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
a well-known genetic focal epilepsy.1 CHRNA4 and CHRNB2 gene mutations encoding the alfa4 and beta2 subunits are most frequently reported. More recently, an additional nAChR gene (CHRNA2), encoding the alfa2 subunit, was mutated in a large family with nocturnal frontal lobe epilepsy with atypical features such as wandering and ictal fear.2 As other nAChRs, CHRNA2 is a neurotransmitter-gated ion channel that has been finetuned through evolution to transduce a chemical signal into an electrical signal. It is a pentameric ligand-gated ion channel distributed widely throughout the nervous system, and each subunit spans the plasma membrane with four transmembrane domains (M1–M4) giving rise to several different nAChR subtypes in the brain.3 The description of the CHRNA2-mutated family reported by Aridon et al.2 remained unique and isolated. Different
e53
e54 M. Trivisano et al. independent studies explored the CHRNA2 gene role in ADNFLE, without significant results.4–6 In the present study we report a family with the benign familial infantile seizure (BFIS) phenotype, segregating a mutation in the cytoplasmic region of CHRNA2. BFIS is a condition characterized by onset during the first 2 years of life in otherwise normal children. Seizures have an autosomal dominant trait of inheritance and a typical onset around the sixth month. Seizures are typically grouped in cluster and are characterized by psychomotor arrest, slow deviation of the head and eyes to one side, diffuse hypertonia, cyanosis, and unilateral limb jerks, which become bilateral.7 Outcome is always excellent with a normal psychomotor development. In a series described by Caraballo,8 benign infantile seizures have been listed as the third most common type of epilepsy in the first 2 years of life. This is the second family segregating a CHRNA2 mutation and the first association between neuronal nAChRs subunit and BFIS phenotype. BFIS families are genetically heterogeneous: genetic studies led to the identification of mutations in KCNQ2 and KCNQ3, in SCN2A and in PRRT2 genes. In about 10% of cases, a causative gene mutation is still lacking.9
Methods Family We reviewed medical charts of all the affected members of this family to obtain clinical and genetic information. All family members were personally examined by M.T., F.V., and N.S. Electroencephalography (EEG), brain magnetic resonance imaging (MRI), and neuropsychological assessments were reviewed. All individuals included in the study gave written informed consent. The ethical committee of the institution approved this study. Target parallel resequencing Blood samples were obtained from five individuals, and genomic DNA was isolated. For proband DNA analysis, we used TrueSeq Custom amplicon sequencing (TSCA) (Illumina, San Diego, CA, U.S.A.) to amplify 10 voltage-gated ion channel genes: SCN1A, SCN2A, SCN3A, SCN8A, SCN1B, KCNQ2, KCNQ3, CHRNA2, CHRNA4, and CHRNB2. CHRNA2 c.1126 C>T; p.Arg376Trp mutation was found, and analysis was expanded to the whole family by direct Sanger sequencing. PRRT2 gene was analyzed by Sanger sequencing. One hundred eighty-eight ethnically matched controls were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis. Further details are reported in Supporting Information. Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
Results Patients and pedigree The investigated family includes parents and their three daughters (Fig. 1). The mother (I-1) is a healthy woman with no neurologic diseases in her family. The father (I-2) is 35-year-old man. He was born at term after uneventful pregnancy and delivery and had normal psychomotor development. At the age of 8 months he started to present with febrile and afebrile focal seizures, sometimes followed by secondarily generalization. He was treated with phenobarbital until the age of 10 years, when it was withdrawn without seizure recurrence. Brain MRI was normal and EEG performed during infancy was reported to show rare bilateral posterior slow waves. Among three daughters, the first is a 9-year-old (II-1) healthy girl. At the age of 8 months she presented with a benign myoclonus of Fejerman-Lombroso that remitted spontaneously. She has a normal psychomotor development and she attends the primary school with good results. Patient II-2 is a 5-year-old girl, born after a normal pregnancy. She had normal psychomotor development: she started walking at the age of 16 months and saying words at the age of 13 months. At the age of 2 years she presented with a cluster of afebrile seizures within a few hours. Seizures were characterized by loss of consciousness, diffuse hypotonus, cyanosis, and up-gaze deviation for about 40– 60 s, followed by postictal sleep. Interictal EEG revealed rare frontocentral and parietooccipital sharp waves, increasing during sleep. Brain MRI was normal. She was diagnosed as having focal cryptogenic epilepsy and treated with valproic acid (10 mg/kg/day). She was seizure-free for 2 years, until the age of 4, when she presented with a single seizure characterized by staring, loss of consciousness, right head and eye deviation, and right leg clonic jerks lasting about 40 s. She is still taking valproate, and she did not experience more seizures. Neuropsychological assessment performed at the age of 5 years with Griffiths Mental Development Scales (GMDS), was normal (Griffiths Quotient 102). Patient II-3 is a 2 years and 3 months old girl who was born at term after a normal pregnancy and delivery. She had normal psychomotor development. At the age of 6 months she was admitted to the hospital because of five episodes characterized by psychomotor arrest, slow deviation of the head and eyes to one side, diffuse hypertonia, cyanosis, and unilateral limb jerks, which became bilateral: seizures lasted about 30–40 s and recurred every 10 min. Episodes were stopped with endorectal diazepam. After 2 days the patient experienced a second cluster of three seizures with semiology identical to that previously reported. The interictal EEG was normal except for the presence of rare slow waves. Brain MRI was normal. She was not treated. She continues to present brief focal seizures (two to three per night) characterized by unilateral upper or lower limbs jerking lasting
e55 BFIS and CHRNA2 Gene Mutation
A
B
C
Figure 1. (A) Pedigree structure and disease-mutation segregation. The arrow indicates the proband. Blackened symbols denote individuals affected by benign infantile epilepsy; unblackened symbols denote unaffected individuals. Circles and squares indicate females and males, respectively. Mut: c.1126C>T; p.Arg376Trp. (B) Structural effects of mutation. The model of CHRNA2 in the position of interest is shown with a cartoon representation and involved residues interacting with residue 376 are represented by stick and labeled with CHRNA2 numbering. Noncovalent interaction of Arg376 with Gly372, Met380, and Asp484 is visualized by yellow dashed lines. The residue is predicted to H-bond the glycine 372 and methionine 380 residues: these interactions are required to stabilize the helix formation. (C) Mutation R376W causes clashes with neighboring residues. The arginine side chains are predicted to be exposed to solvent, according to ConSurf server results. Small green disks are shown when atoms are almost in contact or slightly overlapping, whereas large red disks indicate significant van der Waals overlap. The indole functional group is slightly overlapping the Met380, Val373, and Asp484 residues, whereas there is significant van der Waals overlap with His474 and Ala477 residues. Epilepsia ILAE
30–40 s; seizures tend to recur during sleep every 3 months. The last two episodes were isolated. Last follow-up EEG and neuropsychological assessment were normal (GMDS; Griffiths Quotient 105). Mutational analysis The 98.5% of 382 total exons included in TSCA were covered by the amplicon design. TSCA revealed a heterozygous single-nucleotide substitution (c.1126C>T; pArg376Trp) in the CHRNA2 gene; the variant was identified with a coverage of 250 and a Q-score = 38, and then confirmed by Sanger sequencing. Mutation analysis had been expanded to the rest of the family (Fig. 1A). The father I-2, the sister II-2, and the proband II-3 segregated the heterozygous mutation c.1126 C>T;p. Arg376Trp. The mother I-1 and the elder sister II-1 reported a wild-type genotype. PRRT2 gene mutations were excluded. The p.Arg376Trp change is not reported in Single Nucleotide Polymorphism database (dbSNP), was absent from 188 healthy ethnically matched controls and was never observed in the NHLBI Exome Variant Server database (http://evs.gs.washington.edu/EVS/).
The CHRNA2 three-dimensional structure model shows that Arg376 is located in the cytoplasmic domain of receptor subunit. In particular, mutated residue (Fig. 1B) is involved in an a-helix secondary structure element close to the M3 transmembrane domain. As shown in Figure 1C (best “rotamer” fitting the structure), although the mutation does not appear to affect the helix stability, the Trp side chain bumps with other surrounding residues.
Discussion CHRNA2 gene together with the other two nicotinic genes, CHRNA4 and CHRNB2, are considered responsible for ADNFLE. CHRNA2 mutation was described only once, in 2006, in a family with nocturnal epilepsy with wandering and ictal fear.2 Later on, other mutations were searched in three large cohorts of families with ADNFLE, but the absence of mutations in all of them, led to consideration of a minor role of CHRNA2 in this epileptic syndrome.4 When the first CHRNA2-mutated family was described, some differences with the typical ADNFLE were highlighted such
Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
e56 M. Trivisano et al. Table 1. Clinical features of benign familial infantile seizures Family history of seizures (similar age at onset, autosomal dominant trait) Normal development before onset Onset between 4 and 8 months of age Seizures in clusters Partial seizures localized in the occipitoparietal areas Semiology: psychomotor arrest, cyanosis, head/eye deviation to one side (variable), tonic contraction, bilateral clonic jerks Normal interictal EEG Ictal EEG: fast activity originating in the occipitoparietal area Normal developmental outcome
as the presence of seizures with prominent fear and motor behaviors without hypermotor manifestations.2 Nevertheless, the occurrence of seizures exclusively during sleep was typical of the ADNFLE, as cholinergic neurons are known to exert a modulating effect on sleep and arousal oscillations at thalamic and cortical levels.10 The family described herein seems to be not typical of an ADNFLE because of the presence of seizures not related to sleep, the early age at onset, and the absence of hypermotor manifestations. Otherwise, the occurrence of clusters of focal seizures in healthy children is more suggestive of BFIS. Moreover, seizure semiology, characterized by psychomotor arrest, cyanosis, head/eye deviation, tonic contraction, and bilateral clonic jerks is typical of this epileptic syndrome. The outcome was excellent for the father who had complete seizure remission and normal psychomotor development. In addition, the two daughters had a good outcome characterized by rare and well-controlled seizures in one of them and normal psychomotor development. A summary of clinical findings in BFIS has been shown in Table 1. We looked for differences in clinical features (including seizure semiology) between our cases and PRRT2 mutated patients, without detecting significant differences. Furthermore, in previous reports, statistically significant differences in clinical findings among BFIS patients with mutations in KCNQ2, KCNQ3, SCN2A, and PRRT2 genes were not pointed out. The only distinctive feature was the occurrence of paroxysmal dyskinesia in some patients with PRRT2 mutations.9 We found a novel c.1126c>T:p.Arg376Trp missense mutation of CHRNA2 gene, segregating in a cytoplasmic a-helix structure. The quite conserved arginine is closely related to M1 and M4 transmembrane helices, previously described as site of pathogenic ADNFLE mutations.11 Arginine 376, involved in hydrogen bonds, leads to the stabilization of the a-helix structure, also supported by the formation of salts bonds with the very high conserved asp484, in a range of 6.5 A. Based on these features, the pathogenic conversion of arginine 376 to tryptophan alters all the interactions. Even if the cytoplasmic domain Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
has never been reported to be involved in epileptogenic mechanism, the chemical interactions impaired by the CHRN2A mutation may account for the pathogenic role of the variant. Following this genetic result is important to reconsider the role of CHRNA2 gene in ADNFLE and to test it in other types of epileptic syndromes such as BFIS. Further functional tests will be necessary to strongly relate CHRNA2 mutation with BFIS phenotype. However, the pathogenic bioinformatics prediction, the effects of this mutation on the channel helix structure, and the cosegregation of mutation12 allow us to speculate on a deleterious effect of this variant in our family. The absence of the mutation in SNP/variant databases, and in our control population also supports this hypothesis. Considering that about 10% of BFIS families still remain undiagnosed, this description can represent an important starting point to explore the role of CHRNA2 subunit channel in other epileptic syndromes such as BFIS. Other genetic mutations that were not tested could also exist in this family and could also contribute to the observed phenotype. The extent of CHRNA2 mutation on the reported phenotype is still not proven, and further studies will be necessary.
Acknowledgment We thank the family for the kind collaboration.
Disclosure 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.
References 1. De Fusco M, Becchetti A, Patrignani A, et al. The nicotinic receptor beta 2 subunit is mutant in nocturnal frontal lobe epilepsy. Nat Genet 2000;26:275–276. 2. Aridon P, Marini C, Di Resta C, et al. Increased sensitivity of the neuronal nicotinic receptor alpha 2 subunit causes familial epilepsy with nocturnal wandering and ictal fear. Am J Hum Genet 2006;79: 342–350. 3. Gotti C, Clementi F, Fornari A, et al. Structural and functional diversity of native brain neuronal nicotinic receptors. Biochem Pharmacol 2009;78:703–711. 4. Gu W, Bertrand D, Steinlein OK. A major role of the nicotinic acetylcholoine receptor gene CHRNA2 in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is unlikely. Neurosci Lett 2007;422:74–76. 5. Liu H, Lu C, Li Z, et al. The identification of a novel mutation of nicotinic acetylcholine receptor gene CHRNB2 in a Chinese patient: its possible implication in non-familial nocturnal frontal lobe epilepsy. Epilepsy Res 2011;95:94–99. 6. Combi R, Ferini-Strambi L, Tenchini ML. CHRNA2 mutations are rare in the NFLE population: evaluation of a large cohort of Italian patients. Sleep Med 2009;10:139–142. 7. Specchio N, Vigevano F. The spectrum of benign infantile seizures. Epilepsy Res 2006;70:S156–S167. 8. Caraballo RH, Cersosimo RO, Espeche A, et al. Benign familial and non-familial infantile seizures: a study of 64 patients. Epileptic Disord 2003;5:45–49.
e57 BFIS and CHRNA2 Gene Mutation 9. Zara F, Specchio N, Striano P, et al. Genetic testing in benign familial epilepsies of the first year of life: clinical and diagnostic significance. Epilepsia 2013;54:425–436. 10. Steriade M, McCormick DA, Sejnowski TJ. Thalamo-cortical oscillations in the sleeping and aroused brain. Science 1993;262:679–685. 11. Hoda JC, Gu W, Friedli M, et al. Human nocturnal frontal lobe epilepsy: pharmocogenomic profiles of pathogenic nicotinic acetylcholine receptor beta-subunit mutations outside the ion channel pore. Mol Pharmacol 2008;74:379–391. 12. Waxman SG, Merkies ISJ, Gerrits MM, et al. Sodium channel genes in pain-related disorders: phenotype–genotype associations
and recommendations for clinical use. Lancet Neurol 2014;13: 1152–1160.
Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Functional and structural analysis.
Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
Mutation of CHRNA2 in a family with benign familial infantile seizures: Potential role of nicotinic acetylcholine receptor in various phenotypes of epilepsy *1Marina Trivisano, †1Alessandra Terracciano, ‡Teresa Milano, §Simona Cappelletti, *¶Nicola Pietrafusa, †Enrico Silvio Bertini, *Federico Vigevano, and *Nicola Specchio Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
SUMMARY
Marina Trivisano is researcher at the Neuroscience Department of Bambino Gesu Children’s Hospital, Rome.
Nicotinic acetylcholine receptor genes are involved mainly in nocturnal frontal epilepsy. Despite extensive studies, to date, the a2 subunit did not show a strong association with this peculiar epileptic phenotype. We report CHRNA2 missense mutation in a family with benign familial infantile seizures (BFIS). TrueSeq Custom Amplicon (TSCA) sequencing approach was used to screen 10 ion channel genes in patients with idiopathic epilepsies. TSCA revealed a heterozygous single-nucleotide substitution in CHRNA2 gene (c.1126 C>T; p. Arg376Trp) that segregated in a family with BFIS; based on bio-informatics inspection, the change was predicted to be pathogenic. The investigated family includes parents and their three daughters. In affected individuals, seizures started between 6 and 24 months of age. Seizures were mainly in cluster and well-controlled. Outcome was good in all subjects. Even if nicotinic acetylcholine receptor genes are traditionally associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), this single-family description can open new possibilities in the genetic diagnosis, molecular characterization, and management of CHRNA2-related epilepsy. The pathogenic conversion of arginine 376 to tryptophan alters all of these interactions in the cytoplasmic domain, never reported to be involved in epileptogenic mechanism. Further functional tests will be necessary to strongly relate CHRNA2 mutation with BFIS phenotype. KEY WORDS: Benign familial infantile epilepsies, CHRNA2 gene, Acetylcholine receptor, Benign epilepsies, Autosomal dominant nocturnal frontal lobe epilepsy.
Mutations in genes encoding the neuronal acetylcholine receptor (nAChRs) are typically associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), Accepted February 11, 2015; Early View publication April 3, 2015. *Division of Neurology, Bambino Gesu Children’s Hospital, IRCCS, Rome, Italy; †Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, Bambino Gesu Children’s Hospital, IRCCS, Rome, Italy; ‡Department of Biochemical Science “Rossi Fanelli,” Sapienza University of Rome, Rome, Italy; §Unit of Clinical Psychology, Bambino Ges u Children’s Hospital, IRCCS, Rome, Italy; and ¶Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari, Bari, Italy 1 Both authors equally contributed to this paper. Address correspondence to Nicola Specchio, Division of Neurology, Bambino Ges u Children’s Hospital, IRCCS, P.zza S. Onofrio 4, 00165 Rome, Italy. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
a well-known genetic focal epilepsy.1 CHRNA4 and CHRNB2 gene mutations encoding the alfa4 and beta2 subunits are most frequently reported. More recently, an additional nAChR gene (CHRNA2), encoding the alfa2 subunit, was mutated in a large family with nocturnal frontal lobe epilepsy with atypical features such as wandering and ictal fear.2 As other nAChRs, CHRNA2 is a neurotransmitter-gated ion channel that has been finetuned through evolution to transduce a chemical signal into an electrical signal. It is a pentameric ligand-gated ion channel distributed widely throughout the nervous system, and each subunit spans the plasma membrane with four transmembrane domains (M1–M4) giving rise to several different nAChR subtypes in the brain.3 The description of the CHRNA2-mutated family reported by Aridon et al.2 remained unique and isolated. Different
e53
e54 M. Trivisano et al. independent studies explored the CHRNA2 gene role in ADNFLE, without significant results.4–6 In the present study we report a family with the benign familial infantile seizure (BFIS) phenotype, segregating a mutation in the cytoplasmic region of CHRNA2. BFIS is a condition characterized by onset during the first 2 years of life in otherwise normal children. Seizures have an autosomal dominant trait of inheritance and a typical onset around the sixth month. Seizures are typically grouped in cluster and are characterized by psychomotor arrest, slow deviation of the head and eyes to one side, diffuse hypertonia, cyanosis, and unilateral limb jerks, which become bilateral.7 Outcome is always excellent with a normal psychomotor development. In a series described by Caraballo,8 benign infantile seizures have been listed as the third most common type of epilepsy in the first 2 years of life. This is the second family segregating a CHRNA2 mutation and the first association between neuronal nAChRs subunit and BFIS phenotype. BFIS families are genetically heterogeneous: genetic studies led to the identification of mutations in KCNQ2 and KCNQ3, in SCN2A and in PRRT2 genes. In about 10% of cases, a causative gene mutation is still lacking.9
Methods Family We reviewed medical charts of all the affected members of this family to obtain clinical and genetic information. All family members were personally examined by M.T., F.V., and N.S. Electroencephalography (EEG), brain magnetic resonance imaging (MRI), and neuropsychological assessments were reviewed. All individuals included in the study gave written informed consent. The ethical committee of the institution approved this study. Target parallel resequencing Blood samples were obtained from five individuals, and genomic DNA was isolated. For proband DNA analysis, we used TrueSeq Custom amplicon sequencing (TSCA) (Illumina, San Diego, CA, U.S.A.) to amplify 10 voltage-gated ion channel genes: SCN1A, SCN2A, SCN3A, SCN8A, SCN1B, KCNQ2, KCNQ3, CHRNA2, CHRNA4, and CHRNB2. CHRNA2 c.1126 C>T; p.Arg376Trp mutation was found, and analysis was expanded to the whole family by direct Sanger sequencing. PRRT2 gene was analyzed by Sanger sequencing. One hundred eighty-eight ethnically matched controls were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis. Further details are reported in Supporting Information. Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
Results Patients and pedigree The investigated family includes parents and their three daughters (Fig. 1). The mother (I-1) is a healthy woman with no neurologic diseases in her family. The father (I-2) is 35-year-old man. He was born at term after uneventful pregnancy and delivery and had normal psychomotor development. At the age of 8 months he started to present with febrile and afebrile focal seizures, sometimes followed by secondarily generalization. He was treated with phenobarbital until the age of 10 years, when it was withdrawn without seizure recurrence. Brain MRI was normal and EEG performed during infancy was reported to show rare bilateral posterior slow waves. Among three daughters, the first is a 9-year-old (II-1) healthy girl. At the age of 8 months she presented with a benign myoclonus of Fejerman-Lombroso that remitted spontaneously. She has a normal psychomotor development and she attends the primary school with good results. Patient II-2 is a 5-year-old girl, born after a normal pregnancy. She had normal psychomotor development: she started walking at the age of 16 months and saying words at the age of 13 months. At the age of 2 years she presented with a cluster of afebrile seizures within a few hours. Seizures were characterized by loss of consciousness, diffuse hypotonus, cyanosis, and up-gaze deviation for about 40– 60 s, followed by postictal sleep. Interictal EEG revealed rare frontocentral and parietooccipital sharp waves, increasing during sleep. Brain MRI was normal. She was diagnosed as having focal cryptogenic epilepsy and treated with valproic acid (10 mg/kg/day). She was seizure-free for 2 years, until the age of 4, when she presented with a single seizure characterized by staring, loss of consciousness, right head and eye deviation, and right leg clonic jerks lasting about 40 s. She is still taking valproate, and she did not experience more seizures. Neuropsychological assessment performed at the age of 5 years with Griffiths Mental Development Scales (GMDS), was normal (Griffiths Quotient 102). Patient II-3 is a 2 years and 3 months old girl who was born at term after a normal pregnancy and delivery. She had normal psychomotor development. At the age of 6 months she was admitted to the hospital because of five episodes characterized by psychomotor arrest, slow deviation of the head and eyes to one side, diffuse hypertonia, cyanosis, and unilateral limb jerks, which became bilateral: seizures lasted about 30–40 s and recurred every 10 min. Episodes were stopped with endorectal diazepam. After 2 days the patient experienced a second cluster of three seizures with semiology identical to that previously reported. The interictal EEG was normal except for the presence of rare slow waves. Brain MRI was normal. She was not treated. She continues to present brief focal seizures (two to three per night) characterized by unilateral upper or lower limbs jerking lasting
e55 BFIS and CHRNA2 Gene Mutation
A
B
C
Figure 1. (A) Pedigree structure and disease-mutation segregation. The arrow indicates the proband. Blackened symbols denote individuals affected by benign infantile epilepsy; unblackened symbols denote unaffected individuals. Circles and squares indicate females and males, respectively. Mut: c.1126C>T; p.Arg376Trp. (B) Structural effects of mutation. The model of CHRNA2 in the position of interest is shown with a cartoon representation and involved residues interacting with residue 376 are represented by stick and labeled with CHRNA2 numbering. Noncovalent interaction of Arg376 with Gly372, Met380, and Asp484 is visualized by yellow dashed lines. The residue is predicted to H-bond the glycine 372 and methionine 380 residues: these interactions are required to stabilize the helix formation. (C) Mutation R376W causes clashes with neighboring residues. The arginine side chains are predicted to be exposed to solvent, according to ConSurf server results. Small green disks are shown when atoms are almost in contact or slightly overlapping, whereas large red disks indicate significant van der Waals overlap. The indole functional group is slightly overlapping the Met380, Val373, and Asp484 residues, whereas there is significant van der Waals overlap with His474 and Ala477 residues. Epilepsia ILAE
30–40 s; seizures tend to recur during sleep every 3 months. The last two episodes were isolated. Last follow-up EEG and neuropsychological assessment were normal (GMDS; Griffiths Quotient 105). Mutational analysis The 98.5% of 382 total exons included in TSCA were covered by the amplicon design. TSCA revealed a heterozygous single-nucleotide substitution (c.1126C>T; pArg376Trp) in the CHRNA2 gene; the variant was identified with a coverage of 250 and a Q-score = 38, and then confirmed by Sanger sequencing. Mutation analysis had been expanded to the rest of the family (Fig. 1A). The father I-2, the sister II-2, and the proband II-3 segregated the heterozygous mutation c.1126 C>T;p. Arg376Trp. The mother I-1 and the elder sister II-1 reported a wild-type genotype. PRRT2 gene mutations were excluded. The p.Arg376Trp change is not reported in Single Nucleotide Polymorphism database (dbSNP), was absent from 188 healthy ethnically matched controls and was never observed in the NHLBI Exome Variant Server database (http://evs.gs.washington.edu/EVS/).
The CHRNA2 three-dimensional structure model shows that Arg376 is located in the cytoplasmic domain of receptor subunit. In particular, mutated residue (Fig. 1B) is involved in an a-helix secondary structure element close to the M3 transmembrane domain. As shown in Figure 1C (best “rotamer” fitting the structure), although the mutation does not appear to affect the helix stability, the Trp side chain bumps with other surrounding residues.
Discussion CHRNA2 gene together with the other two nicotinic genes, CHRNA4 and CHRNB2, are considered responsible for ADNFLE. CHRNA2 mutation was described only once, in 2006, in a family with nocturnal epilepsy with wandering and ictal fear.2 Later on, other mutations were searched in three large cohorts of families with ADNFLE, but the absence of mutations in all of them, led to consideration of a minor role of CHRNA2 in this epileptic syndrome.4 When the first CHRNA2-mutated family was described, some differences with the typical ADNFLE were highlighted such
Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
e56 M. Trivisano et al. Table 1. Clinical features of benign familial infantile seizures Family history of seizures (similar age at onset, autosomal dominant trait) Normal development before onset Onset between 4 and 8 months of age Seizures in clusters Partial seizures localized in the occipitoparietal areas Semiology: psychomotor arrest, cyanosis, head/eye deviation to one side (variable), tonic contraction, bilateral clonic jerks Normal interictal EEG Ictal EEG: fast activity originating in the occipitoparietal area Normal developmental outcome
as the presence of seizures with prominent fear and motor behaviors without hypermotor manifestations.2 Nevertheless, the occurrence of seizures exclusively during sleep was typical of the ADNFLE, as cholinergic neurons are known to exert a modulating effect on sleep and arousal oscillations at thalamic and cortical levels.10 The family described herein seems to be not typical of an ADNFLE because of the presence of seizures not related to sleep, the early age at onset, and the absence of hypermotor manifestations. Otherwise, the occurrence of clusters of focal seizures in healthy children is more suggestive of BFIS. Moreover, seizure semiology, characterized by psychomotor arrest, cyanosis, head/eye deviation, tonic contraction, and bilateral clonic jerks is typical of this epileptic syndrome. The outcome was excellent for the father who had complete seizure remission and normal psychomotor development. In addition, the two daughters had a good outcome characterized by rare and well-controlled seizures in one of them and normal psychomotor development. A summary of clinical findings in BFIS has been shown in Table 1. We looked for differences in clinical features (including seizure semiology) between our cases and PRRT2 mutated patients, without detecting significant differences. Furthermore, in previous reports, statistically significant differences in clinical findings among BFIS patients with mutations in KCNQ2, KCNQ3, SCN2A, and PRRT2 genes were not pointed out. The only distinctive feature was the occurrence of paroxysmal dyskinesia in some patients with PRRT2 mutations.9 We found a novel c.1126c>T:p.Arg376Trp missense mutation of CHRNA2 gene, segregating in a cytoplasmic a-helix structure. The quite conserved arginine is closely related to M1 and M4 transmembrane helices, previously described as site of pathogenic ADNFLE mutations.11 Arginine 376, involved in hydrogen bonds, leads to the stabilization of the a-helix structure, also supported by the formation of salts bonds with the very high conserved asp484, in a range of 6.5 A. Based on these features, the pathogenic conversion of arginine 376 to tryptophan alters all the interactions. Even if the cytoplasmic domain Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
has never been reported to be involved in epileptogenic mechanism, the chemical interactions impaired by the CHRN2A mutation may account for the pathogenic role of the variant. Following this genetic result is important to reconsider the role of CHRNA2 gene in ADNFLE and to test it in other types of epileptic syndromes such as BFIS. Further functional tests will be necessary to strongly relate CHRNA2 mutation with BFIS phenotype. However, the pathogenic bioinformatics prediction, the effects of this mutation on the channel helix structure, and the cosegregation of mutation12 allow us to speculate on a deleterious effect of this variant in our family. The absence of the mutation in SNP/variant databases, and in our control population also supports this hypothesis. Considering that about 10% of BFIS families still remain undiagnosed, this description can represent an important starting point to explore the role of CHRNA2 subunit channel in other epileptic syndromes such as BFIS. Other genetic mutations that were not tested could also exist in this family and could also contribute to the observed phenotype. The extent of CHRNA2 mutation on the reported phenotype is still not proven, and further studies will be necessary.
Acknowledgment We thank the family for the kind collaboration.
Disclosure 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.
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e57 BFIS and CHRNA2 Gene Mutation 9. Zara F, Specchio N, Striano P, et al. Genetic testing in benign familial epilepsies of the first year of life: clinical and diagnostic significance. Epilepsia 2013;54:425–436. 10. Steriade M, McCormick DA, Sejnowski TJ. Thalamo-cortical oscillations in the sleeping and aroused brain. Science 1993;262:679–685. 11. Hoda JC, Gu W, Friedli M, et al. Human nocturnal frontal lobe epilepsy: pharmocogenomic profiles of pathogenic nicotinic acetylcholine receptor beta-subunit mutations outside the ion channel pore. Mol Pharmacol 2008;74:379–391. 12. Waxman SG, Merkies ISJ, Gerrits MM, et al. Sodium channel genes in pain-related disorders: phenotype–genotype associations
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Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Functional and structural analysis.
Epilepsia, 56(5):e53–e57, 2015 doi: 10.1111/epi.12967
