ORIGINAL CONTRIBUTION A Splice-Site Mutation in GABRG2 Associated With Childhood Absence Epilepsy and Febrile Convulsions Colette Kananura, MS; Karsten Haug, MD; Thomas Sander, MD; Uwe Runge, MD; Wenli Gu, MS; Kerstin Hallmann, MS; Johannes Rebstock, MD; Armin Heils, MD; Ortrud K. Steinlein, MD Context: Missense mutations in the GABRG2 gene, which mutation, which is predicted to lead to a nonfunctional encodes the ␥2 subunit of central nervous ␥-aminobu- protein, cosegregates with the disease status in a family tyric acid (GABA)A receptors, have recently been de- with childhood absence epilepsy and febrile convul- scribed in 2 families with idiopathic epilepsy. In one of these sions. The association study did not find any significant families, the affected individuals predominantly exhibited differences in the allele and genotype frequencies of the childhood absence epilepsy and febrile convulsions. common exon 5 polymorphism (C588T) between pa- tients with idiopathic absence epilepsy and controls Objective: To assess the role of GABRG2 in the ge- (PϾ.35). netic predisposition to idiopathic absence epilepsies. Conclusions: Our study identified a splice–donor-site Design: The GABRG2 gene was screened by single- mutation that was probably causing a nonfunctional strand conformation analysis for mutations. Further- GABRG2 subunit. This mutation occurred in heterozy- more, a population-based association study assessing a gosity in the affected members of a single nuclear fam- common exon 5 polymorphism (C588T) was carried out. ily, exhibiting a phenotypic spectrum of childhood ab- sence epilepsy and febrile convulsions. The GABRG2 gene Patients: The sample was composed of 135 patients with seems to confer a rare rather than a frequent major sus- idiopathic absence epilepsy and 154 unrelated and eth- ceptibility effect to common idiopathic absence epi- nically matched controls. lepsy syndromes. Results: A point mutation (IVS6+2T→G) leading to a splice–donor site mutation in intron 6 was found. The Arch Neurol. 2002;59:1137-1141 HILDHOOD ABSENCE epi- lap between both of these disorders has lepsy (CAE) is one of the been suggested.6 Such a common molecu- most common subtypes of lar basis is most obvious in the syndrome idiopathic generalized of “generalized epilepsy with febrile sei- epilepsy (IGE). It is char- zures plus” (GEFS+), a monogenic or oli- acterized by daily clusters of absence sei- gogenic epilepsy that was first described From the Institute of Human C 7 zures at an age of onset between 2 and 12 in 1997. Generalized epilepsy with fe- Genetics (Mss Kananura, Gu, years.1 brile seizures plus is characterized by FCs and Hallmann, and Drs Haug, Heils, and Steinlein), and Febrile convulsions (FCs) are the that may persist beyond the age of 6 years Department of Epileptology most common seizure subtypes, affect- and are often followed by generalized sei- (Drs Rebstock and Heils), ing about 3% to 5% of children younger zures, including myoclonic and absence University Hospital Bonn, than 6 years.2-4 While CAE is often fol- seizures.8 Rheinische Friedrich lowed by other IGE syndromes, includ- While the molecular basis of com- Wilhelms-University Bonn, ing generalized tonic-clonic seizures and mon forms of seizure disorders, includ- Bonn; Department of myoclonic seizures, only 3% to 7% of chil- ing FC and CAE has been elusive so far, Neurology, University Hospital dren suffering from FCs develop epi- disease-causing GEFS+ mutations have Charite´, Humboldt University lepsy later in life.5 already been identified in SCN1B, of Berlin, Berlin (Dr Sander); and Department of Neurology, A genetic basis for both CAE and FC SCN1A, SCN2A, and GABRG2, genes is well established.5 Because the inci- encoding neuronal sodium channel sub- University Hospital Greifswald, ␥ Ernst Moritz Arndt-University, dence of FC is significantly increased in types and the 2-subunit of central ner- ␥ Greifswald (Dr Runge), patients with CAE (10%-15%) compared vous -aminobutyric acid (GABA)A Germany. with the general population, a genetic over- receptors, respectively.9-13 A potential (REPRINTED) ARCH NEUROL / VOL 59, JULY 2002 WWW.ARCHNEUROL.COM 1137 ©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 PATIENTS AND METHODS 154 unrelated and ethnically matched controls. All con- trols were healthy volunteers of German descent. PATIENTS FAMILY 510 The 15-year-old index patient (II-1) had exhibited typical The study sample included 135 unrelated German pa- pyknoleptic absence seizures starting at the age of 4 years tients with IAE at the University Hospital Rudolf Virchow (syndrome diagnosis: CAE) and experienced 3 uncompli- at the Free University of Berlin (Berlin, Germany) and at cated FCs at age 4 years. In addition, he had 1 generalized the University Hospital of Bonn (Bonn, Germany). The tonic-clonic seizure at age 10 and 1 at age 11 years. His in- sample consisted of 59 patients with juvenile AE and 46 terictal EEG results showed 3/s generalized spike-wave dis- patients with CAE who had at least 1 first-degree family charges during resting as well as photosensitivity. At age member affected by IGE. In addition, 19 patients with ju- 12 years, he began daily treatment with 1800 mg of val- venile AE and 11 with CAE were included as sporadic cases. proic acid and had been free of seizures since that time. His The study protocol was approved by the local ethics com- 13-year-old sister (II-2) had 4 uncomplicated FCs at age 3 mittees, and written informed consent was obtained from years. She exhibited typical absence seizures and several all participants. Diagnostic criteria for IAE (CAE or juve- generalized tonic-clonic seizures at age 4 years (syndrome nile AE) were: (1) onset with typical absence seizures; (2) diagnosis: CAE). Results of her interictal EEG showed 3/s age at onset of typical absence seizures between 3 and 20 generalized spike-wave discharges while resting. Valproic years; (3) electroencephalographic (EEG) findings of nor- acid treatment was started and she had been seizure-free mal background activity and paroxysmal generalized spike- since then. The 42-year-old father (I-1) had experienced wave EEG discharges; and (4) normal intellectual and neu- 20 uncomplicated FCs between the ages of 3 and 6 years. rological status apart from seizures. Exclusion criteria From ages 6 to 15 years, he was treated with phenobarbi- included (1) evidence for structural lesions or metabolic tal and ethosuximide and remained seizure-free without an- or degenerative diseases of the brain; (2) atonic/astatic or tiepileptic treatment. He had no siblings and his parents tonic seizures; (3) complex partial seizures; (4) epilepsy had no known history of seizures. The 43-year-old mother with myoclonic absences; and (5) exclusively stimulus- (I-2) reported no history of epileptic seizures. induced seizures.17 In case of a rare mutation in the sample of patients with IAE, we assessed the presence of the latter MUTATION SCREENING in a sample comprising 88 unrelated and ethnically matched controls. For the association study, we obtained the geno- Genomic DNA was extracted either from 10-mL aliquots type and allele frequencies in all 135 patients as well as in of EDTA-anticoagulated blood samples or from lympho- role of the GABAergic system has often been implicated sition 770, which is located at the boundary between ex- in epileptogenesis14-16; however, genetic evidence for ons 6 and 7. Compared with the previous GABRG2 this hypothesis has been obtained only recently by the sequence, the additional guanine would result in an in- discovery of different GABRG2 mutations identified in 2 frame stop codon following amino acid position 227 up- families. The phenotype in one of these families was stream of the first transmembrane domain. Reverse tran- described to be compatible with GEFS+, but no further scriptase PCR amplification of the respective region could details regarding the seizure types observed in the not verify the extra guanine. These results were further affected pedigree members were given.12 In the second confirmed by amplification from genomic DNA and di- family affected, individuals predominantly had CAE rect sequencing of both the exon 6/intron 6 and intron and FC.13 Accordingly, these findings raise the question 6/exon 7 boundaries (data not shown). whether genetic variation of the GABRG2 gene confers susceptibility to the epileptogenesis of common sub- DETECTION OF types of idiopathic absence epilepsies (IAEs). We there- A SPLICE–DONOR SITE MUTATION fore systematically searched for mutations and common IN A FAMILY WITH CAE AND FC sequence variants in a sample comprising a total of 135 patients with IAE and performed a population-based The coding region and exon/intron boundaries of GABRG2 association study assessing a frequent silent polymor- were screened by single-strand conformation analysis for phism (C588T) in exon 5 of the GABRG2 gene. mutations in genomic DNA samples derived from 135 patients. The DNA of the index patient (II-1) from fam- RESULTS ily 510 showed aberrant electrophoresis patterns indica- tive of a sequence variation in the respective PCR frag- VERIFICATION OF ment containing exon 6. Direct sequencing revealed a THE GABRG2 WILD TYPE SEQUENCE single base pair exchange at the splice–donor site of intron 6, substituting a thymine with a guanine (IVS6+2T→G) The GABRG2 cDNA sequence (XM_003986.2), which had (Figure 1). No further mutations were found by screen- been deposited into Genbank on April 6, 2001, differed ing the entire GABRG2 coding region. from the previous sequence (XM_003986.1) by the pres- For evaluating the segregation of the IVS6+2T→G ence of an additional guanine following nucleotide po- mutation, exon 6 and the adjacent part of intron 6 were (REPRINTED) ARCH NEUROL / VOL 59, JULY 2002 WWW.ARCHNEUROL.COM 1138 ©2002 American Medical Association. All rights reserved.