Confirmation of R82Q Mutation in g2 Subunit of Gamma Amino Butyric Acid Receptor in an Iranian Family
Majid Sadeghizadeh ,
In this study, 46 patients with Febrile Seizures were selected and total DNA was extracted from white blood cells. All exons of GABRG2 gene were amplified by standard PCR. After which the PCR products were screened by Single Stranded Conformational Polymorphism (SSCP) analysis. Selected samples in SSCP analysis that showed new conformer were sequenced. In order to verify the sequencing data, a restriction analysis was carried out. We report R82Q mutation in GABRG2 gene in an Iranian family. Proband was a 12 years old boy. SSCP and Sequencing of exon-2 of GABRG2 gene in proband demonstrated an exonic G245A mutation leading to R82Q substitution. The RFLP of PCR product showed heterozygosity in patients. Arg82 is a conserved amino acid in the benzodiazepine binding site at the N-terminus of the g2 subunit. The R82Q mutation does not alter the response of the receptor to Gamma Amino Butyric Acid but impairs the potentiation by benzodiazepine. Result of this research and several other lines of evidence support complex inheritance, heterozygosity and diverse phenotypes for epilepsy.
Febrile Seizures (FS) are the most common convulsive syndrome in childhood.
Genetic components are significantly involved in the etiology of the majority
of FS (Hirose et al., 2000; Hirose et al., 2002a, b; Kaneko et
al., 2002). The incidence of FS reaches 6-9% in the Japanese population
while it is 5% in Caucasians (Kugler and Johnson, 1998). This racial difference
clearly suggests that genetic components are involved in the etiology of FS.
Several genetic loci for FS have been mapped and genetic defects have been identified
in Autosomal Dominant Epilepsy and Febrile Seizures (ADEFS) (Ito et al.,
2002) and Generalized Epilepsy with Febrile Seizures (GEFS) (Scheffer and Berkovic,
1997). Gamma Amino Butyric Acid receptors are ligand-gated ion channels that
have always been an inviting target in the etiology of epilepsy. Gamma Amino
Butyric Acid (GABA) is the major inhibitory neurotransmitter in the CNS. Mutations
in the gene encoding the g2 subunit of Gamma Amino Butyric Acid-A receptors,
GABRG2 have been identified in four families with generalized epilepsy syndromes
associated with febrile seizures, including GEFS and classical Childhood Absence
Epilepsy (CAE) (Baulac et al., 2001; Wallace et al., 2001; Harkin
et al., 2002; Kananura et al., 2002). Moreover, mutations in GABRG2
have been found as a cause of both ADEFS and FS followed by CAE. The missense
mutation 983A.T: K328M was first identified in a French family and affected
individuals showed the ADEFS phenotype (Baulac et al., 2001). A second
missense mutation (c.245G.A: R82Q) was found in an Australian family in which
affected individuals showed FS and CAE (Wallace et al., 2001). In this
study we searched for these previously identified mutations in Iranian patients
with FS and related epileptic disorders. Approximately seven hundred thousand
individuals are affected by epilepsy in Iran, making this disorder a very economically
and socially important issue to address. In addition, there is no definite statistical
data about portion of FS and related epileptic disorders. The main objective
of this study is the optimization of a method for screening of genetic alterations
involved in pathogenesis of FS. Molecular screening of previously identified
mutations in the patients with epileptic disorders is a sound approach for differential
diagnosis in future.
MATERIALS AND METHODS
Patients and samples: In this study, a total of 46 patients with FS, registered in Iran Epilepsy Association, were selected. The purpose of the study was explained to the subjects and information recorded with their consent. The Institutional Ethics Committee had approved the study protocol. Genetic counseling was carried out with patients and their families. Family pedigree for patients was designed by personally interviewing the subjects and their family members (parents or siblings). For each patient; clinical and informative data was collected and recorded. Epilepsy was diagnosed and classified according to the definitions and terminologies of ILAE classification of seizures and epileptic syndromes (CCTILAE, 1989). All patients were examined neurologically for detection of abnormalitities such as mental and cranial nerves as well as long tracts and cerebellar functions. Patients who had post-ictal motor paralysis were not included in this study. Laboratory tests showed no signs of any serious viral or microbial infections such as meningitis. Electroencephalography was carried out for each patient to detect special causes of epilepsy. Patients with abnormal neurological findings compatible with brain disorders and mentally-retarded children were excluded. Epileptic patients whose conditions were affected by meningitis were also excluded. Magnetic Resonance Imaging (MRI) was requested for some patients in situations where doubtful brain malformations were suspected and those detected with any were subsequently excluded. Finally, peripheral blood sample was collected from patients and their families.
DNA extraction and PCR amplification: Total DNA was extracted from white blood cells. Forward and reverse primers were designed for all exons of GABRG2 gene and DNA amplification was carried out in standard conditions. For the design of primers and definition of mutations, we used the nomenclature of the cDNA (refer to the full length GABRG2 cDNA in GenBank with accession number NM_198904). Sequences of primers are available upon request.
SSCP and sequencing: The amplified DNA fragments were initially screened by analysis of Single Stranded Conformational Polymorphism (SSCP). Ten microliter PCR products were added to 10 μL SSCP buffer containing 2 μL TBE 1X and 7 μL formamide loading buffer (Sambrook and Russell, 2001) and 2 μL NaOH 0.05 M. The reactions were denatured at 96EC for 10 min, ice-chilled and separated by electrophoresis in 10% acrylamide-bisacrylamide (29:1) gels containing 5% glycerol without urea. TBE 1X was used as running buffer. Voltage conditions were optimized as described by previous studies (Sunnucks et al., 2000). The samples selected in SSCP analysis were sequenced by ABI 3700 sequencer (Gene Fanavaran, Macrogen, South Korea).
Restriction fragment length polymorphism: In order to confirm of sequencing data and detection of R82Q mutation Restriction Fragment Length Polymorphism (RFLP) was carried out. Five microliter of the PCR product was incubated with 4 units of HaeIII in presence of 2 μL enzyme specific buffer (10x) and 1 μL BSA for 16 h at 37EC. Digestion mixture was studied in a poly acrylamide gel electrophoresis.
R82Q mutation was detected in a 12 years old male proband with Febrile and
Afebrile seizures from a family with febrile seizure. He had experienced three
generalized tonic-clonic seizures and was on AED. The historical study of the
proband and his parents showed some symptoms in his mother. She had suffered
from eyelid myoclonia and minor epileptic seizures. PCR-SSCP showed a new conformer
for DNA samples of patient and his mother (Fig. 1). Sequencing
of the forward and reverse strands of these fragment demonstrated an exonic
G245A mutation leading to R82Q substitution (Fig. 2). The
nucleotide substitution (G6A) at this position changes a restriction site.
||(a) Pedigree of family; solid symbols in the pedigree refer
to patients with epilepsy (I1 for mother, I2 for father and II1 for son).
(b) SSCP pattern of the proband and his parents for exon-2 of GABRG2. SSCP
analysis showed new conformers in patients (I1 and II1). Denatured products
were run in 10% acrylamide gel containing 5% glycerol and revealed by silver
||Chromatogram of the G245A mutation: Traces of the sense DNA
strands of the proband (a), his mother (b) and his father (c). The mutated
nucleotide sequences are indicated over the sense strand by arrow
||Restriction Fragment Length Polymorphism (RFLP). PCR products
were analyzed using Hae III endonuclease for digestion as described
in Methods. The fragments were separated in an 8% polyacrylamide gel and
bands were detected by silver staining method. The non-digested PCR product
(lane 4) is 250 bp in length and the fragments separated from the GGCC restriction
site are 205 and 45 bp long. G245A changes this restriction site and interferes
with the action of enzyme. Therefore, heterozygous individuals harbouring
the G245A mutation (lane 1: mother, lane 2: son) have three fragments of
250, 205 and 45 bp. The father of family (lane 3) with homozygous wild type
alleles showed normal pattern of RFLP. (M: DNA size marker)
This site is recognized in wild type sequence by the HaeIII endonuclease
that splits the 250 bp PCR product into 205 and 45 bp fragments. The RFLP of
PCR product showed heterozygosity in mother and her son and generated 250, 205
and 45 bp fragments (Fig. 3). Analysis of this region in father
of family did not show any mutation and the enzymatic digestion produced 205
and 45 bp fragments. These results showed a typically autosomal dominant inheritance
for G245A mutation in this family.
We report a family with FS with indications of a generally autosomal dominant inheritance. On the other hand, clinical symptoms were different between the proband and his mother, as the latter did not experience febrile convulsions during childhood. Thus, it remains possible that either two different epilepsy phenotypes cluster in this small family or it is a clinical heterogeneity (two phenotypes with one genotype). R82Q mutation was detected in this family; however, the origin of this mutation in mother is unknown. Unfortunately, the grandparents of proband were inaccessible. the sequencing and RFLP analysis showed that R82Q mutation in this family follows an autosomal dominant inheritance and this mutation has been inherited to the boy from his mother.
The GABAA receptor is a ligand-gated Cl¯ channel and serves as the main
inhibitory system in the brain. The receptor functions as a heteropentamer consisting
of several subunits including a, b, g, 1, u, p and r. Furthermore, each subunit
has several subtypes. Since a1, b2 and g2 are expressed widely in the brain,
(a1)2(b2)2(g2) is hypothesized to be the main configuration of GABAA receptors
in the brain (Hirose et al., 2002b). The GABAA receptor is the target
of many neuronal inhibitory drugs such as benzodiazepines and barbiturates.
The g subunit of GABAA receptors has binding sites for benzodiazepine at the
N-terminus and spans the plasma membrane four times (Wong and Snead, 2001).
Arg82 is a conserved amino acid in the benzodiazepine binding site at the N-terminus
of the g2 subunit. The electrophysiological properties of GABAA receptors bearing
R82Q mutations have been characterized; this mutation does not alter the response
of the receptor to GABA but impairs the potentiation by benzodiazepine (Baulac
et al., 2001). GABAA receptors with the R82Q mutant g2 subunit are considered
to be insensitive to endozepine, a putative endogenous benzodiazepine-like substance.
Mutant GABAA receptors bearing the R82Q mutation may not be able to stabilize
neuronal excitabilities, thereby loosing control by endozepine. In addition,
functional studies in oocytes in vitro showed that this mutation is associated
with either reduction or abolition of the GABA-activated Cl- current
or loss of sensitivity to benzodiazepines (Baulac et al., 2001; Wallace
et al., 2001; Harkin et al., 2002). We have reported a family
with two types of epilepsy in which members are affected by the same mutation.
Despite previously identifying of R82Q mutation in large families with FS, such
families still provide support for complex inheritance in more complicated phenotypes.
For example, in one GEFS family in which the proband has Severe Myoclonic Epilepsy
type I (SME-I), there is clinical genetic evidence to indicate that at least
three epilepsy genes could be inherited (Baulac et al., 2001; Harkin
et al., 2002). Incomplete penetrance in addition to variable expressivity
and polygenic inheritance are main features of epilepsy (Szepetowski and Monaco,
1998). Interestingly, affected individuals in one of the two families with GEFS+
presented typical CAE symptoms together with the occurrence of febrile convulsions
(Wallace et al., 2001). In the light of these findings and the uncertainties,
which are sometimes associated with the diagnosis of FS, it is possible that
the R82Q mutation reported here has a pivotal role in epilepsy in this family.
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