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An idiopathic epilepsy syndrome linked to 3q13.3-q21 and missense mutations in the extracellular calcium sensing receptor gene

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ORIGINAL ARTICLE
An Idiopathic Epilepsy Syndrome Linked to
3q13.3-q21 and Missense Mutations in the
Extracellular Calcium Sensing Receptor Gene
Ashish Kapoor, PhD,1 Parthasarathy Satishchandra, MBBS, DM, FAMS,2 Rinki Ratnapriya,1
Ramesh Reddy, MSc,1 Jayaram Kadandale, PhD,3 Susarla K. Shankar, MD,4 and Anuranjan Anand, PhD1
Objective: To identify the disease locus in a three-generation south Indian family having several of its members affected with
idiopathic epilepsy.
Methods: Genome-wide parametric linkage analysis was performed with 382 autosomal markers. Mutational analysis of the
positional candidate genes in linked interval was performed by direct sequencing of genomic DNA from the proband in the
family. Expression analysis in human adult brain was performed by Western blotting.
Results: A novel epilepsy genetic locus on chromosome 3q13.3-q21 was identified by linkage analysis. This locus comprises
about 12 megabases of the genomic interval, with its proximal and distal genetic boundaries defined by microsatellite markers,
D3S3675 and D3S1551, respectively. In this interval, we found a novel, patient-specific, missense variant, Arg898Gln, at the
extracellular calcium sensing receptor (CASR), a gene belonging to the G-protein–coupled receptor family. CASR expression was
detected in the temporal lobe, frontal lobe, parietal lobe, cerebellum, and hippocampus. Four additional, potentially pathogenic,
missense CASR variants, Glu354Ala, Ile686Val, Ala988Val, and Ala988Gly, were observed in five individuals affected with
idiopathic generalized epilepsy.
Interpretation: A novel idiopathic epilepsy locus has been mapped on chromosome 3q13.3-q21, as evident by presence of
significant genetic linkage. Identification of novel, rare missense CASR variants at evolutionary-conserved residues in epilepsy
patients and CASR expression in various subregions of human brain raises an interesting possibility of involvement of CASR in
pathophysiology of epileptic disorders.
Ann Neurol 2008;64:158 –167
Epilepsy is one of the most common and heterogeneous groups of neurological disorders, characterized
by recurrent, synchronous, and usually unprovoked seizures, affecting nearly 3% of people during their lifetimes.1 Based on the clinical features such as age of
onset, seizure type, seizure frequency, electroencephalographic (EEG) characteristics, and associated neurological symptoms, epilepsies are classified into several clinical categories as per the International League Against
Epilepsy guidelines.2 The category of idiopathic generalized epilepsies, with its common subtypes including
juvenile myoclonic epilepsy (JME), juvenile absence
epilepsy, and childhood absence epilepsy, accounts for
about 40% of all epilepsies.3 Idiopathic generalized epilepsies display a complex inheritance pattern suggesting that several genetic factors contribute to the cause
of idiopathic generalized epilepsies.4,5 In the past few
years there has been significant progress in mapping
and characterization of genes for rare monogenic forms
of idiopathic epilepsies,6 establishing importance of genetic approaches to generate better understanding of
molecular mechanisms involved in causation of these
disorders.
Here, we describe identification of a novel epilepsy
genetic locus on chromosome 3q13.3-q21 in a study
involving genetic linkage analysis of a three-generation
epilepsy family, Family 1, from south India (Fig 1).
On sequence analysis of several potential epilepsy candidate genes mapping at 3q13.3-q21, we identified a
novel, patient-specific, rare variant at the calcium sensing receptor (CASR) gene. Four additional novel, rare
CASR variants were observed in 5 out of 96 individuals
affected with JME, ascertained from the same population. We found CASR expression in various subregions
From the 1Molecular Biology and Genetics Unit, Jawaharlal Nehru
Centre for Advanced Scientific Research; 2Department of Neurology, National Institute of Mental Health and Neurosciences; 3Centre for Human Genetics, International Technology Park Bangalore;
and 4Department of Neuropathology, National Institute of Mental
Health and Neurosciences, Bangalore, India.
Additional Supporting Information may be found in the online version of this article.
Received Aug 9, 2007, and in revised form Apr 21, 2008. Accepted
for publication Apr 25, 2008.
158
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/ana.21428
Address correspondence to Dr Anand, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India. E-mail: anand@jncasr.ac.in
© 2008 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Fig 1. Pedigree of Family 1. Solid symbols represent clinically affected individuals; open symbols represent unaffected individuals.
Myoclonic jerks (MJ), generalized tonic-clonic seizures (GTCS), complex partial seizures (CPS), absences (ABS), and febrile seizures
(FS) indicate the seizure types observed in the affected individuals. Roman numerals to the left of the pedigree denote generations,
and Arabic numerals above the symbols denote individuals. Haplotypes, based on the seven markers in the 3q13-q21 interval, are
indicated below the symbols. Alleles in parentheses are inferred. Key recombinants (Individuals II:2 and II:9) are marked by arrows. Solid bar indicates the disease haplotype. The maximum logarithm of odds (LOD) score (Zmax) obtained for the markers in
the haplotype are shown.
of the human brain, suggesting a brain-specific, functional role of the gene in susceptibility to idiopathic
epilepsies.
Subjects and Methods
Family Ascertainment and Clinical Characterization
A three-generation family from south India, Family 1 (see
Fig 1), in whom idiopathic epilepsy was found to segregate
in an autosomal dominant mode, was ascertained at the Department of Neurology, National Institute of Mental Health
and Neurosciences (NIMHANS), Bangalore, through a 17year-old proband (III:3) affected with JME. Since age 8, he
has a history of frequent myoclonic seizures, appearing early
in the morning. He later developed absence seizures at the
age of 9 and generalized tonic-clonic seizures (GTCSs) when
he was 14. Scalp EEG showed generalized bursts of spikes
with polyspikes, characteristic of generalized epilepsy. General physical and medical examination of the proband was
found to be normal. Magnetic resonance imaging of the
brain showed no apparent abnormalities.
A clinical follow-up of the family was performed by neurological examination of all family members and interviews
with relatives. The institutional ethics committee approved
this study, and all family members provided written informed consent to participate in the study. The family mem-
bers were interviewed by two neurologists independently,
and diagnosis was established following the International
League Against Epilepsy guidelines.2 For the members who
were deceased or could not be examined, the diagnosis was
arrived at from information provided by surrogates. Family 1
has six additional affected members available for the study.
Individual III:1 has JME with a history of myoclonic seizures
and absence seizures. Individual III:2 had a history of nocturnal myoclonic seizures disturbing her sleep. Subsequently,
she experienced development of frequent early morning
myoclonic seizures. Individual III:4 had episodes of febrile
convulsions and GTCSs. Individual II:2 has a history of
myoclonic seizures and GTCSs. Individual II:3, father of
proband, has history of complex partial seizures with secondary GTCSs. Individual II:7 has history of complex partial
seizures with secondary GTCSs. As described by family
members, Individuals II:5 and II:6 had histories of GTCSs.
The clinical profile of Individual II:10 was unclear; hence,
this individual was considered to be of unknown clinical status for genetic analysis. Individual II:12 had abnormal mental developmental milestones, without any history of seizures.
Individual I:2 had a history of seizures whose specific types
could not be ascertained from the description provided. The
remaining family members had no history of epilepsy or any
other paroxysmal disorder (see Fig 1). The clinical picture
Kapoor et al: CASR and Epilepsy
159
was compatible with an idiopathic epilepsy syndrome, with
affected individuals in the family manifesting myoclonic seizures, absence seizures, GTCSs, febrile seizures, and complex
partial seizures with secondary GTCSs.
Genetic Analysis
Twenty milliliters of peripheral venous blood sample was
collected from each member of the family. Genomic DNA
was extracted using the phenol-chloroform method.7 A
genome-wide linkage study of Family 1 was performed using
382 fluorescent-labeled polymorphic microsatellite markers
covering 22 autosomes at an average density of about 10 centimorgan (cM) (ABI Prism Linkage Mapping Set v2.5; Applied Biosystems, Foster City, CA). The marker order and
intermarker distances were obtained from the Genethon
linkage maps8 (1996 Genethon Microsatellite Maps at Genlink Database). Individual markers were amplified on GeneAmp PCR System 9700 (Applied Biosystems), and sets of
the pooled amplified products were electrophoresed using an
ABI3100 Genetic Analyzer (Applied Biosystems). The output data were fragment sized using Genescan software3.7
(Applied Biosystems) followed by allele calling using Genotyper software3.7 (Applied Biosystems). In addition, DNA
from a reference individual, CEPH 1347-02 of the known
genotype, was analyzed for each marker as an internal allele
size control. All markers were tested for Mendelian segregation in the family.
For fine mapping at 3p13-q24, 19 additional markers,
D3S1284, D3S3653, D3S3633, D3S1595, D3S3671,
D3S3556, D3S1603, D3S3632, D3S3654, D3S3695,
D3S3675, D3S3720, D3S1551, D3S3607, D3S3637,
D3S1576, D3S3612, D3S3627, and D3S1555, were analyzed in the family to further confirm linkage and to refine
the linked region. These 19 markers covered genetic distance
of about 67cM and provided resolution of less than 3cM
along with the panel markers from the linked region. Polymerase chain reaction (PCR) amplification and genotyping
for each marker was performed in the manner similar to the
genome-wide scan approach described earlier.
Parametric linkage analysis was conducted using autosomal dominant mode of inheritance, 90% penetrance, 1%
phenocopy, mutant allele frequency of 0.0001, equal marker
allele frequencies, and no difference in the male and female
recombination rates. Two-point (LOD) logarithm of odds
scores were calculated using MLINK of LINKAGE 5.2.9
Multipoint LOD scores were calculated using GENEHUNTER 2.1.10 Haplotypes were assigned manually based
on the genotyping data and were inferred when possible,
whereas a minimum number of intermarker recombination
events were maintained. These haplotypes were confirmed
with those generated using MaxProb option in GENEHUNTER 2.1.10
Positional Candidate Gene Sequence Analysis
From known or speculated function, expression in central
nervous system, or homology to a relevant human or mouse
gene, candidate genes were selected for sequence analysis.
Gene structures, including 5⬘- and 3⬘-untranslated regions,
exons, and intron-exon boundaries, were delineated from the
Human Genome Map Viewer database (Human Genome
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Map Viewer Build 36 database at National Center for Biotechnology Information [NCBI], National Institute of
Health [NIH], Bethesda, MD). Primers pairs to amplify exonic and flanking intronic regions of the candidate genes
were designed using the Primer3 software.
The candidate genes were analyzed in the proband of the
family by direct genomic DNA sequencing of PCR amplicons. DNA sequencing was conducted by cycle sequencing
using BigDye Terminator Cycle Sequencing kit v3.0 (Applied Biosystems) and was analyzed on an ABI3100 Genetic
Analyzer (Applied Biosystems). To detect sequence variants
in the candidate genes, we compared the sequences obtained
in the proband with the sequences available in the GenBank
database, using SeqMan software5.01 (DNASTAR, Madison, WI). Novel variants observed in the proband in a heterozygous state were analyzed in the family members by direct sequencing to identify the segregation pattern, and the
ones found to segregate appropriately were analyzed in the
south Indian, population-matched, unaffected control subjects by direct sequencing to determine their respective allele
frequencies.
Expression Analysis of CASR in Human Brain
CASR protein expression in adult human brain tissue was
analyzed by Western blotting. The tissue samples from temporal lobe, parietal lobe, frontal lobe, hippocampus, and cerebellum were collected from Human Brain Tissue Repository
at NIMHANS, Bangalore. Aliquots of 50␮g of protein from
tissue lysates were resolved electrophoretically on 8% sodium
dodecyl sulfate polyacrylamide gel. The proteins were electrotransferred to a nitrocellulose membrane, and the blot was
incubated with rabbit anti-CASR affinity purified polyclonal
antibody (1␮g/ml; Chemicon International, Temecula, CA).
A 1:7,500 dilution of horseradish peroxidase–conjugated,
goat anti–rabbit IgG (Bangalore Genei, Bangalore, India)
was used as secondary antibody, and specific protein bands
were detected using an enhanced chemiluminescent substrate
for horseradish peroxidase (Pierce, Rockford, IL). Actin was
used as a loading control, and a 1:10,000 dilution of antiactin mouse monoclonal antibody (Calbiochem, San Diego,
CA) and 1:10,000 dilution of horseradish peroxidase–conjugated, goat anti–mouse IgM (Calbiochem) were used for detection.
Protein Sequence Analysis
Multiple protein sequence alignment analysis of CASR
protein from various species was done using ClustalW
(ClustalW at European Bioinformatics Institute, Cambridge,
United Kingdom). Conserved domains of CASR were predicted using Pfam database (Pfam database at Wellcome
Trust Sanger Institute, Cambridge, United Kingdom).
Results
To explore the underlying genetic basis for epilepsy
phenotypes segregating in the multiple-affected family
(see Fig 1), we performed a genome-wide linkage
study. In this study, the greatest two-point LOD score
obtained was 3.05 at ␪ ⫽ 0 for the microsatellite
marker D3S1267 at 3q21 (Table 1). The neighboring
Table 1. Two-Point Parametric Logarithm of Odds Score Analysis in Family 1 for 3p14-q25 Markers at Different
␪ Values
LOD at the ␪ Value
Markera
0
D3S1289
D3S1300
D3S1566
D3S1284b
D3S3653b
D3S3633b
D3S1595b
D3S3632b
D3S3695b
D3S3675b
D3S1278c
D3S3720b,c
D3S1267c
D3S1551b
D3S1292
D3S3612b
D3S1569
D3S1279
D3S3677b
⫺3.1758
⫺5.0924
1.3263
2.0573
1.6040
1.3575
2.0573
2.0573
1.5018
2.0573
2.4974
1.6779
3.0529
1.0989
1.0991
1.3928
1.0991
⫺5.2304
⫺4.6755
0.1
0.3854
⫺0.2786
1.1103
1.8480
1.2628
1.1395
1.8480
1.8480
1.3799
1.8493
2.0582
1.3411
2.5275
1.6142
1.6202
1.1959
1.6202
⫺0.0779
0.3844
0.2
0.3
0.4
0.5846
0.1913
0.8818
1.4769
0.8915
0.9035
1.4769
1.4769
1.1079
1.4816
1.5728
0.9561
1.9464
1.3633
1.3768
0.9667
1.3768
0.1951
0.5363
0.5062
0.2784
0.6317
1.0012
0.5006
0.6432
1.0012
1.0012
0.7447
1.0108
1.0326
0.5350
1.2987
0.9494
0.9680
0.6988
0.9680
0.2178
0.4418
0.2967
0.1933
0.3443
0.4667
0.1492
0.3474
0.4667
0.4667
0.3352
0.4784
0.4487
0.1525
0.5919
0.4578
0.4739
0.3817
0.4739
0.1357
0.2462
a
Marker order is as per the human genome physical map (Human Genome Map Viewer Build 36 database, National Center for
Biotechnology Information, National Institutes of Health, Bethesda, MD).
b
Fine mapping markers.
c
Markers within the disease haplotype.
LOD ⫽ logarithm of odds.
marker D3S1278 at 3q13.3 also showed a positive
two-point LOD score of 2.50 (see Table 1). No significant linkage was found at markers elsewhere in the
genome.
Additional markers at 3q13.3-q21, which provided
the average resolution of less than 3cM over a 67cM
genetic distance, were used for fine mapping and refining the linked genetic interval (see Table 1). Extended
haplotypes were constructed according to the order of
the Genethon linkage map8 (1996 Genethon Microsatellite Maps at Genlink database). Examination of the
marker haplotypes confirmed that the marker alleles in
the linked interval cosegregated with the epilepsy phenotype in the family. The disease haplotype segregating
in the family is shown in Figure 1. The distal flanking
marker was defined by a recombination event in Individual II:2 (affected) occurring between D3S1267 and
D3S1551. A recombination event in Individual II:9
(unaffected) between markers D3S3675 and D3S1278
defined the proximal flanking marker. Individual II:9
has no history of seizures, no EEG abnormality, and is
40 years of age. Given his clearly defined clinical status,
recombination event in him was considered for definition of the linked interval. From these two recombination events, the critical linkage interval, flanked by
D3S3675 (centromeric) and D3S1551 (telomeric), was
localized to the 12.6cM region at 3q13.3-q21. This
corresponds to a physical length of about 12 megabases
(Mb) (Human Genome Map Viewer Build 36 database
at NCBI, NIH). Parametric multipoint LOD scores
and information content were calculated for all the
markers analyzed, and are shown in Figure 2, for the
markers from the linked interval. The greatest multipoint LOD score of 3.05 was obtained for the same
genomic region at 3q13.3-q21 (see Fig 2) where we
had found the greatest two-point LOD score.
Substantial genomic DNA from the 3q13.3-q21 region has been sequenced during the Human Genome
Project.11 The 12Mb subgenomic interval harbors 74
known validated or provisional genes (Human Genome
Map Viewer Build 36 database at NCBI, NIH). From
known or speculated expression or function in the central nervous system, 13 candidate genes were selected
for sequence analysis, with a hope to identify sequence
variant(s) cosegregating exclusively with the disease
phenotype in the family. These genes were SLC15A2
(GenBank accession number, NM_021082; Mendelian
Inheritance in Man [MIM] 602339), SLC12A8
(NM_024628), GAP43 (NM_002045; MIM 162060),
LSAMP (NM_002338; MIM 603241), DRD3
Kapoor et al: CASR and Epilepsy
161
Fig 2. Parametric multipoint logarithm of odds (Lod) score and information content graph of the markers spanning the linked genetic interval on chromosome 3. The x-axis represents the genetic length of a part of chromosome 3. The left-hand y-axis represents
multipoint LOD scores (thick line), and the right-hand y-axis represents information content (thin line) for markers.
(NM_000796; MIM 126451), GPR156 (NM_153002),
CASR (NM_000388; MIM 601199), SEC22L2
(NM_012430), TRAD (NM_007064; MIM 604605),
STXBP5L (XM_045911), ATP6V1A (NM_001690;
MIM 607027), GSK3B (NM_002093; MIM 605004),
and SEMA5B (NM_018987). Exons and flanking intronic regions of these genes were analyzed by direct sequencing in the affected individual (III:3). Predicted regulatory elements of GAP43 were also analyzed because
GAP43 overexpression has been linked to mossy fiber
sprouting in murine models of epilepsy.12 Several sequence variants were found during this analysis (see
Supplementary Table 1) but were excluded from being
pathogenic mutations from their allele frequencies in the
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normal populations as reported in the single nucleotide
polymorphism (SNP) database (ENTREZ SNP database
at NCBI, NIH) or allelic frequencies (⬎2%) calculated
in analysis of 96 control chromosomes of unaffected individuals from south India (see Supplementary Table 1)
and the lack of segregation with the disease phenotype
in Family 1 (data not shown).
One of the genes in the critical interval is CASR
(NM_000388; MIM 601199). CASR is known to code
for an extracellular CASR belonging to family C of Gprotein–coupled receptors (GPCRs).13 Human CASR
protein contains 1,078 amino acid residues.14 The receptor protein has an extracellular domain of 612 amino
acids, 7 transmembrane domains of 250 amino acids,
and a rather large intracellular domain of 216 amino acids. We found a novel putative mutation, g.80709G⬎A
(GenBank accession number AC068754.16), or
c.2693G⬎A (GenBank accession number NM_000388)
in CASR to be cosegregating with the clinical phenotype
in Family 1. c.2693G⬎A was not found in the unaffected members of Family 1. This putative mutation was
not found in at least 504 control chromosomes from
apparently normal individuals who were studied (Table
2). c.2693G⬎A transition changes an arginine to a glutamine at the 898 residue in the protein (Fig 3). Arg898
is located close to three potential phosphorylation sites
for protein kinase C at residues Thr888, Ser895, and
Ser915 in the intracellular tail of the protein that are
known to modulate the coupling of CASR to intracellular signaling pathways.15
Guided by these observations, we analyzed CASR in
an additional 96 apparently unrelated JME patients
from southern parts of India. Genomic DNA from
these patients was analyzed by direct sequencing of
the seven exons and their flanking intronic sequences
of this gene. Sequence comparison showed 12 variants, 8 of which were known SNPs (see Supplementary Table 1), and the remaining 4 rare variants were
novel (see Table 2). These rare variants were not detected in 504 normal control chromosomes analyzed
from the south Indian populations (see Table 2). These
four nucleotide variants, g.58158A⬎C, g.80072A⬎G,
g.80979C⬎T, and g.80979C⬎G (GenBank accession
number AC068754.16), lead to Glu354Ala, Ile686Val,
Ala988Val, and Ala988Gly amino acid alterations, respectively, in the CASR protein (see Fig 3). We could
not perform segregation analysis of these variants to
strengthen their possible roles in pathogenicity. However, rarity and evolutionary conservation of these variants support their possible roles in clinical pathophysiology.
In addition, we sequenced complete open reading
frame of CASR in 504 normal control chromosomes
from the south Indian populations. Eight coding polymorphisms were observed in the control group: Ser18Ser
(minor allele frequency [maf]: 0.002), Gln179Gln (maf:
0.005), Glu191Glu (maf: 0.009), Ser388Ser (maf:
0.002), Ile967Ile (maf: 0.005), Ala986Ser (maf: 0.19),
Arg990Gly (maf: 0.23), and Glu1011Gln (maf: 0.05).
Unlike in the case of CASR sequence analysis in
epilepsy-affected individuals described earlier, where four
rare sequence variants were found, no such change
(maf ⱕ 0.01), except for the synonymous changes
Ser18Ser, Gln179Gln, Glu191Glu, Ser388Ser, and
Ile967Ile, was detected in the normal control individuals
analyzed.
Multiple protein sequence alignment analysis of
CASR, done using ClustalW (ClustalW at European
Bioinformatics Institute), found Arg898, Glu354,
Ile686, and Ala988 to be conserved in human, cow,
dog, mouse, rat, and fowl (see Fig 3). This evolutionary conservation across six species over approximately
300 million years is suggestive of a functional role for
these amino acid residues in the regulation of CASR
activity. We used the Pfam database at Wellcome Trust
Sanger Institute to view the conserved domain organization of the CASR protein and found that Glu354 is
located in the extracellular domain of CASR belonging
to the ANF_receptor (atrial natriuretic factor receptor)
domain family that includes extracellular ligand binding domains of a wide range of receptors. Ile686 is situated in the third transmembrane domain of CASR
belonging to 7tm_3, seven-transmembrane receptor
domain specific to metabotropic glutamate receptors
(mGluRs) or family C of GPCRs. Ala988 is located in
the intracellular domain of CASR and is a part of
Pfam-B_29858 domain family, every member of which
is associated with ANF_receptor, 7tm_3, and NCD3G
(nine-cysteine domain of family 3 GPCR) domains.
Relatively little is known about the expression of
CASR in the human brain. We cloned the full-length
coding CASR transcript from human adult brain
Table 2. Epilepsy Families/Individuals, Missense Mutations at CASR, and Allele Frequencies
Family/Individual
Variation
Predicted Effect
on Protein
Allele Frequency
in Control Population
Family 1
AC068754.16 g.80709G⬎A
p.Arg898Gln
0/504
Family 80
AC068754.16 g.58158A⬎C
p.Glu354Ala
0/504
Family 93
AC068754.16 g.80072A⬎G
p.Ile686Val
0/504
Individual 17
AC068754.16 g.80979C⬎T
p.Ala988Val
0/504
Individual 50
AC068754.16 g.80979C⬎T
p.Ala988Val
0/504
Individual 45
AC068754.16 g.80979C⬎G
p.Ala988Gly
0/504
Families 80, 93, and 17 reported positive histories of epilepsy with one first-degree relative affected with epilepsy. No family history was
reported for Individuals 50 and 45 up to three previous generations. Family 1 is described in Subjects and Methods. Individuals
available for this study exhibited early-morning myoclonic jerks, generalized tonic-conic seizures, and generalized polyspike and wave
discharges in electroencephalographic recordings. In addition, febrile seizures were reported in the proband of Family 80.
Kapoor et al: CASR and Epilepsy
163
Fig 3. Calcium sensing receptor gene (CASR) mutations associated with epilepsy. (A–E) Electropherograms of the amplicons from
the genomic DNA showing homozygosity of the wild-type allele in unaffected individuals (WT/WT) and heterozygosity of each of
the five CASR mutations (E354A, I686V, R898Q, A988V, and A988G) identified in juvenile myoclonic epilepsy (JME) families/
individuals. Amino acid changes are underlined, and arrows indicate the heterozygous gene variations. (F–J) Amino acid sequence
alignment of the region near the locations of CASR mutation from cow, dog, human, mouse, rat, and fowl. Multiple amino acid
sequence alignment was done using ClustalW. The conserved E354, I686, R898, and A988 residues are boxed, and additional
residues conserved across species are indicated by asterisks.
Marathon-Ready complementary DNA (ClonTech,
Palo Alto, CA) using gene-specific primers, and on sequencing found it to be identical to the isoform reported from human parathyroid cells14 (data not
shown). We analyzed expression of CASR in the human brain regions by reverse transcriptase PCR and
Western blotting. CASR expression was observed in cerebral cortex, hypothalamus, hippocampus, whole adult
brain, and fetal brain by reverse transcriptase PCR using exon 4 – and exon 5–specific intron-spanning
primers (data not shown). Western blot analysis performed using 50␮g of total protein resulted in an immunoreactive CASR protein doublet of approximately
120kDa molecular mass observed in temporal lobe,
frontal lobe, parietal lobe, hippocampus, and cerebellum (Fig 4).
Discussion
In this study, we have found significant evidence of
linkage for a novel locus on 3q13.3-q21 in a family
from south India, with its members exhibiting generalized and focal seizures. At the linked locus, a novel
G3 A transition at position 2693 of CASR was found
to cosegregate with the disorder in the family. Although both generalized and focal idiopathic epilepsies
are clinically well defined, have characteristic EEG
features, and are known to be present in distinct large
kindreds, there is emerging evidence for coexistence
of generalized and focal seizures in a given affected
individual in an age-dependent manner, as well as
clustering of the two subtypes in families with multiple affected individuals,16,17 thus supporting the neurobiological concept that idiopathic epilepsies may
share an overlapping genetic predisposition.
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CASR is a member of the family C of GPCRs.
mGluRs and type B GABA receptors are two additional members of this family, which are involved
in neuronal excitation and inhibition, respectively.
Cloned from the bovine parathyroid gland,13 CASR
has been studied extensively for its role in the regulation of systemic Ca2⫹ homeostasis.18 CASR senses
small changes in extracellular calcium concentrations
and integrates this information to intracellular signaling pathways, permitting it to play its biological roles.
The physiological importance of CASR in regulating
systemic Ca2⫹ concentration has been established
based on studies of human disorders resulting from the
CASR activating and inactivating mutations that lead
to hypocalcemia and hypercalcemia,19 respectively. Seizures are, indeed, occasionally observed among clinical
features of hypocalcemia. Interestingly, CASR is also
known to be expressed in many other tissue types,
most notably in the brain.18 These findings have led to
suggestions that CASR regulates additional biological
processes that are quite independent of its role in systemic Ca2⫹ homeostasis. Indeed, CASR has been
shown to regulate membrane excitability, cellular differentiation, apoptosis, and gene expression.18 Although it remains to be elucidated as to when and how
these receptors become functionally active during the
development of brain, the expression and potential
functional role of CASR in the brain has been of considerable scientific interest since the time it was first
reported.13
Sporadic reports of deranged homeostasis of calcium
and parathyroid gland physiology concurring with seizures have led to suggestions that calcium-controlled
mechanisms may be involved in causing seizures.20,21
Fig 4. Expression analysis of calcium sensing receptor gene (CASR) in human brain. Western blot analysis of tissue lysates from
different regions of human brain: temporal lobe (TL), frontal lobe (FL), parietal lobe (PL), hippocampus (HC), and cerebellum
(CB). Rabbit anti-CASR affinity purified polyclonal antibody (Chemicon International, Temecula, CA) and mouse anti-actin
monoclonal antibody (Calbiochem, San Diego, CA) were used in these experiments. Expected molecular mass of CASR is 120kDa.
Our study provides genetic evidence for participation
of CASR in subjects from families with epilepsy. Considering the involvement of CASR in regulation of systemic Ca2⫹ and parathyroid hormonal levels, we examined the proband (III:3) of Family 1 for the levels of
serum calcium, phosphate, sodium, potassium, and
parathyroid hormone, and found all of these to be
within reference range (calcium: 9.3mg/dl; phosphate:
4.2mg/dl; sodium: 133mEq/L; potassium: 4.4mEq/L;
parathormone: 42pg/ml). Moreover, additional clinical
features of hypocalcemia or hypercalcemia were also
not present in the affected members of the family, suggesting that the effect of CASR for epilepsy phenotypes
is allele and tissue specific.
It is reasonable to expect that extracellular Ca2⫹ levels have an important role in regulating brain-specific
functions of CASR. Several modeling studies and measurements have shown that extracellular Ca2⫹ fluctuations, manifesting as a result of ongoing neuronal activity, constitute an important information-bearing
signal in the brain.22,23 CASR may couple these fluctuations in the levels of extracellular Ca2⫹ to certain
specific intracellular signaling responses mediated by its
intracellular tail. CASR expression has been detected in
bovine brain,13 rat neurons,24 human embryonic primary astrocytes,25 and human adult brain regions (see
Fig 4). It is known that in the cultured rat and mouse
hippocampal neurons, CASR agonists activate Ca2⫹permeable, nonselective cation channels.26,27 In cultured mouse hippocampal neurons, CASR agonists
have been shown to activate Ca2⫹-activated K⫹ channels.28 This activation was not observed when hippocampal cells were isolated and examined from the
mice carrying a targeted disruption of the gene, indicating that CASR regulates the activity of Ca2⫹-
permeable nonselective cation channels and Ca2⫹activated K⫹ channels in hippocampal neurons.
Therefore, the role of CASR in modulating neuronal
excitability and neurotransmission, via membrane depolarization and repolarization, respectively,27,28 is already established. CASR is also expressed in human astrocytoma cell lines U87 and U373, where it has been
found to modulate an outward K⫹ channel and a nonselective cation channel.29,30 Activation of these channels by CASR could regulate membrane excitability,
and these biological functions of the gene may be crucial in relation to epilepsy.
CASR expression in several rat brain regions has
been found to overlap with that of group I mGluRs31
and the G proteins, G␣q and G␣11,32 implicated in
CASR- and mGluR-mediated activation of downstream cellular signaling pathways. In bovine brain extracts, mGluR and CASR have been reported to form
functional heterodimers.33 In the rat brain, CASR and
group I mGluRs colocalize throughout the hippocampus and cerebellum, suggesting the potential for
heterodimerization.33 Studies conducted in cultured
hippocampal neurons, mouse brains, and human embryonic kidney 293 cells show that complex formation
between CASR and type B GABA receptors affects the
signaling of CASR.34 Collectively, these studies point
at possible roles of CASR in modulating neuronal function.
Although a detailed immunohistochemical analysis
of CASR expression in the human brain regions needs
to be conducted, our initial results of its expression in
the brain are in line with the expected involvement of
the gene in susceptibility to epilepsy. We consider the
role of CASR as a modulator of ion channels in response to extracellular Ca2⫹ concentrations to be quite
Kapoor et al: CASR and Epilepsy
165
crucial in the pathophysiology of epilepsy. However, it
is possible that the role of CASR in biological processes
of cellular proliferation, differentiation, or apoptosis in
the brain underlie susceptibility to epilepsy. That regulation of apoptosis may have a role in the cause of
epilepsy has been suggested by identification of
EFHC135 variants in epilepsy families.
Our results, based largely on the genetic evidence
obtained, raise an interesting possibility that disruption
of a cellular signaling regulatory network involving
CASR as its genetic component in the human brain
could lead to epilepsy. However, to establish the mechanistic link of CASR to the seizure phenotypes, it is
necessary to identify the molecular pathways affected
by the potential mutations found. Analysis of mitogenactivated protein kinase pathways known to regulate
ion channels36 will be worth exploring in the context
of seizure phenotypes. Detailed gene expression studies
and identification of proteins interacting with CASR in
the human brain will be useful in establishing a causal
role for the gene. A systematic genetic epidemiology
should help demonstrate a detailed picture of the nature of a functional relationship between epilepsy and
CASR gene sequence variants.
Data in this article were obtained from the following databases:
1996 Genethon Microsatellite Maps at Genlink database (http://
www.genlink.wustl.edu/genethon_frame/), Human Genome Map
Viewer Build 36.1 database at National Center for Biotechnology
Information, National Institute of Health (http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid⫽9606), GenBank database
at National Center for Biotechnology Information, National Institute of Health (http://www.ncbi.nlm.nih.gov/Genbank/index.html),
OnlineMendelianInheritanceinMan(OMIM;(http://www.ncbi.nlm.
nih.gov/entrez/query.fcgi?db⫽OMIM), ENTREZ SNP database at
National Center for Biotechnology Information, National Institute
of Health (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db⫽snp),
ClustalW at European Bioinformatics Institute (http://www.
ebi.ac.uk/clustalw/), and Pfam database at Wellcome Trust Sanger
Institute (http://www.sanger.ac.uk/Software/Pfam/).
This work was supported by the DBT (BT/PR1414/Med/13/050/
98, A.A.), DAE (2005/21/10-BRNS/1166, A.A.), JNCASR (JNC/
PC-19, A.A.), and CSIR (A.K., R.P.).
We are grateful to the families for participation in the study. We
thank Dr S. Girirajan for assistance in sample collection, A. Mahedarkar for assistance in computational analysis, and Prof S. Chandra
for critical reading of the manuscript. We thank Drs P. Basu and R.
Ramesh for assistance in clinical studies.
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