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Association between genetic variation of CACNA1H and childhood absence epilepsy.

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Association between Genetic
Variation of CACNA1H and
Childhood Absence Epilepsy
Yucai Chen, PhD,1,2 Jianjun Lu, PhD,1,2 Hong Pan, MD,1
Yuehua Zhang, PhD,1 Husheng Wu, MD,3
Keming Xu, MD,4 Xiaoyan Liu, MD,1 Yuwu Jiang, PhD,1
Xinhua Bao, PhD,1 Zhijian Yao, PhD,2
Keyue Ding, PhD,5 Wilson H. Y. Lo, PhD,5
Boqin Qiang, PhD,2,5 Piu Chan, MD, PhD,2,6
Yan Shen, PhD,2,5 and Xiru Wu, MD1
Direct sequencing of exons 3 to 35 and the exon–intron
boundaries of the CACNA1H gene was conducted in 118
childhood absence epilepsy patients of Han ethnicity recruited from North China. Sixty-eight variations have
been detected in the CACNA1H gene, and, among the
variations identified, 12 were missense mutations and
only found in 14 of the 118 patients in a heterozygous
state, but not in any of 230 unrelated controls. The identified missense mutations occurred in the highly conserved residues of the T-type calcium channel gene. Our
results suggest that CACNA1H might be an important
susceptibility gene involved in the pathogenesis of childhood absence epilepsy.
Ann Neurol 2003;54:239 –243
Absence epilepsy is defined as a paroxysmal loss of consciousness of sudden onset and offset associated with
bursts of bilaterally synchronous spike-and-wave discharges. Studies have shown that the spike wave is inherited as a complex trait. The basic underlying mechanism of generalized absence seizures appears to involve
thalamocortical circuitry and the generation of abnormal
oscillatory rhythms from that particular neuronal network.1,2 It has been proposed that the low-threshold
T-type Ca2⫹ channels might be involved in the genesis
of absence seizures in the thalamocortical network.3–5
From the 1Department of Pediatrics, First Hospital of Peking University; 2National Center of Human Genome Research (Beijing);
3
Beijing Children’s Hospital. 4Capital Institute of Pediatrics; 5National Laboratory of Medical Molecular Biology, Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences/Peking
Union Medical College; and 6Xuanwu Hospital of Capital University of Medical Sciences, Beijing, China.
Received Jan 16, 2003, and in revised form Mar 19. Accepted for
publication Mar 20, 2003.
Address correspondence to Dr Wu, Department of Pediatrics, First
Hospital of Peking University, Beijing, China 100034, e-mail:
wxrwwn@public.bta.net.cn, or Dr Shen, National Center of Human
Genome Research, Beijing, China 100176; E-mail: sheny@cdm.
imicams.ac.cn
We have conducted direct sequencing of exons 3 to 35
and the exon–intron boundaries of the CACNA1H
gene6 in 118 consecutive childhood absence epilepsy
(CAE) patients of Han ethnicity from North China to
investigate whether CACNA1H could be one of the important candidate genes for CAE.
Patients and Methods
Patients
The patients were recruited from several hospitals in Beijing and were of Han ethnicity. Informed consent was obtained from their parents. The inclusion criteria for the diagnosis of CAE were as follows: (1) absence seizures as
the initial seizure type at 3 to 12 years of age, (2) absence
seizures occurring multiple times per day, (3) absence seizures associated with bilateral, symmetric, and synchronous
discharge of regular 3Hz spike-and-wave discharges with
normal background activity on electroencephalogram
(EEG), (4) normal general physical and neurological examinations, and (5) normal neuroradiological examination including computed tomography or magnetic resonance imaging7 All patients were sensitive to valproic acid and were
from one-child families. The 230 normal controls, also of
Han ethnicity, were normal adults with matched gender
from North China, with no history or family history of
epilepsy.
Variation Detection
Genomic DNA was extracted from peripheral blood leukocytes.8 Polymerase chain reaction (PCR) amplification (primers available on request) was performed in a reaction volume
of 15␮l, containing 50ng of genomic DNA, 10mM TrisHCl (pH 8.4), 50mM KCl, 3.0mM MgCl2, 200␮M of each
dNTP, 0.6U HotStar Taq DNA Polymerase, and 0.3 ␮M of
each primer (synthesized by Aoke, Hehan, China). PCR
thermocycles were conducted in a Perkin Elmer GeneAmp
PCR System 9700 (Oak Brook, IL) using a touchdown algorithm. The first 15 cycles consisted of denaturation for 30
seconds at 94°C, annealing for 60 seconds at 63°C (the annealing temperature was decreased by 0.5°C after each cycle)
and extension for 90 seconds at 72°C. For the following 25
cycles, the annealing temperature was held at 56°C for 40
seconds, with an extension of 60 seconds at 72°C. A final
extension was performed at 72°C for 10 minutes. The PCR
products were purified with 96 Multiscreen filter plates (Millipore, Bedford, MA) and sequenced on an ABI 3700 DNA
sequencer (Applied Biosystems, Foster City, CA).
Data Analysis
Analysis of the sequence chromatograms was carried out using Phred/Phrap/Consed software9 and by visually examining
printed chromatograms to detect sequence changes and heterozygous nucleotides. All variations were verified by PCR
and sequencing reactions with forward and reverse primers
for at least two more times.
Results
The mean age of the 118 patients was 8.5 years (range,
3.9 –12 years). The mean age at onset was 7.2 years
© 2003 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
239
Table 1. CACNA1H Missense Mutations Found Only in Childhood Absence Epilepsy Patients
Exon
Nucleotide
Substitution
1
2
3
4
7
9
562C3A
923G3A
1445T3A
F161L
E282K
C456S
IS2–IS3
IS5–SS1
Linker I–II
4
5
6
7
8
9
10
11
12
13
14
9
9
10
10
10
10
10
10
11
11
23
1574G3A
2022C3T
2310G3A
2310G3A
2322C3T
2397G3A
2397G3A
2429G3A
2570G3A
2621G3A
4466G3A
G499S
P648L
R744Q
R744Q
A748V
G773D
G773D
G784S
V831M
G848S
D1463N
Linker I–II
Linker I–II
Linker I–II
Linker I–II
Linker I–II
Linker I–II
Linker I–II
Linker I–II
IIS2
IIS2
IIIS5–SS1
Case No.
Amino Acid
Substitution
Structural
Location
Comments
Conserved
Conserved/change of change status
Conserved/CK2
Phosphorylation sitea
N-myristoylation sitea
—
Change of charge status
Change of charge status
—
Change of charge status
Change of charge status
PKC phosphorylation sitea
Conserved
Conserved
Conserved/change of charge status
The numbering of the nucleotide acid and amino acids is according to the system used in AF0519461.
a
PROSITE database of protein families and domain (http://www.expasy.ch/prosite).
F ⫽ phenylalanine; L ⫽ leucine; C ⫽ cysteine; G ⫽ glycine; S ⫽ serine; P ⫽ proline; R ⫽ arginine; Q ⫽ glutamine; A ⫽ alanine; V ⫽ valine;
D ⫽ aspartic acid; M ⫽ methionine; PKC ⫽ protein Kinase C.
(range, 3.9 –11 years). There were 3 patients with a
positive family history of seizures and 12 patients with
febrile seizures before the onset of CAE. In the first
screening of exon 3 to 35 in all 118 cases, 68 sequence
variations have been detected in the CACNA1H gene
in our study. After the initial screening for these mutations in 96 gender-matched normal controls, there
were 29 of the 68 variations found only in 118 patients
but not the controls, among which 12 were missense
mutations (Tables 1–3). To further investigate whether
the 12 missense mutations were more specific for CAE,
we screened an additional 134 unrelated control subjects, and none of them was found to carry the missense mutations.
All together, 14 CAE cases were found to be heterozygous for the 12 missense mutations including 2
cases with the same R744Q mutation, and 2 cases with
the same G773D mutation. The mean age of the 14
Fig 1. Locations of mutations found in CACNA1H. (blue star) F161L; (blue circle) E282K; (red diamond) C456S; (green diamond) G499S; (red star) P648L, (blue diamond) R744Q and A748V; (purple diamond) G773D; (green star) G784S; (red
square) V831M; (green square) G848S; (pink triangle) D1463N.
240
Annals of Neurology
Vol 54
No 2
August 2003
Fig 2. Clustal W alignment of T-type calcium channel gene products. Blue arrows indicate heterozygous point mutation sites. Green
arrows point to the highly conserved amino acids. Red shading indicates the transmembrane areas. (A–C, E, F) Sequence alignment
of linker I–II, IS2–IS3, IIS2, IIIS5–SS1, and IS5–SS1 portion of human a1H with orthologs from other species. (D) Sequence
alignment of IIS2 portion of human a1H with all known voltage-gated calcium channel gene products.
patients was 7.5 years, and the mean age at onset was 7
years. None of these patients in 14 cases had family
history of epilepsy. There appeared no difference in
clinical phenotypes between the CAE patients with and
without the missense variations (Table 4).
Through genotyping the parents, we found that all
patients received his or her missense mutations from
one of his or her parents. The parents of 3 of the 14
CAE patients were willing to undergo EEG examinations, but their EEG results were normal. Two other
parents of CAE patients who did not carry the missense mutations also underwent EEG examination, and
the EEG results were normal.
Discussion
We have for the first time to our knowledge identified
12 missense mutations in the coding regions of the
CACNA1H gene that exist only in CAE patients but
not controls, suggesting a close relationship between
the CACNA1H gene and CAE susceptibility. This is
supported by the fact that (1) none of these missense
mutations were found in 230 control subjects, indicating a very low frequency in the population; and (2)
most of these missense mutations were located in the
conserved sequences and important domains of the
protein6,10 (see Table 1, Fig 1, Fig 2).
Although the missense mutations identified only in
Chen et al: Genetic Variation of CACNA1H
241
CAE patients were not found in 230 controls, they
were inherited from their parents. However, none of
these parents reported a history of CAE; neither do
they express the characteristic abnormal EEG trait at
least in the three parents consented. The possible explanations may include the following. First, CAE only
affects children, and most clinical symptoms of CAE
can remit or disappear with age. The parents might
have CAE in their childhood but they were unaware of
it. Second, current evidence suggests that CAE is a
multifactorial disease, and many factors might be involved in the mechanisms of the complexity of CAE.2
We would be able to answer these questions by testing
the siblings of the patients with the mutations. Unfortunately, all families of the 14 CAE patients collected
were nuclear families with only one child because of
China’s one-child policy. As a result we could not determine whether all children with the missense mutation had CAE as compared with healthy siblings who
do not have the mutation. In summary, our data suggest that the CACNA1H gene missense mutations
might be an important susceptibility risk factor for abTable 2. CACNAIH Variations Found Only in Childhood
Absence Epilepsy in Patients
Location
Exon 4
Exon 7
Intron 7
Exon 8
Intron 8
Exon 9
Exon 10
Exon 11
Intron 11
Exon 15
Exon 23
Exon 31
Exon 36
Nucleotide
Substitution
Amino Acid
Substitution
63103C3A
67460G3A
67504C3T
67651C3T
67798G3A
67948C3T
68036G3A
68128G3A
68755G3T
68980T3A
69109G3A
69504C3T
69557C3T
71402G3A
71414C3T
71481C3T
71489G3A
71522G3A
72316C3T
72317G3A
72368G3A
73210C3T
75028C3G
75075G3A
75079G3T
78681G3A
84147G3A
88169T3A
88280G3A
F161L
E282K
D296D
N345N
F396F
C456S
G499S
P630P
P648L
R744Q
A748V
G770G
G773D
G784S
I830I
V831M
G848S
D1463N
A1765A
The numbering of the nucleotide acid is according to the system
used in NT_010540. The numbering of the amino acids is according to the system used in AF051946.1.
242
Annals of Neurology
Vol 54
No 2
August 2003
Table 3. CACNA1H Variations Found in Both Cases and
Controls
Location
Polymorphism
Intron 2
62439A3T
62516G3A
63070C3T
63468C3T
66472G3A
67553G3A
67723C3T
67858A3C
68726A3G
69967G3T
69282C3T
69423G3A
69438G3A
69533C3T
69605C3T
69901C3G
70966G3C
71221C3T
71533C3T
73249C3T
75104C3T
80969C3G
81104G3A
81173T3C
85650T3C
85659C3T
85707G3A
87220T3C
87276A3G
87327G3A
87421C3T
87514C3T
87614C3T
88260T3C
88340C3T
88354G3A
88364G3T
88513G3A
88636T3C
Exon 4
Intron 4
Intron 5
Exon 7
Intron 7
Intron 8
Exon 9
Intron 9
Exon 10
Exon 15
Exon 23
Intron 27
Exon 33
Exon 35
Amino Acid
Change
Y166Y
V331M
I369I
S451S
P556P
R603R
L608L
P640L
V664A
P684S
R788C
A1601A
G1907G
D1910D
S1926S
G2041G
H2060R
R2077H
A2108A
D2139D
P2173S
The numbering of the nucleotide acid is according to the system
used in NT_010540. The numbering of the amino acids is according to the system used in AF051946.1.
sence epilepsy. Further studies with a large sample are
warranted to confirm our results.
This research was supported by grants from the Beijing Natural Science Foundation (7001003, X.W.), the China National High-Tech
R & D Program (863-102-10, Y.S.; 2002AA223011, X.W.), the
Peking University Human Disease Gene Research Center Foundation (2000-A-8, X.W.), the Beijing Municipal Commission for Science and Technology (H010210230119, X.W.), the China National Key Program on Basic Research (G1998051003, Y.S.), and
the National Natural Science Foundation of China (39625007,
39993420, Y.S.).
We are grateful to members of the families who participated in this
work; to the many clinicians who contributed cases to the study; to
Table 4. Clinical Characteristics of Childhood Absence Epilepsy Patients with Mutations of CACNA1H Gene
Case
Mutation
Onset Age
1
F161L
5 yr
2
E282K
6 yr 7 mo
3
C456S
4 yr
4
G499S
5 yr 4 mo
5
P648L
7 yr 1 mo
6
R744Q
7 yr
7
R744Q
8 yr
8
A748V
6 yr 5 mo
9
G773D
9 yr 5 mo
10
G773D
5 yr 6 mo
11
G784S
8 yr
12
V831M
8 yr
13
G848S
7 yr 5 mo
14
D1463N
9 yr
Clinical Features
Electroencephalogram
Frequent absence seizures (staring) can be induced by HV
Frequent absence seizures (staring) can be induced by HV, occasionally with chewing
Frequent absence seizures (staring) can be induced by HV, occasionally with chewing
Frequent absence seizures (staring) can be induced by HV
Frequent absence seizures (staring) can be induced by HV, occasionally with chewing
Frequent absence seizures (staring) can be induced by HV, occasionally with chewing and
incontinence
Frequent absence seizures (staring) can be induced by HV, with once generalized tonicclonic seizure
Frequent absence seizures (staring) can be induced by HV
Frequent absence seizures can be induced by HV
Generalized, symmetric, 2.5–4Hz
SWDs with normal background
Generalized, symmetric, 3Hz SWDs
with normal background
Generalized, symmetric, 3Hz SWDs
with normal background
Generalized, symmetric, 2.5–3.5Hz
SWDs with normal background
Generalized, symmetric, 3Hz SWDs
with normal background
Generalized, symmetric, 2.5–3.5Hz
SWD with normal background
Frequent absence seizures (staring) can be induced by HV
Frequent absence seizures (staring) can be induced by HV
Frequent absence seizures (staring) can be induced by HV, occasionally with chewing
Frequent absence seizures (staring) can be induced by HV, occasionally with eye blinking
Frequent absence seizures can be induced by HV
Generalized, symmetric, 3–4 Hz
SWDs with normal background
Generalized, symmetric, 3Hz SWDs
with normal background
Generalized, symmetric, 3Hz SWDs
with normal background
Generalized, symmetric, 3–3.5Hz
SWDs with normal background
Generalized, symmetric, 3Hz SWDs
with normal background
Generalized, symmetric, 3Hz SWD
with normal background
Generalized, symmetric, 3–3.5Hz
SWD with normal background
Generalized, symmetric, 3Hz SWD
with normal background
HV ⫽ hyperventilation; SWD ⫽ spike-and-wave discharge.
Drs A. V. Delgado-Escueta and D. Ma for their valuable discussions.
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Chen et al: Genetic Variation of CACNA1H
243
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