Cell, Vol. 119, 19–31, October 1, 2004, Copyright 2004 by Cell Press
CaV1.2 Calcium Channel Dysfunction
Causes a Multisystem Disorder
Including Arrhythmia and Autism
ultimate signaling molecule for organisms ranging from
ates processes as diverse as synaptic transmission,
muscle contraction, insulin secretion, fertilization, and
lized, cells have evolved complex mechanisms for regu-
lating intracellular Ca2?levels, which are 10,000-fold
lower than extracellular levels.Many proteins have been
adapted to bind and transport Ca2?, in some cases to
reduce Ca2?levels and in others to trigger second-mes-
senger pathways. Excitable cells contain voltage-
dependent calcium channels that can dramatically in-
crease cytosolic Ca2?. In heart and brain, the L-type
calcium channel CaV1.2 (CACNA1C, ?1C, ?11.2) mediates
et al., 1993). By contrast with physiology, the role of
Ca2?signaling in development is poorly understood.
Previously characterized calcium channel disorders,
however, have been marked by dysfunction of a distinct
organ system, particularly the membrane excitability of
neurons and skeletal muscle. For example, calcium
channel syndromes like hypokalemic periodic paralysis
and malignant hyperthermia affect skeletal muscle
plegic migraine affects vascular smooth muscle (Ophoff
(Bech-Hansen et al., 1998; Strom et al., 1998). None of
these disorders has illustrated the full extent of Ca2?
signaling in human development and physiology.
Cardiac arrhythmias cause sudden loss of conscious-
ness and sudden death in approximately 1 million Euro-
peans and North Americans every year (Priori et al.,
2002; Zheng et al., 2001). Over the last decade, we and
others have identified arrhythmia susceptibility genes
by studying familial syndromes (Antzelevitch, 2003;
Keating and Sanguinetti, 2001). These genes include
SCN5A, KVLQT1, and HERG, which encode important
cardiac sodium and potassium channels. In the vast
majority of arrhythmia syndromes, individuals appear
normal except for subtle electrocardiographic abnor-
Here, we describe the phenotypic characterization of
Timothy syndrome (TS), an arrhythmia disorder associ-
ated with dysfunction in multiple organ systems, includ-
ing congenital heart disease, syndactyly, immune defi-
ciency, and autism. We show that this disorder results
from a recurrent, de novo missense mutation in the
CaV1.2 L-type calcium channel gene. The CaV1.2 gene is
expressed in multiple tissues. We demonstrate through
functional expression in heterologous systems that the
disease-associated mutation causes abnormal Ca2?
current. This gain-of-function mechanism is mediated
through failed channel inactivation, suggesting that cal-
cium channel blockers may be useful for treating this
and related disorders.
Igor Splawski,1,* Katherine W. Timothy,2
Leah M. Sharpe,1Niels Decher,3Pradeep Kumar,3
Raffaella Bloise,4Carlo Napolitano,4
Peter J. Schwartz,5,6Robert M. Joseph,7
Karen Condouris,7Helen Tager-Flusberg,7
Silvia G. Priori,4,5Michael C. Sanguinetti,3
and Mark T. Keating1
1Department of Cardiology
Departments of Pediatrics and Cell Biology
Harvard Medical School and
Howard Hughes Medical Institute
Boston, Massachusetts 02115
2Department of Human Genetics and
3Department of Physiology and Nora Eccles Harrison
Cardiovascular Research and Training Institute
University of Utah
Salt Lake City, Utah 84112
4Department of Molecular Cardiology
IRCCS Fondazione Salvatore Maugeri
5Department of Cardiology
University of Pavia
6IRCCS Policlinico San Matteo
7Department of Anatomy and Neurobiology
Boston University School of Medicine
Boston, Massachusetts 02118
CaV1.2, the cardiac L-type calcium channel, is impor-
tant for excitation and contraction of the heart. Its role
in other tissues is unclear. Here we present Timothy
syndrome, a novel disorder characterized by multior-
gan dysfunction including lethal arrhythmias, webbing
of fingers and toes, congenital heart disease, immune
deficiency, intermittent hypoglycemia, cognitive ab-
normalities, and autism. In every case, Timothy syn-
drome results from the identical, de novo CaV1.2 mis-
sense mutation G406R. CaV1.2 is expressed in all
affected tissues. Functional expression reveals that
G406R produces maintained inward Ca2?currents by
causing nearly complete loss of voltage-dependent
channel inactivation. This likely induces intracellular
Ca2?overload in multiple cell types. In the heart, pro-
longed Ca2?current delays cardiomyocyte repolariza-
tion and increases risk of arrhythmia, the ultimate
cause of death in this disorder. These discoveries es-
tablish the importance of CaV1.2 in human physiology
and development and implicate Ca2?signaling in
“Ja, Kalzium, das ist alles!” So stated Nobel laureate
Otto Loewi in 1959, and it is now clear that Ca2?is the
syndrome. By contrast, the complexity of the TS pheno-
type suggested a second possibility, a contiguous gene
deletion syndrome or a chromosomal rearrangement.
Karyotypic analysis of metaphase chromosomes from
analysis, however, would not exclude small deletions.
A consistent feature of TS was severe QT interval
prolongation. Therefore, to define the genetic basis of
this disorder, we screened known long QT syndrome
and KCNJ2, by single-strand conformation polymor-
phism (SSCP) and/or DNA sequence analyses. We also
examined several other genes encoding channel or
channel-associated subunits, including FKBP1A, KCNA4,
KCNA5, KCND3, KCNE3, KCNIP2, KCNJ4, KCNJ9,
KCNJ10, KCNJ12, NCX1, SCN1B, DNAJB1, and TRPC3.
The transcription factors TFAP2B, FOG2, NKX2.5, and
GATA4 were tested because they have been implicated
in one or more phenotypic features of TS. No mutations
The abnormal electrocardiographic morphology ob-
served in TS patients was similar to that of individuals
with long QT syndrome caused by gain-of-function mu-
tations in SCN5A, the cardiac sodium channel (Zhang
et al., 2000). The cardiac L-type calcium channel CaV1.2,
like SCN5A, mediates an inward depolarizing current in
cardiomyocytes. Thus, in 1996, we examined the CaV1.2
gene as a candidate. SSCP analyses of 47 exons and
promoter regions did not show mutations (Table 2).
However, new alternatively spliced forms of the CaV1.2
gene were subsequently identified (Abernethy and Sol-
datov, 2002). Analysis of one CaV1.2 splice variant re-
for whom DNA samples were available (Figures 2A and
2B). This transition caused a substitution of glycine with
arginine at residue 406 (G406R). This amino acid is com-
pletely conserved in other voltage-dependent calcium
channels of multiple species, ranging from worms to
humans (Figure 2C). G406 is located at the C-terminal
180 ethnically matched control samples (360 chromo-
somes, p ? 1.8 ? 10?20).
Mutational analysis of additional family members, in-
cluding parents, failed to reveal mutations at this site.
Thus, the phenotype in all probands resulted from the
same de novo mutation. However, in one family, two
siblings had the syndrome (Figure 2B). Both parents
were phenotypically unaffected. The probability of the
mutation event happening twice in the same family was
minimal. To explain this apparent paradox, we hypothe-
sized that one parent was mosaic for the mutation. Mo-
cally different cell types in the same organism. To test
this hypothesis, we sequenced DNA samples from the
father’s sperm and blood, but the mutation was not
observed. In the mother, we sequenced DNA samples
from blood and oral mucosa. Although the blood DNA
contained only wild-type sequences, we were able to
detect a small peak for the missense mutation in DNA
from the oral mucosa (Figure 2B). These findings indi-
cate that the mother is mosaic and transmitted this mu-
tation to her two affected children. To further test this
hypothesis, we performed genotypic analysis in this
Timothy Syndrome, a Multisystem Disorder
In 1992, cases of a novel arrhythmia syndrome associ-
ated with syndactyly (webbing of fingers and toes) were
described (Marks et al., 1995; Reichenbach et al., 1992).
We named this disorder Timothy syndrome (TS). Be-
cause therapy is extending lives of affected children,
we have observed that TS manifests major phenotypic
abnormalities of multiple organ systems, including
heart, skin, eyes, teeth, immune system, and brain (Fig-
ure 1, Table 1).
The inheritance pattern of TS was sporadic in all but
one family. In that family, two of three siblings were
affected. None of the parents in any of the families was
affected. Ten of 17 children with TS died. The average
age of death was 2.5 years. All affected individuals had
severe prolongation of the QT interval on electrocardio-
gram, syndactyly, and abnormal teeth and were bald at
birth. Arrhythmias were the most serious aspect of this
disorder, as 12 of 17 children had life-threatening epi-
sodes. Arrhythmic death in two children was triggered
by sepsisand intwo otherchildren byepisodic hypogly-
cemia. Individuals with this syndrome also had congeni-
tal heart disease including patent ductus arteriosus, pa-
tent foramen ovale, ventricular septal defects, and
tetralogy of Fallot. Some children had dysmorphic facial
features, including a flat nasal bridge, small upper jaw,
low-set ears, or small and misplaced teeth. Episodic
serum hypocalcemia was described in four individuals.
delay and delay in other motor skills. Many did not pro-
duce speech sounds (babbling) during infancy. Testing
revealed significant problems in language skills includ-
ing articulation, reception, and expression. Children
children were formally evaluated for autism. Three met
the criteria for this disorder, one met criteria for autism
spectrum disorders, and one had severe delays in lan-
guage development. We could not evaluate additional
children because they were deceased or unavailable.
ordersand Timothysyndromewas significant(p? 1.2?
10?8). Taken together, the diversity of these phenotypic
Recurrent De Novo Missense Mutation
in CaV1.2 Causes TS
The TS phenotype suggested two possible genotypes.
The severity of arrhythmias in this disorder suggested
a recessive gene knockout similar to Jervell and Lange-
Nielsen syndrome, which is caused by homozygous
loss-of-function mutations of KVLQT1 or KCNE1 potas-
sium channel genes (Neyroud et al., 1997; Schulze-Bahr
et al., 1997; Splawski et al., 1997a; Tyson et al., 1997).
Consistent with this thesis, all parents were unaffected,
and one of the families had two children with TS. How-
ever, none of the parents from any family were related,
Calcium Channel Mutation in Arrhythmia and Autism
Figure 1. Timothy Syndrome Is Characterized by Multisystem Dysfunction and Developmental Defects
(A–C) TS individuals exhibiting dysmorphic facial features including round face, flat nasal bridge, receding upper jaw, and thin upper lip.
(D and E) Webbing of the toes and fingers (syndactyly).
(F) Left panel electrocardiogram shows severe QT interval prolongation causing 2:1 atrioventricular block seen as two atrial beats (P-waves)
for each ventricular beat (QRS complex). Right panel electrocardiogram shows alternating T-wave polarity (arrows), indicating severe cardiac
(G) Ventricular tachycardia recorded from a TS patient by an implanted automatic defibrillator.
Exons 8and 8Aare mutuallyexclusive asthey encode
the same structural domain (DI/S6), but one must be
present to encode a functional channel. To quantify the
relative expression of exons 8 and 8A in human heart
that 23 of 101 clones (22.8%) from heart cDNAs con-
tained exon 8A, and 78 clones (77.2%) contained exon
8. In the brain, 13 of 56 clones (23.2%) contained exon
expression of exon 8A is consistent in heart and brain
and is expressed at significantly lower levels than
To define the cellular distribution of CaV1.2 gene ex-
pression, we performed in situ hybridization experi-
ments in mice. CaV1.2 was expressed throughout the
brain (Figure 4A), including hippocampus, cerebellum,
and amygdala. Abnormalities of these brain regions
have been implicated in autism (Allen and Courchesne,
2003; Brambilla et al., 2003). CaV1.2 showed highest
expression in the granular layers of hippocampal den-
tate gyrus (Figures 4A and 4B) and cerebellum (data not
(Figures 4C and 4D) and the vascular system, including
ductus arteriosus (Figure 4E). In the eye, CaV1.2 was
expressed in the retina and sclera (Figure 4F). CaV1.2
was also expressed in developing digits and teeth (Fig-
ures 4G and 4H). Thus, the expression pattern of CaV1.2
in humans and mice is consistent with the phenotypic
abnormalities associated with TS.
Table 1. Phenotypic Features of Timothy Syndrome
(1) Ventricular tachyarrhythmia
(2) Bradycardia, AV blockb
Patent ductus arteriosus
Patent foramen ovale
Ventricular septal defects
Tetralogy of Fallot
Autism spectrum disorders
Bald at birth
G406R Mutation Impairs Channel Inactivation
mutation, we heterologously expressed wild-type (wt)
and mutant (G406R) forms of the CaV1.2 channel in Chi-
nese hamster ovary (CHO) cells and Xenopus oocytes.
The biophysical properties of the channel were first
niques using Ca2?(15 mM) as a charge carrier in CHO
cells cotransfected with CaV1.2 and its accessory sub-
units, CaV?2band CaV?2?1. The most striking difference
between wt and G406R channelswas the extent of inac-
tivation. Inactivation of wt channel current was nearly
nels only partially inactivated during the same time pe-
riod (Figure 5B). Next, we assessed the voltage depen-
dence of Ca2?current inactivation. Wild-type channel
inactivation was complete at ?20 mV and slightly de-
creased at more positive potentials, as expected for
vation (Lee et al., 1985) (Figure 5C). In contrast, the
maximum attained inactivation was only 56% for G406R
channels. Relief of inactivation was greater for G406R
comparedto wtchannelsat potentials ??30 mV(Figure
5C). This observation suggests that mutant channels
have lost voltage-dependent Ca2?current inactivation.
The time constant for inactivation (?) was a U-shaped
function of voltage for wt channels but increased with
membrane voltage for mutant channels (Figure 5D). The
current amplitudes measured at the peak of the current-
voltage (I/V) relationship were similar (p ? 0.32) and
averaged 70 ? 12 pA for wt (n ? 11) and 94 ? 20 pA
for G406R (n ? 9). The shape of the normalized I/V
relationship (Figure 5E) was only slightly altered by the
mutant channels, consistent with a mere ?3 mV shift in
aNine affected individuals were male, and eight were female.
family. We found that the G406R mutation resided on
the maternal chromosome. Thus, in one case, G406R
arose de novo in a parent during development, leading
to mosaicism. Taken together, these data indicate that
a recurrent, de novo G406R mutation of the CaV1.2 gene
Cav1.2 Is Widely Expressed
Previous studies indicate that the CaV1.2 gene was ex-
pressed in heart, brain, smooth muscle, and pituitary
and adrenal glands (Ertel et al., 2000). However, the TS
phenotype suggested a broader expression pattern of
the alternatively spliced form of CaV1.2 containing exon
8A. To determine the pattern of expression in humans,
we used exon 8A as a probe for Northern and dot blot
analyses (Figure 3). This exon was highly expressed in
containing exon 8A was also expressed in multiple adult
and fetal tissues, including brain, gastrointestinal sys-
tem, lungs, immune system, smooth muscle, and testis.
These data indicate that exon 8A of the CaV1.2 gene is
widely expressed in humans.
Calcium Channel Mutation in Arrhythmia and Autism
Table 2. Oligonucleotide Pairs Used to Amplify Exons of CaV1.2 Gene
Exon Forward Oligonucelotide Reverse OligonucleotideSize (C)a
a(C), condition; two different conditions were used for PCR, see Experimental Procedures.
bExons 2, 44, 49, and 50 are long, and each was amplified with two overlapping oligonucleotide pairs.
cOligonucleotides for exon 47 also amplify an apparent noncoding duplication of the genomic sequences encompassing exons 45–49 found
3? of the CaV1.2 gene. The duplicated exon 47 differs at several nucleotide positions, which should not be mistaken for mutations.
the voltage dependence of activation (Figure 5F). These
data indicate that G406R substantially impairs voltage-
dependent channel inactivation.
a loss of voltage-dependent inactivation, we recorded
Ba2?(40 mM) as a charge carrier. Ba2?currents inacti-
vate much slower than Ca2?currents because, in the
absence of extracellular Ca2?, channel inactivation is
almost entirely dependent on transmembrane voltage
(Lee et al., 1985). The time-dependent inactivation was
dramatically reduced by the mutation (Figures 5G and
Figure 2. Identical De Novo Cav1.2 Missense Mutation Causes Timothy Syndrome
(A) TS pedigree showing sporadic occurrence of the disease phenotype and de novo G1216A missense mutation. This mutation leads to the
substitution of glycine 406 with arginine (G406R). Circles and squares indicate females and males, respectively. Filled and empty symbols
denote affected and unaffected individuals. Sequence tracings were derived from blood DNA samples unless otherwise indicated.
(B) TS family with two affected children. A small mutant peak (green, arrow) in the mother’s sequence from oral mucosa DNA is apparent.
This peak is not seen in the sequence of her blood DNA, indicating mosaicism. Germline mosaicism explains the presence of two affected
children in this family. The individual with a slash is deceased.
(C) Amino acid sequence alignment showing conservation of glycine 406 from multiple species. Bracket indicates the end of the sixth
transmembrane segment of domain I (DI/S6).
(D) Predicted topology of CaV1.2, showing the location of the mutation.
5H). Next, we determined the voltage dependence of
inactivation. Whereas wt channels inactivated ?90%
after a conditioning pulse to ?30 mV, G406R channels
inactivated ?20% at the same potential (Figure 5I).
These data demonstrate that the G406R mutation pro-
duces maintained inward Ca2?currents by causing
Block of L-type calcium channels by dihydropyridines
is enhanced by inactivation (Bean, 1984; Sanguinetti
and Kass, 1984). To determine if mutant channels with
defective inactivation were still affected by these drugs,
pus oocytes. The IC50(the drug concentration at which
50% of the current is inhibited) for block of peak current
by nisoldipine was 74 ? 7 nM for wt channels (two to
six cellsper concentration). TheIC50forG406R channels
the end of a 1 s pulse. These data indicate that mutant
channels remain sensitive to dihydropyridines and sug-
gest that these drugs or other calcium channel blockers
may be useful to treat TS.
G406R Prolongs Simulated Action Potentials
A prominent feature of TS is prolongation of the QT
interval and lethal arrhythmias. An important function of
CaV1.2 channels is mediating the plateau phase of the
Calcium Channel Mutation in Arrhythmia and Autism
Figure 3. The Cav1.2 Gene Is Widely Expressed
(A) Human Northern blot analyses show expression of CaV1.2 mRNA containing exon 8A in brain, heart, bladder, prostate, uterus, stomach,
and other tissues.
(B) mRNA dot blot demonstrates expression of CaV1.2 mRNA containing exon 8A in multiple tissues, including many regions of the brain.
cardiac action potential. We predicted that a slowed
rate of channel inactivation would prolong the inward
delay cardiomyocyte repolarization. We determined
that, in the heart, 23% of CaV1.2 channels contained
exon 8A. Thus, in the heterozygous state, only 11.5%
of CaV1.2 channels carry the G406R mutation. To simu-
late the effect of the TS mutation, we assumed these
ratios in a dynamic model of a mammalian ventricular
myocyte (Faber and Rudy, 2000). We altered the voltage
dependence of L-type calcium channel inactivation to
mimic the expected biophysical effects of the mutation
in heterozygous condition (Figure 6A, blue triangles).
The net effect on total CaV1.2 channel inactivation was
small. However, this resulted in a maintained inward
Ca2?current that prolonged action potential duration by
17% (Figure 6B, blue traces). Simulations also indicated
that 35% reduction of the abnormal L-type Ca2?current
could restore normal action potential duration (Figure
tials, consistent with the QT interval prolongation and
increased risk of arrhythmia in TS.
We conclude that the G406R mutation of the CaV1.2
and developmental defects in TS. Several lines of evi-
dence support this conclusion. First, we identified an
Figure 4. The Cav1.2 Gene Is Expressed in Multiple Mouse Tissues
(A) In the brain, the CaV1.2 gene is expressed in cortex (C), hippocampus (H), thalamus (T), hypothalamus (HT), caudate putamen (CP), and
amygdala (A). For all tissues, in situ hybridization experiment with antisense probe is shown in the top panel, and sense probe (control) is
(B) Magnification shows expression in the granular (GrDG) and polymorph (PoDG) layers of the hippocampal dentate gyrus.
(C) CaV1.2 gene is expressed throughout adult heart.
(D) Higher magnification of heart ventricle.
(E–H) Expression in ductus arteriosus ([E], E12.5), retina (left arrows), and sclera (right arrows) of eye ([F], E16.5), developing digits ([G], E12.5),
and tooth papilla ([H], P0).
identical, de novo missense mutation of the CaV1.2 gene
in 13 of 13 TS individuals. This mutation was not present
in controls, and the affected amino acid G406 was com-
pletely conserved across species. Second, expression
of the CaV1.2 gene in brain, teeth, digits, lungs, ductus
arteriosus, and the immune system was consistent with
the TS phenotype. Third, the CaV1.2 gene was strongly
expressed in the heart. Gain-of-function mutations of
and cardiac arrhythmias. Fourth, functional expression
of mutant channels demonstrated that G406R had a
dramatic effect on channel inactivation, causing pro-
Calcium Channel Mutation in Arrhythmia and Autism
Figure 5. Timothy Syndrome Mutation Reduces Cav1.2 Channel Inactivation
Inactivation is a time-, voltage-, and Ca2?-dependent decrease of channel current. (A and B) Wild-type (A) and G406R (B) CaV1.2 channel
currents recorded from CHO cells in response to voltage pulses applied in 10 mV increments from ?40 to ?60 mV. External solution contained
15 mM CaCl2. Note that inward Ca2?current is markedly prolonged by the mutation. (C) Voltage dependence of Ca2?current inactivation in
CHO cells for wt channels (circles; V1/2? ?8 mV, k ? 6.9 mV; n ? 5) and G406R channels (squares; V1/2? 4 mV, k ? 10.6 mV; n ? 5). Note
that the overall extent of G406R channel inactivation is reduced and that the Ca2?-dependent component of inactivation (ascending limb of
the relationship) is accentuated. These data suggest reduced voltage-dependent inactivation for G406R channels. Reduced inactivation results
in a maintained inward current and delayed repolarization of the action potential.
(D) Time constants of Ca2?current inactivation as a function of voltage (n ? 5–9).
(E) G406R channels have similar Ca2?current-voltage (I/V) relationship compared to wt.
(F) Voltage dependence of Ca2?current activation is not significantly altered by the mutation (n ? 9–11).
(G and H) G406R causes nearly complete loss of voltage-dependent channel inactivation. Wild-type (G) and G406R (H) CaV1.2 channel currents
were recorded from Xenopus oocytes in response to voltage pulses applied in 10 mV increments from ?70 to ?40 mV. External solution
contained 40 mM BaCl2to eliminate Ca2?-dependent inactivation.
(I) Voltage dependence of Ba2?current inactivation in oocytes for wt (V1/2? ?8 mV, k ? 8.9 mV; n ? 7) and G406R channels (V1/2? 0 mV, k ?
15.9 mV; n ? 6).
indicated that the G406R effect on channel function
tent with the TS phenotype.
It is remarkable that all TS individuals carry the identi-
cal de novo mutation. One reason that G406R always
arose de novo is the early fatality caused by the muta-
tion, making its inheritance rare. Two factors may ex-
plain the unusual recurrence of this mutation. First, our
physiological studies indicate that arginine at this posi-
Figure 6. Computer Modeling Shows Prolonged Action Potentials in G406R Heterozygotes
(A) Simulated voltage dependence of calcium channel current inactivation for wt (circles), G406R (squares), and wt/G406R heterozygotes (blue
triangles). The relative voltage-dependent inactivation gate term fssis plotted as a function of transmembrane voltage. In the heart and brain,
mRNA analysis indicated that ?23% of CaV1.2 channels contain exon 8A. As a result, heterozygotes are predicted to express 11.5% mutant
channels, leading to small effect on total CaV1.2 channel inactivation.
(B) Although the net effect on total CaV1.2 channel inactivation is small, cardiac action potentials (upper panel) are prolonged by 17%. L-type
Ca2?currents for wt (black) and heterozygous (blue) channels are shown in the lower panel. Hetrozygous Ca2?currents must be reduced by
35% to simulate normal action potentials (red trace).
tion has a profound gain-of-function effect. Gain-of-
function mutations are uncommon and may be domain
specific. The DI/S6 segment is known to be important
for voltage-dependent inactivation of calcium channels
(Herlitze et al., 1997; Shi and Soldatov, 2002; Zhang et
al., 1994). Second, the mutated nucleotide (G1216) is
located in a CpG dinucleotide on the noncoding strand.
Deamination of a methylated cytosine causes the muta-
tion of CpG to TpG, the most common mechanism for
mutations (Cooper and Youssoufian, 1988; Vitkup et al.,
G to A change on the coding strand in a subsequent
cycle of DNA replication. Thus, this is a mutational hot-
spot. Together, these factors explain the unusual recur-
rence of G406R.
How does a single amino acid substitution of CaV1.2
cause the striking phenotypic abnormalities of TS? Ex-
function and the wide tissue expression of CaV1.2. In
the pancreas, Ca2?mediates insulin secretion by pan-
creatic ? cells. Episodic dysfunction of CaV1.2 signaling
likely accounts for the intermittent hypoglycemia that
led to the death of two affected children. In the heart,
maintained depolarizing Ca2?current through mutant
CaV1.2 channels causes lengthening of cardiac action
and life-threatening arrhythmias. Now that we know the
molecular basis of TS, prenatal and neonatal diagnosis
is feasible. Early diagnosis is important, as cardiac
arrhythmias and other features of this disorder are
By contrast with these physiological defects, many
phenotypic abnormalities of TS, such as syndactyly and
congenital heart disease, are developmental. CaV1.2 is
highly expressed in apical ectodermal ridge cells of de-
veloping digits. It has been shown that destruction of
these cells causes syndactyly (Hurle and Ganan, 1986).
It is likely that Ca2?-induced cell death (Orrenius et al.,
2003) in the apical ectodermal ridge is the mechanism
of syndactyly in TS. Abnormalities in cell death may also
lead to the failure of the developing ductus arteriosus
to properly close (Imamura et al., 2000; Tananari et al.,
2000). Thus, TS demonstrates the importance of Ca2?
signaling in human development. Genetically modified
mice harboring G406R may address mechanistic ques-
tions raised by these findings.
The fact that CaV1.2 is associated with autism is of
difficulties in social interaction, communication deficits,
notypes varies considerably in autism spectrum disor-
childhood disintegrative disorder, Rett syndrome, and
pervasive developmental disorder not otherwise speci-
fied (PDD NOS). In the general population, autism spec-
morbidity(Bryson etal.,2003; Volkmarand Pauls,2003).
Epidemiologic studies estimate that 200,000–400,000
children are affected in the United States alone (Fom-
bonne, 2003). Despite their importance, very little is
als with TS met the criteria for autism or had severe
deficits of language and social development suggest
that abnormal Ca2?signaling may contribute to these
disorders. As autism is a uniquely human phenotype,
future work will focus on the genetic analysis of CaV1.2
and other calcium channels in nonsyndromic forms of
autism and autism spectrum disorders. Future studies
will also determine if features of TS are amenable to
calcium channel blocker therapy.
Calcium Channel Mutation in Arrhythmia and Autism
(Promega). Transformed colonies were screened by PCR for the
presence of exons 8 or 8A using the original oligonucleotide pair.
To identify colonies carrying only exon 8A, the same colony collec-
tion was then tested using a forward oligonucleotide specific to
exon 8A (8AF5?-CTGGGTCAATGATGCCGTAG) and the 9R reverse
oligonucleotide. DNA from 30 of 157 clones was sequenced to con-
firmthe results.To controlfor thecloning efficiencyof thefragments
carrying each exon, we cloned PCR fragments amplified from a
template mixture of cDNA containing exon 8 and cDNA containing
8A in a 1:1 ratio and screened colonies as detailed above. No differ-
ence in cloning efficiency was observed.
Nonradioactive in situ hybridization was performed as described
(Berger and Hediger, 2001), using a digoxigenin (DIG)-labeled ?800
nucleotide cRNA probe from the C-terminal region of the mouse
CaV1.2 gene. The probe was derived from a PCR fragment amplified
from mouse heart Marathon-Ready cDNA (BD Biosciences Clon-
tech) using MF5?-AGGCTGGCTTGCGCACCTT and MR5?-GAGA
GATGTCTCCCCCTTGA oligonucleotides. Frozen sections (10 ?m)
slides (Fisher). Sections were then fixed, acetylated, and hybridized
70?C. Hybridized probe was visualized using alkaline phosphatase-
conjugated anti-DIG Fab fragments (Roche) and 5-bromo-4-chloro-
3-indolyl-phosphate/nitro-blue tetrazolium (BCIP/NBT) substrate
(Kierkegard and Perry Laboratories). Sections were rinsed several
times in 100 mM Tris, 150 mM NaCl, and 20 mM EDTA (pH 9.5) and
coverslipped with glycerol gelatin (Sigma). Control sections were
Digital imagesfor antisenseand sense probesfor eachsection were
captured using identical microscope settings.
Subject Ascertainment and Phenotypic Analysis
guardians according to standards established by local institutional
review boards. Phenotypic analyses included history, physical ex-
amination, electrocardiography, and echocardiography. Five chil-
dren were evaluated forbehavioral phenotypes and cognitive devel-
opment. These tests included the Diagnostic Criteria for Autistic
Disorder, DSM-IV, Autism Screening Questionnaire, Autism Diag-
nostic Interview—Revised, the Child Behavior Checklist, Vineland
Adaptive Behavior Scales, and the Autism Diagnostic Observation
Schedule (Berument et al., 1999; Lord et al., 1994, 2000; Task Force
Ability Scales, Clinical Evaluation of Language Fundamentals—III,
Goldman-Fristoe Test of Articulation, and NEPSY (Korkman et al.,
Fisher’s exact test and data from the study with highest prevalence
of autism and autism spectrum disorders (60 of 8,896 individuals)
were used to determine the p value for the association of these
disorders and TS (Bertrand et al., 2001). This comparison gave the
most conservative estimate of the p value. Fisher’s exact test was
also used to assess the p value for the association of G406R with
the TS phenotype.
Genotypic and Sequence Analyses
Genomic DNA from peripheral blood lymphocytes or cell lines de-
rived from Epstein-Barr virus-transformed lymphocytes was pre-
pared using Puregene DNA isolation kit (Gentra Systems). Genomic
DNA from buccal swabs and sperm was prepared using QIAamp
DNA Mini Kit (Qiagen). Oligonucleotides to all known exons of the
CaV1.2 gene were designed to genomic sequences found in the
Celera database using Oligo6.6 (Molecular Biology Insights, Table
2). PCR amplification of DNA samples and mutational analyses were
carried out as previously described (Splawski et al., 1997b). Two
different conditions were used for PCR: (1) 94?C for 2 min, 35 cycles
of 10 s at 94?C, 20 s at 58?C, and 20 s at 72?C, followed by 5 min
extension at 72?C; and (2) same as (1), but annealing was done at
and 4% formamide (Table 2).
Oligonucleotides OriF5?-TACACTAATCATCATAGGGTCAT and
Ori2R5?-TAGCGATTCCCAGTTTAGGTAC were used to amplify a
fragment of 1122 nucleotides containing part of exon 8A and adja-
cent intron 8A sequences. PCR products were obtained with Pfu
Ultra HF DNA polymerase (Stratagene), purified, and sequenced.
An intronic polymorphism (C to G) was identified 344 nucleotides
downstream from exon 8A. PCR fragments were then cloned using
the PCR-Script Amp Cloning Kit (Stratagene) to separate the prod-
for each individual was sequenced to determine the parent chromo-
some on which the mutation arose.
DNA Constructs for Functional Expression
Full-length human wt CaV1.2 cDNAs (accession number Z34815),
sion vector systems, were a generous gift from Dr. N. Soldatov. The
G406Rmutation wasintroducedbysite-directed mutagenesisusing
QuikChange (Stratagene) into the Xenopus expression clone. Sub-
sequently, an AfeI/SgrAI fragment containing the introduced muta-
tion was cloned into the AfeI and SgrAI sites of the wt pcDNA3
clone to obtain the mammalian G406R expression construct.
CaV?2bis the ? subunit splice variant associated with CaV1.2 in
the human heart (Colecraft et al., 2002). We obtained a cDNA clone
containing the 5? end sequence of CaV?2b from RZPD (clone
DKFZp313F1242,RZPD, Germany)andpurchasedthe splicevariant
CaV?2a(the 3? sequences of CaV?2aand CaV?2bare identical) from
Genecopoeia (clone GC-T4617, Genecopoeia). EcoRI/PshAI frag-
ment from DKFZp313F1242 and PshAI/NotI fragment from GC-
T4617 were cloned into the EcoRI and NotI sites of pcDNA3.1 (In-
vitrogen) to obtain the full-length cDNA clone for human CaV?2b
(accession number AAG01473). The rabbit CaV?2bclone (accession
number CAA45575, amino acid sequence 96% identical to human
CaV?2b) for expression in Xenopus oocytes was a kind gift from Dr.
CaV?2?1 is the ?2? subunit associated with CaV1.2 in the heart
(Arikkath and Campbell, 2003). Full-length clone for the human
CaV?2?1subunit (accession number NP_000713) was obtained by
ligation of a NotI/Bpu10I fragment from IMAGE clone 2006073 and
a Bpu10I/XbaI fragment from a PCR product, amplified from human
heart Marathon-Ready cDNA (BD Biosciences Clontech) using oli-
gonucleotides A2D1F5?-TGAATGTAGCTTCATTTAACAGCA-3? and
site underlined), into the NotI and XbaI sites of pcDNA3.1 (In-
vitrogen). The rabbit CaV?2?1clone (accession number AAA81562,
amino acid sequence 96% identical to human CaV?2?1) for expres-
sion in Xenopus oocytes was a kind gift from Dr. N. Dascal.
The full-length clones for all eight expression constructs de-
scribed above were sequenced inforward and reverse direction and
compared to genomic DNA to ensure that no unintended mutations
were present or introduced.
mRNA Expression and cDNA Analyses
Blot analyses were performed using human 12-lane multiple-tissue
Northern blots I and III (BD Biosciences Clontech). mRNA dot blot
Array 3 (BD Biosciences Clontech). A 115 base pair PCR fragment,
amplified using HF5?-TGGGTCAATGATGCCGTAGG and HR5?-GAA
AACTCTCCGCTAAGCACA oligonucleotides, was used as a probe
for exon 8A containing mRNAs. The fragment was labeled with the
otide instead of the provided random 9-mers. Hybridization and
washing conditions followed manufacturer’s suggestions. The blots
were exposed to film for 3 days.
Human heart marathon-ready cDNA (BD Biosciences Clontech)
and human heart and brain race-ready cDNAs (Ambion) were ana-
lyzed to estimate the ratio of CaV1.2 transcripts containing exon 8A
to transcripts containing exon 8. Briefly, PCR products amplified
using the 7F and 9R oligonucleotides (forward from exon 7, 7F5?-
TCACGGTGTTCCAGTGCATC and reverse from exon 9, 9R5?-CAG
GTAGCCTTTGAGATCCTC) were ligated into pGEM-T Easy Vector
Transfection and Solutions for CHO Cells
CHO cells were cultured in Ham’s F-12 Media and transiently trans-
fected using Lipofectamine 2000 (GIBCO). Cells were transfected
for 18 hr in 35 mm dishes containing 18 ?l Lipofectamine, 242 ?l
lecular Probes), 4 ?g of either wt or mutant human CaV1.2, and
1 ?g each of human CaV?2band human CaV?2?1subunit cDNAs. The
extracellular solution contained the following, in mM: 130 NMDG,
15 CaCl2, 5 KCl, and 10 HEPES (pH 7.4 with HCl, 22?C–25?C). The
intracellular pipette solution contained the following, in mM: 120 Cs
methanesulfonate, 5 CaCl2, 2 MgCl2, 10 EGTA, 2 MgATP, and 10
HEPES (pH 7.3 with CsOH). This solution results in an [Ca2?]iof
?110 nM as calculated with WinMaxc (Bers et al., 1994).
Fondazione Cariplo (S.G.P.) is gratefully acknowledged.
Received: July 9, 2004
Revised: August 9, 2004
Accepted: August 17, 2004
Published: September 30, 2004
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Injection and Solutions for Oocytes
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capped polyA cRNA from linearized cDNA templates were per-
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cRNAs encoding wt or mutant human CaV1.2 subunit (11 ng) plus
rabbit CaV?2b(2.7 ng) and rabbit CaV?2?1(2.7 ng) subunits. The extra-
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Voltage Clamp and Data Analysis
Whole-cell Ca2?currents in fluorescent CHO cells were recorded
using standard techniques (Hamill et al., 1981) and an Axopatch 200
patch-clamp amplifier (Axon Instruments) 2–3 days after transfec-
tion with cDNA. Voltage dependence of Ca2?current inactivation in
CHO cells was determined with a two-pulse protocol. The relative
mV) was plotted as a function of the variable voltage of the first
pulse (0.8s). Ba2?currentsthrough calcium channelswere recorded
from oocytes using standard two microelectrode voltage clamp
techniques (Stuhmer, 1992) 2–10 days after injection of cRNA. Volt-
age dependence of Ba2?current inactivation in oocytes was deter-
mined with a two-pulse protocol. The relative magnitude of inward
current elicited during the second pulse (to ?10 mV) is plotted as
a function ofthe variable voltage ofthe first pulse (2 s).Data acquisi-
tion and analyses were performed using pCLAMP8 (Axon Instru-
ments). Currents were filtered at 2 kHz and digitized at 10 kHz.
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the voltage dependence of CaV1.2 inactivation. Data are presented
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Action Potential Modeling
A dynamic model of mammalian ventricular myocytes (Faber and
Rudy, 2000) was used to simulate the effect of the TS mutation in
heterozygotes. For these simulations, the stimulation rate was set
at 60 per min. Action potential waveforms and L-type Ca2?currents
were computed after 100 stimulations. Channel properties were
term fssto a heterozygous condition in which the exon 8A containing
CaV1.2 protein represents 23% (11.5% wt and 11.5% G406R) of the
by the G406R mutation (as measured in CHO cells) were reduced,
assuming only 0.115 of the total channels were mutated (V1/2was
shifted by ?1.2 mV, and the minimal value for fsswas set at 0.106).
We are grateful to all of the individuals with TS and their families
for donated time and samples. We would also like to express our
gratitude to C. Badame, K. Braegger, S. Etheridge, T. Carson, D.
Goldman, T. Klitzner, J. Skinner, A. Moss, H. Stalker, G.M. Vincent,
M. Marks, J. Towbin, M. Pun, C-L. Lien, GCRC Children’s Hospital
Boston, SADS Foundation, and the UK SADS Foundation. We thank
N. Soldatov and N. Dascal for expression constructs and D. Clap-
ham, S. Orkin, L. Kunkel, K. Thomas, and F. Engel for critically
reviewing the manuscript. Funding from NIH (HL46401 and HL52338
for M.T.K. and M.C.S., DC03610 and MH66398 for H.T.-F.), Donald
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