Download (F193L) in the KCNQ1 gene associated with long

Clinical Science (2003) 104, 377–382 (Printed in Great Britain)
Clinical and electrophysiological
characterization of a novel mutation
(F193L) in the KCNQ1 gene associated
with long QT syndrome
Masato YAMAGUCHI*, Masami SHIMIZU*, Hidekazu INO*, Hidenobu TERAI*,
Kenshi HAYASHI*, Hiroshi MABUCHI*, Naoto HOSHI† and Haruhiro HIGASHIDA†
*Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science,
Kanazawa University, Takara-machi 13-1, Kanazawa 920-8641, Japan, and †Biophysical Genetics, Division of Neuroscience,
Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8641, Japan
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KCNQ1 is a gene encoding an α subunit of voltage-gated cardiac K+ channels, with properties
similar to the slowly activating delayed rectifier K+ current, and one of the genes causing long
QT syndrome (LQTS). However, genotype–phenotype correlations of the KCNQ1 gene
mutations are not fully understood. The aims of this study were to identify a mutation in the
KCNQ1 gene in patients with LQTS, and to characterize the clinical manifestations and
electrophysiological properties of the mutation. We screened and identified mutations by PCR,
single-strand conformational polymorphism analysis and DNA sequencing. We identified a novel
mutation [Phe193Leu (F193L)] in the KCNQ1 gene in one family with LQTS. The patients with
this mutation showed a mildly affected phenotype. The proband was a 17-year-old girl who had
a prolonged QT interval. Her elder brother, father and paternal grandmother also had the
mutation. None of them had any history of syncope. Sudden death was not found in this family.
Next, we studied the electrophysiological characteristics of the F193L mutation in the KCNQ1
gene using the expression system in Xenopus oocytes and the two-microelectrode voltage-clamp
technique. Co-expression of F193L KCNQ1 with the K+ channel minK suppressed peak (by
23.3 %) and tail (by 38.2 %) currents compared with those obtained by the combination of wildtype (WT) KCNQ1 and minK. Time constants of current activation in F193L KCNQ1 and F193L
KCNQ1jminK were significantly slower than those of WT KCNQ1 and WT KCNQ1jminK.
This electrophysiological study indicates that F193L causes less severe KCNQ1 current
suppression, and thereby this mutation may result in a mildly affected phenotype.
INTRODUCTION
The long QT syndrome (LQTS) is a cardiac disease
characterized by a prolongation of ventricular repolarization and recurrent episodes of life-threatening
ventricular tachyarrhythmias, specifically torsades de
pointes, leading to sudden death [1]. Inherited LQTS is
represented by the autosomal dominant Romano–Ward
syndrome [2,3] and the autosomal recessive Jervell and
Lange-Nielsen syndrome ( JLNS) [4]. In addition to the
cardiac phenotype, JLNS patients have severe bilateral
congenital deafness. Mutations in five genes are known to
Key words : long QT syndrome, KCNQ1 gene mutation, mild phenotype.
Abbreviations : ECG, electrocardiogram ; I , slow component of the delayed rectifier K+ current ; LQTS, long QT syndrome ;
Ks
F193L, Phe193Leu ; JLNS, Jervell and Lange-Nielsen syndrome ; QTc, QT interval corrected for heart rate ; RFLP, restriction
fragment length polymorphism ; SSCP, single-strand conformational polymorphism ; WT, wild-type.
Correspondence : Dr Masato Yamaguchi (e-mail yonken2!med.kanazawa-u.ac.jp).
# 2003 The Biochemical Society and the Medical Research Society
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cause LQTS. These genes code for cardiac ion channels
participating in the control of the action potential. Two
of the genes, KCNQ1 and HERG, code for voltage-gated
K+ channels [5,6], whereas SCN5A codes for an Na+
channel [7] and KCNE1 and KCNE2 code for the K+
channels minK and MiRP1 [8,9]. minK and MiRP1 are
known to associate with the KCNQ1 and HERG
channels, giving rise to the cardiac delayed rectifier K+
currents IKs (slow component) and IKr (rapid component).
Priori et al. [10] reported that LQTS may appear with
a very low penetrance in some families, and the family
members considered to be normal may be silent gene
carriers and are unexpectedly at risk of developing
torsades de pointes when they are exposed to repolarization-prolonging drugs. Recently, we found a case
similar to that reported by Priori et al. [10], which was
detected by large screening. In the present study, we
describe a 17-year-old woman who had a long QT
interval on 12-lead electrocardiogram (ECG) without
symptoms such as syncope and faintness. A novel
missense mutation [Phe193Leu (F193L)] in the S2–S3
linker protein encoded by the KCNQ1 gene was identified. In order to test the assumption of the relationship
between the genotype and phenotype of this category of
LQTS with in vitro evidence, we characterized the effect
of the F193L mutation in the KCNQ1 gene on outward
currents in Xenopus oocytes.
Site-directed mutagenesis and cRNA in vitro
transcription
The KCNQ1 and KCNE1 (minK K+ channel) cDNAs in
the pSP64 vector were generously provided by Dr
Michael C. Sanguinetti (Eccles Program in Human
Biology and Genetics, University of Utah, Salt Lake
City, UT, U.S.A.) and Dr Richard Swanson (Department
of Pharmacology, Merck Research Laboratories, West
Point, PA, U.S.A.) respectively. The F193L KCNQ1
cDNA was constructed by an overlap-extension strategy
[15]. Wild-type (WT) KCNQ1 cDNA and F193L
KCNQ1 cDNA were linearized by digestion with NotI,
and cRNAs were prepared with the mMESSAGE
mMACHINE kit (Ambion, Austin, TX, U.S.A.) using
SP6 RNA polymerase.
Oocyte preparation and injection
Defolliculated Xenopus laevis oocytes (stage V–VI) were
isolated as described previously [16]. Each oocyte was
injected with 50 nl of cRNA containing 8.0 ng of WT
KCNQ1 cRNA, 8.0 ng of F193L KCNQ1 cRNA, a
combined cRNA composed of 8.0 ng of WT KCNQ1
and 1.0 ng of KCNE1, or a combined cRNA composed
of 8.0 ng of F193L KCNQ1 and 1.0 ng of KCNE1. The
oocytes were incubated at 16 mC in modified Barth’s
solution (87.4 mM NaCl, 1 mM KCl, 2.4 mM NaHCO ,
$
10 mM Hepes, 0.82 mM MgSO , 0.66 mM NaNO and
%
$
0.74 mM CaCl , pH 7.5) supplemented with penicillin
#
(100 µg\ml) and streptomycin (100 µg\ml). The oocytes
were studied 2 days after injection.
METHODS
Electrophysiological experiments
DNA isolation and mutation analysis
Genetic analysis was performed after obtaining written
informed consent. Genomic DNA was purified from
subjects’ white blood cells after which in vitro amplification was performed by PCR [11]. Single-strand
conformational polymorphism (SSCP) analysis of the
amplified DNA was performed to screen for mutations
in the KCNQ1, HERG, SCN5A, KCNE1 and KCNE2
genes, as described previously [12] with a slight modification [13]. Normal and aberrant SSCP products were
isolated and sequenced by ABI PRISM 310 (PerkinElmer,
Foster City, CA, U.S.A.). For the KCNQ1 gene, we
sequenced all 16 exons in the proband using 16 primer
pairs [14]. To confirm the missense mutation, which
serves as the basis of this study, restriction fragment
length polymorphism (RFLP) analysis was performed.
Using a mutagenic primer, gene amplification by PCR
introduced an artificial MvaI site into the PCR product
only for the T C allele (F193L). Digestion of the PCR
products derived from the mutant allele with MvaI gave
rise to fragments of 180 bp instead of 200 bp when
resolved on a polyacrylamide gel.
# 2003 The Biochemical Society and the Medical Research Society
Membrane currents were studied using the twomicroelectrode voltage-clamp technique with an amplifier AXOCLAMP-2A (Axon Instruments, Foster City,
CA, U.S.A.) at 23–25 mC, as described previously [17].
During recording, oocytes were perfused continuously
with ND 96 solution (96 mM NaCl, 2 mM KCl, 5 mM
MgCl , 0.3 mM CaCl and 5 mM Hepes, pH 7.6). Data
#
#
acquisition and analysis were performed with a Digi
DATA 1200 A\D converter (Axon Instruments) and
pCLAMP (version 5.5.1 ; Axon Instruments).
Voltage-clamp protocols and data analysis
All pulse protocols used are described in the Figure
legends. Data analysis was carried out using Clampfit
(version 6.1 ; Axon Instruments). The voltage-dependence of KCNQ1 current activation was determined for
each oocyte by fitting peak values of tail current (Itail)
versus test potential to a Boltzmann function in the
following form : Itail l Itail−max\o1jexp[(V"/#kVt)\k]q,
where V"/# is the voltage at which I is half of Imax, Vt is the
prepulse of the test potential, and k is the slope factor.
Characterization of an F193L mutation in the KCNQ1 gene
Statistical analysis
All values are expressed as meanspS.E.M. Differences
within these values were evaluated by ANOVA and
unpaired Student’s t test when appropriate. P 0.05 was
considered statistically significant.
the proband’s family members revealed that her grandmother, father and one elder brother had the identical
mutation (Figure 1B). This mutation was not identified in
over 100 Japanese control subjects or 140 probands with
LQTS without known genetic defects.
Phenotypic characterization
RESULTS
Genetic analysis
An abnormal migration pattern in the PCR-SSCP assay
was identified in the proband in one fragment encompassing exon 3 of the KCNQ1 gene. Sequence analyses
revealed a heterozygous mutation leading to a single base
substitution (T C) at position 739 in the KCNQ1 gene,
resulting in an amino acid change from phenylalanine to
leucine (F193L) (Figure 1A). PCR-RFLP analysis from
Figure 1
The proband was a 17-year-old girl. A long QT interval
was revealed by ECG during a routine medical check-up.
Her audiometrics were normal, and serum K+, Mg#+ and
Ca#+ concentrations were also normal. Among the
carriers of the mutation, the proband and her grandmother exhibited long QT intervals on ECG (QTc
intervals of 520 and 525 ms"/# respectively, where QTc is
the QT interval corrected for heart rate), but her father
and elder brother had normal QT intervals. None of the
carriers took any medication that might have affected
Genetic analysis
(A) Direct sequencing revealed a heterozygous nucleotide exchange (T739C) in the KCNQ1 gene that caused an amino acid exchange from phenylalanine to leucine at
codon 193 (F193L). (B) Pedigree and RFLP analysis. I, II and III indicate generations. #, no F193L mutation ; $, contain the F193L mutation. The arrow
indicates the proband. Upper numbers show age (in years) and lower numbers show QTc intervals. The blot from the PCR-RFLP analysis shows the heterozygous mutation
in the KCNQ1 gene, which leads to the change in susceptibility of DNA to MvaI, as shown by the appearance of the 180 bp band. The ECG for the proband, showing
a prolonged QTc interval, is also presented. (C) Upper panel, the topology of KCNQ1. The arrow shows the position of F193L. Lower panel, multiple sequence alignment
analysis of KCNQ1 and homologous proteins. Sequence alignment denotes the S2–S3 linker, and the asterisk indicates the location of F193.
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M. Yamaguchi and others
Figure 2
Expression study
(A) Functional expression of WT KCNQ1 and F193L KCNQ1 alone or co-expressed with minK subunits in Xenopus oocytes. Current traces represent typical examples of
# 2003 The Biochemical Society and the Medical Research Society
Characterization of an F193L mutation in the KCNQ1 gene
the characteristics of the ECG, or had previously experienced an episode of palpitation or syncope. We did not
detect ventricular arrhythmia on the proband’s Holter
ECG monitoring or treadmill exercise test.
F193L KCNQ1 expression assay
In the topology of the KCNQ1 subunit, the mutation
identified was located in the intracellular linker of the
S2–S3 domains (Figure 1C). Although the charge of
the amino acids did not change as a result of the substitution, we hypothesized that the missense mutation
would result in some alterations of KCNQ1 channel
function.
To test this hypothesis, we examined the functional
effect of the F193L mutation on the KCNQ1 subunit by
using the Xenopus oocyte expression system. Figure 2(A)
shows the representative current traces recorded
from those cells transiently transfected with 8 ng of
WT KCNQ1 or F193L KCNQ1 without minK. WT
KCNQ1 displayed rapidly activating and non-inactivating outward currents on depolarizing test pulses.
F193L KCNQ1 was able to form functional channels
that resulted in a macroscopic outward current greater
than non-injected oocytes, and smaller than WT
KCNQ1. Consistent with previous studies [18,19], coexpression of WT KCNQ1 with minK dramatically
altered the characteristics of the KCNQ1 channels,
producing IKs. Although co-expression of F193L
KCNQ1 with minK also elicited similar outward
currents, the peak and tail currents of mutants were
smaller than those of WT KCNQ1 (Figure 2B). The peak
and tail currents were reduced by 23.3 and 38.2 %
respectively, which were not statistically significantly
different from the values obtained with WT KCNQ1.
The onset of current activation was estimated by fitting
current traces to a two-exponential function. The current
activations in both WT KCNQ1 and WT
KCNQ1jminK were voltage-dependent, and the time
constants were decreased at more positive potentials.
Time constants of F193L KCNQ1 and F193L
KCNQ1jminK were significantly slower than those of
WT KCNQ1 and WT KCNQ1jminK (Figure 2C).
DISCUSSION
The present study describes a novel missense mutation in
the intracellular linker of the S2–S3 domain of KCNQ1
(F193L) (Figure 1C), which did not show the dominantnegative effect on IKs functions. The proband had a long
QT interval on 12-lead ECG, but she had no symptoms.
PCR-RFLP analyses revealed the same mutation in her
grandmother, father and one elder brother, but not in her
grandfather, mother or another elder brother. However,
none of her family members had sudden cardiac death or
syncope. Although genotypical mutations were found,
only her grandmother displayed a long QT interval on
ECG. Conrath et al. [20] reported that adult males with
LQTS1 had shorter QTc intervals than adult females, and
that this difference did not exist in LQTS2 patients. Our
present results, together with their findings, suggest that
the disease penetrance in the patients with a mutation in
the KCNQ1 gene may be affected by gender differences.
The present study is the first report of an evaluation of
the expression of a mutation in the S2–S3 linker. By
analysing functionality with WT or mutant clones in the
absence or presence of minK in oocytes, we found that
the F193L mutation in the KCNQ1 gene evoked an
outward current with smaller amplitude than WT
KCNQ1 gene. Wollnik et al. [21] have reported that the
mutation found in the putative channel pore abolishes
channel activity and reduces the activity of WT KvLQT1
by a dominant-negative mechanism. Donger et al. [22]
found a missense mutation [Arg555Cys (R555C)] in the
C-terminal domain of KCNQ1 in 44 patients from three
congenital LQTS families. R555C is associated with a
‘ forme fruste ’ phenotype in which there is a significantly
less pronounced QT prolongation or lower percentages
of symptomatic carriers and sudden death than those in
the transmembrane domains of KCNQ1. Thus, analogous with the C-terminal mutant, the mutant in the
cytoplasmic site of the S2–S3 linker might show a mild
phenotype. Yoshida et al. [23] reported that a missense
mutation in the S2–S3 inner loop of HERG caused
bradycardia inducing LQTS, whereas the electrophysiological data showed that the mutation affected the HERG
channel less severely and did not show dominantnegative suppression ; data very similar to those in the
present study.
On the other hand, it has been reported [24,25] that the
degree of IKs dysfunction caused by many KCNQ1 gene
mutations does not always correlate with that of QT
prolongation or of other cardiac symptoms. In addition,
the autosomal recessive LQTS (JLNS) in allelic diseases
results from mutations in the KCNQ1 and KCNE1
genes. A significant proportion of patients with heterozygous KCNQ1 gene mutations might have a mild or
normal phenotype, and incomplete penetrance therefore
appears frequent for KCNQ1 gene mutants. Even if the
phenotype is mild, the clinical importance is that these
carriers may easily develop cardiac events after exposure
to antiarrhythmic drugs.
the outward currents recorded. All currents were elicited by 2-s voltage steps from a holding potential of k80 mV to test potentials ranging from k40 to j60 mV
in 20-mV increments (inset). Recordings were made 2 days after cRNA injection. (B) Current–voltage relationships of the peak (upper) and tail (lower) currents measured
at the end of, or after, the 2-s pulses for WT KCNQ1 (WT) and F193L KCNQ1 (F193L) in the absence or presence of minK. (C) Time constants (τ) for slow and fast
components of activation for WT KCNQ1 (WT) and F193L KCNQ1 (F193L) in the absence or presence of minK. *P 0.05 compared with WT KCNQ1.
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In conclusion, we have identified at the molecular level
and functionally characterized a novel mutation (F193L)
in the intracellular S2–S3 linker of KCNQ1 that is
associated with a mild phenotype in a family. It remains
unclear why those having this mutation of the KCNQ1
gene express different phenotypes (low penetrance) in
males and females. One reason may be that the manifestation of the LQTS phenotype could be influenced by
multiple factors in each case of congenital LQTS.
Although the patient has a mild phenotype at present, we
assume she has a potential risk, making strict follow-up
mandatory. The present case suggests that patients having
a long QT interval on ECG may require genetic analyses,
which might be a better way of preventing acquired
LQTS.
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ACKNOWLEDGMENTS
We thank Dr M. C. Sanguinetti (University of Utah) and
Dr R. Swanson (Merck Research Laboratories) for
supplying the KCNQ1 and KCNE1 cDNAs respectively.
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Received 7 June 2002/25 November 2002; accepted 21 January 2003
# 2003 The Biochemical Society and the Medical Research Society