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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 A B S T R A C T 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 377 378 M. Yamaguchi and others 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. # 2003 The Biochemical Society and the Medical Research Society 379 380 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. # 2003 The Biochemical Society and the Medical Research Society 381 382 M. Yamaguchi and others 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. 9 10 11 12 13 14 15 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. 16 17 REFERENCES 1 Vincent, M. G., Timothy, K. W., Leppert, M. and Keating, M. (1992) The spectrum of the symptoms and QT intervals in carriers of the gene for the long-QT syndrome. N. Engl. J. Med. 327, 846–852 2 Romano, C., Gemme, G. and Pongiglione, R. (1963) Artimie cardiach rare delleta pediatrica. II. Accessi sincopali per fibrillazione ventricolare parossitica. Clin. Pediatr. 45, 656–683 3 Ward, O. (1964) A new familial cardiac syndrome in children. J. Ir. Med. Assoc. 54, 103–106 4 Jervell, A. and Lange-Nielsen, F. (1957) Congenital deafmutism, functional heart disease with prolongation of the QT interval, and sudden death. Am. Heart J. 54, 59–68 5 Wang, Q., Curran, M. E., Splawski, I. et al. (1996) Positional cloning of a novel potassium channel gene : KvLQT1 mutations cause cardiac arrhythmias. Nat. Genet. 12, 17–23 6 Curran, M. E., Splawski, I., Timothy, K. W., Vincent, G. 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