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Cardiovascular Research 2004 63(1):60-68; doi:10.1016/j.cardiores.2004.02.011
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Copyright © 2004, European Society of Cardiology

New KCNQ1 mutations leading to haploinsufficiency in a general population

Defective trafficking of a KvLQT1 mutant

Laetitia Gouasa,1, Chloe Bellocqb,1, Myriam Bertheta, Franck Potetb, Sophie Demolombeb, Anne Forhanc, Rachel Lescassea, Françoise Simond, Beverley Balkauc, Isabelle Denjoye, Bernard Hainqued, Isabelle Barób, Pascale Guicheney*,a and The D.E.S.I.R. Study Group

aINSERM U582, Institut de Myologie, IFR no. 14, Groupe Hospitalier Pitié-Salpêtrière, 47 Boulevard de l'Hôpital, 75651 Paris Cedex 13, France
bINSERM U533, Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, Faculté de Médecine, Nantes, France
cINSERM U258, IFR no. 69, Villejuif, France
dService de Biochimie B, IFR no. 14, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Faculté de Pharmacie-Université Paris V, Paris, France
eService de Cardiologie, Groupe Hospitalier Lariboisière, Paris, France

*Corresponding author. Tel: +33-1-42-16-57-50; fax: +33-1-42-16-57-00. Email address: p.guicheney{at}myologie.chups.jussieu.fr

Received 21 October 2003; revised 6 February 2004; accepted 18 February 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: KCNQ1 mutations lead to the long QT syndrome (LQTS), characterized by a prolonged QT interval, syncopes and sudden death. However, some mutations are associated with non-penetrant phenotype (no symptoms, QTc normal or borderline). The objective of this study was to determine whether KCNQ1 variants are associated with borderline QTc prolongation in a general population and to evaluate the frequency of carriers. Methods: We selected 2008 unrelated and untreated healthy individuals from a non-patient population. The KCNQ1 gene was screened by denaturing high-performance liquid chromatography (dHPLC) in 50 men and 50 women presenting the longest QTc intervals (403 to 443 ms). Results: We identified a nonsense mutation, Y148X, and an in-frame deletion of the serine residue 276 ({Delta}S276), in S2 and S5 transmembrane domains, respectively. {Delta}S276 KvLQT1 channels expressed in COS-7 cells failed to conduct any K+ current in the homozygous state. Besides, a slight reduction in channel activity was observed when coexpressed with WT KvLQT1 and IsK. Confocal microscopy performed on transfected COS-7 cells revealed that {Delta}S276 KvLQT1 was retained in the endoplasmic reticulum, whereas WT KvLQT1 was localized in the cell membrane. The two mutation carriers presented borderline QTc interval prolongation at slow heart rate but a 24-h ECG recording revealed a marked QTc prolongation at higher heart rate for the Y148X carrier. Conclusions: In this population, two subjects with borderline QTc prolongations (438 and 443 ms) were carriers of KCNQ1 mutations leading to haploinsufficiency and are potentially at risk of developing drug-induced arrhythmia. The study provides the first demonstration of a defective cell surface localization of a KvLQT1 mutant missing one amino acid in a transmembrane domain.

KEYWORDS Ion channels; K-channel; Long QT syndrome

Abbreviations: D.E.S.I.R., Data from an Epidemiological Study on the Insulin Resistance Syndrome • dHPLC, denaturing high-performance liquid chromatography • DNA, deoxyribonucleic acid • ECG, electrocardiogram • JLNS, Jervell and Lange-Nielsen syndrome • LQTS, Long QT syndrome • PCR, polymerase chain reaction • QTc, QT interval corrected for heart rate with Fridericia formula (ms) • RWS, Romano-Ward syndrome


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The long QT syndrome (LQTS) is an inherited cardiac disorder characterized by a prolonged QT interval on the electrocardiogram (ECG), associated with syncope and a high risk of sudden death. The long QT syndrome occurs as an autosomal dominant inherited form (Romano-Ward Syndrome—RWS), or as an autosomal recessive form (Jervell and Lange-Nielsen Syndrome—JLNS) combining syncopal heart arrhythmias and profound congenital deafness [1]. Drug-induced arrhythmias with QTc prolongation have also been associated with cardiac or noncardiac drug absorption in the context of bradycardia, cardiac ischemia or with defective electrolytic metabolism [2].

The RWS is the most common form of inherited LQTS, although its incidence is unknown. It is estimated to be about one gene carrier in 5000 live births in certain areas, such as in Utah, USA and in Finland [3,4]. The JLNS is a rare disorder, initially estimated at 1.6 and 6 per million (children aged 4 to 15 years) in England, Wales and Ireland [5]. Norwegian data, however, suggested a higher incidence ranging from 1:55,000 to 1:200,000 [6,7].

So far, mutations in six different genes, coding the cardiac potassium channel {alpha} subunits, KCNQ1 (KvLQT1) and KCNH2 (HERG), the β subunits, KCNE1 (IsK or MinK) and KCNE2 (MiRP1), as well as the sodium channel, SCN5A and, recently, ANK2 coding the ankyrin-B protein have been found to underlie the LQTS [8,9].

The {alpha} subunit KvLQT1 is a six spanning-membrane domain protein that homotetramerises and associates with IsK, a one spanning-membrane domain protein, to form the delayed rectifier potassium current (IKs) involved in ventricular repolarisation and endolymph K+ homeostasis of the inner ear [10–12]. Some mutations identified in the KvLQT1 protein have been associated with milder LQTS phenotypes, with borderline QTc prolongation and a low risk of cardiac arrhythmias in the absence of repolarization prolonging drugs. Mild missense mutations have been described either in the C-terminus region of KvLQT1 [13–15], or in the pore region [16,17]. In addition, nonsense and frameshift mutations associated with non-penetrant phenotype (no symptoms, QTc normal or borderline) have been identified in heterozygous carriers in JLNS families [7,18–20].

In this study, we searched for variants in the KCNQ1 gene that could be responsible for a borderline QTc prolongation and to estimate the frequency of carriers in a general population. We screened the KCNQ1 gene in the 100 healthy subjects presenting the longest QT intervals in the French D.E.S.I.R. cohort. We identified two new KCNQ1 mutations: a nonsense mutation, Y148X, in the S2 spanning-membrane domain and an in-frame 3-bp deletion leading to the deletion of the serine 276 residue, {Delta}S276, in the S5 spanning-membrane domain, in two individuals with borderline QTc intervals.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Study population
The Data from an Epidemiological Study on the Insulin Resistance syndrome (D.E.S.I.R.) cohort included Caucasian men and women aged 30–64 years. Participants were recruited from volunteers who agreed to be followed for 9 years and to have a clinical examination every 3 years. This study involved 10 health examination centres from the western and the central parts of France. All individuals gave informed consent to the clinical and genetic study, which was approved by an ethics committee. The investigation conforms with the principles outlined in the Declaration of Helsinki.

The first examination occurred in 1994 (D0) and the second in 1997 (D3). Blood pressure was measured after at least 5-min rest and venous blood samples were collected to determine glucose, triglyceride and HDL-cholesterol concentrations. Historical health data (diabetes, hypertension, cardiovascular disease, etc.), treatment, physical activity, alcohol and tobacco consumption of each subject were collected.

Subjects underwent 12-lead resting ECGs which were analyzed by the Cardionics® software. The QT and RR intervals were automatically measured from all recorded sinus complexes except the first one and two last ones in each lead and then averaged. The QT values were corrected for heart rate according to the Fridericia formula [21]. Subjects with an ECG recording at D0 and D3 (n=3478) form the basis of this study. All subjects with known or detected cardiac pathology, diabetes, treated or fasting plasma glucose (≥7.0 mmol/l), or treated or untreated high blood pressure and subjects taking medication known to prolong the QT interval such as neuroleptics, antiarrhythmics or antihistaminics were excluded. Subjects with a difference ≥30 ms in QTc between the two examinations were also excluded. After correction for age, the DNA samples from the 50 men and 50 women presenting the longest mean QTc intervals of the 2008 remaining subjects were studied.

2.2. Genomic DNA amplification
Genomic DNA was prepared from peripheral blood lymphocytes by standard procedures. Previously designed primers were used to amplify the KCNQ1 exons, except for exon 13 (forward primer 5' GTC AAG CTG TCT GTC CCA CA 3'; reverse primer 5'CGG GAGAGT CCA TTG CAG 3') [22].

2.3. Denaturing high-performance liquid chromatography analysis (dHPLC)
dHPLC analysis was carried out using automated instrumentation (DNA Fragment Analysis System, Transgenomic). PCR products were separated (flow rate of 0.9 ml/min) through a linear acetonitrile gradient determined on the size of the amplicon. Between each sample analysis, the column was regenerated with 75% acetonitrile.

2.4. Sequence analysis
Each exon with an abnormal dHPLC pattern was amplified by a new PCR reaction. The PCR products were sequenced by the dideoxynucleotide chain termination method with fluorescent dideoxynucleotides on an ABI PRISM 377 DNA sequencer (Applied Biosystem, Perkin Elmer).

2.5. In vitro expression studies
2.5.1. Intranuclear injection of plasmids
COS-7 cells, obtained from the American Type Culture Collection (Manassas, VA), were cultured and plasmids were microinjected into cell nuclei at day 1 after plating using the Eppendorf ECET microinjector 5246 system protocol as previously reported [23]. Briefly, plasmids were diluted in a buffer made of (mmol/l): NaCl 40, HEPES 50, NaOH 50, pH 7.4 supplemented with 0.5% fluorescein isothiocyanate-dextran (150 kDa). Human cardiac KvLQT1 isoform 1, KvLQT1 isoform 2 and human IsK cDNA were subcloned into the mammalian expression vector pCI (Promega, Madison, VI) and pRC under the control of a cytomegalovirus enhancer/promoter, respectively. A green fluorescence protein pCI plasmid (a gift from Dr. Rainer Waldmann, Sophia-Antipolis, France) was used as an inert plasmid to ensure that cells were always injected with a constant 15 µg/ml plasmid concentration.

2.5.2. Electrophysiological analysis
Whole-cell currents were recorded as previously described [23]. Cells were placed on the stage of an inverted microscope and continuously superfused with the standard extracellular solution. Stimulation data recording and analysis were performed through a patch-clamp amplifier (Axopatch 200A; Axon instruments, Foster City, CA) and an analog-to-digital converter (Tecmar TM100 Labmaster, Scientific Solutions, Solon, OH) and Acquis1 software (Bio-Logic, Claix, France). A microperfusion system consisted in different tubings conducted at the close vicinity of the recorded cell, allowed local application of experimental solution at 35 °C through gravity. Current measurements were normalised using the cell capacitance. Patch-clamp measurements were presented as the mean±S.E.M. Statistical significance of the observed effects was assessed by means of parametric Student's t-test or nonparametric Mann–Whitney rank sum test. A value of P<0.1 was considered significant [24].

2.5.3. Solutions
The standard extracellular medium contained (mmol/l): NaCl 145; KCl 4; MgCl2 1; CaCl2 1; HEPES 5; glucose 5; pH adjusted to 7.4 with NaOH. The intracellular medium contained (mmol/l): K-gluconate 145, HEPES 5, EGTA 2, hemi Mg-gluconate 2 (free-Mg2+: 0.1), K2ATP 2, pH 7.2 with KOH, whereas the extracellular medium used to record K+ currents contained (mmol/l): Na-gluconate 145, K-gluconate 4, hemi Ca-gluconate 7 (free-Ca2+: 1), hemi Mg-gluconate 4 (free-Mg2+: 1), HEPES 5, glucose 5, pH 7.4 with NaOH. Free activities were calculated using software designed by GL Smith (University of Glasgow, Scotland).

2.6. Confocal microscopy experiments
Green fluorescent protein (GFP) optimized for maximal fluorescence (pEGFP-N2, Clontech Laboratories, Palo Alto, CA) was used for tagging to the C-terminus of WT and mutant KCNQ1 on the pCI-KCNQ1 vector. The pDsRed-ER localization vector, containing the KDEL sequence of the calreticulin protein cloned in frame with the DsRed sequence of pDsRed, was used to visualize the endoplasmic reticulum [25]. The pCI-KCNQ1-GFP plasmid and the pDsRed-ER vector were co-tranfected into COS-7 cells by the PEI method [26]. At 12-h post-transfection, cells were dissociated by enzymatic treatment and seeded on glass in Petri dishes bottomed with a coverslip for 48 h. Cells were then washed three times with phosphate-buffered saline (PBS) and fixed for 10 min with 4% (v/v) paraformaldehyde in PBS. After rinsing another three times with PBS, cells were mounted upside down on a slide glass with a drop of Mowiol (Calbiochem) for confocal microscopic study. The localization of GFP-tagged proteins was pursued using a Leica TCS NT confocal microscope equipped with a krypton-argon laser beam. Cells were imaged using 488- and 647-nm illuminations (for GFP and rhodamine, respectively) and an oil-immersion 100x lens.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Subjects with the longest QTc intervals
The D.E.S.I.R. cohort included 2008 healthy and untreated subjects with two ECG recordings. The mean QTc intervals (D0 and D3) ranged from 332.6 to 442.8 ms. From this subpopulation, we selected the 50 men and 50 women presenting the longest age adjusted QTc intervals. The QTc intervals in men ranged from 403.5 to 438.9 ms (mean±S.D.=412.9±8.8 ms) and varied from 407.7 to 442.8 ms (mean±S.D.=415.8±7.6 ms) in women (Fig. 1). The mean heart rate (beats/min) was 61.1±9.7 (D0) and 61.5±8.2 (D3) for men and 65.7±11.4 (D0) and 66.4±9.1 (D3) for women. The mean age was 46.4±9.2 years for men and 49.5±11.0 years for women.


Figure 1
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  		Fig. 1 Distribution of QTc intervals according to gender (men bullet, women {blacksquare}). Mean QTc interval for men=376.9±15.7 ms (n=972); mean QTc interval for women=383.0±15.4 ms (n=1036). The 50 men and 50 women with the longest QTc intervals are shown as grey and dotted areas, respectively.

 
3.2 Identification of two novel KCNQ1 mutations
In addition of known polymorphisms, we identified two novel mutations (Fig. 2A) in KCNQ1: a substitution of a thymine by an adenine at position 444, leading to a stop codon in exon 1, Y148X, and an in-frame 3-bp deletion, 828–830delCTC, in exon 5 of KCNQ1 (Fig. 2B). Both these mutations were absent on 400 chromosomes from unrelated control subjects. The 3-bp deletion results in an in-frame deletion of serine 276 ({Delta}S276) within the spanning-membrane domain S5, bordering the channel pore. This serine residue is highly conserved throughout evolution, from Caenorhabditis elegans to Homo sapiens and is also conserved among the KCNQ protein family (Fig. 3).


Figure 2
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  		Fig. 2 (A) dHPLC analysis of KCNQ1 exons 1 and 5: abnormal patterns observed for two subjects compared to controls. (B) Sequence analysis of KCNQ1 exon 1 and 5. Upper lane: normal homozygous control. Lower lane: Subjects 50909 and 53000 heterozygous for the T>A transition in exon 1 and for a 3-bp deletion (CTC) in exon 5.

 

Figure 3
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  		Fig. 3 KCNQ1 exon 5 sequence alignments. Serine 276 is a highly conserved residue through evolution from C. elegans to H. sapiens and among the human KCNQ protein family.

 
The Y148X mutation was identified in a 38-year-old man with a mean QTc interval (D0–D3) of 438.9 ms and the {Delta}S276 mutation in a 54-year-old woman with a mean QTc interval of 442.8 ms. Interestingly, these two subjects presented the longest mean QTc intervals of the men and women, respectively.

3.3. Clinical re-evaluation of mutation carriers
The Y148X male carrier had no family history of syncope nor sudden death. Nevertheless, he experienced one syncope during acute hepatitis at age 16 without any documented arrhythmia and the diagnosis of LQTS was not suspected. Since this age, he remained asymptomatic although he practiced sports regularly (jogging twice a week). On three ECGs, the heart rate was slow and QTc was borderline at rest (Fig. 4). The 24-h ECG recording showed a marked QT prolongation at higher heart rate (Fig. 4).


Figure 4
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  		Fig. 4 Electrocardiograms of the male KCNQ1 Y148X and female {Delta}S276 carriers. QT and RR intervals were measured on three consecutive sinus complexes in lead II. QT were corrected for heart rate both by Fridericia (QTcF) and Bazett's formulas (QTcB). (A) ECGs at D0 and D3. (B) Additional ECGs recorded independently. (C) Holter recording (between 13:00 and 21:00) of the Y148X carrier at RR intervals of 600 and 1000 ms. The QTcF given in this figure differed slightly from the QTcF automatically calculated by Cardionics, which were at D0 and D3: 433 and 444 ms for the Y148X carrier, and 440 and 445 ms for the {Delta}S276 carrier.

 
The {Delta}S276 carrier had no family history of syncope or sudden death and never experienced any symptoms. She also presented a slow heart rate on all ECGs recordings (Fig. 4). The QTc values varied from 435 to 462 ms according to Bazett or Fridericia corrections. No Holter recording was available to document ECG modifications at higher heart rates.

3.4. Electrophysiological properties of the {Delta}S276 KvLQT1 isoforms
To determine the effects of the {Delta}S276 mutation on the KvLQT1 channel protein activity, we expressed the wild type (WT) and the mutant channels in COS-7 cells in the presence of the regulator protein IsK. {Delta}S276 KvLQT1/IsK channels failed to produce any K+ current (n=5) in contrast to WT KvLQT1/IsK channels (n=10). When WT and mutated KvLQT1 channels were co-expressed in the presence of IsK, the amplitude of the K+ current was slightly decreased compared with the K+ current measured in cells transfected with the WT KvLQT1/IsK (6.72 ± 2.02 pA/pF, n=10 versus 10.58±2.31 pA/pF, n=10, respectively, P=0.093) (Fig. 5A).


Figure 5
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  		Fig. 5 Current–voltage (I–V) relation for KvLQT1/IsK channels; (A) cells expressing WT KvLQT1 isoform 1 plus IsK(bullet), {Delta}S276 KvLQT1 isoform 1 plus IsK ({blacksquare}) and {Delta}S276 KvLQT1 isoform 1 with KvLQT1 isoform 1 plus IsK ({blacktriangleup}). Inset left: typical current traces elicited by a +40-mV depolarizing pulse in each condition (scales 5 pA/pF, 200 ms); right: voltage protocol. (B) Cells expressing WT KvLQT1 isoform 1 plus IsK in presence of WT KvLQT1 isoform 2 ({bigtriangleup}) or {Delta}S276 KvLQT1 isoform 2 ({circ}). (C) Average tail amplitude of the current related to WT KvLQT1 isoform 1/IsK expression in the presence of {Delta}S276 KvLQT1 isoform 1, WT KvLQT1 isoform 2 or {Delta}S276 KvLQT1 isoform 2. Respective concentrations of plasmids (µg/ml) in the injection mixture are given on the x-axis. NS: non significant, P values when compared to cells injected with WT KvLQT1 isoform 1 plus IsK. Number in parentheses indicates n values.

 
Two different isoforms of KvLQT1 have been described in the human heart [27]. The long isoform, isoform 1, constitutes the pore of the K+ channel. We have shown that the alternative spliced isoform, isoform 2, is shorter than isoform 1 and exerts a dominant-negative activity toward isoform 1 channel activity. The dominant-negative effect of the {Delta}S276 N-terminus truncated KvLQT1 isoform 2 on isoform 1 was studied. The {Delta}S276 KvLQT1 isoform 2 has no effect on the WT KvLQT1 isoform 1/IsK, while co-expression of WT KvLQT1 isoform 2 with WT KvLQT1 isoform 1/IsK generated a smaller K+ current (7.58±2.07 pA/pF, n=11, 3.68±0.91 pA/pF, n=7 P<0.05, respectively) (Fig. 5B). Average tail currents are given in Fig. 5C. Thus, the {Delta}S276 variant (i) induces non-functional homomeric isoform 1 channels, (ii) exerts a weak dominant-negative effect on WT protein, and (iii) abolishes the isoform 2 dominant-negative activity.

3.5. Subcellular localization of WT and {Delta}S276 KvLQT1
To test the possibility of defective cellular trafficking of the {Delta}S276 KvLQT1 mutant, GFP-tagged WT and {Delta}S276 KvLQT1 were transfected in COS-7 cells. Cells transfected with GFP-fused WT KvLQT1 showed a plasma membrane fluorescence pattern (Fig. 6A), whereas the GFP-tagged mutant showed an intracellular fluorescence pattern (Fig. 6B). Furthermore, {Delta}S276 KvLQT1 was co-localized with fluorescence for the ER resident protein DsRed reflecting a processing defect of the mutant (Fig. 6B, right panel). This result correlates with the absence of current in patch-clamp recordings.


Figure 6
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  		Fig. 6 Confocal laser scanning images of COS-7 cells transfected with DsRed ER plasmid and (A) WT KvLQT1-GFP isoform1; (B) {Delta}S276 KvLQT1-GFP isoform 1. Left panels: localization of KvLQT1 channels. Middle panels: localization of DsRed ER. Right panels: superimposed images.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this report, we identified two novel KvLQT1 variants in a non-patient population. A heterozygous nonsense mutation, Y148X, was identified in the S2 spanning-membrane domain of the KvLQT1 protein in a 38-year-old man with QTc intervals of 433.8 and 443.9 ms at a 3-year interval. We also identified a heterozygous in-frame 3-bp deletion, leading to the deletion of a conserved serine residue in the S5 spanning-membrane domain, {Delta}S276, in a 54-year-old woman with QTc intervals of 440.4 and 445.2 ms at 3-year intervals.

Y148X is expected to lead to truncated proteins lacking the P loop, constituting the pore domain, and the C-terminus of the protein containing the assembly domain (CAD), necessary for multimerization [28]. Whether the mutated protein is expressed or not in vivo remains unknown. Theoretically, even expressed, the mutated protein lacking the C-terminus domain should not heteromerize with the WT protein and form functional channels, leading to haploinsufficiency. Such mutations leading to a premature stop codon have been described in some carriers presenting normal or borderline QTc intervals in RWS [15,29] or JLNS families [7,18–20].

Concerning the {Delta}S276 KvLQT1 mutation, whole-cell patch clamp experiments showed that {Delta}S276 KvLQT1 isoforms 1 expressed in COS-7 cells failed to conduct any K+ current in the homozygous state, while in the heterozygous model where {Delta}S276 KvLQT1 and WT KvLQT1 were co-expressed, K+ channel activity was slightly reduced. Confocal microscopy of COS-7 cells transfected with tagged channel subunits showed that {Delta}S276 KvLQT1 was retained in the endoplasmic reticulum. The reason for loss of cell surface localization of the {Delta}S276 KvLQT1 channels is not understood. It is possible that {Delta}S276 KvLQT1 mutant protein results in an abnormally misfolded protein recognized by ER quality control mechanisms, leading to mutants retention in the ER [30]. As for some HERG mutants, {Delta}S276 KvLQT1 proteins might display increased and prolonged association with chaperone proteins in an attempt to refold them into a correct native conformation [31]. Besides, the slight decrease of the K+ current observed when {Delta}S276 mutant and WT KvLQT1 protein were coexpressed in COS-7 cells raised the possibility of a weak dominant-negative suppression of WT currents by {Delta}S276 KvLQT1 proteins. As {Delta}S276 KvLQT1 proteins are trafficking-deficient, the weak dominant-negative suppression of WT current might be explained by an impeded trafficking of WT KvLQT1 due to a co-assembly with {Delta}S276 KvLQT1 proteins for which the multimerization CAD domain is still present.

In JLNS, most of the mutations are frameshift deletions, insertions, nonsense, or missense mutations in the C-terminal assembly domain [7,18–20]. They lead to RNA decay or truncated proteins that cannot form functional channels. Patch clamp experiments have confirmed that JLNS mutant proteins display no functional homomeric channels. Moreover, it has been shown that JLNS mutants exerted no or a weak dominant-negative activity on WT proteins [32,33]. Thus, JLNS patients suffer from severe QTc prolongation with loss of hearing in contrast to heterozygous carriers who in the great majority never experience spontaneous cardiac events but may present drug-induced arrhythmias. In RWS, most of the mutations are missense mutations located in cytoplasmic loops, transmembrane domains or the pore region [8]. The mutated proteins display no functional homomeric channels but can assemble with WT subunits to form channels with modified properties [32,33]. Thus, RWS patients have a severe phenotype and suffer from syncope or sudden death with a prolonged QTc interval on the ECG. However, several studies highlighted that some RWS mutations led to LQTS with low penetrance: borderline QTc interval prolongation and no symptoms [34]. Some rare KCNQ1 mutations have also been shown to lead to prolonged QTc intervals and cardiac symptoms at homozygous or compound heterozygous state without hearing loss [15,17,20]. In this study, the two mutations we identified induced a complete loss of function of the mutated protein. Nevertheless, the macroscopic WT K+ current produced by WT channels was sufficient to maintain a ventricular repolarization compatible with an apparently normal phenotype in carriers. Thus, we identified two new KCNQ1 mutations in a non-patient population compatible with KCNQ1 mutations leading to LQTS with low penetrance as observed in most of JLNS heterozygous carriers or in rare RWS mutation carriers.

Mutations in various ionic channels have been reported to cause defective trafficking and congenital arrhythmias, such as T65P, N470D, A561V, G601S, Y611H, R752W, F805C, V822M and R823W in HERG potassium channels in LQT2 [35–40], L51H in IsK in LQT5 [41] and R1432G, R1232W/T1620M in SCN5A in Brugada syndrome [42,43]. So far, in KCNQ1, only the T587M mutation has been described as causing a defective cell surface localization [44]. In this study, we report that the deletion of a neutral amino acid residue, serine 276, also leads to a defective protein trafficking. {Delta}S276 mutation is localized in the S5 transmembrane domain of KvLQT1 close to the pore-forming extracellular loop between the S5 and S6 domains. Other mutations for which ion channel mutants are retained in perinuclear regions such as A561V and G601S, G611Y in HERG, are also localized in the S5 transmembrane domain and in the pore-forming extracellular loop, respectively [35,36]. The R1432G SCN5A mutation, also inducing protein retention within the endoplasmic reticulum, is similarly localized in the pore-forming extracellular loop between the S5 and S6 domains of the domain III [42].

Two KvLQT1 mutations, {Delta}S276 and T587M, induce a complete loss of function of the mutant protein because of a defective cell surface localization. As a consequence, one would expect that {Delta}S276 and T587M KvLQT1 would lead to a poorly penetrant phenotype in the heterozygous state. However, the phenotype of heterozygous carriers of the T587M mutation was not previously known because Neyroud et al. [22] identified this mutation as a de novo mutation associated with a splice mutation in a compound heterozygous JLN proband. Yamashita et al. [44] also identified the T587M mutation in three unrelated Japanese RW patients. Unexpectedly, the three patients were clearly symptomatic with a significant QTc interval prolongation (508–532 ms) and a family history of sudden cardiac death. Such a severe phenotype in these three Japanese patients, as seen in some rare heterozygous carriers of JLN mutations [29], may be explained by environmental or additional undetermined genetic factors.

In the present study, the two carriers with a KCNQ1 mutation leading to haploinsufficiency exhibited a slow heart rate and borderline QTc interval prolongation. However, a 24-h ECG recording for the male patient revealed a marked QT interval prolongation at higher heart rate. Such significant prolongation of QT interval during adrenergic stimulations has been previously described for LQT1 carriers in comparison to LQT2 carriers [45]. Besides, LQT1 patients with QTc <460 ms have been shown to be sensitive to epinephrine challenge, which has been proposed as a provocative test in silent LQT1 mutation carriers [46]. However, it appears difficult to indicate primarily epinephrine challenge or genetic screening of LQTS genes in every subjects with QTc interval at the upper limit without any individual or family history of cardiac events. Non-penetrant LQTS or acquired LQTS might be involved in these subjects who should first undergo Holter monitoring, a non-invasive and safe technique, to detect abnormalities of duration and morphology of ventricular repolarization, allowing orientation of the genetic screening of LQTS genes [47].

In conclusion, this report is the first to explore whether variants in a cardiac ionic channel could explain mild QTc interval prolongation in a general population and to estimate the frequency of carriers. Although other major cardiac ion channel-encoding genes were not studied, we identified two KCNQ1 mutations leading to haploinsufficiency. The pathogenic consequences of these mutations should be subtle as seen in some heterozygous carriers of JLNS and recessive RWS KCNQ1 mutations. Thus, our data are in favour of the hypothesis that the frequency of carrying a KCNQ1 mutation responsible for a borderline QTc prolongation in a non-patient population could not be so rare in subjects with QTc intervals at the upper limits of the normality. Carriers of such variants could be at risk of developing arrhythmias when exposed to repolarization prolonging drugs. These data should be confirmed by other population studies. Moreover, our study describes for the first time a defective cell surface localization in a KvLQT1 mutant channels missing one amino acid in a transmembrane domain.


   Acknowledgements
 
This work was supported by the Fondation de France, the Fondation Leducq and the Fédération des Maladies Orphelines-Association Française de Recherche Génétique. CB is financially supported by INSERM/Pays-de-Loire. We thank Dr. D.J. Snyders for his kind gift of the pDsRed-ER vector.


   Notes
 
1 These authors contributed equally to the work. Back

{star} Laetitia Gouas and Myriam Berthet performed the genetic analysis and identified the KCNQ1 mutations. Rachel Lescasse had worked on this project before leaving the laboratory U582. They were under the leadership of Dr. Pascale Guicheney. Françoise Simon was very helpful in introducing to the dHPLC technology under the leadership of Bernard Hainque. Chloe Bellocq performed the electrophysiological studies, Franck Potet performed the cellular localization of the {Delta}S276 KvLQT1 mutant and Sophie Demolombe, the site-directed mutagenesis, under the leadership of Isabelle Baró. Anne Forhan and Beverley Balkau, from the D.E.S.I.R. study group, participated in the selection of the study population. Dr. Isabelle Denjoy organized a medical consultation for the two mutation carriers.

Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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