Author + information
- Received July 6, 1999
- Revision received December 13, 1999
- Accepted February 9, 2000
- Published online June 1, 2000.
- Kirsi Piippo, PhD∗,
- Päivi Laitinen, PhD∗,
- Heikki Swan, MD∗,
- Lauri Toivonen, MD∗,
- Matti Viitasalo, MD∗,
- Michael Pasternack, PhD†,
- Kristian Paavonen, MD∗,
- Hugh Chapman, PhD†,‡,
- Kenneth T. Wann, PhD‡,
- Eeva Hirvelä, MD†,
- Antti Sajantila, MD§ and
- Kimmo Kontula, MD∗,* ()
- ↵*Reprint requests and correspondence: Prof. Kimmo Kontula, University of Helsinki, Department of Medicine, Haartmaninkatu 4, 00290 Helsinki, Finland
We studied the clinical characteristics and molecular background underlying a severe phenotype of long QT syndrome (LQTS).
Mutations of cardiac ion channel genes cause LQTS, manifesting as increased risk of ventricular tachycardia and sudden death.
We studied two siblings showing prolonged QT intervals corrected for heart rate (QTc), their asymptomatic parents with only marginally prolonged QTc intervals and their family members. The potassium channel gene HERG was screened for mutations by deoxyribonucleic acid sequencing, and the electrophysiologic consequences of the mutation were studied in vitro using the whole-cell patch-clamp technique.
A novel missense mutation (L552S) in the HERG channel, present in the homozygous state in the affected siblings and in the heterozygous state in their parents, as well as in 38 additional subjects from six LQTS families, was identified. One of the homozygous siblings had 2:1 atrioventricular block immediately after birth, and died at the age of four years after experiencing unexplained hypoglycemia. The other sibling had an episode of torsade de pointes at the age of two years. The mean QTc interval differed significantly (p < 0.001) between heterozygous symptomatic mutation carriers (500 ± 59 ms), asymptomatic mutation carriers (452 ± 34 ms) and noncarriers (412 ± 23 ms). When expressed in vitro, the HERG-L552S formed functional channels with increased activation and deactivation rates.
Our data demonstrate that homozygosity for a HERG mutation can cause a severe cardiac repolarization disorder without other phenotypic abnormalities. Absence of functional HERG channels appears to be one cause for intrauterine and neonatal bradycardia and 2:1 atrioventricular block.
The long QT syndrome (LQTS) is an inherited cardiac channelopathy typically manifesting with ventricular tachycardias, such as torsade de pointes, syncopal attacks and sudden death at a young age (1). The clinical phenotype is due to distorted myocardial repolarization and varies markedly both within affected families and between families (2). On the basis of inheritance and phenotypic characteristics, LQTS was originally subclassified into an autosomal dominant form (Romano-Ward syndrome) (3,4) and a rare and more malignant recessive form associated with congenital deafness (Jervell and Lange-Nielsen syndrome) (5). Recent molecular genetic studies have revealed that these two diseases are, in fact, allelic, resulting from mutations of two potassium channel genes—KVLQT1 and minK (6–10). The KVLQT1 and minK proteins co-assemble to form the slowly activating delayed rectifier potassium (K+) channel (IKs) present in cardiac myocytes and the inner ear (11,12). In addition, mutations of the potassium channel gene HERG (13) or sodium channel gene SCN5A (14) are established causes of Romano-Ward syndrome. The HERG gene encodes an alpha subunit of a voltage-gated, rapidly activating delayed rectifier potassium channel that induces significant repolarizating current IKr in cardiac myocytes (15). Until now, mutations of the HERG gene have been reported in heterozygous patients only.
The LQTS displays marked heterogeneity at the molecular level, and typically each family is affected by a unique mutation of a particular ion channel gene. In the present study, we describe the occurrence and molecular characteristics of an apparent founder mutation of the HERG gene, designated as HERG-Fin, which manifests both in the heterozygous and homozygous state in the Finnish population. Our data demonstrate that homozygosity for a HERG mutation causes a severe cardiac repolarization disorder without other phenotypic abnormalities. The prevalence of the HERG-Fin mutation defines a milieu in which the role of genetic and extrinsic factors, as modifiers of the LQTS phenotype, can be effectively examined.
Patients and control subjects
The proband in family 041090 was a 10-year-old girl (Fig. 1; III/5) who had experienced tachycardia at the age of two years. Her younger sister (Fig. 1; III/6) had intrauterine bradycardia and a prolonged QT interval immediately after birth, and she died at the age of four years. The paternity in the index family was tested using highly polymorphic microsatellite markers, and subject II/8 was interpreted to be father of both III/5 and III/6.
The individuals originally chosen as probands (n = 88) to search for ion channel mutations underlying LQTS were all of Finnish origin, had LQTS without deafness and a prolonged QT interval corrected for heart rate (QTc) (>440 ms) and were symptomatic. Although they originated from different parts of the country, most of them were from Eastern Finland. Specific mutations in the KVLQT1 gene have been characterized in 30% of Finnish subjects with LQTS (16,17), but the disease-causing mutation is still unknown in 57 probands. Informed consent was obtained from all patients, and the study was approved by the Ethical Review Committee of the University of Helsinki. Control deoxyribonucleic acid (DNA) samples were obtained from 100 apparently healthy adult individuals.
The QT interval, defined according to the tangent method, was presented as the mean value of two consecutive QRST complexes, usually in lead II, and corrected for heart rate (QTc) according to Bazett’s formula. Five symptomatic and five asymptomatic heterozygous carriers underwent an exercise stress test, as described earlier (18).
Mutation and haplotype analyses
Genomic DNA was extracted from peripheral leukocytes using standard methods. Exons 4 and 7 of the HERG gene were sequenced after their amplification by polymerase chain reaction (PCR), using primers 5′-ACGACCACGTGCCTCTCCTCTC-3′ and 5′-GGCTGGGGCGGAACGGGTCC-3′ for exon 4 (19) and primers 5′-TGCCCCATCAACGGATGTGC-3′ and 5′-GCCCGCCCCTGGGCACACTCA-3′ for exon 7 (13). The PCR reactions were performed in a standard way (16), and DNA sequencing was carried out using the dye terminator, cycle-sequencing procedure and the ABI Prism 377 automatic DNA sequencer (PE Biosystems, Foster City, California). A comparison of the HERG sequence with its homologues was performed using the BLAST2 sequence alignment program. The nucleotide numbering in the HERG gene is started from the initiation methionine.
A primer-induced restriction assay (PIRA) was applied to develop a simple and specific test for the detection of the L552S mutation, essentially as described earlier (16). In PCR, the PIRA-primer 5′-ACGGCGCGGCCGTGCTGTCCT-3′ was used as the forward primer and 5′-CAGCCAGCCGATGCGTGAGTC-3′ as the reverse primer. The 125–base pair PCR amplicon was digested with the enzyme Mnl I (New England Biolabs, Beverly, Massachusetts), with subsequent analysis of the PCR products by 12% polyacrylamide gel electrophoresis. The mismatch in the PIRA-primer generates an extra Mnl I restriction site in the PCR product of the mutant allele, thus permitting its separation from the normal allele.
Four highly polymorphic microsatellite markers, D7S505, D7S1826, D7S688 and D7S483, flanking the HERG gene were used for haplotype analysis. After PCR, the amplified fragments were separated using the ABI Prism 377 automatic DNA sequencer (PE Biosystems) and analyzed with the Genotyper program (PE Biosystems).
Functional expression of the mutant and wild type HERG channels in COS7 cells
The HERG complementary DNA (cDNA) (U04270) in pcDNA3 plasmid was kindly provided by Dr. Gail Robertson. The in vitro mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California). Transfections were made using the Effectene Transfection Reagent (Qiagen, Valencia, California). An enhanced green fluorescent protein plasmid was transfected with experimental constructs to identify the transfected cells. Whole-cell membrane currents were measured using an EPC-9 amplifier and Pulse/Pulsefit software (HEKA, Lambrecht, Germany). The extracellular solution used in the recordings contained (in mmol/liter): NaCl 150, KCl 5.4, CaCl2 1.8, MgCl2 1 and HEPES 5 (pH 7.4 with NaOH), and the recording pipettes were filled with a solution containing (in mmol/liter): KCl 150, MgCl2 2, Bapta 10 and HEPES 10 (pH 7.2 with KOH). The recordings were made 48 to 72 h after transfection. The activation and deactivation rates (τact and τdeact) were calculated by fitting a single exponential to the activation and tail currents. The half-activation voltage (V0.5) was calculated from the immediate tail currents at −60 mV using the Bolzmann equation:
All data are presented as the mean value ± SD for clinical analysis and the mean value ± SEM for electrophysiologic measurements. Analysis of variance was used to compare data between the different groups. A p value <0.05 was considered statistically significant.
Phenotypic characteristics of the index family
A family (pedigree 041090, Fig. 1) with two severely affected children but phenotypically normal parents was examined for the first time in 1992. The proband was a girl who had an episode of torsade de pointes tachycardia at the age of two years, which coincided with the eruption of diabetes mellitus (III/5). Her actual QT interval was 480 ms at the heart rate 100 beats/min (QTc 677 ms) (Fig. 2). Intravenous administration of lidocaine and magnesium did not abolish ventricular arrhythmias, but the arrhythmias disappeared with amiodarone infusion. Since then, she has been treated with beta–anti-adrenergic medication and insulin, with no recurrence of symptoms (QTc 510 ms). Her audiogram showed normal hearing bilaterally. Her sister (III/6) had intrauterine bradycardia and was found to have a prolonged QTc interval (513 ms) after birth. In addition, immediately after delivery, 2:1 atrioventricular block and frequent ventricular premature complexes occurred (Fig. 2) and were still present five months after birth. Infusion of isoprenaline abolished these ventricular arrhythmias during the early phase. The patient was subsequently treated with propranolol and remained symptomless (QTc 470 ms). However, at the age of four years, an unexplained hypoglycemic episode occurred while the patient had an acute respiratory infection. No medications, except propranolol, were used at the time of hypoglycemia. The severe illness led to aspiration and death soon thereafter. The parents of the siblings had borderline QTc intervals (436 and 441 ms, respectively) (Fig. 2) and were free of any cardiac symptoms. Also, the youngest child of the family (III/7) was phenotypically normal (QTc 420 ms).
Identification of the HERG-Fin mutation
The coding sequence of the HERG gene (U04270) was directly sequenced, and a nucleotide change (T1655C) was detected in exon 7 (data not shown). This mutation is predicted to result in substitution of a conserved leucine by serine at amino acid position 552, located in the cytoplasmic end of the transmembrane domain S5 of the HERG subunit. The L552S mutation was present in a heterozygous state in DNA samples of both the father and mother of the family 041090 and in a homozygous state in DNA samples of the two affected daughters (Fig. 1). In addition, the mutation was present in the heterozygous form in four other families. Altogether, the L552S mutation was found in the heterozygous form in 40 individuals, but it was absent in 63 unaffected family members and in all 100 control subjects.
Six carriers of the L552S mutation (designated as HERG-Fin) of the 88 unrelated Finnish LQTS probands correspond to a prevalence rate of 7% for this mutation among Finnish LQTS families. Four microsatellite markers adjacent to the HERG gene locus were analyzed in all families to construct disease-associated haplotypes; a common haplotype was present in all affected probands. The affected grandparents of the homozygous individuals originated from the same commune in Lapland, whereas the place of birth of the other ancestors was in Eastern Finland.
Clinical characteristics of the HERG-Fin mutation carriers
Ten of the 35 heterozygous carriers of the HERG-Fin mutation were symptomatic and 25 were asymptomatic. Ninety percent of the former patients had their symptoms at rest, whereas there was only one patient in whom concomitant physical exercise was associated with a syncopal spell. There was no instance of an association of a QT-prolonging drug therapy with cardiac events. In one heterozygous carrier, failure with beta–anti-adrenergic medication led to implantation of a cardiac defibrillator.
The mean QTc value of nonaffected family members (412 ± 23 ms [range 370 to 475]) was significantly lower than that of all heterozygotes (466 ± 47 ms [range 400 to 620]; p < 0.001) (Fig. 3). There was a trend toward a longer mean QTc interval in heterozygous female mutation carriers (471 ± 47 ms) than in male carriers (447 ± 43 ms; p = NS). Mean QT and QTc intervals of the symptomatic mutation carriers were significantly (p < 0.001) longer than those of asymptomatic carriers. However, there was a marked female preponderance among the symptomatic carriers (Table 1). None of the mutation carriers included in these calculations had any medication or other disease, except for the ventricular repolarization abnormality at the moment of QTc measurement.
In ergometer exercise testing, the mean maximal heart rate was 99% of the expected age-related maximal heart rate in both the symptomatic (n = 5) and asymptomatic (n = 5) heterozygous carriers studied. Three of five symptomatic carriers exhibited frequent ventricular premature complexes, whereas none of the asymptomatic carriers had any arrhythmias during the exercise stress test (data not shown).
Functional properties of the L552S mutation in vitro
To characterize the properties of the L552S mutation in vitro, it was introduced into a HERG-wild type (HERG-wt) cDNA construct (HERG-L552S). The HERG-wt and HERG-L552S were transiently expressed in COS7 cells and used in whole-cell, patch-clamp experiments. The HERG-L552S construct produced functional channels in COS7 cells, with robust currents similar in amplitude to those generated by the HERG-wt construct (Fig. 4). The current–voltage relationships of the peak tail currents were slightly different for HERG-wt and HERG-L552S, and HERG-L552S displayed a marked increase in the rate of activation and deactivation (Fig. 4). The activation rate constants (τact) at an activation voltage of 0 mV were 891 ± 116 ms and 151 ± 28 ms for HERG-wt and HERG-L552S, respectively, whereas the corresponding deactivation rate constants at −60 mV (τdeact) were 623 ± 101 ms and 120 ± 19 ms, respectively (n = 8). The HERG-L552S was activated at slightly lower voltages than was HERG-wt. The half-activation voltage (V0.5) was −5.4 ± 2.1 mV for HERG-wt and −13.8 ± 1.6 mV for HERG-L552S. The increase in the HERG-L552S deactivation rate is expected to result in a decreased channel activity at the end of the cardiac action potential repolarization, making the cell more vulnerable to early afterdepolarizations (EADs).
The clinical presentation of a homozygous state in type 2 long QT syndrome (LQT2) is described for the first time, to our knowledge. Although a novel missense mutation of the HERG potassium channel gene was found to cause typical Romano-Ward syndrome in its heterozygous carriers, homozygosity for this mutation resulted in a severe cardiac phenotype, similar to that described earlier for homozygous KVLQT1 and minK mutation carriers (Jervell and Lange-Nielsen syndrome), but no other abnormalities. The present study also suggests that homozygosity for a HERG channel defect may be associated with abnormalities in atrioventricular conduction and glucose homeostasis.
The HERG-Fin mutation affects a structure conserved in evolution and important in channel deactivation
The HERG-Fin (L552S) mutation is located in the intracellular amino terminal end of the S5 domain. This region, together with the pore and S4 domains, appears to be the most conserved part of the eag gene family (20). Furthermore, leucine 552 is highly conserved in the homologous genes of several distinct species, including those of Drosophila, Mus musculus, Rattus norwegicus, Oryctolagus cuniculus and Bos taurus, as well as in genes of the Shaker gene family.
In whole-cell patch-clamp experiments, the L552S mutation displayed a marked increase in both activation and deactivation rates, as compared with wild type channels. The amplitude and timing of the HERG channel conductance during the cardiac action potential are critically dependent on the fast inactivation and slow deactivation properties of the channel. During the cardiac action potential, the rapid inactivation properties of HERG result in only a small conductance at depolarized membrane potentials, and the conductance reaches a maximum at the end of the repolarization phase at voltages well below −60 mV (21). Although the peak HERG current occurs during the action potential repolarization, the HERG conductance normally peaks during the terminal action potential repolarization phase, effectively preventing the occurrence of EADs. An increase in the HERG deactivation rate, as seen here for the HERG-Fin mutation, is expected to decrease the HERG conductance during terminal action potential repolarization. This could prolong the repolarization and reduce the stability of the membrane in the face of depolarizing currents, subjecting the cell to EADs.
In voltage-gated potassium channels Kv2.1 and Kv3.1, specific residues in the S5 region are shown to regulate deactivation of the channel (22). The S5 domain (codons 549 to 572) of the HERG subunit could have a similar function, as the adjacent S4–S5 linker, corresponding to amino acids 539–548, is suggested to interact directly with the N-terminus of the protein, and thus modulate both deactivation and inactivation of the HERG channel (23). Indeed, our present data show that the amino acid at position 552 is important in channel deactivation, suggesting that the intracellular NH2 domain may also interact with L552 when the channel is in the open conformation (23).
Phenotype in HERG-Fin homozygotes and heterozygotes and influence of gender
Siblings homozygous for HERG-Fin had markedly prolonged QTc intervals and experienced ventricular tachycardias at a very young age. The cardiac phenotype therefore resembles that present in the Jervell and Lange-Nielsen syndrome. A neonatal bradycardia resulting from 2:1 atrioventricular block was observed in one of the homozygous siblings. Such arrhythmias have been observed infrequently in neonates, often with a poor prognosis (24). An interesting feature in both homozygous patients was the association between cardiac events and disturbances of serum glucose levels. Although the pathophysiologic relation between the HERG channel defect and disorder of glucose homeostasis, if any, remains to be explored, it is tempting to suggest that the arrhythmias of the HERG-Fin homozygotes were related to rapid changes in serum potassium concentration, possibly arising from an imbalance between circulating insulin and glucose levels. Although there appear to be no data on expression of the HERG gene in the inner ear, the present findings suggest that the HERG channel, unlike KVLQT1 and minK, has no critical auditory function.
The spectrum of the phenotype of the heterozygous HERG-Fin carriers was wide, ranging from asymptomatic patients to those who had experienced recurrent syncopal spells. It is of particular interest that only one heterozygous individual experienced syncopes related to physical exercise, whereas the symptoms of the others occurred at rest. The low incidence of exercise-related cardiac events in this group of patients with LQT2 is in congruence with our previous findings demonstrating that the QT interval of the patients with LQT2 shortens efficiently during exercise-induced acceleration of heart rate (18).
In the present series of genetically homogeneous patients with LQTS, all but one of the 10 symptomatic patients were women. Our data are in accordance with those derived from the LQTS International Registry (25,26). Earlier findings in acquired LQTS due to pharmacologic blockade of HERG, likewise, suggest that women are more prone to QT interval prolongation and risk of cardiac arrhythmias than are men (27,28). The proarrhythmic effect of female sex hormone in the presence of IKr-blocking agents has also been demonstrated in animal studies (27,29).
Evidence for a HERG-Fin founder gene
Six apparently unrelated families of Finnish origin were shown to carry the identical HERG-Fin mutation. We propose that this mutation has been enriched in Finland as a consequence of a founder gene effect, by virtue of the relatively young age of the Finnish population and its historically isolated nature owing to geographic, cultural and linguistic reasons (30). Ancestors of all families appeared to be clustered in Northern and Eastern Finland. On the basis of studies on Y-chromosomal haplotypes, the province of Lapland is supposed to be a genetic isolate possibly inhabited by people living in Sweden about 2,000 years ago (31,32). The geographic distribution of affected grandparents, combined with Finnish population history, predicts a founder gene effect that was confirmed by haplotype analysis. Previously, a founder mutation of the KVLQT1 gene was tentatively suggested to occur in a South African population (33).
Clinical implications and conclusions
This study describes the first patients with LQTS who were homozygous for a HERG potassium channel gene mutation, presenting with a severe cardiac phenotype but no other phenotypic characteristics. Our findings, based on a genetically homogeneous cohort of patients with LQTS, also suggest that female gender may be an additional risk factor for QT prolongation and possibly for occurrence of cardiac events in LQT2.
We warmly thank Dr. Gail Robertson, PhD (University of Wisconsin, Madison, Wisconsin) for providing the HERG cDNA. We also thank Ms. Susanna Tverin, Ms. Tuula Soppela-Loponen, Ms. Hanna Ranne and Ms. Mari Palviainen for their expert technical assistance.
Note: While this study was in press, Hoorntje et al. (Circulation 1999;100:1264–7) described an infant and a stillborn child, both homozygous for a HERG gene mutation.
☆ This work was supported by grants from the Medical Council of the Finnish Academy, the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation, the Instrumentarium Science Foundation and the Aarne Koskelo Foundation.
- deoxyribonucleic acid
- early afterdepolarization
- rapidly activated delayed rectifier potassium channel
- slowly activated delayed rectifier potassium channel
- long QT syndrome
- type 2 long QT syndrome
- polymerase chain reaction
- primer-induced restriction assay
- QT interval corrected for heart rate
- Received July 6, 1999.
- Revision received December 13, 1999.
- Accepted February 9, 2000.
- American College of Cardiology
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