1
The Phenotypic Spectrum of the Most
Frequent Mutation Responsible for the Short
QT Syndrome
Running Title - Hu et al: Most Frequent Mutation in Short QT Syndrome
Dan Hu, MD, PhD,1,2,a,* Jiancheng Zhang, MD, PhD,3,a Yang Li, MD, PhD, 4 Ryan Pfeiffer, BS,1 Michael H. Gollob, MD, 5 Jeff Healey, MD,6 Daniel Toshio Harrell, BS,7 Naomasa Makita, MD, PhD,7 Haruhiko Abe, MD, PhD,8 Yaxun Sun, MD,9 Jihong Guo, MD,10 Li Zhang, MD,11 Ganxin Yan, MD,11 Douglas Mah, MD,12 Edward P. Walsh, MD,12 Harris B. Leopold, MD,13 Carla Giustetto, MD,14 Fiorenzo Gaita, MD,14 Andrea Mazzanti, MD,15 Silvia G. Priori, MD, PhD,15,16 Charles Antzelevitch, PhD,11 Hector
Barajas-Martinez, PhD1,*
1 Molecular Genetics Department, Masonic Medical Research Laboratory, Utica, New York, USA
2 Department of Cardiology and Cardiovascular Research Institute, Renmin Hospital of Wuhan University, Wuhan, China
3 Department of Cardiology, Provincial Clinical Medicine College of Fujian Medical University, Fujian, China
4 Institute of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, China
5 Department of Cardiology, Toronto General Hospital, University of Toronto, Toronto, ON, Canada 6 Population Health Research Institute, McMaster University, Hamilton, ON, Canada
7 Department of Molecular Physiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
8 Department of Heart Rhythm Management, University of Occupational and Environmental Health, Fukuoka,Japan
9Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China
10 Department of Cardiac Electrophysiology, Division of Cardiology, People’s Hospital, Peking University, Beijing, China
11 Lankenau Institute for Medical Research and Jefferson Medical College, Philadelphia, PA, USA 12 Cardiac Electrophysiology Division, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
2
13 Connecticut Children’s Medical Center, University of Connecticut School of Medicine, Hartford, CT, USA
14 Division of Cardiology, University of Torino, Department of Medical Sciences, "Città della Salute e della Scienza” Hospital, Torino, Italy
15 Molecular Cardiology, IRCCS Salvatore Maugeri Foundation, Pavia, Italy 16
Department of Molecular Medicine, University of Pavia, Pavia, Italy
a Authors contributed equally.
Funding
This study was supported by grants from NIH/NHLBI HL47678 (CA); CONACYT #FM201866 (DH and HBM); the Masons of New York, Florida, Massachusetts, Connecticut, Maryland, Wisconsin and Rhode Island; and National Natural Science Foundation of China #81100067(YXS).
Conflict of Interest: None.
* Address for correspondence: Dan Hu, MD. PhD. FHRS.
Research Scientist II, Clinical Laboratory Consultant
Molecular Genetics Department
Masonic Medical Research Laboratory 2150 Bleecker St, Utica, NY, 13501 Phone: (315) 735-2217
FAX: (315) 735-5648 Email: [email protected]
Hector Barajas-Martinez, PhD. FHRS. Research Scientist II, Clinical Laboratory Director
Molecular Genetics Department
Masonic Medical Research Laboratory 2150 Bleecker St, Utica, NY, 13501 Phone: (315) 735-2217
FAX: (315) 735-5648 Email: [email protected]
Word Count: 6,201 words (Text – 4,280; References – 1,085; Figure Legends – 836) Abstract: 248 words; Condensed Abstract: 100 words;
3 Abstract
Objectives: This study sought to evaluate the phenotypic and functional expression of an apparent hotspot mutation associated with SQTS.
Background: The short QT syndrome (SQTS) is a rare channelopathy associated with a high risk of life-threatening arrhythmias and sudden cardiac death (SCD).
Methods: Probands diagnosed with SQTS and their family members were evaluated clinically and genetically. KCNH2 wild-type (WT) and mutant genes were transiently expressed in HEK293 cells and currents were recorded using whole-cell patch clamp and action potential (AP) clamp techniques.
Results: KCNH2-T618I was identified in 18 members of 7 unrelated families (10 males,; Median age, 24.0 years). All carriers showed 100% penetrance with variable expressivity. Eighteen members in 7 families had SCD. The average QTc of probands and all carriers was 294.1±23.8 and 313.2±23.8 ms, respectively. Seven carriers received an ICD, Quinidine with adequate plasma levels was effective in prolonging QTc among 5 cases, but 3 remained with premature ventricular contraction (PVC) or nonsustained ventricular tachycardia (nsVT). Bepridil successfully prevented drug-refractory ventricular fibrillation (VF) with only 19 ms prolongation of QTc in one case. Functional expression studies revealed a significant gain-of-function of IKr tail-current density compared with WT (207.5±25.8 vs. 122.7±12.2 pA/pF; n=10 respectively, P<0.01). AP clamp recordings showed IKr was larger and peak repolarizing current occurred earlier in mutant vs. WT channels.
Conclusions: We report the clinical characteristics and biophysical properties of the most frequent mutation contributing to genetically-identified SQTS probands. These findings extend our understanding of the spectrum of KCNH2 channel defects in SQTS.
Key Words: Short QT Syndrome; Sudden Cardiac Death; Genetics; Channelopathy; Therapy
4 Condensed Abstract
We find that KCNH2-T618I is the most frequent mutation associated with SQTS worldwide, with a high incidence of VT/VF or SCD and complete penetrance. ICD implantation is still the first-choice therapy, although the technical difficulties and a high rate of complications are also encountered. Quinidine is effective in prolonging QTc, but whether this translates into decreased SCD is still unknown. Bepridil may be the new alternative to prevent VT/VF in SQTS. The mutation causes a major gain of function in IKr, leading to abbreviation of APs and QT, which in turn is thought to developing a reentrant substrate for arrhythmias.
5 ABBREVIATIONS AND ACRONYMS
AF = atrial fibrillation AP = action potential ECG = electrocardiogram ER = early repolarization
GWAS = genome-wide association studies ICD = implantable cardioverter defibrillator MAF = minor allele frequency
PRI = PR interval
QTc = Bazett corrected QT interval SCD = sudden cardiac death
SD = sudden death
SQTS = Short QT Syndrome
VT/VF = ventricular tachycardia/fibrillation WT = wild type
6 Introduction
Short QT Syndrome (SQTS) is a rare genetic disease characterized by an abnormally short QT interval in subjects with structurally normal hearts. It is a recognized cause of cardiac rhythm disorders, including both atrial and ventricular arrhythmias, and sudden cardiac death (SCD) (1-4). As an inherited ion-channelopathy, the molecular basis for SQTS has been associated with mutations in 6 genes: KCNH2 (IKr, SQTS1), KCNQ1 (IKs, SQTS2), and KCNJ2 (IK1, SQTS3), which encode different
potassium channels; CACNA1C, CACNB2b and CACNA2D1 (SQTS4-6), which encode the L-type calcium channel (ICa)(5). The latter are reported to induce a combined
Brugada-SQTS phenotype(6-11). When expressed in heterologous experimental systems, SQTS mutations display an increase in the potassium currents involved or a decrease in the calcium current, thus resulting in acceleration of the repolarization process and an abnormally short QT interval.
The precise QTc cutoff that define SQTS remain controversial, similar to Long QT syndrome. To improve a diagnostic approach, Gollob and colleagues proposed a diagnostic scorecard for SQTS incorporating both clinical and genetic criteria(12). Later, modified Gollob Score was applied in some studies as a guide to risk stratification(4). To date, few mutations have been identified to cause SQTS and attempts of genotype-phenotype correlation are limited by small numbers of mutation carriers. Herein, we report the data from the largest cohort of SQTS patients carrying the hotspot mutation T618I in KCNH2, and provide genotype-phenotype correlation on 18 mutation carriers
7
from 7 unrelated families. The functional characteristics and electrophysiological observations of this hotspot mutation are described.
8 Methods
Clinical Investigation and Follow-up
SQTS index cases were defined as those with QTc ≤340 ms even if they were asymptomatic. Those with QTc between 340 ms and 360 ms associated with one or more of the following, were also considered SQTS cases: (a) A confirmed pathogenic mutation, (b) A family history of SQTS, (c) A family history of SD at age < 40 years, (d) Survival from a ventricular tachycardia / fibrillation (VT/VF) episode in the absence of heart disease(13). Structural heart disease was excluded by echocardiography and/or magnetic resonance imaging (MRI). The final study group from the North America, Europe and Asia, was comprised of 7 proband-identified families [4 males, 57.1%; median age, 30.0 years; interquartile range (IQR), 27.0 years; the 25th and 75th quartiles, 16.0 and 43.0 years]. There were 18 cases of SCD or SD in the families. Family 4-7 have been partially reported previously(14-17).
Demographic data of probands and their family members were collected. In all patients, at least one 12-lead ECG was obtained at a stable heart rate. ECG parameters, including RR, PR, QRS, QT, QTc, J-Tpeak and early repolarization (ER), were manually measured. QT was measured using the tangent method and QTc was calculated using Bazett’s Formula. The J point was defined as the end of the QRS interval and the beginning of the ST segment. ER was defined as an elevation of more than 0.1 mV of the J point from baseline. Tpeak was measured at the highest point of the T-wave. U wave was best visualized in the precordial lead presenting highest amplitude, which was best visualized in the precordial leads V2-V4. The “Gollob Diagnostic Score”(12) was calculated in those cases in which all parameters needed
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were available. Long-term follow-up (60.2±17.0 month -median, 56.0 month; IQR, 28.0 month) was available for 6 probands and 10 family members, a total of 16 SQTS patients.
Molecular Genetic Analysis
After informed consent letters were obtained, blood samples were collected with IRB approvals. Genomic DNA was extracted from peripheral blood leukocytes using a commercial kit (Gentra System, Puregene, Valencia, CA, USA). All exons and intron borders of cardiac ion channel genes, including all isoforms of 6 known short QT candidate genes, were amplified and analyzed by direct sequencing from both directions usingan ABI PRISM 3100-Avant Automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA). Genomic DNA from 430 ethnically-matched healthy reference alleles was used as controls. The KCNH2 primers of exon 7 used for screening is 5’-CTTGCCCCATCAACGGAATG-3’ (sense) and 5’-CTAGCAGCCTCAGTTTCCTC-3’ (antisense).
Mutagenesis and Transfection in HEK 293 cells
The KCNH2-T618I mutation was engineered into wild-type (KCNH2-WT) cDNA cloned in pcDNA3.1 (Invitrogen, CA, USA) by overlap extension using mutation-specific primers and a Quick Change Site-Directed Mutagenesis Kit (Stratagene, CA, USA). The presence of mutations was confirmed by sequence analysis. HEK293 cells were transfected with 2.0 µg
KCNH2 cDNA (WT or T618I, respectively) using Lipofectamine (Life Technologies, MD, USA).
CD8 cDNA was co-transfected as a reporter gene. CD8-positive cells identified by Dynabeads (Dynal, M-450 CD8) were studied in 48-72 hours after transfection.
10 Electrophysiological Study and Analysis
Membrane currents were measured using whole-cell patch-clamp procedures with Axopatch 700B amplifiers (Axon Instruments Foster City, CA). Patch electrodes were pulled from borosilicate glass (Hilgenberg) on a Sutter P-97 horizontal puller (Sutter Instruments, Novato, CA, USA). All signals were acquired at 0.33 kHz (Digidata 1440A, Axon Instruments) and analyzed with pCLAMP version 10.2 (Axon Instruments). Series resistance and capacitive transients were compensated using standard techniques. Membrane currents were low pass filtered at 10 kHz and digitized at 100 kHz. Recordings were made at room temperature using an internal solution containing (mmol/L) 140 KAsp, 10 EGTA, 4 MgATP, 1 MgCl2, 10 HEPES, (pH = 7.2 with KOH). External solution contained (mmol/L) 140 NaCl, 1 CaCl2, 1 MgCl2, 4 KCl. 10 HEPES, and 5 Glucose (pH = 7.4 with NaOH). In order to ensure reproducibility, after whole-cell conditions were established by rupturing the cell membrane, we allowed a dialysis period of 4 minutes before beginning measurements. During the dialysis period, we monitored current-voltage relationships to ensure stability and consistency of recordings.
Statistical Analysis
Data are presented as percentages, mean ± SD, median with interquartile range (IQR) for clinical values; and mean±SEM for experimental values. P<0.05 is considered statistically significant according to Student’s t-test or ANOVA analysis for continuous variables; Chi-square or Fisher exact tests for categorical data, as appropriate. pClamp 10.2 (Axon
Instruments, Inc.), Excel (Microsoft), and Origin (Microcal Software) software are used for data acquisition and analysis.
11 Results
Clinical and demographic profiles of probands and their families
The KCNH2-T618I mutation was identified in 18 cases of 7 unrelated families (10 males, 55.6%; Median age, 24.0 years; IQR, 23.0 years) throughout the world. All carriers were clinically diagnosed as SQTS in the absence of an identifiable etiology and showed 100% penetrance with variable expressivity (Figure 1, Table 1). Flat QT-HR relationship was observed in all 8 mutation carriers (Family 3, 5, 6 and 7) and absent in healthy relatives who underwent exercise test. Combined information in our database with reported results, suggests that the proportions of KCNH2-T618I among genetic identified SQTS patients and probands were 29.0% and 25.9%. The second most frequent mutation of SQTS is KCNH2-N588K (Table 2 / Online Table 1).
In all, there was a high incidence of SCD (18 members in 7 families; median age of death, 40.0 years; range from 1 month to 65 years). The average QTc of probands and all mutant carriers were 294.1 ± 23.8 ms and 313.2 ± 23.8 ms, respectively (range: 260 - 344 ms), which were significantly shorter than the QTc of 12 mutation-negative first-degree relatives (426.2 ± 26.4 ms, P < 0.05). ECGs typically showed tall, peaked and symmetrical T waves, preceded by a short or absent ST segment in most affected individuals. There was no gender preference among mutant carriers. Despite of tendency of longer QTc in female carriers (323.7 ± 16.3 ms vs. 304.8 ± 26.2 ms in female and male, P = 0.095), female prone to be symptomatic [62.5% (5) vs. 10.0% (1), P < 0.05], which was not observed in N588K carriers [symptomatic, 66.7% (6) vs. 85.7% (6) in female and male, P > 0.05]. Similar to previous observation(4), there was no
12
statistical difference of QTc between symptomatic and asymptomatic cases (310.9 ± 27.0 ms vs. 314.3 ± 23.3 ms, P > 0.05). There was also no difference in age at diagnosis between probands and affected family members (29.4 ± 14.1 years vs. 25.3±11.3 years, P>0.05), but differences were observed with respect to presentation of symptoms (57.1% vs. 18.2%, P < 0.05) and QTc (294.1 ± 23.8 ms vs. 325.3 ± 13.9 ms, P < 0.05, Table 3). Arrhythmias were common in symptomatic carriers with KCNH2-T618I, including ventricular ectopic beats, VT, and VF. In contrast with a higher incidence of atrial fibrillation (AF) in KCNH2-N588K (60.0% for probands and 45.5% for affected relatives), none of the KCNH2-T618I carriers suffered from AF (0.0% for both groups). Among all 18 KCNH2-T618I carriers, half (50.0%) presented with minor ER pattern in lead V2-V4. A distinct U wave was evident in precordial leads (most in V2 to V4) in 72.2% cases.
Demographic and ECG characteristics of the probands are shown in Table 1 and Figure 2. Gollob Diagnostic Score for SQTS ranged from 7 to 11 for all mutation carriers, indicating a high sensitivity and clinical accuracy of the scorecard for cases independently diagnosed as SQTS.
Treatment and Follow-up
During 69.3 ± 20.9 months of follow-up, an ICD was implanted in 7 patients: 3 probands and 1 family member were implanted after presenting with symptoms (such as unexplained syncope, documented VT/VF), and 2 asymptomatic probands and 1 asymptomatic family member for primary prophylaxis. The proband of family 2 and the other 2 family carriers only experienced occasional ventricular premature beats during follow-up. Proband 5 experienced 5 inappropriate shocks 10 days after ICD implantation (Epic 296 by St. Jude Inc.) due to T wave over-sensing (sinus rhythm at 106 bpm which
13
the ICD recognized as VF of 212 bpm). After programming to decrease R-wave sensing threshold, no further T-wave oversensing or ICD shocks were recorded over a period over 5 years. Proband 6 received 1 inappropriate shock during fast sinus rhythm (200 bpm), which triggered ICD shock (Atlar VR V-193). One member of family 7 with an ICD and 2 other carriers with loop recorders only have nsVT during follow-up.
The proband of family 3 and her son were treated with Quinidine (900 mg/d). The increments of QTc after medication were 65ms and 90 ms (Figure 3A and 3B), and the effective refractory period (ERP) was also normalized during EP study. Quinidine failed to normalize QTc in proband 7 (333 ms after 1000mg), however the ventricular ectopy resolved. It also showed reduced efficacy for the other 2 carriers of this family 7, since NSVT was observed during the follow-up in both, even though the QTc had been normalized by Quinidine (Table 4).
Sotalol showed poor clinical efficacy in all 3 carriers who received it with unchanged or even reduced QTc. The proband of family 4 experienced spontaneous VF, and the implanted ICD appropriately terminated the arrhythmia three times. Bisoprolol (Maximum 5 mg/day) failed to prevent his VF recurrence. Oral Bepridil (150mg/day) was then administered to her after last VF episode. QTc was slightly prolonged (341 ms) and no VF has been observed for 32 months follow-up (Figure 3C, Table 4).
Molecular Genetic Analysis
PCR-based sequencing analysis revealed a double peak in the sequence of the
KCNH2 gene among all SQTS subjects, but not in relatives with a normal ECG. The
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a substitution of isoleucine for threonine at residue 618 (p.Thr618Ile, T618I, rs199472947) of Kv11.1 (Figure 4A). This nucleotide change was observed in 0 of 200 healthy controls (400 alleles, frequency, 0.0%) in our database; a frequency of 0.0% is reported in the 1000 genome project database; and 0.0% in Exome Sequencing Project (ESP). It is predicted to be damaging and probably damaging in Sift and PolyPhen tools.
Alignment of the amino acid sequence of Kv11.1 proteins showed that threonine at position 618 is highly conserved among different species (Figure 4B). Residue T618 is located at the pore helix (Intramembrane) region of Kv11.1 channel (Figure 4C). Figure 4D presents a plot of QTc values for all known KCNH2 mutations in SQT1 carriers. N588K and T618I, both in the pore region are associated with lower QTc and higher frequency of carriers, whereas mutations in other regions (E50D in N-terminal and R1135H in C-terminal) have reported much higher QTc’s in only single individuals to date, suggesting the KCNH2 P-loop may be the critical region for a more extreme SQTS phenotype.
All T618I mutation carriers in family 2 also carried a KCNH2-R1047L rare polymorphism. Both the proband and positive carriers in family 3 and 7 also had KCNH2-K897T polymorphism (Table 1). These SNPs have been reported to exert a modifying effect to prolong QTc (18,19).
Functional Characterization
Figure 5A and B presents current traces for WT (top panel) and T618I mutant (lower panel) KCNH2 channels heterologously expressed in HEK293 cells.
Current-15
Voltage relationship revealed that step current density of T618I significantly increased at more positive potentials than 0 mV of WT (Figure 5C). Tail current densities of at -40 mV and -110 mV were larger for T618I vs. WT (Figure 5D and 5E). Current densities of T618I and WT at test potential of -40 mV after depolarizing from +50 mV were 207.5 ± 25.8 and 122.7±12.2 pA/pF (n = 10, P < 0.01); Current densities of T618I and WT at -110 mV after depolarizing from +50 mV were -267.4 ± 33.2 vs. -164.5 ± 27.6 pA/pF (n = 10, P < 0.05, Figure 5F and 5G).
The envelope test of KCNH2 tail current was applied to measure the rates of the channel activation. The protocol used and original WT and T618I current traces are shown in Figure 6A. Figure 6B and 6C showed the time dependence of fractional tails fitted with a single exponential association. Activation was considerably faster for T618I than for WT (Tau of T618I and WT: 121.58 ± 10.5 vs. 240.98 ± 7.16, n = 11 respectively, P=0.0156). The voltage dependence of current activation was assessed using standard tail current analysis. The steady-state V1/2 of activation amounted to -29.59 ± 3.12 mV
and -13.13 ± 2.18 mV of T618I and WT channel (n = 10 respectively, P＜0.05), with the similar slope factors (k = 10.4 ± 1.16 vs. 10.92 ± 2.18, P＞0.05, Figure 6D). The T618I mutation caused a negative shift in the voltage dependence of channel activation and increased activation availability of KCNH2 channel, accounting for the increase in current densities.
To determine how the mutation altered the kinetics of the current during an action potential (AP), we elicited T618I and WT currents by a stimulus generated by a previously recorded AP. WT current displayed a waveform with slow activation kinetics and a rapid increase at the phase 3 repolarization, due to the rapid recovery of
16
inactivated channels. In sharp contrast, T618I current displayed fast activation kinetics and a rapid increase during the early phase of AP repolarization, thus indicating a more important contribution to repolarization during the early phases (Figure 6E).
The inhibitory effects of 10 nM Dofetilide on KCNH2 channel with repolarizing pulse of -40 mV were shown in Figure 7. At test potential of +50 mV, the WT tail current reduced by 46.2% after exposure to 10 nM dofetilide, while T618I tail currents only reduced by 27.9% after same treatment (P < 0.05).
17 Discussion
In 1993, a 2-year follow-up study reported that people with an abbreviated mean QTc interval over a period of 24 hours (<400 ms) have as high a risk of SCD (∼2.4 fold increased risk), similar to that of individuals with long QT intervals (>440 ms, ∼2.4 fold increased risk)(20). However, it was not until 2000 that Gussak et al suggested a new inherited arrhythmia syndrome characterized by abbreviated QT intervals, AF and SCD, which they termed SQTS (1). In the present study, we report the data from the largest cohort of SQTS patients carrying the hotspot mutation T618I in KCNH2. We present the data collected over an average period of 5 years, providing new insights into the natural history, genetics and response to therapy of these SQT1 patients.
Demographics and ECG characteristics
Nucleotide positions with an exceptionally high mutation frequency, which also has ≥ 95% probability, are called “hotspots”(21). As shown in Table 2, we summarized all 27 SQTS probands with genetic mutation. Five cases carry KCNH2-N588K (18.5%), and 7 have KCNH2-T618I (25.9%). Familial clustering of the SQTS phenotype is present in all of the SQTS probands with KCNH2-T618I, similar to probands identified with KCNH2-N588K, which indicates 100% inherited probability. Within the familial cases, all family members with a high probability Gollob diagnostic score were subsequently determined to be KCNH2-T618I gene positive, consistent with an autosomal dominant pattern and 100% phenotype penetrance. This percentage is higher than a previous report (~50%) in SQTS (4). KCNH2-T618I was discovered from unrelated SQTS families across the world (Table 1), suggesting the mutation does not
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arise from a recent “founder effect”. Our data indicates that KCNH2-T618I is a hotspot mutation in SQTS.
The QTc of healthy males is known to be less than that of females(22,23), and similar gender distinctions were observed in the present study. Contrary to previous reports of overwhelming predominance of males in the phenotype of SQTS (91.0%)(4),
KCNH2-T618I carriers were fairly evenly distributed (55.6% in males). Our data are
consistent with previous findings that no clear gender difference are observed in
KCNH2-N588K carriers (43.8% in males) or in all 37 carriers with SQTS1 (54.1% in
males). Interestingly, the phenotype appears to be more severe in KCNH2-T618I females, with 3 of 3 female but only 1 of 4 male probands presenting with ventricular arrhythmias, or aborted SCD. In general, the risk of cardiac event and lethal arrhythmias in affected SQTS1 females was not less than males. Similar to other SQTS cohorts, symptoms in the KCNH2-T618I carriers included palpitations, syncope, SCD across the family.
A family history of SCD was commonly reported in our KCNH2-T618I families, however only a small portion of living carriers screened were symptomatic. It may suggest lethal cardiac events and SCD are common in KCNH2-T618I families, and could be the first clinical symptom. In comparison, 75.0% of family carriers with the
KCNH2-N588K mutation were reported to be symptomatic, which could be explained by
slightly but significantly longer QTc in KCNH2-T618I family carriers than those with
KCNH2-N588K (Table 3). The longer QTc could be due to different genetic background
and environmental modifier in those cases. Some genetic variants (K897T and R1047L) were previously reported to be associated with drug-induced or acquired LQTS and
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some have been shown to reduce IKr (24,25). These QT interval modifiers may also contribute to atypical ECG morphology in several cases. (21)
SQTS1 is generally characterized by the appearance of an abbreviated QTc interval, often associated with high amplitude and symmetrical T waves (7). The Tpeak to Tend interval (Tp-e) reflects spatial, including transmural, dispersion of repolarization. In our study, Tp-e interval was relatively prolonged as compared to other SQTS cases. Poor rate adaption of the QT interval is also common in our KCNH2-T618I cases
(Figure 1). These factors are suggested to produce a substrate for reentry that leads to VF (Online Figure 1)(26).
As expected, a distinct U wave is commonly observed in our cohort. Schimpf and coworkers provided evidence for dissociation between ventricular repolarization and the end of mechanical systole in SQTS patients. Coincidence of the U wave with termination of mechanical systole provides support for the mechano-electrical hypothesis for the origin of the U wave(27). This concept is also supported by a recent report by Suzuki and coworkers(28).
Watanabe et al. reported a high prevalence of ER in patients with SQTS associated with arrhythmic events (65%) when compared with a short QT control cohort (30%) or a normal QT control cohort (10%)(29). In the EuroShort Registry, this percentage is 33%(30). In the present study, 50.0% of KCNH2-T618I SQTS carriers presented with an ER pattern. AF incidence is higher in subjects with a short QT interval, or SQTS than in the general population(1,3,9,31). Electrophysiological studies in these cases are characterized by significant shortening of atrial and ventricular refractory periods and inducibility of both atrial and ventricular arrhythmias(32). Interestingly, in contrast to the 50.0% incidence of AF in KCNH2-N588K cases, we observed no
20
documented cases or family history of AF, but multiple episodes of VT/VF in the
KCNH2-T618I SQTS cases. The significant diversity among these studies may stem
from different modifier genes or simply be due to the unique effects of the respective mutations.
Therapy of SQTS patients with KCNH2-T618I
Therapy of SQTS has met with several challenges. Patients with the KCNH2-N588K mutation are known to be resistant to traditional IKr blockers, such as sotalol, dofetilide, flecainide and ibutilide, that have a higher affinity for the inactivated state of IKr channel, which has been lost due to the effect of the mutation (33,34). Quinidine, presumably due to its interaction with the activated state of the channel, has been shown to be effective in normalizing the QT interval and preventing inducibility of VT/VF in patients with SQTS, including KCNH2-N588K carriers(35,36). Disopyramide may be a suitable alternative(37,38). Intravenous administration of nifekalant is useful for rapid restoration of extremely short QT intervals to normal ranges, such as during frequent discharge of ICD due to incessant VF(38).
In the present study, we provide further information for the effectiveness of quinidine to SQTS cases. Quinidine administration prolonged QTc in all 5 KCNH2-T618I cases receiving this drug, but only suppressed arrhythmia in 3 of them during follow-up. Although previous in vitro research has been indicated that sotalol (in high dosage) may be effective for T618I-linked SQTS1 patients(15,39), our results show the ineffectiveness of sotalol and bisoprolol in KCNH2-T618I carriers. Current experimental study also first indicates the poor effect of another class III drug, dofetilide. Bepridil (a
21
class IV antiarrhythmic drug with potassium and sodium blocking properties) effectively terminated drug-refractory VF in 1 carrier in our study, providing the first line evidence for the use of this drug in SQTS. This drug has also been reported as effective in preventing VF, including electric storms, on several long-term reports with BrS patients(40,41). However, the clinical efficacy of quinidine and bepridil are still under investigation.
Implantation of an ICD is first line therapy for high-risk patients. However, long-term follow-up of patients with SQTS indicates that 58% of patients receiving ICDs have device-related complications, especially inappropriate shocks secondary to T-wave over-sensing in SQTS1(42). We encountered one case of this in our cohort which was corrected by re-programming ventricular sensitivity for VF detection. In such patients, careful assessment of QT interval and T-wave amplitude is indispensable to avoid the problem.
Genetic analysis and biophysical features of KCNH2-T618I mutation
Since our group identified the first SQTS mutation in 2004 (KCNH2-N588K)(7), mutations in six genes have now been associated with SQTS. The genes responsible for SQT1-4 are the same responsible for the congenital LQT2, LQT1, LQT7 and LQT8. In SQTS1, KCNH2-N588K results in reduced inactivation and greater current flow during the plateau potentials of the cardiac AP(5) . Hence the ventricular and atrial AP has a shorter duration and thence a shorter QT in ECG. The other 3 KCNH2 mutations associated with SQTS are I560T, R1135H and E50D. The patient harboring the R1135H also demonstrated a type 1 Brugada ECG pattern. Functional studies of the
22
mutant channels consistently show prolonged deactivation time constants as compared to WT channels, without significant changes in any other gating parameters(43). The I560T mutant shows an increase in peak current density, and a positive shift of the inactivation curve(14). Our unpublished data on E50D shows a gain of function of IKr tail density, slower deactivation and a modest positive shift in the voltage dependence of inactivation. Our result with KCNH2-T618I shows a significant gain-of-function in IKr, particularly in the tail density, which plays an important role in phases 2 and 3 of ventricular APs. Then, we confirm KCNH2-T618I has fast activation kinetics, and a rapid increase using AP wave forms. Similar effects of KCNH2-T618I have been reported at room or body temperature (15,39). Compared with a 6.0 times increase of IKr of
KCNH2-T618I mutation on the peak steady density (15), present study displays a 6.25
times increase of IKr in the similar in vitro condition. Additionally, our study shows that the gain-of-function is also caused by a negative shift in the voltage dependence of activation, which allows more potassium channels to open.
Study Limitation and Future Scope
Although it is the largest collection of SQTS mutation carriers thus far reported, the sample size of our study is necessarily small because of the rarity of the syndrome. We performed functional analysis with only homozygous expression, which may not accurately mimic the phenotypes of patients presenting with heterozygous mutations. Further long-term follow-up of patients are needed to clarify the prognosis of KCNH2-T618I mutation carriers and the most efficacious drug approaches to therapy.
23 Summary and Conclusions
KCNH2-T618I is the most frequent mutation associated with SQTS worldwide,
with a high incidence of VT/VF or SCD and complete penetrance. Female carriers appear prone to have more symptoms and cardiac events. Together with the 2nd most prevalent hotspot (KCNH2-N588K), it accounts for 85.0% of SQT1 and 54.8% of all genetically identified SQTS cases. ICD implant is still the first-choice therapy in KCNH2-T618I patients, although the technical difficulties and a high rate of complications are also encountered. This study demonstrates quinidine is effective in prolonging QTc, but whether this translates into decreased SCD is still unknown. Bepridil may be the new alternative to prevent VT/VF, which has not been reported previously. The mutation causes a major gain of function in IKr, leading to acceleration of repolarization and abbreviation of APs. Shortening of the ventricular AP underlies the shortening of the QT interval. This in turn is thought to be responsible for heterogeneous abbreviation of refractoriness, leading to the development of a reentrant substrate giving rise to arrhythmias.
24 Perspectives
Carriers with the most frequent SQTS mutation (KCNH2-T618I) have complete penetrance, and high incidence and family history of VT/VF and/or sudden cardiac death (SCD) after long term follow-up. It accounts for 25.9% of genetically identified SQTS probands without clear gender preference. Our data describes the clinical characteristics of patients with this ‘hotspot’ mutation and provides guidance on treatment options. Although quinidine is highly effective on prolonging QTc among
25 Central Illustration
Distribution and Therapy of KCNH2-T618I Mutation in Short QT Syndrome. The proportion of KCNH2-T618I among genetic identified SQTS probands is the highest (25.9%, A), and the mutation is discovered in unrelated SQTS families across the world (B). KCNH2-T618I mutation leads to an increase in the repolarizing potassium current (IKr) and a decrease in action potential duration, which shortens the QT interval. With the IKr blocking effect by certain drugs, such as Quinidine and Bepridil, the action potential duration and QT/QTc get ameliorative, and VT/VF could be suppressed (C).
26 Acknowledgments
The authors are grateful to Judy Hefferon for preparing figures and Susan Bartkowiak for maintaining the MMRL genetic database.
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33
Table 1. Summary of the Clinical and Genetic Properties of Probands carriers of the KCNH2 - T618I Mutant.
Probands' No
1
2
3
4
5
6
7
Average/Percentage
General Properties
Region Canada USA Italy Japan China Poland Italy -
FH SCD(1) SD(1) SCD(1) SCD(1) SCD(4) SD(7) SD(3) 100.0% (7/7)
Gender (Male/Female) M F F F M M M M, 57.1% (4/7)
Age at Diagnosis (year) 30 9 46 39 43 23 16 Median, 30; IQR, 27
Symptom N Y Y Y N Y N 57.1% (4/7) ER N N Y Y Y Y N 57.1% (4/7) AF N N N N N N N 0.0% (0/7) Treatment Y N Y Y Y Y Y 85.7%(6/7) Follow-up (month) 52 64 86 43 78 103 59 69.3 ± 20.9 Gollob Score 9 11 10 10 9 10 9 9.7±0.8
Modified Gollob Score 9 9 9 8 9 8 9 8.7±0.5
ECGs HR (ms) 60 74 88 74 59 67 80 71.7 ± 10.6 PR (ms) 160 88 150 130 190 137 150 143.6 ± 31.2 P wave (ms) 80 50 100 90 110 80 80 84.3 ±19.0 QT (ms) 260 270 260 290 300 250 260 270.0 ± 18.2 QTc (ms) 260 300 315 322 297 264 300 294.1 ± 23.8 QRS (ms) 100 80 90 80 90 82 80 86.0 ± 7.7 Jpoint-Tpeak 90 110 110 130 110 106 110 109.4 ± 11.6
Tp-e/QT ratio (II) 0.27 0.3 0.23 0.28 0.33 0.25 0.27 0.27 ± 0.03
Tp-e/QT ratio (V5) 0.29 0.3 0.27 0.29 0.37 0.28 0.29 0.30 ± 0.03 U wave Y Y N Y Y N Y 71.4% (5/7) Genetics Other variants in KCNH2 may affect QT N R1047 L (+/-) K897T (+/+) N N N K897T (+/+) 42.9% (3/7)
Y, Yes; N, No; -, Not Available. FH, family history; ER, early repolarization; AF, atrial fibrillation; SCD, sudden cardiac death; SD,
34
Table 2. Summary of reported SQTS Mutations.
Gene Mutation Carriers Proportion in carriers (%) Probands Proportion in proband (%) Genetic Traits (Penetrance) Sources
KCNH2 T618I 18 29.0% 7 25.9 % Inherited (100.0 %) Current study and Reference
N588K 16 25.8% 5 18.5 % Inherited (100.0 %) Reference
R1135H 3 4.8% 1 3.7 % Inherited (33.0 %) Reference
E50D 2 3.2% 1 3.7 % Inherited (50.0 %) Reference
I560T 1 1.6% 1 3.7 % Unknown Reference
KCNQ1 V141M 2 3.2% 1 3.7 % Inherited (50.0 %) Reference
R259H 1 1.6% 1 3.7 % Unknown/Adopted Reference
V307L 1 1.6% 1 3.7 % Unknown Reference
KCNJ2 D172N 4 6.5% 2 7.4 % Inherited (75.0 %) Current study* and Reference
E299V 1 1.6% 1 3.7 % De novo Reference
M301K 1 1.6% 1 3.7 % Unknown Reference
CACNA1C A39V 1 1.6% 1 3.7 % Unknown Reference
G490R 1 1.6% 1 3.7 % Unknown Reference
R1977Q 1 1.6% 1 3.7 % De novo Reference
CACNB2b S481L 6 9.7% 1 3.7 % Inherited (66.7 %) Reference
CACNA2D1 S755T 3 4.8% 1 3.7 % Inherited (33.0 %) Reference
Total 16 62 100% 27 100%
35
Table 3. Comparison of demographic parameters of KCNH2-N588K and KCNH2-T618I carriers.
N588K
T618I
Probands Affected
Relatives Total Probands
Affected
Relatives Total
No. 5 11 16 7 11 18
Male (%) 3 (60.0%) 4 (36.4%) 7 (43.8%) 4 (57.1%) 6 (54.5%) 10 (55.6%)
No. of families history of
SCD/SD 5 (100.0%) 7 (100.0%)
No. of SCD/SD in relatives 10 18
Average age at Dx (y/o) 24.0±18.3 25.8±24.3 25.2±21.6 29.4±14.1 25.3±11.3 26.9±12.4
Symptom positive (%) 4 (80.0%) 8 (72.7%) 12 (75.0%) 4 (57.1%) 2 (18.2%)* 5 (27.7%)*
QTc (ms) 285.2±15.9 284.5±18.0 284.7±16.7 294.1±23.8 325.3±13.9*# 313.2±23.8*
AF 3 (60.0%) 5 (45.5%) 8 (50.0%) 0 (0.0%)* 0 (0.0%)* 0 (0.0%)*
Treatment - ICD (%) 3 (60.0%) 5 (45.5%) 8 (50.0%) 5 (71.4%) 2 (18.2%) 7 (38.9%)
Treatment - Quinidine (%) 2 (40.0%) 1(9.1%) 3 (18.8%) 2 (28.6%) 3 (27.3%) 5 (27.8%)
*P<0.05 for comparison of same subgroup between N588K and T618I carriers. #P<0.05 for comparison between probands and affected relatives
36
Table 4. The effect of antiarrhythmic drugs on KCNH2-T618I carriers. Drug Patient No. Dosage Plasma levels
(µg/mL) * QTc before medication (ms) QTc after medication (ms) Effect on QTc Effect on arrhythmia
Quinidine Family 3, II-2 300mg, Tid 0.7 315 380 Good normalizing ERP
Family 3, III-1 300mg, Tid 1.2 320 410 Good normalizing ERP
Family 7, IV-1 250mg, Tid 500mg, Bid 0.6 0.9 300 340 333 Fine Fine remaining PVC suppressing PVC Family 7, IV-2 250mg, Bid
250mg, Tid 0.3 1.0 340 376 389 Good Good remaining slow nsVT remaining slow nsVT Family 7, III-3 250mg, Bid
250mg, Tid 0.6 N/A 344 Unchanged 411 Poor Good remaining nsVT remaining nsVT
Sotalol Family 6, IV-6 N/A N/A 320 306 Poor -
Family 7, IV-1 40mg, Tid 80mg, Bid N/A N/A 300 Unchanged 280 Poor Poor
remaining ventricular bigeminy remaining ventricular bigeminy Family 7, III-3 40mg, Tid
80mg, Bid N/A N/A 344 340 343 Poor Poor remaining nsVT remaining nsVT
Bisoprolol Family 4, II-3 5 mg/day N/A 322 Unchanged Poor remaining VF
Bepridil Family 4, II-3 150 mg/day N/A 322 341 Fine suppressing VF
*Therapeutic range of Quinidine is 0.6-2.2 µg/mL. ERP, PVC, VF, and NSVT indicate effective refractory period, premature ventricular contraction, ventricular fibrillation, and nonsustained ventricular tachycardia. N/A, not available.
37 Figure Legend
Figure 1. Pedigree of families identified with T618I-KCNH2 mutation. Circles represent female subjects and squares represent male subjects. The arrow denotes the proband. Diagonal bars indicate deceased family members. +/- means the mutation positive/negative. SCD = Sudden Cardiac Death; SD = Sudden Death. (4)Inset panel shows the HR-QT relationship of the family 5. The relation between QT and HR was evaluated by linear regression analysis according to the formula: QT = β×HR + α, β is the slope of the linear relationship, and α is the intercept expressed in ms.
Family 1: The proband was asymptomatic and had an ICD implanted 4 years earlier. Family 2: the first child (II-1) experienced at least 4 life-threatening arrhythmic episodes prior to SCD. The proband presented with symptoms at very young age, but her brother and father with SQTS were asymptomatic. Family 3: Symptomatic proband and her asymptomatic son had very short QT/QTc interval. Both have a very flat QT/HR slope (0.49 ms/bpm of the proband, -0.47 ms/bpm of the son). Family 4: Symptomatic proband and 2 asymptomatic family members had SQTS. Family 5: A total of 11 members were enrolled. The proband and his 3 SQTS children were asymptomatic, despite a flat QT/HR slope (-0.61, -0.69, -0.89 ms/bpm of the proband, his eldest and youngest daughter). Family 6: Proband had short QTc and a very flat QT/HR slope. He and his mother were symptomatic with 7 male SD relatives in the family. Family 7: Proband and his sister were asymptomatic with short QTc and poor QT/HR adaption. Their maternal aunt suffered from aborted SCD caused by VF. Two family members had SD, including one (the mother) with QTc 300 ms.
38
Figure 2. ECGs of Seven Affected Probands. Note the narrow and peaked T wave in affected individuals with short QT intervals.
Figure 3. Before and after ECGs of 3 mutation carriers who received medication with (A & B) Quinidine and (C) Bepridil.
Figure 4. Genetic analysis of KCNH2-T618I mutation and the comparison of QTc among all SQT1 carriers.
A: Electropherograms of wild type (WT) and mutant KCNH2 gene showing heterozygous transition c.1853c>t predicting replacement of isoleucine by threonine at position 618. B: Amino acid sequence alignment showing that threonine at position 618 is highly conserved among different mammal’s species. C: Predicted topology of the KCNH2 gene encoded Kv11.1 α potassium channels subunit showing the location of the T618I and other reported mutants (red circle). I560T, N588K, T618I are at the at the P-loop linker S5-S6 region (the pore region of KCNH2). D: Scatter plot of QTc in SQT1 mutant carriers. Blue square/red circle indicate male/female. Mean ± Sem is presented as black lines.
Figure 5. Current densities and rectification of KCNH2-WT and KCNH2-T618I channels.
A and B: Representative current traces for WT (top panel) and T618I (lower panel). From a holding potential (HP) of -90 mV, channel activation was obtained by a step protocol from -50 mV to +50 mV in 10 mV increments for 2000 ms. Tail current was recorded at different potential of -40 mV (A) and -110 mV (B) for 3000 ms. C: I-V
39
relation curve revealed T618I step current densities significantly increased at potentials more positive than 0 mV. D and E: Comparison of the tails current densities of WT and T618I repolarizing at -40 mV and -110 mV. When test potential is more positive than +20 mV, the tails current of T618I were significantly larger than those of WT at both voltages. F and G: Bar graphs of current densities of T618I and WT at test potential of -40 mV and -110 mV after depolarizing at +50 mV. *P < 0.05.
Figure 6. Steady-state activation and KCNH2 channel current with AP clamp of KCNH2-WT and T618I. A: Representative traces of time course of activation in
KCNH2-WT and T618I. An envelope of tails protocol illustrated at the top was used to
determine the rate of activation. Cells were held at -90 mV and stepped to +50 mV for increasing durations between 40 and 800 ms, after that KCNH2 tails were evoked on repolarization to -40 mV. This protocol permitted the investigation of progressive
KCNH2 activation. B and C: Time course of activation curves in WT and mutant by an
envelope of tails protocol. Peak current measurements were normalized by dividing all currents by the current after prepulse and then averaged. These averaged current values were fitted with a single exponential association. D: Steady-state activation of 2 currents. Cells were depolarized to potentials in the range -60 mV to +30 mV (4s) and tail current recording at -110 mV. Tail current data were normalized to the maximum current value and fitted with Boltzmann function. E: Representative currents (red/black trace for WT/T618I) elicited during an AP clamp using pre-recorded ventricular AP (right insert).
40
Fig 7. Inhibition effect of Dofetilide in WT and T618I KCNH2 channels on step and tail currents. A: Representative current traces recorded under control conditions and after superfusion with 10 nM dofetilide (Dof). B: Summary of dofetilide on current-voltage relationship of tail currents.
41 Figure 1
42 Figure 2
43 Figure 3
44 Figure 4
45 Figure 5
46 Figure 6
47 Figure 7
