Matteo Pedrazzini

Prevalence of the Congenital Long-QT Syndrome

Background— The prevalence of genetic arrhythmogenic diseases is unknown. For the long-QT syndrome (LQTS), figures ranging from 1:20 000 to 1:5000 were published, but none was based on actual data. Our objective was to define the prevalence of LQTS. Methods and Results— In 18 maternity hospitals, an ECG was performed in 44 596 infants 15 to 25 days old (43 080 whites). In infants with a corrected QT interval (QTc) >450 ms, the ECG was repeated within 1 to 2 weeks. Genetic analysis, by screening 7 LQTS genes, was performed in 28 of 31 (90%) and in 14 of 28 infants (50%) with, respectively, a QTc >470 ms or between 461 and 470 ms. A QTc of 451 to 460, 461 to 470, and >470 ms was observed in 177 (0.41%), 28 (0.06%), and 31 infants (0.07%). Among genotyped infants, disease-causing mutations were found in 12 of 28 (43%) with a QTc >470 ms and in 4 of 14 (29%) with a QTc of 461 to 470 ms. One genotype-negative infant (QTc 482 ms) was diagnosed as affected by LQTS on clinical grounds. Among family members of genotype-positive infants, 51% were found to carry disease-causing mutations. In total, 17 of 43 080 white infants were affected by LQTS, demonstrating a prevalence of at least 1:2534 apparently healthy live births (95% confidence interval, 1:1583 to 1:4350). Conclusions— This study provides the first data-based estimate of the prevalence of LQTS among whites. On the basis of the nongenotyped infants with QTc between 451 and 470 ms, we advance the hypothesis that this prevalence might be close to 1:2000. ECG-guided molecular screening can identify most infants affected by LQTS and unmask affected relatives, thus allowing effective preventive measures.

Prevalence of Long-QT Syndrome Gene Variants in Sudden Infant Death Syndrome

Background— The hypothesis that some cases of sudden infant death syndrome (SIDS) could be caused by long-QT syndrome (LQTS) has been supported by molecular studies. However, there are inadequate data regarding the true prevalence of mutations in arrhythmia-susceptibility genes among SIDS cases. Given the importance and potential implications of these observations, we performed a study to more accurately quantify the contribution to SIDS of LQTS gene mutations and rare variants. Methods and Results— Molecular screening of 7 genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2, CAV3) associated with LQTS was performed with denaturing high-performance liquid chromatography and nucleotide sequencing of genomic DNA from 201 cases diagnosed as SIDS according to the Nordic Criteria, and from 182 infant and adult controls. All SIDS and control cases originated from the same regions in Norway. Genetic analysis was blinded to diagnosis. Mutations and rare variants were found in 26 of 201 cases (12.9%). On the basis of their functional effect, however, we considered 8 mutations and 7 rare variants found in 19 of 201 cases as likely contributors to sudden death (9.5%; 95% CI, 5.8 to 14.4%). Conclusions— We demonstrated that 9.5% of cases diagnosed as SIDS carry functionally significant genetic variants in LQTS genes. The present study demonstrates that sudden arrhythmic death is an important contributor to SIDS. As these variants likely modify ventricular repolarization and QT interval duration, our results support the debated concept that an ECG would probably identify most infants at risk for sudden death due to LQTS either in infancy or later on in life.

KCNH2-K897T Is a Genetic Modifier of Latent Congenital Long-QT Syndrome

Background—Clinical heterogeneity among patients with long-QT syndrome (LQTS) sharing the same disease-causing mutation is usually attributed to variable penetrance. One potential explanation for this phenomenon is the coexistence of modifier gene alleles, possibly common single nucleotide polymorphisms, altering arrhythmia susceptibility. We demonstrate this concept in a family segregating a novel, low-penetrant KCNH2 mutation along with a common single nucleotide polymorphism in the same gene. Methods and Results—The proband is a 44-year-old white woman with palpitations associated with presyncope since age 20, who presented with ventricular fibrillation and cardiac arrest. Intermittent QT prolongation was subsequently observed (max QTc, 530 ms), and LQT2 was diagnosed after the identification of a missense KCNH2 mutation (A1116V) altering a conserved residue in the distal carboxyl-terminus of the encoded HERG protein. The proband also carried the common KCNH2 polymorphism K897T on the nonmutant allele. Relatives who carried A1116V without K897T were asymptomatic, but some exhibited transient mild QTc prolongation, suggesting latent disease. Heterologous expression studies performed in cultured mammalian cells and using bicistronic vectors linked to different fluorescent proteins demonstrated that coexpression of A1116V with K897T together resulted in significantly reduced current amplitude as compared with coexpression of either allele with WT-HERG. Thus, the presence of KCNH2-K897T is predicted to exaggerate the IKr reduction caused by the A1116V mutation. These data explain why symptomatic LQTS occurred only in the proband carrying both alleles. Conclusions—We have provided evidence that a common KCNH2 polymorphism may modify the clinical expression of a latent LQT2 mutation. A similar mechanism may contribute to the risk for sudden death in more prevalent cardiac diseases.

Cardiac Sodium Channel Dysfunction in Sudden Infant Death Syndrome

Background— Mutations in genes responsible for the congenital long-QT syndrome, especially SCN5A, have been identified in some cases of sudden infant death syndrome. In a large-scale collaborative genetic screen, several SCN5A variants were identified in a Norwegian sudden infant death syndrome cohort (n=201). We present functional characterization of 7 missense variants (S216L, R680H, T1304M, F1486L, V1951L, F2004L, and P2006A) and 1 in-frame deletion allele (delAL586-587) identified by these efforts. Methods and Results— Whole-cell sodium currents were measured in tsA201 cells transiently transfected with recombinant wild-type or mutant SCN5A cDNA (hH1) coexpressed with the human &bgr;1 subunit. All variants exhibited defects in the kinetics and voltage dependence of inactivation. Five variants (S216L, T1304M, F1486L, F2004L, and P2006A) exhibited significantly increased persistent sodium currents (range, 0.5% to 1.7% of peak current) typical of SCN5A mutations associated with long-QT syndrome. These same 5 variants also displayed significant depolarizing shifts in voltage dependence of inactivation (range, 5 to 14 mV) and faster recovery from inactivation, but F1486L uniquely exhibits a depolarizing shift in the conductance-voltage relationship. Three alleles (delAL586-587, R680H, and V1951L) exhibited increased persistent current only under conditions of internal acidosis (R680H) or when expressed in the context of a common splice variant (delQ1077), indicating that they have a latent dysfunctional phenotype. Conclusions— Our present results greatly expand the spectrum of functionally characterized SCN5A variants associated with sudden infant death syndrome and provide further biophysical correlates of arrhythmia susceptibility in this syndrome.

Calmodulin Mutations Associated With Recurrent Cardiac Arrest in Infants

Background— Life-threatening disorders of heart rhythm may arise during infancy and can result in the sudden and tragic death of a child. We performed exome sequencing on 2 unrelated infants presenting with recurrent cardiac arrest to discover a genetic cause. Methods and Results— We ascertained 2 unrelated infants (probands) with recurrent cardiac arrest and dramatically prolonged QTc interval who were both born to healthy parents. The 2 parent-child trios were investigated with the use of exome sequencing to search for de novo genetic variants. We then performed follow-up candidate gene screening on an independent cohort of 82 subjects with congenital long-QT syndrome without an identified genetic cause. Biochemical studies were performed to determine the functional consequences of mutations discovered in 2 genes encoding calmodulin. We discovered 3 heterozygous de novo mutations in either CALM1 or CALM2, 2 of the 3 human genes encoding calmodulin, in the 2 probands and in 2 additional subjects with recurrent cardiac arrest. All mutation carriers were infants who exhibited life-threatening ventricular arrhythmias combined variably with epilepsy and delayed neurodevelopment. Mutations altered residues in or adjacent to critical calcium binding loops in the calmodulin carboxyl-terminal domain. Recombinant mutant calmodulins exhibited several-fold reductions in calcium binding affinity. Conclusions— Human calmodulin mutations disrupt calcium ion binding to the protein and are associated with a life-threatening condition in early infancy. Defects in calmodulin function will disrupt important calcium signaling events in heart, affecting membrane ion channels, a plausible molecular mechanism for potentially deadly disturbances in heart rhythm during infancy.

Cardiac Potassium Channel Dysfunction in Sudden Infant Death Syndrome

Life-threatening arrhythmias have been suspected as one cause of the sudden infant death syndrome (SIDS), and this hypothesis is supported by the observation that mutations in arrhythmia susceptibility genes occur in 5-10% of cases. However, the functional consequences of cardiac potassium channel gene mutations associated with SIDS and how these alleles might mechanistically predispose to sudden death are unknown. To address these questions, we studied four missense KCNH2 (encoding HERG) variants, one compound KCNH2 genotype, and a missense KCNQ1 mutation all previously identified in Norwegian SIDS cases. Three of the six variants exhibited functional impairments while three were biophysically similar to wild-type channels (KCNH2 variants V279M, R885C, and S1040G). When co-expressed with WT-HERG, R273Q and K897T/R954C generated currents resembling the rapid component of the cardiac delayed rectifier current (I(Kr)) but with significantly diminished amplitude. Action potential modeling demonstrated that this level of functional impairment was sufficient to evoke increased action potential duration and pause-dependent early afterdepolarizations. By contrast, KCNQ1-I274V causes a gain-of-function in I(Ks) characterized by increased current density, faster activation, and slower deactivation leading to accumulation of instantaneous current upon repeated stimulation. Action potential simulations using a Markov model of heterozygous I274V-I(Ks) incorporated into the Luo-Rudy (LRd) ventricular cell model demonstrated marked rate-dependent shortening of action potential duration predicting a short QT phenotype. Our results indicate that certain potassium channel mutations associated with SIDS confer overt functional defects consistent with either LQTS or SQTS, and further emphasize the role of congenital arrhythmia susceptibility in this syndrome.

Identification of a KCNQ1 Polymorphism Acting as a Protective Modifier against Arrhythmic Risk in Long QT Syndrome

Background—Long-QT syndrome (LQTS) is characterized by such striking clinical heterogeneity that, even among family members carrying the same mutation, clinical outcome can range between sudden death and no symptoms. We investigated the role of genetic variants as modifiers of risk for cardiac events in patients with LQTS. Methods and Results—In a matched case–control study including 112 patient duos with LQTS from France, Italy, and Japan, 25 polymorphisms were genotyped based on either their association with QTc duration in healthy populations or on their role in adrenergic responses. The duos were composed of 2 relatives harboring the same heterozygous KCNQ1 or KCNH2 mutation: 1 with cardiac events and 1 asymptomatic and untreated. The findings were then validated in 2 independent founder populations totaling 174 symptomatic and 162 asymptomatic patients with LQTS, and a meta-analysis was performed. The KCNQ1 rs2074238 T-allele was significantly associated with a decreased risk of symptoms 0.34 (0.19–0.61; P<0.0002) and with shorter QTc (P<0.0001) in the combined discovery and replication cohorts. Conclusions—We provide evidence that the KCNQ1 rs2074238 polymorphism is an independent risk modifier with the minor T-allele conferring protection against cardiac events in patients with LQTS. This finding is a step toward a novel approach for risk stratification in patients with LQTS.

Malignant Perinatal Variant of Long-QT Syndrome Caused by a Profoundly Dysfunctional Cardiac Sodium Channel

Background—Inherited cardiac arrhythmia susceptibility contributes to sudden death during infancy and may contribute to perinatal and neonatal mortality, but the molecular basis of this risk and the relationship to genetic disorders presenting later in life is unclear. We studied the functional and pharmacological properties of a novel de novo cardiac sodium channel gene (SCN5A) mutation associated with an extremely severe perinatal presentation of long-QT syndrome in unrelated probands of different ethnicity. Methods and Results—Two subjects exhibiting severe fetal and perinatal ventricular arrhythmias were screened for SCN5A mutations, and the functional properties of a novel missense mutation (G1631D) were determined by whole-cell patch clamp recording. In vitro electrophysiological studies revealed a profound defect in sodium channel function characterized by ≈10-fold slowing of inactivation, increased persistent current, slowing of recovery from inactivation, and depolarized voltage dependence of activation and inactivation. Single-channel recordings demonstrated increased frequency of late openings, prolonged mean open time, and increased latency to first opening for the mutant. Subjects carrying this mutation responded clinically to the combination of mexiletine with propranolol and survived. Pharmacologically, the mutant exhibited 2-fold greater tonic and use-dependent mexiletine block than wild-type channels. The mutant also exhibited enhanced tonic (2.4-fold) and use-dependent block (≈5-fold) by propranolol, and we observed additive effects of the 2 drugs on the mutant. Conclusions—Our study demonstrates the molecular basis for a malignant perinatal presentation of long-QT syndrome, illustrates novel functional and pharmacological properties of SCN5A-G1631D, which caused the disorder, and reveals therapeutic benefits of propranolol block of mutant sodium channels in this setting.

A KCNH2 branch point mutation causing aberrant splicing contributes to an explanation of genotype-negative long QT syndrome

BACKGROUND Genetic screening of long QT syndrome (LQTS) fails to identify disease-causing mutations in about 30% of patients. So far, molecular screening has focused mainly on coding sequence mutations or on substitutions at canonical splice sites. OBJECTIVE The purpose of this study was to explore the possibility that intronic variants not at canonical splice sites might affect splicing regulatory elements, lead to aberrant transcripts, and cause LQTS. METHOD Molecular screening was performed through DHPLC and sequence analysis. The role of the intronic mutation identified was assessed with a hybrid minigene splicing assay. RESULTS A three-generation LQTS family was investigated. Molecular screening failed to identify an obvious disease-causing mutation in the coding sequences of the major LQTS genes but revealed an intronic A-to-G substitution in KCNH2 (IVS9-28A/G) cosegregating with the clinical phenotype in family members. In vitro analysis proved that the mutation disrupts the acceptor splice site definition by affecting the branch point (BP) sequence and promoting intron retention. We further demonstrated a tight functional relationship between the BP and the polypyrimidine tract, whose weakness is responsible for the pathological effect of the IVS9-28A/G mutation. CONCLUSIONS We identified a novel BP mutation in KCNH2 that disrupts the intron 9 acceptor splice site definition and causes LQT2. The present finding demonstrates that intronic mutations affecting pre-mRNA processing may contribute to the failure of traditional molecular screening in identifying disease-causing mutations in LQTS subjects and offers a rationale strategy for the reduction of genotype-negative cases.

Vagal reflexes following an exercise stress test: A simple clinical tool for gene-specific risk stratification in the long QT syndrome

OBJECTIVES The study assessed whether heart rate (HR) reduction following an exercise stress test (ExStrT), an easily quantifiable marker of vagal reflexes, might identify high- and low-risk long QT syndrome (LQTS) type 1 (LQT1) patients. BACKGROUND Identification of LQTS patients more likely to be symptomatic remains elusive. We have previously shown that depressed baroreflex sensitivity, an established marker of reduced vagal reflexes, predicts low probability of symptoms among LQT1. METHODS We studied 169 LQTS genotype-positive patients < 50 years of age who performed an ExStrT with the same protocol, on and off β-blockers including 47 South African LQT1 patients all harboring the KCNQ1-A341V mutation and 122 Italian LQTS patients with impaired (I(Ks)-, 66 LQT1) or normal (I(Ks)+, 50 LQT2 and 6 LQT3) I(Ks) current. RESULTS Despite similar maximal HR and workload, by the first minute after cessation of exercise the symptomatic patients in both I(Ks)- groups had a greater HR reduction compared with the asymptomatic (19 ± 7 beats/min vs. 13 ± 5 beats/min and 27 ± 10 beats/min vs. 20 ± 8 beats/min, both p = 0.009). By contrast, there was no difference between the I(Ks)+ symptomatic and asymptomatic patients (23 ± 9 beats/min vs. 26 ± 9 beats/min, p = 0.47). LQT1 patients in the upper tertile for HR reduction had a higher risk of being symptomatic (odds ratio: 3.28, 95% confidence interval: 1.3 to 8.3, p = 0.012). CONCLUSIONS HR reduction following exercise identifies LQT1 patients at high or low arrhythmic risk, independently of β-blocker therapy, and contributes to risk stratification. Intense exercise training, which potentiates vagal reflexes, should probably be avoided by LQT1 patients.