Elena Ronchetti

Risk Stratification in the Long-QT Syndrome

BACKGROUND Mutations in potassium-channel genes KCNQ1 (LQT1 locus) and KCNH2 (LQT2 locus) and the sodium-channel gene SCN5A (LQT3 locus) are the most common causes of the long-QT syndrome. We stratified risk according to the genotype, in conjunction with other clinical variables such as sex and the length of the QT interval. METHODS We evaluated 647 patients (386 with a mutation at the LQT1 locus, 206 with a mutation at the LQT2 locus, and 55 with a mutation at the LQT3 locus) from 193 consecutively genotyped families with the long-QT syndrome. The cumulative probability of a first cardiac event, defined as the occurrence of syncope, cardiac arrest, or sudden death before the age of 40 years and before the initiation of therapy, was determined according to genotype, sex, and the QT interval corrected for heart rate (QTc). Within each genotype we also assessed risk in the four categories derived from the combination of sex and QTc (<500 msec or > or =500 msec). RESULTS The incidence of a first cardiac event before the age of 40 years and before the initiation of therapy was lower among patients with a mutation at the LQT1 locus (30 percent) than among those with a mutation at the LQT2 locus (46 percent) or those with a mutation at the LQT3 locus (42 percent) (P<0.001 by Fishers exact test). Multivariate analysis showed that the genetic locus and the QTc, but not sex, were independent predictors of risk. The QTc was an independent predictor of risk among patients with a mutation at the LQT1 locus and those with a mutation at the LQT2 locus but not among those with a mutation at the LQT3 locus, whereas sex was an independent predictor of events only among those with a mutation at the LQT3 locus. CONCLUSIONS The locus of the causative mutation affects the clinical course of the long-QT syndrome and modulates the effects of the QTc and sex on clinical manifestations. We propose an approach to risk stratification based on these variables.

A Novel Form of Short QT Syndrome (SQT3) Is Caused by a Mutation in the KCNJ2 Gene

Short QT syndrome (SQTS) leads to an abbreviated QTc interval and predisposes patients to life-threatening arrhythmias. To date, two forms of the disease have been identified: SQT1, caused by a gain of function substitution in the HERG (IKr) channel, and SQT2, caused by a gain of function substitution in the KvLQT1 (IKs) channel. Here we identify a new variant, “SQT3”, which has a unique ECG phenotype characterized by asymmetrical T waves, and a defect in the gene coding for the inwardly rectifying Kir2.1 (IK1) channel. The affected members of a single family had a G514A substitution in the KCNJ2 gene that resulted in a change from aspartic acid to asparagine at position 172 (D172N). Whole-cell patch-clamp studies of the heterologously expressed human D172N channel demonstrated a larger outward IK1 than the wild-type (P<0.05) at potentials between −75 mV and −45 mV, with the peak current being shifted in the former with respect to the latter (WT, −75 mV; D172N, −65 mV). Coexpression of WT and mutant channels to mimic the heterozygous condition of the proband yielded an outward current that was intermediate between WT and D172N. In computer simulations using a human ventricular myocyte model the increased outward IK1 greatly accelerated the final phase of repolarization, and shortened the action potential duration. Hence, unlike the known mutations in the two other SQTS forms (N588K in HERG and V307L in KvLQT1), simulations using the D172N and WT/D172N mutations fully accounted for the ECG phenotype of tall and asymmetrically shaped T waves. Although we were unable to test for inducibility of arrhythmia susceptibility due to lack of patients’ consent, our computer simulations predict a steeper steady-state restitution curve for the D172N and WT/D172N mutation, compared with WT or to HERG or KvLQT1 mutations, which may predispose SQT3 patients to a greater risk of reentrant arrhythmias.

Evidence for a cardiac ion channel mutation underlying drug-induced QT prolongation and life-threatening arrhythmias.

Ion Channel Mutations and Drug‐Induced TdP. The aim of this study was to test the hypothesis that some cases of drug‐induced arrhythmias depend on genetic predisposition. Excessive prolongation of the QT interval and life‐threatening arrhythmias (torsades de pointes or ventricular fibrillation) may occur in response to a variety of cardiac and noncardiac drugs, with detrimental effects on patient safety and the investments made by the pharmaceutical industry. Moss and Schwartz hypothesized that some drug‐induced arrhythmias might represent cases of “forme fruste” of the congenital long QT syndrome (LQTS). The availability of molecular screening techniques for LQTS genes allowed us to test this hypothesis. An elderly female patient with documented cardiac arrest related to cisapride, a prokynetic drug that blocks Ikr, and transiently prolonged QT interval underwent mutational analysis of the known LQTS‐related genes performed by single‐strand conformational polymorphism and DNA sequencing. Double‐electrode voltage clamp in Xenopus oocytes as the expression system was used to study the in vitro cellular phenotype caused by the genetic defect in coexpression with the wild‐type (WT) gene. Molecular analysis revealed a heterozygous mutation leading to substitution of a highly conserved amino acid in the pore region of KvLQT1. This mutation was present not only in the patient with ventricular fibrillation but also in her two adult asymptomatic sons who have a normal QT interval. In vitro expression of the mutated KvLQT1 protein showed a severe loss of current with a dominant negative effect on the WT‐KvLQT1 channel. Our findings demonstrate that some cases of drug‐induced QT prolongation may depend on a genetic substrate. Molecular screening may allow identification among family members of gene carriers potentially at risk if treated with Ikr blockers. Evolving technology may lead to rapid screening for mutations of candidate genes that cause drug‐induced life‐threatening arrhythmias and allow early identification of individuals at risk.

The Elusive Link Between LQT3 and Brugada Syndrome The Role of Flecainide Challenge

BackgroundDefects of the SCN5A gene encoding the cardiac sodium channel are associated with both the LQT3 subtype of long-QT syndrome and Brugada syndrome (BS). The typical manifestations of long-QT syndrome (QT interval prolongation) and BS (ST segment elevation in leads V1 through V3) may coexist in the same patients, which raises questions about the actual differences between LQT3 and BS. Intravenous flecainide is the standard provocative test used to unmask BS in individuals with concealed forms of the disease, and oral flecainide has been proposed as a treatment option for LQT3 patients because it may shorten their QT interval. Methods and ResultsWe tested the possibility that in some LQT3 patients, flecainide might not only shorten the QT interval, but also produce an elevation of the ST segment. A total of 13 patients from 7 LQT3 families received intravenous flecainide using the protocol used for BS. As expected, QT, QTc, JT, and JTc interval shortening was observed in 12 of the 13 patients, and concomitant ST segment elevation in leads V1 through V3 (≥2 mm) was observed in 6 of the 13. ConclusionsThe data demonstrate that flecainide may induce ST segment elevation in LQT3 patients, raising concerns about the safety of flecainide therapy and demonstrating the existence of an intriguing overlap between LQT3 and BS.

Molecular diagnosis in a child with sudden infant death syndrome

Although sudden infant death syndrome (SIDS) has been associated with long QT syndrome-a genetic disorder that causes arrhythmia-a causal link has not been shown. We screened genomic DNA from a child who died of SIDS and identified a de-novo mutation in KVLQT1, the gene most frequently associated with long QT syndrome. This mutation (C350T) had already been identified in an unrelated family that was affected by long QT syndrome. These results confirm the hypothesis that some deaths from SIDS are caused by long QT syndrome and support implementation of neonatal electrocardiographic screening.

Non-telomeric chromosome localization of (TTAGGG) n repeats in the genus Eulemur

The chromosomal distribution of the (TTAGGG)n telomeric repetitive sequences was studied in the Malagasy species Eulemur fulvus fulvus (2n = 60), Eulemur rubriventer (2n = 50), Eulemur coronatus (2n = 46) and Eulemur macaco (2n = 44). These sequences hybridize to the telomeres of all chromosomes of the four species and also to the pericentromeres of all chromosomes of E. fulvus, E. coronatus and E. macaco, with the exception of the pericentromeres of E. coronatus and E. macaco chromosomes 9, the homeologous E. fulvus chromosomes 2 and E. macaco chromosomes 1. In E. rubriventer only a very weak signal was detected at the pericentromeres of a few chromosomes. In E. fulvus, E. coronatus and E. macaco, non-telomeric (TTAGGG)n sequences collocalize with constitutive heterochromatin. The interspecific differences of the hybridization pattern of (TTAGGG)n sequences at the pericentromeres suggest that E. rubriventer branched off the common trunk before amplification of endogenous (TTAGGG)n sequences occurred in pericentromeric regions.

Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de‐adaptation of EUE cells to a hypertonic medium

Abstract. EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle‐related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/l range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the Go/1 range of DNA contents: this suggests that a G0 block occurs under long‐term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.