John P. Bourke

Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study

Summary Background We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. Method We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5–15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. Findings 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1–10·6) to 16·4% (10·8–22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. Interpretation The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. Funding UK Medical Research Council; AVI BioPharma.

Importance of lead selection in QT interval measurement.

The influence of lead selection on QT estimation in the 12-lead electrocardiogram was assessed in 63 patients (21 control subjects, 21 with anterior myocardial infarction, 21 with inferior myocardial infarction). QT estimates varied between leads. The variation was greater in patients with myocardial infarction than in control subjects (mean dispersion of QT: control subjects, 48 +/- 18 ms [+/- standard deviation]; anterior myocardial infarction, 70 +/- 30 ms; inferior myocardial infarction, 73 +/-32 ms). The maximum QT in any lead (QTmax) was determined and the deviation of each lead from this maximum value calculated. In all 3 groups, anteroseptal leads (V2 or V3) provided the closest approximation to QTmax. Interlead variability was found to be mainly due to variation in timing of the end of the T wave, rather than the onset of the QRS complex. The variability due to leads was considerably greater than the variability due to cycles, observers or measurement error. Implementation of a variety of current lead selection practices resulted in widely divergent estimates of QT interval. It is concluded that there is a need for standardization of lead selection practice for QT measurement. If measurements are confined to one or a few leads, anteroseptal leads provide the closest approximation to QTmax.

Errors in manual measurement of QT intervals.

OBJECTIVE--To quantify the errors associated with manual measurement of QT intervals and to determine the source of the errors. DESIGN--A randomised study of QT measurement by four cardiologists of electrocardiograms plotted on paper in presentations with different noise levels, paper speeds, amplifier gains, and with and without a second QRST complex to indicate the RR interval. SUBJECTS--Four electrocardiograph leads (I, aVR, V1, V5) recorded in eight healthy people relaxing in a semirecumbent position. MAIN OUTCOME MEASURES--Manual measurement of QT interval in 512 electrocardiograms (eight subjects x four leads x eight presentations x two repeats) by each of four cardiologists. RESULTS--QT intervals measured were significantly longer with greater amplifier gain: by 8 ms for a doubling of gain (p < 0.005), equivalent to a doubling of T wave height. QT intervals measured were significantly longer at slower paper speeds: by 11 ms when paper speed was reduced from 100 to 50 mm/s (p < 0.001) and by 16 ms when speed was further reduced from 50 to 25 mm/s (p < 0.001). Neither the presence of noise nor the presence of a second QRST complex altered the mean QT measurements. There were consistent differences in the measurements between cardiologists, amounting to a maximum mean difference of 20 ms. CONCLUSIONS--Manual measurement of QT interval is significantly affected by the paper speed used to plot the electrocardiogram and by electrocardiogram gain, and hence also T wave amplitude. Manual QT measurement also differed consistently with different cardiologists.

Clinical and electrophysiological differences between patients with arrhythmogenic right ventricular dysplasia and right ventricular outflow tract tachycardia

AIMS Radiofrequency catheter ablation is considered first line treatment for symptomatic patients with right ventricular outflow tract tachycardia (RVOT). The role of ablation in arrhythmogenic right ventricular dysplasia (ARVD) is more limited. As such, differentiating between the two conditions is essential. METHODS AND RESULTS This study compared non-invasive findings, magnetic resonance images (MRI), invasive electrophysiological characteristics, results of ablation and long-term outcome in 50 consecutive patients with RVOT (33) or ARVD (17). Structural abnormalities were uniform in the ARVD group; in addition 18 (54%) of the RVOT tachycardia group had MRI abnormalities. At electrophysiological study the tachycardia in the ARVD group displayed features of re-entry in over 80%, but behaved with a triggered automatic basis in 97% with RVOT. Ablation was complete or partial success in 12 (71%) patients with ARVD and ventricular tachycardia (VT) recurred in eight (48%). In the RVOT patients, ablation was a complete success in 97% with recurrent VT in 6%. Long-term success in the RVOT patients was 95% in both patients with and without MRI abnormalities. CONCLUSIONS Electrophysiological characterization can differentiate ARVD from RVOT. The finding of abnormalities on MRI does not have any bearing on arrhythmia mechanism, acute or long-term success of RFA.

The phenotype of limb-girdle muscular dystrophy type 2I

Background: Mutations in the fukutin-related protein gene FKRP cause limb-girdle muscular dystrophy (LGMD2I) as well as a form of congenital muscular dystrophy (MDC1C). Objective: To define the phenotype in LGMD2I. Methods: The authors assessed 16 patients from 14 families with FKRP gene mutations and LGMD and collected the results of mutation analysis, protein studies, and respiratory and cardiac investigations. Results: Thirteen patients, most with adult presentation, were homozygous for the common C826A mutation in FKRP. The three other cases were compound heterozygotes for C826A and two of them presented in childhood, with more progressive disease. The pattern of muscle involvement, frequently including calf hypertrophy, was similar to dystrophinopathy. Complications in patients with LGMD2I were common and sometimes out of proportion to the skeletal muscle involvement. Six patients had cardiac involvement, and 10 had respiratory impairment: five required nocturnal respiratory support. All patients had serum creatine kinase at least 5 to 70 times normal. The most consistent protein abnormality found on muscle biopsy was a reduction of laminin α2 immunolabeling, either on muscle sections or immunoblotting alone. Conclusions: LGMD2I due to FKRP mutations appears to be a relatively common cause of LGMD, with respiratory and cardiac failure as prominent complications.

Sequence of epicardial repolarisation and configuration of the T wave.

Epicardial activation and repolarisation sequences were investigated in patients with upright or inverted T waves in left ventricular leads of the surface electrocardiogram. Fifteen patients were studied: 10 were undergoing coronary artery bypass grafting (upright T waves) and five aortic valve replacement (four patients with T inversion). Monophasic action potentials were recorded intraoperatively from eight to 10 left ventricular sites in each patient. In patients with upright T waves there was an inverse relation between the duration of the monophasic action potential and the activation time (mean slope -1.44). As a consequence, activation and repolarisation proceeded in opposite directions. Dispersion of repolarisation time (14 ms) was less than dispersion of activation time (23 ms). In patients with T wave inversion caused by aortic stenosis there was no relation between the duration of action potential and activation time; the repolarisation sequence resembled the activation sequence, and the dispersion of repolarisation time was greater than the dispersion of activation time (31 and 26 ms respectively). These results show that there are epicardial repolarisation gradients in man and that these are related to the configuration of the T wave. In patients with upright T waves an inverse relation between the duration of the action potential and the activation time reduces the dispersion of the repolarisation time. When the T wave was inverted this relation was no longer found and the dispersion of repolarisation increased.

Prevention of tolerance to nitroglycerin patches by overnight removal

This investigation assesses the extent of tolerance development with nitroglycerin patches and whether tolerance might be prevented by overnight patch removal. On commencing therapy, active patches significantly prolonged exercise time (3.5 hours after patch application) in comparison with placebo, with an accompanying reduction in ST-segment depression at maximal common workload. Patients then received continuous or 12-hour-daily intermittent patch therapy, in a double-blind fashion, for 7 days. Exercise testing was repeated before and after active patch application, on the eighth day of each treatment phase. During continuous therapy, beneficial effects on exercise time and ST depression were abolished. By contrast, during intermittent therapy, prolongation of exercise time and reduction in ST-segment depression still occurred, on testing 3.5 hours after active patch application. These results confirm previous studies showing a high degree of tolerance during continuous therapy with nitroglycerin patches and suggest that tolerance can be prevented by 12-hour-daily intermittent therapy.

Cardiac and respiratory failure in limb-girdle muscular dystrophy 2I.

Mutations in the gene encoding fukutin‐related protein cause limb‐girdle muscular dystrophy 2I. In this multicenter retrospective analysis of 38 patients, 55.3% had cardiac abnormalities, of which 24% had developed cardiac failure. Heterozygotes for the common C826A mutation developed cardiac involvement earlier than homozygotes. All patients initially improved while receiving standard therapy. Independent of cardiac status, forced vital capacity was below 75% in 44.4% of the patients. There was no absolute correlation between skeletal muscle weakness and cardiomyopathy or respiratory insufficiency. These complications are a primary part of this specific type of limb‐girdle muscular dystrophy, with important implications for management. Ann Neurol 2004;56:738–741

Frequency analysis of atrial fibrillation

Atrial fibrillation (AF) is a common arrhythmia which can have serious clinical consequences. Different characteristics of AF may be more amenable to treatment, but it is not easy to assess the characteristics of the atrial rhythm from ECG recordings since the atrial complexes are small relative to the ventricular complexes. AF is usually seen on the ECG as apparently irregular deviations of the baseline. Our aim was to develop a technique which would allow researchers to retrieve information about the atrial rhythm non-invasively from body-surface ECGs and to assess the stability of the atrial rhythm. We recorded 300 s of simultaneous 12-lead ECGs at 500 Hz directly to a computer from six patients with AF for subsequent analysis. For stability analysis, we split the 300 s recordings into two sections of 150 s each. These were then subdivided into sections of approximately 10 s, and principal component analysis was performed on each 12-lead section, generating 12 orthogonal data components. A frequency analysis of each component was carried out using a fast Fourier transform algorithm and the average spectrum calculated for each 150 s section. From this the dominant AF frequency was identified from the peak in the spectrum between 5 and 10 Hz. The atrial waveform was most commonly observed in principal components 6, 7 and 8 with mean (SD) frequency 6.8 Hz (0.9 Hz) and range 5.9 Hz to 8.2 Hz across the subjects. The difference between the paired 150 s sections was 0.3 Hz (0.3 Hz), showing that the dominant atrial frequency could be identified consistently and that there was little short term variability.

Atrial fibrillation associated with ivabradine treatment: meta-analysis of randomised controlled trials

Objective To quantify any risk of atrial fibrillation (AF) associated with ivabradine treatment by meta-analysis of clinical trial data. Methods Medline, Embase, Web of Knowledge and the Cochrane central register of controlled trials were searched for double-blinded randomised controlled trials of ivabradine with a minimum follow-up period of 4 weeks. For studies where AF data were unpublished, safety data were obtained from the European Medicines Agency (EMeA) website and personal communications. Studies were appraised for risk of bias using components recommended by the Cochrane Collaboration. Meta-analyses were performed of relative risk of AF and absolute risk difference of AF per year of treatment. The main outcome measure was incident AF during the follow-up period. Results AF data were available from 11 studies: one from the published report, six from the EMeA and four from personal communications. Ivabradine treatment was associated with a relative risk of AF of 1.15 (95% CI 1.07 to 1.24, p=0.0027) among 21 571 patients in the meta-analysis. From this we estimated that the number needed to harm for ivabradine would be 208 (95% CI 122 to 667) per year of treatment. Conclusions AF is a substantially more common side effect of ivabradine treatment than one patient in 10 000, the risk presently reported in the product literature. The incidence of AF has not routinely been reported in clinical trials of ivabradine.