Summary
Background
In patients with myotonic dystrophy type 1 (DM1), the mechanisms underlying sudden cardiac death, which occurs in up to 1/3 of patients, are unclear.
Aims
To study the potential role of Brugada syndrome in ventricular tachyarrhythmias and sudden death in DM1 patients.
Methods
We screened 914 adult patients included in the DM1 Heart Registry during 2000–2009 for the presence of type 1 Brugada pattern on electrocardiogram (ECG). We also performed direct sequencing of SCN5A in patients with Brugada pattern. Further, we analysed SCN5A splicing on ventricular myocardial specimens harvested during cardiac transplantation in a 45-year-old patient with DM1 and three controls with inherited dilated cardiomyopathy.
Results
A type 1 Brugada pattern was present on the ECG of seven of 914 patients (0.8%), including five with a history of sustained ventricular tachyarrhythmia or sudden death, who fulfilled the criteria for Brugada syndrome. SCN5A sequencing was normal in all patients. Ventricular myocardial specimen analysis displayed abnormal splicing of SCN5A exon 6, characterized by over-expression of the ‘neonatal’ isoform, called exon 6A, in the patient with DM1, but not from the controls.
Conclusion
Our findings suggest a potential implication of Brugada syndrome in sudden death in DM1, which may be related to missplicing of SCN5A. Our findings provide a new insight into the pathophysiology of heart disease in DM1.
ClinicalTrials.gov
Number NCT01136330 .
Résumé
Contexte
Dans la dystrophie myotonique de type 1 (DM1), les mécanismes de la mort subite, qui sont responsables d’un tiers des décès, sont inconnus.
Objectif
Étudier l’implication du syndrome de Brugada dans les troubles du rythme ventriculaires et la mort subite dans la DM1.
Méthodes
Nous avons recherché un aspect de Brugada de type 1 sur les électrocardiogrammes de 914 patients adultes inclus dans le DM1 Heart Registry entre 2000 et 2009. Un séquençage direct de SCN5A a été effectué chez les porteurs d’un Brugada de type 1. Nous avons étudié l’épissage de SCN5A sur du tissu myocardique prélevé lors de transplantations cardiaques chez un patient atteint de DM1 et trois témoins atteints de cardiomyopathies dilatées.
Résultats
Un aspect de Brugada de type 1 a été identifié chez sept patients (0,8 %), dont cinq remplissant les critères pour un diagnostic de syndrome de Brugada, devant l’association à un trouble du rythme ventriculaire soutenu ou une mort subite. Le séquençage de SCN5A était normal chez tous ces patients. L’étude du tissu myocarde du patient DM1 a mis en évidence un épissage anormal de l’exon 6 de SCN5A, caractérisé par la surexpression de l’isoforme 6A, dite isoforme « néonatale », non identifiée dans les tissus témoins.
Conclusions
Ces résultats suggèrent l’implication potentielle du syndrome de Brugada dans certains cas de mort subite chez les patients atteints de DM1, qui pourrait être liée à des anomalies d’épissage de SCN5A. De manière plus générale, ces résultats apportent un éclairage nouveau sur la physiopathologie de l’atteinte cardiaque dans cette pathologie.
Introduction
Myotonic dystrophy type 1 (DM1), also known as Steinert disease, is the most common inherited neuromuscular disease in adults with an incidence of 1/8000 . It is an autosomal dominant disorder mapping to chromosome 19q, caused by an expansion of a (CTG)n triplet repeat in the 3’ region of the gene encoding dystrophia myotonica protein kinase (DMPK) .
The main pathogenetic characteristic of DM1 is the nuclear accumulation of mutant messenger ribonucleic acids (mRNAs), which induces the aberrant alternative splicing of multiple pre-mRNAs responsible for phenotypical abnormalities of multiple systems . Clinical manifestations of DM1 include myotonia, progressive skeletal muscle loss, cataracts, insulin resistance and heart disease.
The most frequent cardiac complications of DM1 are atrioventricular block, sinus node dysfunction, atrial fibrillation and ventricular tachyarrhythmias . Up to 1/3 of DM1 patients die suddenly, most often due to asystole after atrioventricular block or from ventricular fibrillation . Severe ECG conductive abnormalities or a diagnosis of atrial tachyarrhythmia predict sudden death . Prophylactic pacing has been associated with improved survival , but indications for the implantation of a cardioverter defibrillator are not clear, as clinical risk factors specific to ventricular arrhythmias have not yet been identified. In addition, in contrast with other manifestations of the disease , the mechanisms behind the development of cardiac arrhythmias have not been clearly identified .
Brugada syndrome is an autosomal dominant disorder that associates ST-segment elevation in leads V1 to V3 on the electrocardiogram (ECG) and a high incidence of ventricular fibrillation and sudden cardiac death . SCN5A, the gene encoding the Na+ channel α-subunit expressed in the human heart, is the main gene responsible for this syndrome . Other cardiac manifestations of SCN5A mutations causing Na+ channel loss of function, such as familial atrioventricular blocks or atrial fibrillation , are very similar to those observed in DM1.
We tested the hypothesis that Brugada syndrome could be a cause of severe ventricular arrhythmias in DM1. We therefore assessed the prevalence of patients with a Brugada ECG pattern in a large population of DM1 patients and their risk profile for severe ventricular arrhythmias and studied the splicing of SCN5A in myocardial tissue harvested from a patient suffering from DM1.
Methods
Screening for type 1 Brugada ECG pattern among patients included in the DM1 Heart Registry
We screened for the presence of a Brugada pattern on the ECGs of patients included in the DM1 Heart Registry. This Registry was designed and organized by the Neurological Unit of Pitié-Salpêtrière Hospital and the Cardiological Unit of Cochin Hospital (ClinicalTrials.gov no: NCT01136330 ). This registry retrospectively included 914 consecutive patients aged ≥ 18 years admitted to our institutions between January 2000 and December 2009 for the management of DM1, diagnosed by the presence of ≥ 50 CTG triplets in the 3’ un-translated region of the DMPK gene of blood leukocytes. The patients’ medical records were reviewed and the genetic, neurological and cardiac information was entered in a dedicated database. The patients’ ECGs at baseline and during follow-up were screened for type 1 Brugada pattern by two independent experts (HMB and DD) who were unaware of the patients’ information. A Brugada pattern was considered to be present when both investigators shared the same positive interpretation.
This study, which complies with the ethical principles formulated in the declaration of Helsinki, was approved by our local ethics committee, and all patients, except those who died before initiation of the study, granted their informed written consent to participate in the registry.
Cardiac investigations
Patients with type 1 Brugada pattern underwent clinical examination, 12-lead ECG, 24-hour ambulatory ECG, echocardiography and electrophysiological testing. The electrophysiological testing protocol used one bipolar and one quadripolar electrode introduced into the femoral vein and advanced to the right atrium and right ventricle, respectively. Programmed stimulation was performed with three extrastimuli at a strength equal to twice end-diastolic threshold delivered during spontaneous rhythm and after paced trains at rates of 100 and 150 bpm from the high right atrium and from right ventricular apex and pulmonary outflow tract.
SCN5A sequencing in patients with type 1 Brugada pattern
The patients whose ECG was consistent with type 1 Brugada pattern underwent direct sequencing of SCN5A on genomic deoxyribonucleic acid (DNA) from blood lymphocytes. All coding exons and intronic flanking sequences were amplified by polymerase chain reaction (PCR) as previously described .
Statistical analysis
Data are expressed as mean ± standard deviation (SD) for continuous variables, and counts and percentages for categorical variables. The STATA ® statistical software, version 10.1 (StataCorp LP, College Station, TX) was used for all analyses.
Ribonucleic acid (RNA) splicing studies in human ventricular myocardial tissue
Ventricular myocardial specimens were harvested during cardiac transplantation for end stage heart failure in: (1) a 45-year-old patient suffering from DM1 (166 CTG repeats) and dilated cardiomyopathy with conduction system disease and (2) three control patients suffering from familial dilated cardiomyopathy of uncharacterized genetic origin. Total RNA was extracted using the kit RNAPLUS2TM according to manufacturer’s instructions corresponding to an improvement on Chomczynski’s procedure . We used a reverse transcriptase (RT)-PCR assay to study the alternative splicing in right ventricular myocardial tissue of SCN5A and two control genes where abnormal splicing has previously been reported in DM1: troponin T and dystrophin . For the SCN5A splicing study, a set of primers spanning all the genes were selected. PCR was performed using the GeneAmp ® PCR System 9700 (Applied Biosystems, France). Products were analysed by Gel Electrophoresis to detect abnormal splicing. PCR products were sequenced using the “BigDye ® Terminator v.3.1 cycle sequencing kit” on ABI PRISM 3130 analyser (Applied Biosystems). Primers sequences, with their position on the complementary DNA (cDNA) variant Nav1.5 (NM_000335), and technical conditions are available on request. PCR products were subjected to restriction analysis with Bfa I and ApoI, respectively, specific to digestion of exons 6a and 6b of SCN5A. The digestion protocol was carried out in appropriate reaction buffer NEB 3 with BSA (100 mM NaCl; 50 mM Tris-HCl; pH: 7.9; 10 mM MgCl2 SH: 1 mM DTT; BSA: 100x). The resulting fragments were analysed by electrophoresis.
Results
The DM1 Heart Registry: prevalence of type 1 Brugada pattern
The baseline characteristics of the 914 patients included in the DM1 Heart Registry are summarized in Table 1 . Conduction system disease was present in 376 patients (41.1%), a history of supraventricular arrhythmia in 64 (7.0%), left ventricular dysfunction in 50 (5.6%), and sustained ventricular tachyarrhythmia in three patients (0.3%). A type 1 Brugada pattern ( Fig. 1 ) was present on the 12-lead ECG of seven patients (0.8%), whose characteristics are summarized in Table 2 . No patient had a family history of sudden death or Brugada syndrome. The ECG Brugada pattern was intermittent in three patients, without apparent trigger. Programmed ventricular stimulation induced sustained ventricular tachyarrhythmia in four patients, prompting the implantation of a cardioverter defibrillator in three patients and a pacemaker in one patient (no. 1), who had refused to undergo the implantation of a defibrillator and who died suddenly of ventricular fibrillation. Patient no. 6 also died suddenly, though no ECG was recorded at the time of death. Overall, five of the seven patients had a history of sustained ventricular tachyarrhythmia or died suddenly, fulfilling the criteria for Brugada syndrome.
| All patients ( n = 914) | |
|---|---|
| Age (years) | 38 ± 14 |
| Men | 443 (48.5) |
| Heart rate (bpm) | 69 ± 13 |
| Systolic blood pressure (mmHg) | 117 ± 13 |
| Diastolic blood pressure (mmHg) | 67 ± 9 |
| Cytosine-thymine-guanine amplification size (number of triplets) | 604 ± 415 |
| History of | |
| Supraventricular arrhythmia | 64 (7.0) |
| Complete heart block | 4 (0.4) |
| Sinus node dysfunction | 12 (1.3) |
| Sustained ventricular tachyarrhythmia | 3 (0.3) |
| Pacemaker implantation | 67 (7.3) |
| Cardioverter defibrillator implantation | 3 (0.3) |
| Surface and intracardiac ECG observations | |
| First degree atrioventricular block | 287 (31.4) |
| Bundle branch block | |
| Left | 98 (10.7) |
| Right | 63 (6.9) |
| Paced rhythm | 23 (2.5) |
| Brugada pattern | 7 (0.8) |
| Intervals (ms) | |
| PR | 189 ± 31 |
| QRS | 100 ± 19 |
| Corrected QT | 410 ± 29 |
| AH a | 117 ± 47 |
| H a | 18 ± 5 |
| HV a | 66 ± 12 |
| HV interval > 55 ms and < 70 ms a | 115 (46.4) |
| HV interval ≥ 70 ms a | 96 (38.7) |
| Induced ventricular fibrillation or sustained polymorphic ventricular tachycardia a | 21 (8.5) |
| Echocardiographic measurements b | |
| Left ventricular | |
| Dysfunction | 50 (5.6) |
| End-diastolic diameter (mm/m 2 ) | 45 ± 5 |
| Posterior wall thickness (mm) | 7.8 ± 1.4 |
| Mass (g) | 204 ± 61 |
| Ejection fraction (%) | 63 ± 7 |
| Septal wall thickness (mm) | 8.6 ± 1.7 |
| Device therapy at inclusion in the Registry | |
| Pacemaker | 66 (7.2) |
| Implantable cardioverter defibrillator | 8 (0.9) |
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