608 Heart 1997;78:608–612 CASE STUDY Heart: first published as 10.1136/hrt.78.6.608 on 1 December 1997. Downloaded from Dystrophin gene abnormalities in two patients with idiopathic dilated cardiomyopathy Francesco Muntoni, Andrea Di Lenarda, Maurizio Porcu, Gianfranco Sinagra, Anna Mateddu, Gianni Marrosu, Alessandra Ferlini, Milena Cau, Jelena Milasin, Maria Antonietta Melis, Maria Giovanna Marrosu, Carlo Cianchetti, Antonio Sanna, Arturo Falaschi, Fulvio Camerini, Mauro Giacca, Luisa Mestroni Abstract Cardiac involvement is an integral part of Neuromuscular Unit, Two new cases of dilated cardiomyopathy DMD and BMD.4–7 In rare instances, however, Department of Paediatrics and (DC) caused by dystrophinopathy are patients can suVer from dilated cardiomyo- Neonatal Medicine, reported. One patient, a 24 year old man, pathy (DC) as the only manifestation of a dys- Hammersmith had a family history of X linked DC, while trophinopathy. This has been now recognised Hospital, London, UK the other, a 52 year old man, had sporadic in families with typical X linked DC8–11 and in F Muntoni patients with sporadic disease.12–14 Unlike A Ferlini disease. Each had abnormal dystrophin immunostaining in muscle or cardiac patients with DMD or BMD, these patients did International Centre biopsy specimens, but neither had muscle not have symptoms of muscle weakness and the for Genetic weakness. Serum creatine kinase activity only sign of neuromuscular involvement was Engineering and was raised only in the patient with familial raised serum creatine kinase (CK) activity. Biotechnology e disease. Analysis of dystrophin gene mu- Several patients had mutations clustered in the Divisione di 91112 tations showed a deletion of exons 48–49 in 5' end of the gene ; a region not aVected by Cardiologia, Ospedale mutations usually found in DMD and BMD. e Università di Trieste, the patient with familial DC and of exons DC has also been reported in asymptomatic Trieste, Italy 49–51 in the other. Dystrophin transcrip- 15 A Di Lenarda female carriers of DMD and BMD. tion in cardiac tissue from the patient with http://heart.bmj.com/ G Sinagra sporadic disease showed abundant expres- Two cases of familial (X linked) and sporadic J Milasin sion, predominantly of the muscle iso- DC secondary to a dystrophinotrophy are A Falaschi reported here. F Camerini form. This study, together with previous M Giacca reports, suggests that some patients with L Mestroni DC have a dystrophinopathy that can be Patients diagnosed using a combination of bio- The two patients described in this study origi- Divisione di nate from Italy. Ethical approval was obtained Cardiologia, Ospedale chemical and genetic analyses. (Heart 1997;78:608–612) from the committees of the two hospitals Brotzu, Italy on September 29, 2021 by guest. Protected copyright. M Porcu involved in their assessment. Informed consent A Sanna Keywords: dilated cardiomyopathy; dystrophin; Becker was obtained from each patient. muscular dystrophy Istituto di PATIENT 1 Neuropsichiatria The first patient, a 24 year old man, was Infantile, Italy Duchenne’s (DMD) and Becker’s muscular admitted in August 1991 after the recent onset A Mateddu dystrophies (BMD) are allelic X linked neuro- G Marrosu of dyspnoea with mild physical activity. DC was M G Marrosu muscular disorders. They result from muta- diagnosed (left ventricular end diastolic diam- C Cianchetti tions in the dystrophin gene that, when severe eter 73 mm; left ventricular end diastolic as in DMD (nonsense or out of frame volume 107 ml/mq; left ventricular ejection Dipartimento di mutations), lead to lack of expression of the fraction 27%) after exclusion of active myocar- Biologia e Clinica sarcolemmal protein dystrophin.1 Up to one dell’Età Evolutiva, ditis by endomyocardial biopsy and coronary Cagliari, Italy third of all cases of DMD and BMD arise from artery disease by angiography. An electrocar- M Cau de novo mutations, so that a significant diogram showed Q waves in the inferior and M A Merlis proportion of aVected males have no family posterior leads and incomplete right bundle history of the condition.12The absence of dys- brunch block. Sustained ventricular arrhyth- Correspondence to: trophin in DMD causes severe and progressive Dr Muntoni, Department of mias were detected after 24 hour Holter moni- Paediatrics and Neonatal muscle weakness, loss of ambulation before the toring. Among the biochemical investigations, Medicine, Royal age of 13, and death in the late teens or early constantly raised serum CK activity (MM iso- Postgraduate Medical twenties.3 BMD is a milder form of muscular School, Hammersmith form, ranging between 540 and 867 U/l, Hospital, Du Cane Road, dystrophy in which individuals are ambulant normal < 200 U/l) was found. Neurological London W12 ONN, UK; after the age of 16. These individuals usually investigation failed to show any weakness or email: [email protected] have in frame deletions and residual expression muscle wasting or hypertrophy. The patient Accepted for publication of a partially functional dystrophin in the denied symptoms of neuromuscular involve- 22 July 1997 muscle.3 ment such as cramps or myalgias associated Dystrophin in dilated cardiomyopathy 609 venous pressure or peripheral oedema. His liver was enlarged and neurological examin- Heart: first published as 10.1136/hrt.78.6.608 on 1 December 1997. Downloaded from ation failed to show muscle atrophy or pseudo- hypertrophy. His muscle strength was normal, as was serum CK activity (84 U/l). Negative T waves in leads V5–V6 were seen on the electro- cardiogram. Echocardiography showed a di- lated left ventricle (left ventricular end diastolic diameter 70 mm, left ventricular ejection frac- tion 20%). The right ventricle and both atria were also dilated. Moderate mitral and tricus- pidal valve regurgitation was also found. Right heart catheterisation disclosed pulmonary hy- Figure 1 Pedigree of family with X linked cardiomyopathy. Open symbols, unaVected pertension, (pulmonary artery pressure 68/ individuals; closed circles, aVected individuals; barred symbols, deceased (number above is 30 mm Hg, mean 42 mm Hg), and a reduced age at death); dotted circles, obligate carriers; arrow, propositus. cardiac index (2.0 l/min/m2). Despite medical treatment with angiotensin converting enzyme with exercise. He improved following treat- inhibitors, digitalis, and diuretics the patient ment with digitalis, enalapril, and metoprolol deteriorated clinically and he underwent car- and remained in New York Heart Assocation diac transplantation nine months later. In the (NYHA) class I–II. During follow up the left early postoperative period he suVered from ventricular dimension slightly diminished (left multiorgan failure and died shortly after. ventricular end diastolic diameter 70 mm), although the systolic function remained signifi- Methods cantly depressed (left ventricular ejection frac- IMMUNOHISTOCHEMICAL STUDY OF SKELETAL tion 37%) with diVuse wall motion abnormali- AND CARDIAC MUSCLE ties, more evident in the inferoposterior region. A cardiac biopsy specimen obtained at Mild right ventricular dysfunction was ob- cardiac transplantation, and a needle biopsy served at radionuclide angiography (right ven- specimen of skeletal muscle taken from patient tricular ejection fraction 35%). At the last 1 were immediately frozen in liquid nitrogen follow up (1996) the patient was in NYHA cooled isopentane and stored at −70ºC or in class II with a peak consumption of oxygen of liquid nitrogen. The specimens were fully 14.4 ml/kg/min (anaerobic threshold 5.8 ml/ processed for histological, histochemical, and kg/min) and without evidence of electrical ven- immunohistochemical examination.16 In par- tricular instability with Holter monitoring or ticular, unfixed cryostat sections (6 µm) were during exercise. immunostained using a panel of six antidys- Analysis of the pedigree disclosed a strong trophin antibodies, including the monoclonal http://heart.bmj.com/ family history of DC suggestive of an X linked antibody DYS1808 (Ylem, Italy), as already inheritance (fig 1). Individuals II:3, III:6, and described.17 III:11 (fig 1) had been aVected by DC and died at the ages of 36, 42, and 38, respectively. DNA STUDIES Moreover, a maternal aunt (individual II:5, fig DNA was isolated from leucocytes by standard 1), an obligate carrier for the condition, also methods. Multiplex DNA amplification of dys- died of DC at the age of 57. Interestingly, indi- trophin exons was carried out according to vidual III:8 had DMD and died because of res- previously described techniques.18 19 piratory complications at the age of 22. All on September 29, 2021 by guest. Protected copyright. other aVected members did not have signs of REVERSE TRANSCRIPTION AND POLYMERASE skeletal muscle involvement, at least as far as CHAIN REACTION could be assessed from their medical records Total RNA20 was isolated from control frozen and questioning their relatives. A needle skeletal muscle and heart and from the left muscle biopsy specimen from the propositus ventricle of patient 2. cDNA synthesis was (individual III:4, fig 1) was obtained to investi- performed using random hexanucleotide prim- gate the possibility of an underlying dystrophi- ers, following the procedure already nopathy. described.21 22 Polymerase chain reaction was performed using forward primers designed to PATIENT 2 amplify the muscle, brain, and Purkinje cell The second patient, a 52 year old man, died isoforms and an exon 6 reverse primer as shortly after cardiac transplantation in 1994. already described.21 He was employed in the chemical industry and was physically active until the age of 50 when Results he presented with the first symptoms of cardiac IMMUNOHISTOCHEMICAL ANALYSIS failure. There was no family history of cardio- The skeletal muscle biopsy specimen from the vascular disease. The patient smoked 20 propositus showed mild dystrophic changes, cigarettes daily and admitted moderate alcohol characterised by an increase in internal nuclei intake until the age of 50, when he developed a (12%), mild variability of fibre size with hypo- gastric peptic ulcer that eventually required trophic and hypertrophic fibres, and rare fibre surgical excision.