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Expanding the Phenotype of LMNA Mutations in Dilated Cardiomyopathy and Functional Consequences of These Mutations

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Expanding the Phenotype of LMNA Mutations in Dilated Cardiomyopathy and Functional Consequences of These Mutations 560 ORIGINAL ARTICLE J Med Genet: first published as 10.1136/jmg.40.8.560 on 14 August 2003. Downloaded from Expanding the phenotype of LMNA mutations in dilated cardiomyopathy and functional consequences of these mutations P Sébillon, C Bouchier, L D Bidot, G Bonne, K Ahamed, P Charron, V Drouin-Garraud, A Millaire, G Desrumeaux, A Benaïche, J-C Charniot, K Schwartz, E Villard, M Komajda ............................................................................................................................. J Med Genet 2003;40:560–567 Aims: Mutations in the lamin A/C gene (LMNA) have been reported to be involved in dilated cardio- myopathy (DCM) associated with conduction system disease and/or skeletal myopathy. The aim of this study was to perform a mutational analysis of LMNA in a large white population of patients affected by See end of article for dilated cardiomyopathy with or without associated symptoms. authors’ affiliations ....................... Methods: We performed screening of the coding sequence of LMNA on DNA samples from 66 index cases, and carried out cell transfection experiments to examine the functional consequences of the Correspondence to: mutations identified. Dr P Sébillon, Laboratoire Results Génétique et Insuffisance : A new missense (E161K) mutation was identified in a family with early atrial fibrillation and Cardiaque, Pavillon a previously described (R377H) mutation in another family with a quadriceps myopathy associated Rambuteau, Groupe with DCM. A new mutation (28insA) leading to a premature stop codon was identified in a family Hospitalier Pitié-Salpêtrière, affected by DCM with conduction defects. No mutation in LMNA was found in cases with isolated 47 bd de l’Hôpital, 75651 dilated cardiomyopathy. Functional analyses have identified potential physiopathological mechanisms Paris Cedex 13, France; [email protected] involving identified mutations, such as haploinsufficiency (28insA) or intermediate filament disorgani- sation (E161K, R377H). Revised version received Conclusion: For the first time, a specific phenotype characterised by early atrial fibrillation is associ- 14 April 2003 Accepted for publication ated with LMNA mutation. Conversely, mutations in LMNA appear as a rare cause of isolated dilated 1 May 2003 cardiomyopathy. The variable phenotypes observed in LMNA-DCM might be explained by the ....................... variability of functional consequences of LMNA mutations. ilated cardiomyopathy (DCM), the most frequent form ated with variable conduction system disease and DCM with- of cardiomyopathy, is a myocardial disorder character- out skeletal myopathy. LMNA mutations in patients with DCM http://jmg.bmj.com/ ised by ventricular dilatation and impaired systolic without conduction defects or skeletal muscle dystrophy have D 22 23 function leading to congestive heart failure and sudden been reported by Genschel et al, but only in mutation death.1 The aetiology of the disease is variable but one-third of reports without detailed clinical data. More recently, we have cases of idiopathic DCM are inherited.2–4 Various modes of identified a missense mutation in a large family associated inheritance have been reported but the autosomal dominant with DCM with conduction defects and a quadriceps specific form occurs most frequently and exhibits both clinical myopathy.24 Despite a few publications implying LMNA muta- variability and genetic heterogeneity. To date, 11 genes, cardiac tions to be responsible for isolated DCM, the exact prevalence actin,5 desmin,6 δ-sarcoglycan,7 cardiac troponin T,89β-myosin of such mutations remains unknown. on October 1, 2021 by guest. Protected copyright. heavy chain,810α-tropomyosin,11 titin,12 the cardiac MyBP-C,10 Lamins A and C proteins are intermediate filaments, metavinculin,13 muscle LIM protein,14 and phospholamban15 components of the inner layer of the nuclear membrane. They have been associated with autosomal dominant DCM without are assumed to play an important role in maintaining the conduction system disease, skeletal muscle dysfunction, or structural integrity of the nuclear envelope and in organising other characterised phenotypes. Differences in clinical mani- chromatin within the nucleus. However, it remains unknown festations of DCM are apparent among the different families why defects in these ubiquitous proteins can result in abnor- where inherited mutations within the lamin A/C gene have malities of specific and highly differentiated tissues such as been described. The first mutation of LMNA was identified by skeletal and cardiac muscle composed of differentiated cells. Bonne et al16 in a large French pedigree with autosomal domi- Functional studies have investigated the intracellular localisa- nant Emery-Dreifuss muscular dystrophy (AD-EDMD).16 tion of mutant lamin A proteins by an immunofluorescence Interestingly, in this pedigree, five out of the 17 affected mem- microscopy approach.25–28 Among 15 mutant proteins ana- bers had typical AD-EDMD with both skeletal and cardiac lysed, four showed an abnormal localisation and partially dis- symptoms and 12 had isolated cardiac involvement character- rupted the endogenous lamina and three altered the localisa- ised by severe atrioventricular conduction defects and sinus tion of emerin. dysfunction that resulted in DCM.17 Mainly, two laboratories In order to determine the epidemiology of LMNA mutations have identified several missense LMNA mutations; each of in a large white population with isolated DCM or affected by these reported mutations was associated with conduction DCM with conduction defect and/or skeletal muscle myo- defects such as sinus bradycardia, atrioventricular conduction pathy, we screened the entire coding sequence of the lamin block, or atrial arrhythmias.18–20 A family was also reported A/C gene for mutations in 47 unrelated probands from with dilated cardiomyopathy with variable skeletal muscle families and 19 sporadic cases recruited in France. We also involvement where one patient, out of five affected subjects, examined the functional consequences of identified muta- displayed an isolated DCM phenotype. Jakobs et al21 reported tions on heart and muscle biopsies by cell transfection experi- two novel mutations in the rod segment in lamin A/C associ- ments of mutant lamin cDNAs and by standardised RT-PCR. www.jmedgenet.com Lamin A/C gene in dilated cardiomyopathy 561 SUBJECTS AND METHODS epitope of influenza haemagglutinin. In order to obtain the Subjects and clinical evaluation lamin cDNAs containing each of the described mutations J Med Genet: first published as 10.1136/jmg.40.8.560 on 14 August 2003. Downloaded from Patients with at least one first degree relative with docu- (28insA, E161K, and R377H), we performed direct in vitro mented idiopathic DCM were identified as familial cases mutagenesis with the QuikChangeTM Site-Directed Mutagen- (probands). The clinical status was determined in all available esis Kit with the protocol according to the manufacturer’s rec- first degree relatives after clinical examination including ommendations (STRATAGENE). Mutant cDNA was entirely muscular testing, 12 lead ECG, and echocardiography, as pre- sequenced using the ABI PRISM BigDye Terminator cycle viously described.29 30 Holter ECG and serum creatine kinase sequencing ready reactions kit and run on a 3100 Genetic dosages were performed when possible. Clinical status Analyzer (Applied Biosystems). (affected, unknown, or healthy) of subjects was defined 29 31 according to criteria previously described. Written in- Cell culture and transfection formed consent was obtained in accordance with the study COS and C2C12 cell lines (American Type Cell Collection) were protocol approved by the local ethical committee. Inheritance maintained in Dulbecco’s modified Eagle’s medium (DMEM) in familial forms was reviewed and families with X linked or Glutamax supplemented with 10% fetal calf serum (AbCys) mitochondrial inheritance were excluded. A total of 66 and gentamicin at 50 µg/ml. Cells were plated on coverslips at consecutive and unrelated index cases were included in the 300 000 cells per 35 mm plates for 12 to 24 hours before trans- study, including 47 from families with autosomal dominant fection. Transient transfections were performed by lipofection DCM and 19 with sporadic forms of DCM. They were mainly with lipofectamine2000 (Invitrogen) as follows: DNA (7.5 µg) of European origin. Most cases were characterised by isolated and lipofectamine2000 (11 µg) were mixed in a final volume DCM (n=56), 3% of patients had DCM with AV block and of 1 ml of DMEM without serum and left at room temperature muscular dystrophy (n=2), and 6% of cases presented a DCM for 15-20 minutes and then incubated with the cells for five with isolated raised serum creatine level (without clinical hours at 37°C. Then the transfection medium was replaced by muscular dystrophy) (n=4). Finally, four families had 8 ml of DMEM with serum and cells incubated for 24 to 48 frequent atrial fibrillation including one family with early hours before analysis. atrial fibrillation preceding DCM (1.5%). Pathological studies on heart Molecular biology We analysed one post-transplant cardiac biopsy from patient Genomic DNA was extracted from white blood cells by means III.15 (family A). Sections of paraffin embedded cardiac tissue of standard procedures, in collaboration with the Genethon were stained with haematoxylin/eosin and Sirius red, and Bank (Evry, France). Systematic PCR-SSCP sequencing analy- morphometric analysis allowed
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