The Role of Lamin A/C Mutations in Danish Patients with Idiopathic Dilated Cardiomyopathy

Abstract

Idiopathic dilated cardiomyopathy is characterized primarily by left ventricular dilatation and impaired function. More than 25 genes have been shown to be associated with IDC1,2; however, with the exception of the Lamin A/C (LMNA) gene, each of these genes accounts for <2% of cases. Idiopathic dilated cardiomyopathy caused by LMNA mutations is often accompanied by conduction disorders, cardiac arrhythmias, and/or discrete muscle disorders.3,4 The LMNA gene consists of 12 exons and encodes the intermediate filament proteins Lamin A and C, which maintain the structural integrity of the nuclear envelope and organize chromatin within the nucleus, thereby influencing DNA transcription.5–7 To examine a Danish IDC cohort for mutations in the LMNA gene, to assess the extent to which the mutation contributes to the disease and how it may impact on family counselling. The study population consisted of 172 unrelated Caucasian IDC patients [67 familial (FDC) and 105 non-FDC] referred for heart failure assessment at a tertiary heart centre at the University Hospital, Rigshospitalet in Copenhagen, Denmark. Controls comprised 185 Guthrie cards (blood spots from newborns), which had been anonymously selected, and samples from 165 healthy blood-donors, giving a total of 350 controls (700 alleles). The study conforms to the principles outlined in the Declaration of Helsinki and was approved by the Scientific Ethics Committee of Copenhagen and Frederiksberg [No. (KF) 01263289]. All patients gave written informed consent. Intron-specific LMNA primers (available upon request) were used to amplify the 12 coding regions and their intronic boundaries by PCR and were bi-directionally sequenced on an ABI 3730 (Applied Biosystems, Foster City, CA, USA). Sequences were analysed using Sequencher software (Gene Codes, Ann Arbor, MI, USA). The frequency of all identified variants was determined in 350 Danish controls (700 alleles) as well as in available family members of the probands. All probands with suspected disease-causing LMNA mutations, determined by the criteria described previously,8 were also screened for mutations by capillary electrophoresis single-strand conformer polymorphism and subsequent bi-directional DNA sequencing of aberrant conformers in MYH7, MYBPC3, MYL2, MYL3, TPM1, TNNT2, ACTC, CSRP3, PLN, TCAP, and TNNI3. We identified six (3.5%) putative disease-causing variants (five known) in six probands. Only 1 of the 105 non-FDC probands (1%) carried an LMNA mutation compared with 5 of the 67 FDC probands (7.5%). Three of the mutations were nonsense mutations, and three were missense mutations. Identified mutations are described in Table 1. Five of these mutations have been identified previously as disease-causing. Three mutations were found in dilated cardiomyopathy (DCM) patients (R321X,9 R331Q,10 and Q355X11); the remaining two (R644C12 and V440M13) were found in another laminopathy-associated disease. The V440M mutation was found in a Dunnigan-type familial partial lipodystrophy (FPLD) pedigree, containing a compound LMNA mutation carrier (V440M and R482Q) as well as a family member with uncertain phenotype only carrying the V440M mutation. This carrier was clinically without symptoms suggestive of FPLD or DCM, so the potential role of the V440M mutation was uncertain.13 The R644C mutation has been linked to a diversity of phenotypic presentations, including limb girdle muscle weakness, dilated cardiomyopathy, lipodystrophia, and atypical progeria.14 All missense mutations involved evolutionarily highly conserved amino acids as shown in Table 2. No variants were found in the 11 additional genes screened. Of the six LMNA mutations, five were not identified in any of the 350 control samples (700 alleles); however, the R644C mutation was found in 3 of the 185 Guthrie cards (1.6%), but not in any of the samples from the 165 healthy blood donors. Excluding the R644C mutation results in 4 out of 67 FDC probands (6%) carrying a mutation and 1 out of 105 (1%) in the non-FDC group. In five of the six pedigrees with disease-causing mutations, there was a history of sudden cardiac death. Clinical data of probands and their genotype-positive family members are shown in Table 1, and pedigrees are shown in Figure 1. Mutations in the LMNA gene account for up to 7.5% of FDC probands. By comparison, only 1% of non-FDC probands carry an LMNA mutation. This makes the gene responsible for a significant proportion of cases with IDC and suggests that it is one of the most important genes to screen when assessing IDC patients, as shown by recent work performed on a US population by Parks et al.15 Conduction system disorders should not be used to discriminate which patients should undergo LMNA gene analysis, as only two of the six probands carrying a mutation had a conduction disorder in our study; in addition, 17 probands had conduction system disease. The mutations, which are apparently pathological in their own way, may themselves be modifiers of other unknown ‘more pathological’ mutations. This could explain the asymptomatic mutation carriers seen within the pedigrees or could be a sign of reduced penetrance/late expression. This phenomenon appears within the M-176 family, where two apparently healthy daughters carry the same mutation as their affected father. As the pathogenicity of the V440M mutation is doubtful based on the existing literature, this mutation could, in fact, act as a disease modifier in conjunction with a not yet discovered mutation in the affected individual (M176, II-1). This could explain the phenotypic diversity found within the family members. The mutation was not found in any of the 350 controls, and the proband did not carry any alterations in the 11 additional genes screened. The presence of the R644C mutation in 0.8% (allelic frequency) of the Danish population (represented by the Guthrie cards) brings into question the pathogenicity of this mutation, although we did not find it in any of our 165 healthy blood donor controls. Co-segregation analysis within the M-133 family was not possible, as no DNA was available from either the deceased father or the apparently healthy sisters. Thus, LMNA mutations account for between 4.5 and 7.5% of FDC, depending on the disease-causing status assigned to the R664C and V440M mutations. Future functional studies should further elucidate the consequence of these two mutations. In view of the malignant nature of LMNA mutations as reported by Pasotti et al.,16 who found that 62% of LMNA mutation carriers developed lethal ventricular arrhythmias, genetic cascade screening could be used to identify which family members to focus on and in whom prophylactic measures may be required. Future strategies for the management of this controversial group of patients include prospective trials of device therapy, and anti-congestive and anti-arrhythmic medication in asymptomatic mutation carriers. In conclusion, among 172 unrelated cases of IDC we identified six (3.5%) to be caused by mutations in the LMNA gene. Presymptomatic LMNA mutation screening of relatives seems indicated considering the high proportion of ventricular arrhythmias and cardiac arrests associated with LMNA mutation carriers. This work was supported by The Danish Heart Foundation (grant 07–10-R60-A1826-B323–22423), The Edith and Olfert Dines Hansen Foundation (to D.V.M.), and The John and Birthe Meyer Foundation (to M.C.). Conflict of interest: none declared.