Genetic and ultrastructural studies in dilated cardiomyopathy patients: a large deletion in the lamin A/C gene is associated with cardiomyocyte nuclear envelope disruption

Abstract

Major nuclear envelope abnormalities, such as disruption and/or presence of intranuclear organelles, have rarely been described in cardiomyocytes from dilated cardiomyopathy (DCM) patients. In this study, we screened a series of 25 unrelated DCM patient samples for (a) cardiomyocyte nuclear abnormalities and (b) mutations in LMNA and TMPO as they are two DCM-causing genes that encode proteins involved in maintaining nuclear envelope architecture. Among the 25 heart samples investigated, we identified major cardiomyocyte nuclear abnormalities in 8 patients. Direct sequencing allowed the detection of three heterozygous LMNA mutations (p.D192G, p.Q353K and p.R541S) in three patients. By multiplex ligation-dependant probe amplification (MLPA)/quantitative real-time PCR, we found a heterozygous deletion encompassing exons 3-12 of the LMNA gene in one patient. Immunostaining demonstrated that this deletion led to a decrease in lamin A/C expression in cardiomyocytes from this patient. This LMNA deletion as well as the p.D192G mutation was found in patients displaying major cardiomyocyte nuclear envelope abnormalities, while the p.Q353K and p.R541S mutations were found in patients without specific nuclear envelope abnormalities. None of the DCM patients included in the study carried a mutation in the TMPO gene. Taken together, we found no evidence of a genotype-phenotype relationship between the onset and the severity of DCM, the presence of nuclear abnormalities and the presence or absence of LMNA mutations. We demonstrated that a large deletion in LMNA associated with reduced levels of the protein in the nuclear envelope suggesting a haploinsufficiency mechanism can lead to cardiomyocyte nuclear envelope disruption and thus underlie the pathogenesis of DCM.

Fig. 1

Electron micrographs of cardiomyocytes from dilated cardiomyopathy patients with or without LMNA mutation. a, b, c and d Major nuclear abnormalities in, respectively, patient 1, 3, 4 and 5 (Table 2). Patient 1 carried the LMNA deletion exons 3–12, while patients 3, 4 and 5 had no LMNA nor TMPO mutation; a accumulation of mitochondria around the nuclear envelope; b blebbing of the nuclear envelope and separation of the inner and the outer nuclear membrane; c extrusion of nucleoplasm from the cardiomyocyte nucleus into the cytoplasm; d accumulation of cytoplasmic organelles in the nucleoplasm; e non-specific nuclear alteration in patient 10 (Table 2) carrying the p.Q353K LMNA mutation; f non-specific alterations in dilated cardiomyopathy patient 14 (Table 2) with wild-type LMNA and TMPO (original magnifications: a, 20,000×; b, 18,000×; c, 12,000×; d, 10,000×; e, 4,000×; and f, 4,000×)

Fig. 2

Pedigree of the families with LMNA mutations. Arrow indicates proband; black filled symbol dilated cardiomyopathy patient; open symbol asymptomatic individual. a Deletion of exons 3–12; b p.Q353K

Fig. 3

MLPA analysis graph showing the heterozygous LMNA deletion of exons 3–12 in patient 1. Exons 3–12 display a normalized ratio of ~0.5; indicating a loss of genetic materiel. Exons 1, 2 and the remaining control probes display a normalized ration of ~1.0; indicating normal copy number. The control probes are targeted within the same chromosome and to different chromosomes as indicated by their locus

Fig. 4

Confirmation of the deletion of exons 3–12 in LMNA in patient 1 using qPCR. R values were calculated as described in the “Materials and methods”. Patient 14 without the deletion had a R value of 0.955, which indicates a normal copy number of the exon. The R value for patient 1 is 0.476, which indicates the presence of only one copy of the exon (n = 3)

Fig. 5

Indirect immunofluorescence analysis of endomyocardial biopsy from patient 1 carrying the LMNA heterozygous exon 3–12 deletion and from a control patient with no LMNA mutation. Endomyocardial biopsy was taken from the right ventricle. Immunostaining was performed using anti-lamin A+C monoclonal antibodies ACF and antibody A4 which detects lamin A only (see “Materials and methods”) and the goat anti-rabbit IgG secondary antibody (original magnification 1,050×)