Clinical Features of LMNA-Related Cardiomyopathy in 18 Patients and Characterization of Two Novel Variants

Abstract

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed myocardial performance in the absence of hypertension, valvular, congenital, or ischemic heart disease. Mutations in LMNA gene, encoding for lamin A/C, account for 10% of familial DCM. LMNA-related cardiomyopathies are characterized by heterogeneous clinical manifestations that vary from a predominantly structural heart disease, mainly mild-to-moderate left ventricular (LV) dilatation associated or not with conduction system abnormalities, to highly pro-arrhythmic profiles where sudden cardiac death (SCD) occurs as the first manifestation of disease in an apparently normal heart. In the present study, we select, among 77 DCM families referred to our center for genetic counselling and molecular screening, 15 patient heterozygotes for LMNA variants. Segregation analysis in the relatives evidences other eight heterozygous patients. A genotype-phenotype correlation has been performed for symptomatic subjects. Lastly, we perform in vitro functional characterization of two novel LMNA variants using dermal fibroblasts obtained from three heterozygous patients, evidencing significant differences in terms of lamin expression and nuclear morphology. Due to the high risk of SCD that characterizes patients with lamin A/C cardiomyopathy, genetic testing for LMNA gene variants is highly recommended when there is suspicion of laminopathy.

Keywords: LMNA; dilated cardiomyopathy (DCM); lamin A; lamin C; next generation sequencing (NGS).

Figure 1

Schematic representation of lamin A transcript and localization of exonic variants identified in this study. * stands for nonsense mutations while = stands for synonymous mutations.

Figure 2

(A) Segregation of R189Q in Family 6. (B) Segregation of E317K in Family 14. Arrows indicate the proband. Wt: wild type. * stands for positive patients to Lamin A/C gene (LMNA) variants.

Figure 3

(A) Representative immunolabeling for lamin A/C (red) and Prelamin A (red) of WT and DCM HDFs (IV-7, IV-9, and III-2). Nuclei are counterstained with Hoechst 33,342 (blue). Scale bar 20 µm, 100 µm. (B) Percentage of WT and DCM cells with abnormal nuclear irregularities revealed in HDFs patients. Results represent three independent experiments with significant differences between WT and DCM HDFs (** p < 0.01). (C) Bar graphs represent the four parameters relative to nuclear shape (area; circularity; elongation; roundness); they are reported as mean values ± SD (fold DCM vs. WT) of three independent experiments. Significant differences are denoted by the p-value (Student’s two-tailed t-test; * p < 0.05. (D) RT-qPCR of lamin A and C transcripts in WT and DCM HDFs; GAPDH was used as reference gene. Data are from three independent experiments and represented as mean ± SD (* p < 0.05). (E) Densitometric analysis of Western blot performed on WT and DCM HDFs, showing the intensity of the band corresponding to lamin A and C normalized versus β-actin levels. WT densitometric value is the average between two different controls (* p < 0.05; ** p < 0.01). Data are presented as mean ± SD. (F) Representative Western blot of lamin A and C; β-actin is used as housekeeping gene.