Clinical and functional characterization of a novel mutation in lamin a/c gene in a multigenerational family with arrhythmogenic cardiac laminopathy

Abstract

Mutations in the lamin A/C gene (LMNA) were associated with dilated cardiomyopathy (DCM) and, recently, were related to severe forms of arrhythmogenic right ventricular cardiomyopathy (ARVC). Both genetic and phenotypic overlap between DCM and ARVC was observed; molecular pathomechanisms leading to the cardiac phenotypes caused by LMNA mutations are not yet fully elucidated. This study involved a large Italian family, spanning 4 generations, with arrhythmogenic cardiomyopathy of different phenotypes, including ARVC, DCM, system conduction defects, ventricular arrhythmias, and sudden cardiac death. Mutation screening of LMNA and ARVC-related genes PKP2, DSP, DSG2, DSC2, JUP, and CTNNA3 was performed. We identified a novel heterozygous mutation (c.418_438dup) in LMNA gene exon 2, occurring in a highly conserved protein domain across several species. This newly identified variant was not found in 250 ethnically-matched control subjects. Genotype-phenotype correlation studies suggested a co-segregation of the LMNA mutation with the disease phenotype and an incomplete and age-related penetrance. Based on clinical, pedigree, and molecular genetic data, this mutation was considered likely disease-causing. To clarify its potential pathophysiologic impact, functional characterization of this LMNA mutant was performed in cultured cardiomyocytes expressing EGFP-tagged wild-type and mutated LMNA constructs, and indicated an increased nuclear envelope fragility, leading to stress-induced apoptosis as the main pathogenetic mechanism. This study further expands the role of the LMNA gene in the pathogenesis of cardiac laminopathies, suggesting that LMNA should be included in mutation screening of patients with suspected arrhythmogenic cardiomyopathy, particularly when they have ECG evidence for conduction defects. The combination of clinical, genetic, and functional data contribute insights into the pathogenesis of this form of life-threatening arrhythmogenic cardiac laminopathy.

Fig 1. Family pedigree and mutation identification.

(A) Electropherogram of the LMNA gene variant. (B) The 7 duplicated amino acids (LLNSKEA) are highly conserved among LMNA gene homologs in vertebrates. (C) Family pedigree of the index patient with the novel LMNA gene mutation.

Fig 2. Clinical characteristics of index patient and LMNA mutation-positive family members.

Index patient’s electrocardiogram (ECG) showing, at clinical presentation, (A) sinus rhythm, first-degree AV block, and PVC of LBBB morphology and, 13 years later, (B) complete AV block. (C) Asystole documented on implantable loop recorder memory (subject III-8). (D) Sustained VT detected on telemetry monitoring, effectively terminated by internal ICD shock (subject III-1). (E) Episodes of non-sustained VT with LBBB morphology and inferior axis (subject III-5) on 12-lead Holter monitoring. (E) Episodes of non-sustained VT with RBBB morphology and superior axis (subject IV-2) on 12-lead Holter monitoring.

Fig 3. CMR imaging of LMNA mutation-positive family members.

(A) Short-axis cine (a) and LGE sequences in the Short-axis (b) and 2-chambers long-axis views (c). Bulging (arrow in a) and LGE of the RV free wall (arrowheads in b). Linear midwall LGE is localized at the interventricular septum and LV inferior wall (arrowheads in c) (subject III-5). (B) and (C) LGE sequences in the short axis (a) and 4-chambers long axis views (b). LGE with linear midwall pattern is shown on the LV inferior wall and basal interventricular septum (arrowheads) (subjects III-8, and IV-2).

Fig 4. Immunofluorescence confocal analysis of LMNA transfected HL-1 cells.

Cells transfected with both LMNA WT and DUP are depicted. LMNA is visualized in green, Nuclear Pores in red, and colocalization in yellow in the merge panels. In the insets, a merged image of LMNA and Phalloidin-TRITC is shown. Planar XY projections were depicted in each experimental condition.

Fig 5. Analysis of nuclear envelope integrity under stressing condition in LMNA transfected HL-1 cells.

Nuclear WT LMNA (A) and DUP LMNA (B) signals in control (CTR) and stressing conditions (Hyperosmolarity, Hypoxia, Oxidative stress). The merged signals of LMNA proteins and the RFP nuclear marker are shown in the insets. Confocal XY planar projections are depicted in each experimental condition.

Fig 6. Apoptosis assay in LMNA transfected HL-1 cells.

(A) Representative XY confocal planar projections of LMNA transfected cells (green) labelled with EthD-1 (red) in control conditions are depicted. (B) Quantitative analysis of apoptotic cells in control and under hyperosmotic (Hyper), hypoxic (Hypoxia), and oxidative (H₂O₂) conditions. Data are reported as % of apoptotic cells (double-labelled cells) in overall LMNA-expressing cells (green labelled cells). Statistical analysis was performed on 3 independent experiments and significance calculated by Student’s T-test for unpaired samples. *P< 0.0002 is relative to CTR vs. stressing conditions in WT LMNA expressing cells and **P < 0.0001 is relative to CTR vs. stressing conditions in DUP LMNA expressing cells.