Morphological analysis of 13 LMNA variants identified in a cohort of 324 unrelated patients with idiopathic or familial dilated cardiomyopathy

Abstract

Background: Mutations in the LMNA gene, encoding lamins A/C, represent a significant cause of dilated cardiomyopathy. We recently identified 18 protein-altering LMNA variants in a cohort of 324 unrelated patients with dilated cardiomyopathy. However, at least one family member with dilated cardiomyopathy in each of 6 pedigrees lacked the LMNA mutation (nonsegregation), whereas small sizes of 5 additional families precluded definitive determinations of segregation, raising questions regarding contributions by those variants to disease.

Methods and results: We have consequently expressed, in COS7 cells, GFP-prelamin A (GFPLaA) fusion constructs incorporating the 6 variants in pedigrees with nonsegregation (R101P, A318T, R388H, R399C, S437Hfsx1, and R654X), the 4 variants in pedigrees with unknown segregation (R89L, R166P [in 2 families], I210S, R471H), and 3 additional missense variants (R190Q, E203K, and L215P) that segregated with disease. Confocal immunofluorescence microscopy was used to characterize GFP-lamin A localization and nuclear morphology. Abnormal phenotypes were observed for 10 of 13 (77%) variants (R89L, R101P, R166P, R190Q, E203K, I210S, L215P, R388H, S437Hfsx1, and R654X), including 4 of 6 showing nonsegregation and 3 of 4 with uncertain segregation. All 7 variants affecting coil 1B and the lamin A-only mutation, R654X, exhibited membrane-bound GFP-lamin A aggregates and nuclear shape abnormalities. Unexpectedly, R388H largely restricted GFP-lamin A to the cytoplasm. Equally unexpected were unique streaked aggregates with S437Hfsx1 and giant aggregates with both S437Hfsx1 and R654X.

Conclusions: This work expands the recognized spectrum of lamin A localization abnormalities in dilated cardiomyopathy. It also provides evidence supporting pathogenicity of 10 of 13 tested LMNA variants, including some with uncertain or nonsegregation.

Figure 1

Partial pedigrees for families with LMNA variants. Numbering is consistent with tables and figures in this study and with past reports of these families (see , , for clinical data). Probands are indicated with an arrow. Solid symbols represent IDC with or without heart failure. Shaded symbols represent any other cardiovascular abnormality. Open symbols indicate unaffected individuals. Mutation carrier status is shown by a + (presence), (+) (obligate) or − (absence). Absence of any symbol for mutation carrier status indicates lack of available DNA for analysis. Question marks (?) denote insufficient clinical data. Figure 1A: Pedigrees with nonsegregation. Figure 1B: Pedigrees with segregation or unknown segregation.

Figure 1

Partial pedigrees for families with LMNA variants. Numbering is consistent with tables and figures in this study and with past reports of these families (see , , for clinical data). Probands are indicated with an arrow. Solid symbols represent IDC with or without heart failure. Shaded symbols represent any other cardiovascular abnormality. Open symbols indicate unaffected individuals. Mutation carrier status is shown by a + (presence), (+) (obligate) or − (absence). Absence of any symbol for mutation carrier status indicates lack of available DNA for analysis. Question marks (?) denote insufficient clinical data. Figure 1A: Pedigrees with nonsegregation. Figure 1B: Pedigrees with segregation or unknown segregation.

Figure 2

GFP-lamin A (GFPLaA) localization and nuclear morphology in COS7 cells transfected with wildtype/mutant fusion constructs and stained with Hoechst 33258. In all experiments, confocal immunofluorescence microscopy was performed 24 hours post-transfection. Scale bars are 5µm in length. Figure 2A. Representative images for the six LMNA variants in pedigrees with nonsegregation. Figure 2B. Representative images for the three variants with segregation and four variants with unknown segregation.

Figure 2

GFP-lamin A (GFPLaA) localization and nuclear morphology in COS7 cells transfected with wildtype/mutant fusion constructs and stained with Hoechst 33258. In all experiments, confocal immunofluorescence microscopy was performed 24 hours post-transfection. Scale bars are 5µm in length. Figure 2A. Representative images for the six LMNA variants in pedigrees with nonsegregation. Figure 2B. Representative images for the three variants with segregation and four variants with unknown segregation.