Gene expression profiling of fibroblasts in a family with LMNA-related cardiomyopathy reveals molecular pathways implicated in disease pathogenesis

Abstract

Background: Intermediate filament proteins that construct the nuclear lamina of a cell include the Lamin A/C proteins encoded by the LMNA gene, and are implicated in fundamental processes such as nuclear structure, gene expression, and signal transduction. LMNA mutations predominantly affect mesoderm-derived cell lineages in diseases collectively termed as laminopathies that include dilated cardiomyopathy with conduction defects, different forms of muscular dystrophies, and premature aging syndromes as Hutchinson-Gilford Progeria Syndrome. At present, our understanding of the molecular mechanisms regulating tissue-specific manifestations of laminopathies are still limited. Methods: To gain deeper insight into the molecular mechanism of a novel LMNA splice-site mutation (c.357-2A > G) in an affected family with cardiac disease, we conducted deep RNA sequencing and pathway analysis for nine fibroblast samples obtained from three patients with cardiomyopathy, three unaffected family members, and three unrelated, unaffected individuals. We validated our findings by quantitative PCR and protein studies. Results: We identified eight significantly differentially expressed genes between the mutant and non-mutant fibroblasts, that included downregulated insulin growth factor binding factor protein 5 (IGFBP5) in patient samples. Pathway analysis showed involvement of the ERK/MAPK signaling pathway consistent with previous studies. We found no significant differences in gene expression for Lamin A/C and B-type lamins between the groups. In mutant fibroblasts, RNA-seq confirmed that only the LMNA wild type allele predominately was expressed, and Western Blot showed normal Lamin A/C protein levels.

Insights into the SARS-CoV-2-Mediated Alteration in the Stress Granule Protein Regulatory Networks in Humans

“…Lamin family proteins make up the matrix and are thought to be evolutionarily conserved. Any dysregulation in the LMNA gene is known to cause Hutchinson-Gilford progeria syndrome, cardiomyopathy, muscular dystrophy, emeryderifusss muscular dystrophy, and lipodystrophy [34,36,37]. The third gene was DYNC1H1, also known as dynein cytoplasmic-1-heavy chain-1.…”

DNA methylation analysis reveals epimutation hotspots in patients with dilated cardiomyopathy-associated laminopathies

“…To examine this more thoroughly, we first compared our DMR data with the list of differentially expressed genes (DEGs) between patient and their unaffected controls previously obtained from transcriptome-wide expression data from Family A fibroblasts [7] (Fig. 3c).…”

“…Bulk RNA-seq was previously performed on Family A fibroblasts (3 unaffected mutation-negative family members and 3 patients heterozygous for LMNA splicesite (c.357-2A>G)) and 3 healthy, unrelated individuals (Donors 2, 3, and 4). A list of DEGs between patients, control siblings, and the unrelated controls was attained from GSE125990 [7]. DEGs were filtered for FDRadjusted p-value ≤ 0.05.…”

“…We performed reduced representation bisulfite sequencing (RRBS) to explore the regulatory consequences of accumulating DNA methylation aberrancies [14] in patient-biopsied fibroblasts and their induced pluripotent stem cell (iPSCs) derivatives as in vitro culture models for somatic (adult) and early developmental stages, respectively. We identified familyspecific differentially methylated regions (DMRs) in each cell type and performed integrative genomic analysis to compare these DMRs with changes in gene expression and LAD organization observed in previous studies in DCM patient tissues [7,8]. We hypothesize that we will identify differential DNA methylation predictive of a disease mechanism both shared across laminopathies and unique to each family mutation.…”

“…Due to this chromatin structure disturbance, it is unsurprising that LMNA mutation studies related to DCM have focused on identifying differences in gene expression and LADs in diseased cells [7,8]. However, the role of DNA methylation, which works in conjunction with the chromatin to control gene expression, has not been thoroughly investigated in the context of LMNA mutations.…”