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Lamin A/C-Related Dilated Cardiomyopathy: Patient-derived Induced Pluripotent Stem Cells to Study Mutation and Cell Type-specific Pathogenic Mechanisms of Heart Disease

Education and Research Strategies
  • Primary Categories:
    • Genomic Medicine
  • Secondary Categories:
    • Genomic Medicine
Introduction:
Lamin A/C-related Dilated Cardiomyopathy (DCM) is an autosomal-dominant condition with ventricular enlargement, systolic dysfunction, and conduction system defects often resulting in heart failure or sudden death. The condition is caused by mutations in the Lamin A/C (LMNA) gene encoding the Lamin A and C proteins that form the nuclear lamina. Pathogenic mechanisms of LMNA-related DCM may include both nuclear fragility and cell dysfunction involving transcription, signal transduction, and epigenomic regulation; however, specific mutation and cell type mechanisms are poorly understood. Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling because iPSCs can be derived from patient samples, differentiated into relevant cell types, and studied using multiple approaches. Here, I report the generation of a collection of human iPSCs, initial studies, and challenges in modeling cardiac disease using patient-derived iPSCs.

Methods:
This study focuses on three LMNA-related DCM families, each with a different heterozygous mutation: splice-site (Family A), nonsense (Family B), and missense mutation (Family C). Family members were consented to obtain family histories and medical records and to collect saliva for DNA testing. Subsequently, LMNA mutation-positive (PATIENT) and mutation-negative family members (CONTROL) were consented to conduct skin biopsies for fibroblast cultures, iPSC generation using Sendai virus-based reprogramming vectors, and additional DNA/RNA/protein studies including whole exome sequencing (WES). Commercial dermal fibroblast from healthy individuals were included as unrelated controls. Independent iPSC clones were characterized for pluripotency and differentiation capability by immunocytochemistry staining and chromosomal stability by karyotype. In initial studies, several iPSC clones were differentiated into cardiomyocytes (CM) by small molecules modulation of Wnt/Beta catenin signaling, and three iPSC clones from Family A were studied by single-cell RNA-sequencing (scRNA-seq).

Results:
This study includes 56 consented individuals from the three families. Clinical descriptions showed variable presentation, severity, and progression of LMNA-related heart disease that included sick sinus syndrome, atrial fibrillation, DCM, and sudden death within the same family and between families. Non-cardiac features included palmar and plantar fibromatosis (Dupuytren’s and Ledderhose Diseases) in Family B and brachydactyly (Heart-Hand Syndrome IV) in Family C. LMNA Sanger sequencing of saliva DNA identified 27 (48%) mutation-positive and 29 (52%) mutation-negative individuals. WES for 25 fibroblast DNA samples (~8 Gb raw data/sample) also showed the absence of other known, heterozygous dominant or homozygous recessive, pathogenic variants and the presence of rare variants with uncertain significance. Molecular results supported different genetic mechanisms, Lamin A/C haploinsufficiency in Families A and B and dominant-negative or gain-of-function in Family C. Fibroblast reprogramming resulted in 20 different iPSC lines including nine LMNA mutation-positive (PATIENT) and 11 non-mutant (7 related and 4 unrelated CONTROL) iPSC lines. In total, ~140 independent iPSC clones (7 clones/iPSC line) were isolated, passaged, and stored frozen. Of these, 35 iPSC clones were characterized with 22 (63%) iPSC clones having normal results for both karyotype and pluripotency. Thus far, nine iPSC clones (four PATIENT and four CONTROL individuals) were differentiated into CM. For three iPSC clones (one PATIENT and one CONTROL individual from Family A), scRNA-seq analyses (>100,000 total high-quality cells) showed complex cell heterogeneity, and differential expression analyses (PATIENT vs. CONTROL) showed cell type and lineage-specific dysregulation of multiple pathways including X-inactivation and genomic imprinting, cell differentiation, metabolism, and signaling. However, several factors limited the interpretation of these results. 

Conclusion:
From three LMNA-related DCM families with different mutations, this study created and characterized a unique collection of patient-derived iPSC disease models for the investigation of mutation and cell type-specific mechanisms.  Although initial studies provided insight into LMNA-related DCM pathogenesis, results consistent with transcriptome dysregulation and epigenomic failure, study design improvements are required to address weaknesses and limit confounding factors in modeling human cardiac disease using patient-derived iPSCs.

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