Age of heart disease presentation and dysmorphic nuclei in patients with LMNA mutations

Abstract

Nuclear shape defects are a distinguishing characteristic in laminopathies, cancers, and other pathologies. Correlating these defects to the symptoms, mechanisms, and progression of disease requires unbiased, quantitative, and high-throughput means of quantifying nuclear morphology. To accomplish this, we developed a method of automatically segmenting fluorescently stained nuclei in 2D microscopy images and then classifying them as normal or dysmorphic based on three geometric features of the nucleus using a package of Matlab codes. As a test case, cultured skin-fibroblast nuclei of individuals possessing LMNA splice-site mutation (c.357-2A>G), LMNA nonsense mutation (c.736 C>T, pQ246X) in exon 4, LMNA missense mutation (c.1003C>T, pR335W) in exon 6, Hutchinson-Gilford Progeria Syndrome, and no LMNA mutations were analyzed. For each cell type, the percentage of dysmorphic nuclei, and other morphological features such as average nuclear area and average eccentricity were obtained. Compared to blind observers, our procedure implemented in Matlab codes possessed similar accuracy to manual counting of dysmorphic nuclei while being significantly more consistent. The automatic quantification of nuclear defects revealed a correlation between in vitro results and age of patients for initial symptom onset. Our results demonstrate the method’s utility in experimental studies of diseases affecting nuclear shape through automated, unbiased, and accurate identification of dysmorphic nuclei.

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

“…We also found that there were no significant changes in LMNA transcript levels, ratio of Lamin A to Lamin C transcripts, or expression of other NL-genes, LMNB1 and LMNB2 between the Patient group and two control groups. These results are in line with our previous structural studies that found no significant difference in nuclear abnormalities and suggests that there were no compensatory mechanisms at the transcription level for the other proteins that construct the nuclear lamina [63].…”

“…In contrast, there is aberrant upregulation of ERK/MAPK pathway in affected tissues such as LMNA mutant cardiomyocytes which leads to laminopathy phenotype [ 10 , 62 ]. Together, these mechanisms may provide an explanation for the absence of any significant phenotype in fibroblasts [ 63 ].…”

A Study of Gene Expression, Structure, and Contractility of iPSC-Derived Cardiac Myocytes from a Family with Heart Disease due to LMNA Mutation

“…This type of a conclusion would have been impossible with bulk RNA sequencing. In the future, this approach can also be utilized to look at other families with similar mutations 15 , 72 as well as sex as a biological variable. This would require significantly more individuals to be analyzed with the assays detailed here, which was beyond the scope of this study.…”

Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs

“…Substrates were fabricated for structural studies as described previously [5,8,19,24,46]. Briefly, large cover glass (Brain Research Laboratories, Newton, MA) was cleaned by sonicating, then spin coated with 10:1 Polydimethylsiloxane (PDMS; Ellsworth Adhesives, Germantown, WI).…”