ABSTRACT
LMNA encodes the A-type lamins that are part of the nuclear scaffold. Mutations in LMNA can cause a variety of disorders called laminopathies, including Hutchinson-Gilford progeria syndrome (HGPS), atypical Werner syndrome, and Emery-Dreifuss muscular dystrophy. Previous work has shown that treatment of HGPS cells with the mTOR inhibitor rapamycin or with the rapamycin analog everolimus corrects several of the phenotypes seen at the cellular level-at least in part by increasing autophagy and reducing the amount of progerin, the toxic form of lamin A that is overproduced in HGPS patients. Since other laminopathies also result in production of abnormal and potentially toxic lamin proteins, we hypothesized that everolimus would also be beneficial in those disorders. To test this, we applied everolimus to fibroblast cell lines from six laminopathy patients, each with a different mutation in LMNA Everolimus treatment increased proliferative ability and delayed senescence in all cell lines. In several cell lines, we observed that with treatment, there is a significant improvement in nuclear blebbing, which is a cellular hallmark of HGPS and other lamin disorders. These preclinical results suggest that everolimus might have clinical benefit for multiple laminopathy syndromes.
Subject(s)
Everolimus/pharmacology , Fibroblasts/drug effects , Lamin Type A/deficiency , Muscular Dystrophy, Emery-Dreifuss/genetics , Progeria/genetics , TOR Serine-Threonine Kinases/antagonists & inhibitors , Werner Syndrome/genetics , Biomarkers , Cell Division/drug effects , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Cellular Senescence/drug effects , Humans , Lamin Type A/genetics , Muscular Dystrophy, Emery-Dreifuss/pathology , Mutation , Phosphorylation/drug effects , Progeria/pathology , Protein Processing, Post-Translational/drug effects , Ribosomal Protein S6/metabolism , Werner Syndrome/pathologyABSTRACT
Transgenic animals are extensively used to model human disease. Typically, the transgene copy number is estimated, but the exact integration site and configuration of the foreign DNA remains uncharacterized. When transgenes have been closely examined, some unexpected configurations have been found. Here, we describe a method to recover transgene insertion sites and assess structural rearrangements of host and transgene DNA using microarray hybridization and targeted sequence capture. We used information about the transgene insertion site to develop a polymerase chain reaction genotyping assay to distinguish heterozygous from homozygous transgenic animals. Although we worked with a bacterial artificial chromosome transgenic mouse line, this method can be used to analyse the integration site and configuration of any foreign DNA in a sequenced genome.