RESUMEN
BACKGROUND: One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. RESULTS: An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. CONCLUSION: This automated process allows laboratories to discover DNA variations in a short time and at low cost.
Asunto(s)
Análisis Mutacional de ADN/métodos , Distrofias Musculares/genética , Automatización , Análisis Mutacional de ADN/economía , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa/economía , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Within the last few years, it has become evident that the beneficial effect of cell transplantation on ventricular function and myocardial perfusion is in large part mediated through paracrine effects on the host myocardium. Studies in which medium conditioned by cultured cells, usually mesenchymal stem cells, were injected into infarcted animal hearts have provided definitive evidence of this mechanism of action. Paracrine effects of the donor cells include but are not limited to angiogenesis, mobilization of both circulating and bone-marrow-derived stem cells, activation of cardiac-resident stem cells (CRSCs), and stabilization of the extracellular matrix (ECM). These paracrine effects can be augmented by transplantation of cells modified to express therapeutically useful transgenes, or by preconditioning through hypoxic or pharmacologic means. Strategies to enhance the paracrine effects of cell transplantation may thus be employed in the next generation of cell therapies, with greater functional benefit.