RESUMEN
The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples. Here we describe the development of such a methodology based on next-generation sequencing and a new bioinformatics software for data analysis. The diagnostic method was initially developed on an Illumina MiSeq NGS platform using germline-mutated stem cell lines and then adapted for the Ion Torrent PGM NGS platform as well. We also investigated the usability of NGS coverage data for the detection of copy number variations and exon deletions as a replacement of the conventional MLPA technique. Finally, we tested the developed workflow on FFPE samples from breast and ovarian cancer patients. Our method meets the sensitivity and specificity requirements for the genetic diagnosis of breast and ovarian cancers both from germline and FFPE samples.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Reordenamiento Génico , Mutación de Línea Germinal , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Biología Computacional , ADN/genética , Análisis Mutacional de ADN , Femenino , Formaldehído , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/genética , Parafina , Células Madre/metabolismoRESUMEN
The proinflammatory cytokine tumor necrosis factor (TNF) alpha is not encoded in the chicken genome. However, 1 member of the TNF family, TNF-like molecule 1A (TL1A), which is an important immunoregulatory protein, has recently been characterized in chickens. In this study, chicken TL1A (chTL1A) and 1 of its receptors, decoy receptor 3 (DcR3) were found to be expressed in developing bone of 14.5-day-old chicken embryos. Chicken chondrocytes were shown to express TL1A by polymerase chain reaction (PCR) amplification of cDNA and by immunohistochemical studies. Tissue expression was localized to the epiphyeal region of tubular bones, particularly cells of the epiphyseal plate, the outer chondrocytes of the cartilage-interfacing synovia, most of the synovial cells, and the stromal fibroblastic cells of the vascular channels of the femoral head. A tissue-specific developmental function of TL1A was supported by the presence of DcR3 in the embryonic connective tissue.