Genetic screening of the breast and ovarian cancer susceptibility gene BRCA1 has uncovered a large number of variants of uncertain clinical significance. Here, we use biochemical and cell-based transcriptional assays to assess the structural and functional defects associated with a large set of 117 distinct BRCA1 missense variants within the essential BRCT domain of the BRCA1 protein that have been documented in individuals with a family history of breast or ovarian cancer. In the first method, we used limited proteolysis to assess the protein folding stability of each of the mutants compared with the wild-type. In the second method, we used a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a peptide containing a pSer-X-X-Phe motif, a known functional target of the BRCA1 BRCT domain. Finally, we used transcriptional assays to assess the ability of each BRCT variant to act as a transcriptional activation domain in human cells. Through a correlation of the assay results with available family history and clinical data, we define limits to predict the disease risk associated with each variant. Forty-two of the variants show little effect on function and are likely to represent variants with little or no clinical significance; 50 display a clear functional effect and are likely to represent pathogenic variants; and the remaining 25 variants display intermediate activities. The excellent agreement between the structure/function effects of these mutations and available clinical data supports the notion that functional and structure information can be useful in the development of models to assess cancer risk. Cancer Res; 70(12); 4880-90. ©2010 AACR.

Transplantation of human islets is an attractive alternative to daily insulin injections for patients with type 1 diabetes. However, the majority of islet recipients lose graft function within five years. Inflammation is a primary contributor to graft loss, and inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. As mesenchymal stem cells (MSCs) possess numerous immunoregulatory properties, we hypothesized that MSCs could protect human islets from pro-inflammatory cytokines. Five hundred human islets were co-cultured with 0.5 or 1.0×106 human MSCs derived from bone marrow or pancreas for 24 hours followed by 48 hour exposure to interferon-γ, tumor necrosis factor-α and interleukin 1β. Controls include islets cultured alone (± cytokines) and with human dermal fibroblasts (± cytokines). For all conditions, glucose stimulated insulin secretion (GSIS), total islet cellular insulin content, islet β cell apoptosis, and potential cytoprotective factors secreted in the culture media were determined. Cytokine exposure disrupted human islet GSIS based on stimulation index and percentage insulin secretion. Conversely, culture with 1.0×106 bMSCs preserved GSIS from cytokine treated islets. Protective effects were not observed with fibroblasts, indicating that preservation of human islet GSIS after exposure to pro-inflammatory cytokines is MSC dependent. Islet β cell apoptosis was observed in the presence of cytokines; however, culture of bMSCs with islets prevented β cell apoptosis after cytokine treatment. Hepatocyte growth factor (HGF) as well as matrix metalloproteinases 2 and 9 were also identified as putative secreted cytoprotective factors; however, other secreted factors likely play a role in protection. This study, therefore, demonstrates that MSCs may be beneficial for islet engraftment by promoting cell survival and reduced inflammation.