No abstract

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908-321C>G). AMOs were designed to target the newly created 5' splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence-specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild-type levels, we measured the amount of Ras-GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO-dependent decrease in Ras-GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been used successfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders.

Gene expression profiling is an important tool to evaluate genetic heterogeneity in carcinomas and is useful to develop expression-based classifications for many types of cancer, as well as markers of disease outcome. In this study, we have investigated the expression profile of 22 genes involved in the PI3K-AKT pathway in 26 high-grade ovarian carcinomas (19 serous and 7 clear cell carcinomas). Unsupervised hierarchical clustering divided high-grade ovarian carcinomas into three groups. Although all clear cell carcinomas clustered in one group, high-grade serous carcinomas were segregated into two separate groups with different prognosis (P=0.05). High expression of CASP3, XIAP (X-linked inhibitor of apoptosis) , NFKB1, FAS, and GSK3B mRNAs identified high-grade serous carcinomas with better prognosis. In multivariate analysis, these cluster groups were of prognostic significance independent of age, tumor size, and tumor stage (P=0.008). To validate the mRNA expression data, we studied the immunohistochemical expression of caspase-3 and XIAP on a tissue microarray. Immunoreaction for caspase-3 was concordant with the results obtained by mRNA expression analysis (Spearman r=0.762, P=0.000). Caspase-3 was exclusively expressed by the macrophages. Furthermore, co-expression of caspase-3 and XIAP identified high-grade serous carcinomas with different prognosis (P=0.03). Our results suggest that there are different biological subtypes of high-grade serous carcinomas.