ABSTRACT
OBJECTIVE: Authors evaluated the performance of a commercially available next-generation sequencing assay kit; this was based on genomic content from Illumina's TruSight™ Oncology 500 research assay that identifies BRCA variants and proprietary algorithms licensed from Myriad and, with additional genomic content, measures the homologous recombination deficiency (HRD) genomic instability score (GIS) in tumor tissue (TSO 500 HRD assay). METHODS: Data from the TSO 500 HRD assay were compared with data from the Myriad MyChoice®CDx PLUS assay (Myriad assay). Prevalence rates for overall HRD status and BRCA mutations (a deleterious or suspected deleterious BRCA1 or BRCA2 mutation or both) and assay agreement rates for HRD GIS and BRCA analysis were assessed in ovarian tumor samples. Pearson correlations of the continuous HRD GIS and analytic sensitivity and specificity were evaluated. RESULTS: The prevalence of overall HRD positivity was 51.2% (TSO 500 HRD assay) versus 49.2% (Myriad assay) and the prevalence of BRCA mutations was 27.6% (TSO 500 HRD assay) versus 25.5% (Myriad assay). After post-processing optimization, concordance of the HRD GIS was 0.980 in all samples and 0.976 in the non-BRCA mutation cohort; the area under the receiver operating characteristic curve was 0.995 and 0.992, respectively. CONCLUSIONS: Comparison between the Illumina and Myriad assays showed that overall HRD status, the individual components of BRCA analysis, and HRD GIS detection results were highly concordant (>93%), suggesting the TSO 500 HRD assay will approach the analytical accuracy of the FDA-approved Myriad assay.
Subject(s)
Ovarian Neoplasms , Humans , Female , Ovarian Neoplasms/genetics , Ovarian Neoplasms/diagnosis , Homologous Recombination , High-Throughput Nucleotide Sequencing/methods , United States/epidemiology , Mutation , BRCA1 Protein/genetics , Genomic Instability , BRCA2 Protein/genetics , Reagent Kits, Diagnostic/standards , United States Food and Drug Administration , Middle Aged , Genes, BRCA1ABSTRACT
During Drosophila development, translational control plays a crucial role in regulating gene expression, and is particularly important during pre-patterning of the maturing oocyte. A critical step in translation initiation is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the mRNA cap structure, which ultimately leads to recruitment of the ribosome. d4EHP is a translational repressor that prevents translation initiation by out-competing eIF4E on the cap structure for a subset of mRNAs. However, only two examples of mRNAs subject to d4EHP translation repression in Drosophila are known. Here we show that the belle (bel) mRNA is translationally repressed by the d4EHP protein in the Drosophila ovary. Consistent with this regulation, d4EHP overexpression in the ovary phenocopies the bel mutant. We also provide evidence that the Bel protein binds to eIF4E and may itself function as a translation repressor protein, with bruno as a potential target for Bel repression in the oocyte. Bruno is known to repress the mRNA of the key oocyte axis determinant oskar (osk) during oogenesis, and we find that an increase in the level of Bruno protein in bel mutant ovaries is associated with a reduction in Osk protein. Overall, our data suggest that a translational regulatory network exists in which consecutive translational repression events act to correctly pattern the Drosophila oocyte.
Subject(s)
Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Eukaryotic Initiation Factor-4E/metabolism , Oocytes/physiology , RNA Helicases/genetics , Animals , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/biosynthesis , Gene Expression Regulation, Developmental , Oogenesis/genetics , Protein Binding , Protein Biosynthesis/genetics , RNA Caps/metabolism , RNA Helicases/biosynthesis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron-sulfur (Fe-S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe-S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe-S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe-S proteins.
