Validation of a next generation sequencing assay for BRCA1, BRCA2, CHEK2 and PALB2 genetic testing.

BRCA1, BRCA2, CHEK2 and PALB2 genes are associated with hereditary breast and ovarian cancer syndrome. Genetic testing of these genes is of increasing importance to guide therapeutic and management decisions. In this study, we evaluated the performance of a next generation sequencing (NGS) assay for the complete analysis of BRCA1, BRCA2, CHEK2 and PALB2 genes using Agilent's SureMASTR BRCA Screen that enabled the detection of single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variations (CNVs) in a single-tube PCR based library preparation. The results showed 100% sensitivity and specificity on a set of 52 known samples from de-identified patients and external quality assessment program. A concordance rate of 87.5% was achieved in the comparison of variant classification with the external laboratories. The high accuracy of the assay supports the use of SureMASTR BRCA Screen in clinical diagnostic laboratories (SureMASTR BRCA Screen is for research use only, not for use in diagnostic procedures).

The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division.

We have isolated lethal mutations in the Drosophila lkb1 gene (dlkb1), the homolog of C. elegans par-4 and human LKB1 (STK11), which is mutated in Peutz-Jeghers syndrome. We show that these mutations disrupt spindle formation, resulting in frequent polyploid cells in larval brains. In addition, dlkb1 mutations affect asymmetric division of larval neuroblasts (NBs); they suppress unequal cytokinesis, abrogate proper localization of Bazooka, Par-6, DaPKC and Miranda, but affect neither Pins/Galphai localization nor spindle rotation. Most aspects of the dlkb1 phenotype are exacerbated in dlkb1 pins double mutants, which exhibit more severe defects than those observed in either single mutant. This suggests that Dlkb1 and Pins act in partially redundant pathways to control the asymmetry of NB divisions. Our results also indicate that Dlkb1 and Pins function in parallel pathways controlling the stability of spindle microtubules. The finding that Dlkb1 mediates both the geometry of stem cell division and chromosome segregation provides novel insight into the mechanisms underlying tumor formation in Peutz-Jeghers patients.

Metallothionein response following cadmium exposure in the oligochaete Eisenia fetida.

We studied the metallothionein (MT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. MT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg(-1) of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg(-1) of dry soil for 1 day resulted in the production of MT mRNA. This response was maintained for exposure of at least 1 month. Clear differences of MT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg Cd kg(-1) of dry soil). Immunocytochemistry demonstrated that MT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida MT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments.

Comparative effects of a non-steroidal ecdysone agonist RH-5992 and 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2).

The non-steroidal ecdysone agonist, RH-5992, exhibits ecdysteroid activities in vivo as well as in vitro more effectively than 20-hydroxyecdysone (20E). Using the IAL-PID2 cells derived from imaginal wing discs of last larval instar of Plodia interpunctella, we investigated the action of RH-5992 in the control of cell growth. Its effects on the proliferative activity of IAL-PID2 cells, the induction level in G2/M arrest and on the expression rate of Plodia B cyclin (PcycB), ecdysone B1-isoform (PIEcR-B1) and Ultraspiracle-2 isoform (PIUSP-2) were examined. From these cellular and molecular assays, our results brought evidence that RH-5992, like 20E, induced an inhibition on cell proliferation by blocking IAL-PID2 cells in G2/M phase. Moreover, this G2/M arrest was preceded by a decrease in the expression level of PcycB and a high induction of PIEcR-B1, PIUSP-2 mRNAs. Dose-response experiments revealed that RH-5992 was even more potent than 20E. On these parameters, we therefore suggest that the differential observed in the expression level of USP and EcR by RH-5992 and 20E could contribute to the difference observed for the biological potency of these two compounds.

The 20-hydroxyecdysone-induced cellular arrest in G2 phase is preceded by an inhibition of cyclin expression.

We have studied the effect of 20-hydroxyecdysone (20E) on cellular proliferation in IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. Flow cytometry analysis demonstrated that 20E induced an arrest of cells in G2 phase. To determine whether this arrest was due to an effect of 20E on cyclin expression, we cloned two cDNA fragments, named PcycA and PcycB, encoding, respectively, Plodia cyclins A and B. Using PcycA and PcycB probes, we have demonstrated that 20E induced a sharp decrease in the levels of cyclin A and B expression. Studies of induction pattern of Plodia HR3 transcription factor by 20E revealed that its induction preceded the decrease of cyclins transcripts. An exposure of cells to 20E in the presence of juvenile hormone (JH) led to a change in the kinetic of PHR3 induction and prevented both the decline of cyclin A and B expression and the G2 arrest. This effect of JH provides an additional argument for the existence of a correlation between cyclin transcripts level and G2 arrest. For the first time in insects, these findings bring evidence that ecdysteroids regulate cellular proliferation by acting on cell cycle regulators as cyclins.