The functional impact of variants of uncertain significance in BRCA2.

PURPOSE: Genetic testing has uncovered large numbers of variants in the BRCA2 gene for which the clinical significance is unclear. Cancer risk prediction of these variants of uncertain significance (VUS) can be improved by reliable assessment of the extent of impairment of the tumor suppressor function(s) of BRCA2. METHODS: Here, we evaluated the performance of the mouse embryonic stem cell (mESC)-based functional assay on an extensive set of BRCA2 missense variants. RESULTS: Whereas all 20 nonpathogenic (class 1/2) variants were able to complement the cell lethal phenotype induced by loss of endogenous mouse Brca2, only 1 out of 15 pathogenic (class 4/5) variants (p.Gly2609Asp) was able to do so. However, in this variant the major tumor suppressive activity of BRCA2, i.e., homology directed repair (HDR), was severely abrogated. Among 43 evaluated VUS (class 3), 7 were unable to complement the lethal phenotype of mouse Brca2 loss while 7 other variants displayed a more severe reduction of HDR activity than observed for class 1/ 2 variants. CONCLUSION: The mESC-based BRCA2 functional assay can reliably determine the functional impact of VUS, distinguish between pathogenic and nonpathogenic variants, and may contribute to improved cancer risk estimation for BRCA2 VUS carriers.

An efficient pipeline for the generation and functional analysis of human BRCA2 variants of uncertain significance.

The implementation of next-generation sequence analysis of disease-related genes has resulted in an increasing number of genetic variants with an unknown clinical significance. The functional analysis of these so-called "variants of uncertain significance" (VUS) is hampered by the tedious and time-consuming procedures required to generate and test specific sequence variants in genomic DNA. Here, we describe an efficient pipeline for the generation of gene variants in a full-length human gene, BRCA2, using a bacterial artificial chromosome. This method permits the rapid generation of intronic and exonic variants in a complete gene through the use of an exon-replacement strategy based on simple site-directed mutagenesis and an effective positive-negative selection system in E. coli. The functionality of variants can then be assessed through the use of functional assays, such as complementation of gene-deficient mouse-embryonic stem (mES) cells in the case of human BRCA2. Our methodology builds upon an earlier protocol and, through the introduction of a series of major innovations, now represents a practical proposition for the rapid analysis of BRCA2 variants and a blueprint for the analysis of other genes using similar approaches. This method enables rapid generation and reliable classification of VUS in disease-related genes, allowing informed clinical decision-making.

Sensitive DsRed fluorescence-based reporter cell systems for genotoxicity and oxidative stress assessment.

Various in vitro test systems have been developed for genotoxic risk assessment in early drug development. However, these genotoxicity tests often show limited specificity, and provide limited insights into the mode of toxicity of the tested compounds. To identify genes that could serve as specific biomarkers for genotoxicity or oxidative stress, we exposed mouse embryonic stem (ES) cells to various genotoxic and oxidative stress-inducing compounds and performed genome-wide expression profiling. Differentially expressed genes were classified based on the fold-change of expression and their specificity for either genotoxic or oxidative stress. Promoter regions of four selected genes (Ephx1, Btg2, Cbr3 and Perp) were fused to a DsRed fluorescent reporter gene and stably integrated in mouse ES cells. Established stable reporter cell lines displayed significant induction of DsRed expression upon exposure to different classes of genotoxic and oxidative stress-inducing compounds. In contrast, exposure to non-genotoxic carcinogenic compounds did not induce DsRed expression even at cytotoxic doses. Expression of the Cbr3-DsRed reporter was more responsive to compounds that induce oxidative stress while the other three DsRed reporters reacted more specific to direct-acting genotoxic agents. Therefore, the differential response of the Btg2- and Cbr3-DsRed reporters can serve as indicator for the main action mechanism of genotoxic and oxidative stress-inducing compounds. In addition, we provide evidence that inhibition of DNA replication results in preferential activation of the Btg2-DsRed genotoxicity reporter. In conclusion, we have generated sensitive mouse ES cell reporter systems that allow detection of genotoxic and oxidative stress-inducing properties of chemical compounds and can be used in high-throughput assays.

Genome wide molecular analysis of minimally differentiated acute myeloid leukemia.

BACKGROUND: Minimally differentiated acute myeloid leukemia is heterogeneous in karyotype and is defined by immature morphological and molecular characteristics. This originally French-American-British classification is still used in the new World Health Organization classification when other criteria are not met. Apart from RUNX1 mutation, no characteristic molecular aberrations are recognized. DESIGN AND METHODS: We performed whole genome single nucleotide polymorphism analysis and extensive molecular analysis in a cohort of 52 patients with minimally differentiated acute myeloid leukemia. RESULTS: Many recurring and potentially relevant regions of loss of heterozygosity were revealed. These point towards a variety of candidate genes that could contribute to the pathogenesis of minimally differentiated acute myeloid leukemia, including the tumor suppressor genes TP53 and NF1, and reinforced the importance of RUNX1 in this leukemia. Furthermore, for the first time in this minimally differentiated form of leukemia we detected mutations in the transactivation domain of RUNX1. Mutations in other acute myeloid leukemia associated transcriptions factors were infrequent. In contrast, FLT3, RAS, PTPN11 and JAK2 were often mutated. Irrespective of the RUNX1 mutation status, our results show that RAS signaling is the most important pathway for proliferation in minimally differentiated acute myeloid leukemia. Importantly, we found that high terminal deoxynucleotidyl transferase expression is closely associated with RUNX1 mutation, which could allow an easier diagnosis of RUNX1 mutation in this hematologic malignancy. CONCLUSIONS: Our results suggest that in patients without RUNX1 mutation, several other molecular aberrations, separately or in combination, contribute to a common minimally differentiated phenotype.

The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding.

Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general P53-dependent cellular stress response. Extensive validation using the compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals.

Identification of RUNX1/AML1 as a classical tumor suppressor gene.

Based on our previous results indicating the presence of a tumor suppressor gene (TSG), chromosome 21 was analysed for loss of heterozygosity (LOH) in 18 patients with acute myeloid leukemia (17, AML-M0; one, AML-M1). Allelotyping at polymorphic loci was performed on purified material, allowing unequivocal detection of allelic loss and homozygous deletions. Six AML-M0 patients shared a common region of LOH harboring a single gene: RUNX1 (AML1), the most frequent site of translocations in acute leukemia and a well-known fusion oncogene. Fluorescence in situ hybridization allowed the identification of deletions with breakpoints within RUNX1 in two patients as the cause of LOH. In the four others the LOH pattern and the presence of two karyotypically normal chromosomes 21 were in line with mitotic recombination. Further molecular and cytogenetic analyses showed that this caused homozygosity of primary RUNX1 mutations: two point mutations, a partial deletion and, most significantly, a complete deletion of RUNX1. These findings identify RUNX1 as a classical TSG: both alleles are mutated or absent in cancer cells from four of the 17 AML-M0 patients examined. In contrast to AML-M0, the AML-M1 patient was trisomic for chromosome 21 and has two mutated and one normal RUNX1 allele, suggesting that the order of mutagenic events leading to leukemia may influence the predominant tumor type.

The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals.

People are exposed to an ever-increasing number of chemical compounds that are developed by industry for a wide range of applications. These compounds may harmfully react with different cellular components and activate specific defense mechanisms that provide protection against the toxic, mutagenic, and possibly oncogenic consequences of exposure. Monitoring the activation of specific cellular signaling pathways upon exposure may therefore allow reliable and mechanism-based assessment of potential (geno)toxic properties of chemicals, while providing insight into their primary mode of toxicity. By whole-genome transcription profiling of mouse embryonic stem cells, we identified genes that were transcriptionally activated upon exposure to either genotoxic compounds or pro-oxidants. For selected biomarker genes, we constructed reporters encoding C-terminal green fluorescent protein (GFP)-tagged fusion proteins. GFP reporter genes were located on bacterial artificial chromosomes, thereby enabling transcriptional regulation of the reporters by their own physiological promoter. The Bscl2-GFP reporter is selectively activated after exposure to genotoxic agents and its induction is associated with inhibition of DNA replication and activation of the ataxia telangiectasia and Rad3-related protein signaling pathway. The Srxn1-GFP reporter is preferentially induced upon oxidative stress and is part of the nuclear factor (erythroid-derived 2)-like 2-antioxidant response pathway. The novel (geno)toxicity assay (ToxTracker) that utilize the differential responsiveness of various reporter cell lines will enable prediction of the primary reactive properties of known and unknown chemicals.

Lack of genetic and epigenetic changes in meningiomas without NF2 loss.

Approximately 60% of sporadic meningiomas are caused by inactivation of the NF2 tumour suppressor gene. The causative gene for the remaining meningiomas is unknown. Previous studies have shown that these tumours have no recurrent karyotypic abnormalities. They differ from their NF2-related counterparts in that they are more often of the meningothelial subtype and are located preferentially in the anterior skull base. To gain more insight into the aetiology of these tumours, we studied genetic and epigenetic alterations in 25 meningiomas without NF2 involvement. We first established a genome-wide allelotype using 3 microsatellite markers per chromosome arm. Loss of heterozygosity (LOH) was detected at a low frequency and no indication for the location of putative tumour suppressor genes could be established. We next screened the subtelomeric regions by using 2-3 polymorphic markers close to each telomere. Again no evidence for LOH of a particular chromosome arm was obtained, and no LOH was found in the genomic regions containing the NF2-related ERM family members ezrin and radixin, DAL-1, protein 4.1R, and TSLC1. Mutations in the X-chromosome based family member, moesin, were analysed by SSCP and were not detected. Microsatellite instability was studied using 6 commonly used markers but none of these was altered in any meningioma. Methylation was detected in 5 of 16 genes (NF2, p14(ARF), CDH1, BRCA1, RB1) previously shown to be silenced in a variety of tumour types. However, methylation percentages for these genes were generally higher in a group of NF2-related meningiomas, with the exception of the BRCA1 gene. The NF2 gene was methylated in only 1 of 21 tumours. In conclusion, meningiomas with an intact NF2 gene have a normal karyotype and no obvious genetic or epigenetic aberrations, suggesting that the gene(s) involved in the pathogenesis of these tumours are altered by smaller events than can be detected with the techniques used in our study.