Variant Classification Discordance: Contributing Factors and Predictive Models.

An ever-growing catalog of human variants is hosted in the ClinVar database. In this database, submissions on a variant are combined into a multisubmitter record; and in the case of discordance in variant classification between submitters, the record is labeled as conflicting. The current study used ClinVar data to identify characteristics that would make variants more likely to be associated with the conflict class of variants. Furthermore, the Extreme Gradient Boosting algorithm was used to train classifier models to provide prediction of classification discordance for single submission variants in ClinVar database. Population allele frequency, the gene harboring the variant, variant type, consequence on protein, variant deleteriousness score, first submitter identity, and submission count were associated with conflict in variant classification. Using such features, the optimized classifier showed accuracy on the test set of 88% with the weighted average of precision, recall, and f1-score of 0.84, 0.88, and 0.85, respectively. There were pronounced associations between variant classification discordance and allele frequency, gene type, and the identity of the first submitter. The study provides the predicted discordance status for single-submitter variants deposited in ClinVar. This approach can be used to assess whether single-submitter variants are likely to be supported, or in conflict with, future entries; this knowledge may help laboratories with clinical variant assessment.

Future Role of Health Technology Assessment for Genomic Medicine in Oncology: A Canadian Laboratory Perspective.

Genome-based testing in oncology is a rapidly expanding area of health care that is the basis of the emerging area of precision medicine. The efficient and considered adoption of novel genomic medicine testing is hampered in Canada by the fragmented nature of health care oversight as well as by lack of clear and transparent processes to support rapid evaluation, assessment, and implementation of genomic tests. This article provides an overview of some key barriers and proposes approaches to addressing these challenges as a potential pathway to developing a national approach to genomic medicine in oncology.

BRCA1 Variant Assessment Using a Simple Analytic Assay.

BACKGROUND: We previously developed a biological assay to accurately predict BRCA1 (BRCA1 DNA repair associated) mutation status, based on gene expression profiles of Epstein-Barr virus-transformed lymphoblastoid cell lines. The original work was done using whole genome expression microarrays, and nearest shrunken centroids analysis. While these approaches are appropriate for model building, they are difficult to implement clinically, where more targeted testing and analysis are required for time and cost savings. METHODS: Here, we describe adaptation of the original predictor to use the NanoString nCounter platform for testing, with analysis based on the k-top scoring pairs (k-TSP) method. RESULTS: Assessing gene expression using the nCounter platform on a set of lymphoblastoid cell lines yielded 93.8% agreement with the microarray-derived data, and 87.5% overall correct classification of BRCA1 carriers and controls. Using the original gene expression microarray data used to develop our predictor with nearest shrunken centroids, we rebuilt a classifier based on the k-TSP method. This classifier relies on the relative expression of 10 pairs of genes, compared to the original 43 identified by nearest shrunken centroids (NSC), and was 96.2% concordant with the original training set prediction, with a 94.3% overall correct classification of BRCA1 carriers and controls. CONCLUSIONS: The k-TSP classifier was shown to accurately predict BRCA1 status using data generated on the nCounter platform and is feasible for initiating a clinical validation.

Mutually exclusive mutation profiles define functionally related genes in muscle invasive bladder cancer.

Muscle Invasive bladder cancer is known to have an abundance of mutations, particularly in DNA damage response and chromatin modification genes. The role of these mutations in the development and progression of the disease is not well understood. However, a mutually exclusive mutation pattern between gene pairs could suggest gene mutations of significance. For example, a mutually exclusive mutation pattern could suggest an epistatic relationship where the outcome of a mutation in one gene would have the same outcome as a mutation in a different gene. The significance of a mutually exclusive relationship was determined by establishing a normal distribution of the conditional probabilities for having a mutation in one gene and not the other as well as the reverse relationship for each gene pairing. Then these distributions were used to determine the sigma-magnitude of standard deviation by which the observed value differed from the expected, a value that can also be interpreted as the 'p-value'. This approach led to the identification of mutually exclusive mutation patterns in KDM6A and KMT2D as well as KDM6A and RB1 that suggested the observed mutation pattern did not happen by chance. Upon further investigation of these genes and their interactions, a potential similar outcome was identified that supports the concept of epistasis. Knowledge of these mutational interactions provides a better understanding of the mechanisms underlying muscle invasive bladder cancer development, and may direct therapeutic development exploiting genotoxic chemotherapy and synthetic lethality in these pathways.

Identification and characterization of a novel nuclear structure containing members of the homologous recombination and DNA damage response pathways.

The human RAD9A protein is required for successful execution of the G2/M DNA damage checkpoint. Along with RAD1 and HUS1, RAD9A exists in a heterotrimeric ring-shaped complex which is necessary for activation of the CHK1 checkpoint kinase. RAD9A is also required for proper localization of both TopBP1 and the Claspin adaptor protein during the DNA damage response. We have shown large, RAD9A-dense nuclear foci containing several members of the homologous recombination pathway as well as BRCA1 and the DNA damage marker γH2AX. This RAD9A-dense body is closely associated with the inactive X in HeLa cells but not in other cell types analyzed including a Klinefelter's syndrome-derived line containing multiple Xi. We have also shown these foci disappear after cell synchronization but are enriched after treatment with the homologous recombination inhibitor pentoxifylline. We conclude these foci are the result of an active process, suspended in perturbed cells, that involves interaction between the cell cycle checkpoint and homologous recombination machinery.

Eligibility Criteria and Genetic Testing Results from a High-Risk Cohort for Hereditary Breast and Ovarian Cancer Syndrome in Southeastern Ontario.

Germline mutations in breast and ovarian cancer are rare, with approximately 5% to 10% and 13% being hereditary in origin, respectively. In 2001, the Ontario Ministry of Health and Long Term Care, in an effort to contain costs, defined criteria to determine an individual's eligibility for BRCA genetic screening. We studied a cohort of individuals that have undergone genetic testing at Kingston General Hospital between 2001 and late 2013. We focused on determining whether the 13 risk criteria, defined by an expert working group for the Ontario Ministry of Health and Long Term Care, have performed according to expectations in this cohort. Our findings show that all of the criteria perform well by identifying carriers at the expected 10% rate defined by the guidelines. We demonstrate that loose application of the risk criteria does not further enrich for BRCA variant carriers. Our assessment of the established risk criteria that have been in use in Ontario for more than a decade, provide evidence for their effectiveness, and offer insights into how they may be expanded or improved.

Chk1 Activation Protects Rad9A from Degradation as Part of a Positive Feedback Loop during Checkpoint Signalling.

Phosphorylation of Rad9A at S387 is critical for establishing a physical interaction with TopBP1, and to downstream activation of Chk1 for checkpoint activation. We have previously demonstrated a phosphorylation of Rad9A that occurs at late time points in cells exposed to genotoxic agents, which is eliminated by either Rad9A overexpression, or conversion of S387 to a non-phosphorylatable analogue. Based on this, we hypothesized that this late Rad9A phosphorylation is part of a feedback loop regulating the checkpoint. Here, we show that Rad9A is hyperphosphorylated and accumulates in cells exposed to bleomycin. Following the removal of bleomycin, Rad9A is polyubiquitinated, and Rad9A protein levels drop, indicating an active degradation process for Rad9A. Chk1 inhibition by UCN-01 or siRNA reduces Rad9A levels in cells synchronized in S-phase or exposed to DNA damage, indicating that Chk1 activation is required for Rad9A stabilization in S-phase and during checkpoint activation. Together, these results demonstrate a positive feedback loop involving Rad9A-dependend activation of Chk1, coupled with Chk1-dependent stabilization of Rad9A that is critical for checkpoint regulation.

BRCA1 haploinsufficiency leads to altered expression of genes involved in cellular proliferation and development.

The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes.

Tousled-like kinase-dependent phosphorylation of Rad9 plays a role in cell cycle progression and G2/M checkpoint exit.

Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.

EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer.

BACKGROUND: The epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers. METHODS: Agilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. RESULTS: Morphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects. CONCLUSIONS: This work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

Rad9A is required for G2 decatenation checkpoint and to prevent endoreduplication in response to topoisomerase II inhibition.

The Rad9A checkpoint protein interacts with and is required for proper localization of topoisomerase II-binding protein 1 (TopBP1) in response to DNA damage. Topoisomerase II (Topo II), another binding partner of TopBP1, decatenates sister chromatids that become intertwined during replication. Inhibition of Topo II by ICRF-193 (meso-4,4'-(3,2-butanediyl)-bis-(2,6-piperazinedione)), a catalytic inhibitor that does not induce DNA double-strand breaks, causes a mitotic delay known as the G(2) decatenation checkpoint. Here, we demonstrate that this checkpoint, dependent on ATR and BRCA1, also requires Rad9A. Analysis of different Rad9A phosphorylation mutants suggests that these modifications are required to prevent endoreduplication and to maintain decatenation checkpoint arrest. Furthermore, Rad9A Ser(272) is phosphorylated in response to Topo II inhibition. ICRF-193 treatment also causes phosphorylation of an effector kinase downstream of Rad9A in the DNA damage checkpoint pathway, Chk2, at Thr(68). Both of these sites are major targets of phosphorylation by the ATM kinase, although it has previously been shown that ATM is not required for the decatenation checkpoint. Examination of ataxia telangectasia (A-T) cells demonstrates that ATR does not compensate for ATM loss, suggesting that phosphorylation of Rad9A and Chk2 by ATM plays an additional role in response to Topo II inhibition than checkpoint function alone. Finally, we have shown that murine embryonic stem cells deficient for Rad9A have higher levels of catenated mitotic spreads than wild-type counterparts. Together, these results emphasize the importance of Rad9A in preserving genomic integrity in the presence of catenated chromosomes and all types of DNA aberrations.

The Rad9A checkpoint protein is required for nuclear localization of the claspin adaptor protein.

The interaction between the 911 complex, via Rad9A, and Claspin is required for activation of the Chk1-mediated checkpoint response, along with ATR, TopBp1, and the 911 clamp loader complex Rad17/RFC. Despite the importance of the Rad9A-Claspin interaction in the cell cycle, this interaction has yet to be characterized. In this work we show this interaction persists in a variety of different conditions. During the course of this study we also determined the nuclear localization of Rad9A affected the localization of the Claspin protein, leading us to the conclusion that Rad9A is able to affect Claspin cellular localization. This was verified experimentally using a Rad9A-null cell line and reconstitution of Wt Rad9A. We also show that in meS cells the Rad9A paralog, Rad9B, is also capable of affecting Claspin localization. Together, these data suggest that Rad9 plays a role in locating Claspin to sites of DNA damage, facilitating its role during the Chk1-mediated checkpoint response. Since disruption of both Rad9A and Claspin has been shown to abolish Chk1 activation, we postulate that Rad9A-mediated Claspin localization is a vital step during checkpoint activation.

Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex.

The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.

Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair.

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat.

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.

Fission yeast Uve1 and Apn2 function in distinct oxidative damage repair pathways in vivo.

In Schizosaccharomyces pombe, the endonuclease Uve1 functions as the first step in an alternate UV photo-product repair pathway that is distinct from nucleotide excision repair (NER). Based upon the broad substrate specificity of Uve1 in vitro, and the observation that Uve1 mutants accumulate spontaneous mutations at an elevated rate in vivo, we and others have hypothesized that this protein might have a function in a mutation avoidance pathway other than UV photo-product repair. We show here that fission yeast Uve1 also functions in oxidative damage repair in vivo. We have determined the spectrum of spontaneous mutations that arise in uve1 null (uve1 degrees ) cells and have observed that both G-->T(C-->A) and T-->G(A-->C) transversions occur at an increased rate relative to wildtype cells. These mutations are indicative of unrepaired oxidative DNA damage and are very similar to the mutation spectrum observed in 8-oxoguanine glycosylase (OGG1) mutants in Saccharomyces cerevisiae. We have generated an apn2 null (apn2 degrees ) strain and shown that it is mildly sensitive to H(2)O(2). Furthermore we have also shown that apn2 degrees cells have an elevated rate of spontaneous mutation that is similar to uve1 degrees. The phenotype of apn2 degrees uve1 degrees double mutants indicates that these genes define distinct spontaneous mutation avoidance pathways. While uve1 degrees cells show only a modest sensitivity to the oxidizing agent hydrogen peroxide (H(2)O(2)), both uve1 degrees and apn2 degrees cells also display a marked increased in mutation rate following exposure to H(2)O(2) doses. Collectively these data demonstrate that Uve1 is a component of multiple alternate repair pathways in fission yeast and suggest a possible role for Uve1 in a general alternate incision repair pathway in eukaryotes.

hRad9 rapidly binds DNA containing double-strand breaks and is required for damage-dependent topoisomerase II beta binding protein 1 focus formation.

Checkpoint proteins protect the genomic integrity of a cell, repeatedly impaired by DNA damage and normal cellular processes, such as replication. Checkpoint proteins hRad9, hRad1, and hHus1 form a heterotrimeric complex that is thought to act as a genomic surveyor of DNA damage. We show here that, when DNA double-strand breaks (DSBs) are specifically generated in a subnuclear area, hRad9 is rapidly retained at the damaged DNA, within 2 min of damage induction. Rapid localization of hRad9 to regions of DNA containing DSBs is most efficient during replication. Furthermore, hRad9 colocalizes with the phosphorylated form of damage-response protein H2AX (gamma H2AX) after DNA damage. This localization is independent of the damage repair kinase ataxia telangiectasia-mutated kinase (ATM), because hRad9/gamma H2AX colocalization still occurs in ATM(-/-) fibroblasts. Secondly, hRad9 interacts with replication and checkpoint protein topoisomerase II beta binding protein 1 (TopBP1) before and after DNA damage, and this interaction is dependent on the COOH-terminal 17 amino acids of hRad9. Overexpression of a COOH-terminally deleted form of hRad9 abolishes the colocalization of TopBP1 to gamma H2AX, ablating TopBP1 but not gamma H2AX foci formation. The loss of TopBP1 containing foci, but not of gamma H2AX containing foci, indicates that hRad9 is required for TopBP1 focus formation after damage, but is not required for gamma H2AX formation at DSBs. These results are consistent with a model in which the hRad9/hHus1/hRad1 complex acts as a checkpoint sensor during S phase by rapidly localizing to sites of DNA damage and transducing checkpoint responses by facilitating proper localization of downstream checkpoint proteins, including TopBP1.

A role for the phosphorylation of hRad9 in checkpoint signaling.

The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase.