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.

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.

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.

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.

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.

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.

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.

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.

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.

Secreted phospholipases A2 of snake venoms: effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry.

Neuro- and myotoxicological signs and symptoms are significant clinical features of envenoming snakebites in many parts of the world. The toxins primarily responsible for the neuro and myotoxicity fall into one of two categories--those that bind to and block the post-synaptic acetylcholine receptors (AChR) at the neuromuscular junction and neurotoxic phospholipases A2 (PLAs) that bind to and hydrolyse membrane phospholipids of the motor nerve terminal (and, in most cases, the plasma membrane of skeletal muscle) to cause degeneration of the nerve terminal and skeletal muscle. This review provides an introduction to the biochemical properties of secreted sPLA2s in the venoms of many dangerous snakes and a detailed discussion of their role in the initiation of the neurologically important consequences of snakebite.

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.

Neuromuscular toxicology of the venom of Collett's snake (Pseudechis colletti): a histopathological study.

Introduction: A bite by Collett's snake, (Pseudechis colletti) can cause rhabdomyolysis in human victims but no signs of neurotoxicity. The pathology of muscle and peripheral nerve has not been described previously. In this study we investigated neuromuscular toxicity in rats.

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.

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.

Ultrastructural evidence for the uptake of a neurotoxic snake venom phospholipase A2 into mammalian motor nerve terminals.

A mutant form of ammodytoxin A, a neurotoxic phospholipase A(2) from the venom of the long nosed viper Vipera ammodytes ammodytes, was prepared by site-directed mutagenesis, conjugated to a nanogold particle and inoculated into the antero-lateral aspect of one hind limb of female mice. Eight hours later the mice were killed, the soleus muscles of both ipsi- and contra-lateral hind limbs were removed, exposed to a silver enhancing medium and then prepared for transmission electron microscopy. Silver-enhanced particles were subsequently found concentrated in the peri-synaptic area, particularly within the synaptic gutter and the deep synaptic folds, and in many cases had been taken up into the cytoplasm of the terminal boutons of the motor axon.

Venom of Collett's snake (Pseudechis colletti) blocks the binding of alpha-bungarotoxin to acetylcholine receptors at chick but not human neuromuscular junctions: a histochemical study.

Transverse cryosections, 6-8 mum thick, were cut from unfixed biventer cervicis muscles of chicks and quadriceps muscles of humans, mounted on glass slides and incubated for 1h in either isotonic phosphate buffered saline, pH 7.3 (PBS), or crude venom of venom of Pseudechis colletti at concentrations between 2.1 and 210 microgml(-1) in PBS.

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.

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.

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.

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.

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.

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.