Novel genetic predictors of venous thromboembolism risk in African Americans.

Venous thromboembolism (VTE) is the third most common life-threatening cardiovascular condition in the United States, with African Americans (AAs) having a 30% to 60% higher incidence compared with other ethnicities. The mechanisms underlying population differences in the risk of VTE are poorly understood. We conducted the first genome-wide association study in AAs, comprising 578 subjects, followed by replication of highly significant findings in an independent cohort of 159 AA subjects. Logistic regression was used to estimate the association between genetic variants and VTE risk. Through bioinformatics analysis of the top signals, we identified expression quantitative trait loci (eQTLs) in whole blood and investigated the messenger RNA expression differences in VTE cases and controls. We identified and replicated single-nucleotide polymorphisms on chromosome 20 (rs2144940, rs2567617, and rs1998081) that increased risk of VTE by 2.3-fold (P< 6 × 10(-7)). These risk variants were found in higher frequency among populations of African descent (>20%) compared with other ethnic groups (<10%). We demonstrate that SNPs on chromosome 20 are cis-eQTLs for thrombomodulin (THBD), and the expression of THBD is lower among VTE cases compared with controls (P= 9.87 × 10(-6)). We have identified novel polymorphisms associated with increased risk of VTE in AAs. These polymorphisms are predominantly found among populations of African descent and are associated with THBD gene expression. Our findings provide new molecular insight into a mechanism regulating VTE susceptibility and identify common genetic variants that increase the risk of VTE in AAs, a population disproportionately affected by this disease.

Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome.

BACKGROUND: One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility. RESULTS: Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing. CONCLUSIONS: Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Kinase insert domain receptor/vascular endothelial growth factor receptor 2 (KDR) genetic variation is associated with ovarian hyperstimulation syndrome.

BACKGROUND: The objective of this investigation was to determine if kinase insert domain/vascular endothelial growth factor receptor 2 (KDR/VEGFR2) genetic variation was associated with the development of ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation (COH). METHODS: This was a case-control study of 174 patients who underwent controlled ovarian stimulation. Patient blood samples were genotyped for single nucleotide polymorphisms (SNPs) spanning the KDR locus. OHSS development, clinical outcome variables, SNP and haplotype frequencies were compared between control (n = 155) and OHSS (n = 19) groups. RESULTS: Patients who developed OHSS had significantly higher response markers (estradiol levels of the day of hCG administration, number of follicles developed, number of eggs retrieved) than control patients. When adjusted for age and self-identified race, the rs2305945 G/T genotype was associated (P = 0.027) with a decreased risk (OR = 0.30; 95% CI = 0.10, 0.93) of developing OHSS using an overdominant model. The rs2305945 G/T variant was also associated with decreased COH response (number of follicles, number of eggs retrieved) in an overdominant model. The rs2305948, rs1870378, rs2305945 (C-T-G) haplotype was associated with both decreased COH response and OHSS risk (unadjusted OR = 0.10; 95% CI = 0.01, 0.80, P = 0.031). CONCLUSIONS: The KDR receptor is believed to play a central role OHSS development and is a target for pharmacological prevention of OHSS. These results indicate that genetic variation in the KDR gene may impact individual risk of developing OHSS from COH. In addition, the rs2305948 SNP and C-T-G haplotype might serve as potential biomarkers for poor ovarian response to COH.

Association between the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) rs4073366 polymorphism and ovarian hyperstimulation syndrome during controlled ovarian hyperstimulation.

BACKGROUND: The aim of this study was to determine the relationship between a purported luteinizing hormone/chorionic gonadotropin (LHCGR) high function polymorphism (rs4539842/insLQ) and outcome to controlled ovarian hyperstimulation (COH). METHODS: This was a prospective study of 172 patients undergoing COH at the Fertility and IVF Center at GWU. DNA was isolated from blood samples and a region encompassing the insLQ polymorphism was sequenced. We also investigated a polymorphism (rs4073366 G > C) that was 142 bp from insLQ. The association of the insLQ and rs4073366 alleles and outcome to COH (number of mature follicles, estradiol level on day of human chorionic gonadotropin (hCG) administration, the number of eggs retrieved and ovarian hyperstimulation syndrome (OHSS)) was determined. RESULTS: Increasing age and higher day 3 (basal) FSH levels were significantly associated with poorer response to COH. We found that both insLQ and rs4073366 were in linkage disequilibrium (LD) and no patients were homozygous for both recessive alleles (insLQ/insLQ; C/C). The insLQ variant was not significantly associated with any of the main outcomes to COH. Carrier status for the rs4073366 C variant was associated (P = 0.033) with an increased risk (OR 2.95, 95% CI = 1.09-7.96) of developing OHSS. CONCLUSIONS: While age and day 3 FSH levels were predictive of outcome, we found no association between insLQ and patient response to COH. Interestingly, rs4073366 C variant carrier status was associated with OHSS risk. To the best of our knowledge, this is the first report suggesting that LHCGR genetic variation might function in patient risk for OHSS.

Assessment of K-Ras mutant frequency and micronucleus incidence in the mouse duodenum following 90-days of exposure to Cr(VI) in drinking water.

Chronic exposure to high concentrations of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) in drinking water induces duodenal tumors in mice, but the mode of action (MOA) for these tumors has been a subject of scientific debate. To evaluate the tumor-site-specific genotoxicity and cytotoxicity of SDD in the mouse small intestine, tissue pathology and cytogenetic damage were evaluated in duodenal crypt and villus enterocytes from B6C3F1 mice exposed to 0.3-520mg/L SDD in drinking water for 7 and 90 days. Allele-competitive blocker PCR (ACB-PCR) was used to investigate the induction of a sensitive, tumor-relevant mutation, specifically in vivo K-Ras codon 12 GAT mutation, in scraped duodenal epithelium following 90 days of drinking water exposure. Cytotoxicity was evident in the villus as disruption of cellular arrangement, desquamation, nuclear atypia and blunting. Following 90 days of treatment, aberrant nuclei, occurring primarily at villi tips, were significantly increased at ≥60mg/L SDD. However, in the crypt compartment, there were no dose-related effects on mitotic and apoptotic indices or the formation of aberrant nuclei indicating that Cr(VI)-induced cytotoxicity was limited to the villi. Cr(VI) caused a dose-dependent proliferative response in the duodenal crypt as evidenced by an increase in crypt area and increased number of crypt enterocytes. Spontaneous K-Ras codon 12 GAT mutations in untreated mice were higher than expected, in the range of 10(-2) to 10(-3); however no treatment-related trend in the K-Ras codon 12 GAT mutation was observed. The high spontaneous background K-Ras mutant frequency and Cr(VI) dose-related increases in crypt enterocyte proliferation, without dose-related increase in K-Ras mutant frequency, micronuclei formation, or change in mitotic or apoptotic indices, are consistent with a lack of genotoxicity in the crypt compartment, and a MOA involving accumulation of mutations late in carcinogenesis as a consequence of sustained regenerative proliferation.

Prostate cancer cell-platelet bidirectional signaling promotes calcium mobilization, invasion and apoptotic resistance via distinct receptor-ligand pairs.

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbß3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbß3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

Apixaban Concentrations in Routine Clinical Care of Older Adults With Nonvalvular Atrial Fibrillation.

BACKGROUND: Direct-acting oral anticoagulants are first-line agents for prevention of stroke in patients with nonvalvular atrial fibrillation (NVAF), but data are limited for the oldest patients, and with reduced dosing. OBJECTIVES: To determine steady-state apixaban peak and trough concentrations during routine care of older adults with NVAF, compare concentrations to clinical trial concentrations, and explore factors associated with concentrations. METHODS: A cross-sectional study of medically stable older adults with NVAF (≥75 years or ≥70 years if Black) receiving apixaban. Peak (2-4.4 hours post-dose) and trough (before next dose) concentrations were determined by anti-Xa activity calibrated chromogenic assay. Patient characteristics associated with concentrations were determined by multivariate modeling. RESULTS: The median age of patients (n = 115) was 80 (interquartile range: 77-84) years. The cohort comprised 46 women and 69 men; of which 98 are White, 11 Black, and 6 Asian. With 5 mg twice daily per labelling (n = 88), peak concentrations were higher in women: 248 ± 105 vs 174 ± 67 ng/mL in men (P < 0.001) and exceeded expected 95% range in 6 of 30 vs 0 of 55 men (P = 0.002). With 2.5 mg twice daily per label (n = 11), concentrations were <5 mg twice daily (peak: 136 ± 87 vs 201 ± 90 ng/mL, P = 0.026; trough: 65 ± 28 vs 109 ± 56 ng/mL, P < 0.001), but not different than 2.5 mg twice daily without reduction criteria (n = 13; peak: 132 ± 88; trough: 65 ± 31 ng/mL). Covariates associated with concentrations included sex, number of daily medications, and creatinine clearance. CONCLUSIONS: Older women had higher than expected peak apixaban concentrations, and 2.5 mg twice daily produced lower concentrations than standard dosing. Factors not currently included in dosing recommendations affected concentrations. The impact of apixaban concentrations on outcomes needs evaluation.

Race and Drug Toxicity: A Study of Three Cardiovascular Drugs with Strong Pharmacogenetic Recommendations.

The Clinical Pharmacogenetics Implementation Consortium (CPIC®) establishes evidence-based guidelines for utilizing pharmacogenetic information for certain priority drugs. Warfarin, clopidogrel and simvastatin are cardiovascular drugs that carry strong prescribing guidance by CPIC. The respective pharmacogenes for each of these drugs exhibit considerable variability amongst different ethnic/ancestral/racial populations. Race and ethnicity are commonly employed as surrogate biomarkers in clinical practice and can be found in many prescribing guidelines. This is controversial due to the large variability that exists amongst different racial/ethnic groups, lack of detailed ethnic information and the broad geographic categorization of racial groups. Using a retrospective analysis of electronic health records (EHR), we sought to determine the degree to which self-reported race/ethnicity contributed to the probability of adverse drug reactions for these drugs. All models used individuals self-reporting as White as the comparison group. The majority of apparent associations between different racial groups and drug toxicity observed in the "race only" model failed to remain significant when we corrected for covariates. We did observe self-identified Asian race as a significant predictor (p = 0.016) for warfarin hemorrhagic events in all models. In addition, patients identifying as either Black/African-American (p = 0.001) or Other/Multiple race (p = 0.019) had a lower probability of reporting an adverse reaction than White individuals while on simvastatin even after correcting for other covariates. In both instances where race/ethnicity was predictive of drug toxicity (i.e., warfarin, simvastatin), the findings are consistent with the known global variability in the pharmacogenes described in the CPIC guidelines for these medications. These results confirm that the reliability of using self-identified race/ethnic information extracted from EHRs as a predictor of adverse drug reactions is likely limited to situations where the genes influencing drug toxicity display large, distinct ethnogeographic variability.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

Polo-like kinase 1 enhances survival and mutagenesis after genotoxic stress in normal cells through cell cycle checkpoint bypass.

Polo-like kinase 1 (Plk1) is a key regulator of mitosis. Aberrant Plk1 activity is found in tumors, but little is known regarding its role in the DNA damage response of normal cells and its potential contribution to the early stages of carcinogenesis. Inappropriate survival signaling after DNA damage may facilitate clonal expansion of genetically compromised cells, and it is known that protein tyrosine phosphatase (PTP) inhibitors activate key survival pathways. In this study, we employed hexavalent chromium [Cr(VI)], a well-documented genotoxicant, to investigate the mechanism by which survival pathway activation could lead to loss of checkpoint control via a mechanism involving Plk1. We recently reported that PTP inhibition enhances clonogenic survival and mutagenesis after Cr(VI) exposure by overriding Cr-induced growth arrest. Here, we report that checkpoint bypass, facilitated by PTP inhibition, was associated with decreased Cdk1 Tyr15 phosphorylation, as well as increased Plk1 activity and nuclear localization. Plk1 was necessary for increased survival after PTP inhibition and Cr(VI) exposure in normal human fibroblasts via enhanced mitotic progression. In addition, pharmacological inhibition of Plk1 abolished the PTP inhibitor-induced bypass of the G(2)/M checkpoint. Notably, Plk1 overexpression increased survival and mutagenesis after Cr(VI) exposure in wild-type Saccharomyces cerevisiae. Taken together, our data indicate that Plk1 activation and nuclear localization are necessary for PTP-regulated mitotic progression after DNA damage. Our studies highlight a role for Plk1 in the loss of checkpoint control, increased survival and mutagenesis after genotoxic exposure in normal cells, which in turn may lead to genomic instability and carcinogenesis.

DNA polymerase zeta is essential for hexavalent chromium-induced mutagenesis.

Translesion synthesis (TLS) is a unique DNA damage tolerance mechanism involved in the replicative bypass of genetic lesions in favor of uninterrupted DNA replication. TLS is critical for the generation of mutations by many different chemical and physical agents, however, there is no information available regarding the role of TLS in carcinogenic metal-induced mutagenesis. Hexavalent chromium (Cr(VI))-containing compounds are highly complex genotoxins possessing both mutagenic and clastogenic activities. The focus of this work was to determine the impact that TLS has on Cr(VI)-induced mutagenesis in Saccharomyces cerevisiae. Wild-type yeast and strains deficient in TLS polymerases (i.e. Polzeta (rev3), Poleta (rad30)) were exposed to Cr(VI) and monitored for cell survival and forward mutagenesis at the CAN1 locus. In general, TLS deficiency had little impact on Cr(VI)-induced clonogenic lethality or cell growth. rad30 yeast exhibited higher levels of basal and induced mutagenesis compared to Wt and rev3 yeast. In contrast, rev3 yeast displayed attenuated Cr(VI)-induced mutagenesis. Moreover, deletion of REV3 in rad30 yeast (rad30 rev3) resulted in a significant decrease in basal and Cr(VI) mutagenesis relative to Wt and rad30 single mutants indicating that mutagenesis primarily depended upon Polzeta. Interestingly, rev3 yeast were similar to Wt yeast in susceptibility to Cr(VI)-induced frameshift mutations. Mutational analysis of the CAN1 gene revealed that Cr(VI)-induced base substitution mutations accounted for 83.9% and 100.0% of the total mutations in Wt and rev3 yeast, respectively. Insertions and deletions comprised 16.1% of the total mutations in Cr(VI) treated Wt yeast but were not observed rev3 yeast. This work provides novel information regarding the molecular mechanisms of Cr(VI)-induced mutagenesis and is the first report demonstrating a role for TLS in the fixation of mutations induced by a carcinogenic metal.

Bypass of hexavalent chromium-induced growth arrest by a protein tyrosine phosphatase inhibitor: enhanced survival and mutagenesis.

Although the consequences of genotoxic injury include cell cycle arrest and apoptosis, cell survival responses after genotoxic injury can produce intrinsic death-resistance and contribute to the development of a transformed phenotype. Protein tyrosine phosphatases (PTPs) are integral components of key survival pathways, and are responsible for their inactivation, while PTP inhibition is often associated with enhanced cell proliferation. Our aim was to elucidate signaling events that modulate cell survival after genotoxin exposure. Diploid human lung fibroblasts (HLF) were treated with Cr(VI) (as Na(2)CrO(4)), the soluble oxyanionic dissolution product of certain particulate chromates, which are well-documented human respiratory carcinogens. In vitro soluble Cr(VI) induces a wide spectrum of DNA damage, in both the presence and absence of a broad-range PTP inhibitor, sodium orthovanadate (SOV). Notably, SOV abrogated Cr(VI)-induced clonogenic lethality. The enhanced survival of Cr(VI)-exposed cells after SOV treatment was predominantly due to a bypass of cell cycle arrest, as there was no effect of the PTP inhibitor on Cr-induced apoptosis. Moreover, the SOV effect was not due to decreased Cr uptake as evidenced by unchanged Cr-DNA adduct burden. Additionally, the bypass of Cr-induced growth arrest by SOV was accompanied by a decrease in Cr(VI)-induced expression of cell cycle inhibiting genes, and an increase in Cr(VI)-induced expression of cell cycle promoting genes. Importantly, SOV resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of certain types of DNA damage may lead to increased genomic instability, via bypass of cell cycle checkpoints.

The ACCOuNT Consortium: A Model for the Discovery, Translation, and Implementation of Precision Medicine in African Americans.

The majority of pharmacogenomic (PGx) studies have been conducted on European ancestry populations, thereby excluding minority populations and impeding the discovery and translation of African American-specific genetic variation into precision medicine. Without accounting for variants found in African Americans, clinical recommendations based solely on genetic biomarkers found in European populations could result in misclassification of drug response in African American patients. To address these challenges, we formed the Transdisciplinary Collaborative Center (TCC), African American Cardiovascular Pharmacogenetic Consortium (ACCOuNT), to discover novel genetic variants in African Americans related to clinically actionable cardiovascular phenotypes and to incorporate African American-specific sequence variations into clinical recommendations at the point of care. The TCC consists of two research projects focused on discovery and translation of genetic findings and four cores that support the projects. In addition, the largest repository of PGx information on African Americans is being established as well as lasting infrastructure that can be utilized to spur continued research in this understudied population.

Excision repair is required for genotoxin-induced mutagenesis in mammalian cells.

Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Our goal was to investigate the role of excision repair (ER) pathways in Cr(VI)-mediated mutagenesis in mammalian cells. Repair-proficient Chinese hamster ovary cells (AA8), nucleotide excision repair (NER)-deficient (UV-5) and base excision repair (BER)-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. BER was inhibited using methoxyamine hydrochloride (Mx), which binds to apurinic/apyrimidinic sites generated during BER. Notably, we found that both NER-deficient (UV-5 and UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet (UV) radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Moreover, UV-5 cells expressing human xeroderma pigmentosum complementation group D displayed similar sensitivity to Cr(VI) and MMS-induced mutagenesis as AA8 controls, indicating that the genetic loss of NER was responsible for attenuated mutagenesis. Interestingly, Cr(VI)-induced clastogenesis was also attenuated in NER-deficient and BER-inhibited cells. Taken together, our results suggest that NER and BER are required for Cr(VI) and MMS-induced genomic instability. We postulate that, in the absence of ER, DNA damage is channeled into an error-free system of DNA repair or damage tolerance.

Teaching students in clinical programs about pharmacogenomics: do they understand drug-drug interactions?

Teaching the clinical implementation of pharmacogenomics to students in clinical programs first requires careful consideration of their aptitude in basic clinical pharmacologic concepts. Prior to developing training exercises on drug-gene interactions, educators must first assess student competency in identifying and managing drug-drug interactions given the similarities in identifying and managing these sources of medication error.

FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure: activation is not required for repair of Cr(VI)-induced DSBs.

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by gamma-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult.

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease.

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.

Improving nurse practitioners' competence with genetics: Effectiveness of an online course.

PURPOSE: The purpose of this study was to assess the effectiveness of an online genetics course for improving nurse practitioners' knowledge, competence, and comfort with genetic principles and their application to clinical practice. DATA SOURCES: A genetics knowledge test and survey were administered to 232 nurse practitioner students, between 2011 and 2013, before and after completing a 15-week online genetics course taught by a multidisciplinary team of instructors at a private east coast U.S. university. The 65-item survey allowed participants to rate competence regarding genetic principles, diseases, and terminology, as well as comfort performing various clinical tasks related to genetics. The 21-item knowledge test contained multiple choice questions regarding core competencies in genetics. Paired t-tests were used to compare mean pre- and postscores. CONCLUSIONS: Participants significantly increased postcourse knowledge (p < .001) and comfort with genetic core competencies and clinical skills related to genetics (p < .001). This study demonstrates the effectiveness of an online genetics course for increasing nurse practitioners' knowledge, competence, and confidence with genetics and identifies specific topics educators should consider when designing curricula for nurse practitioners. IMPLICATIONS FOR PRACTICE: Findings from this study can improve genetics education for nurse practitioners, which will in turn improve patient health.

Impact of a personal CYP2D6 testing workshop on physician assistant student attitudes toward pharmacogenetics.

AIM: We assessed the impact of personal CYP2D6 testing on physician assistant student competency in, and attitudes toward, pharmacogenetics (PGx). MATERIALS & METHODS: Buccal samples were genotyped for CYP2D6 polymorphisms. Results were discussed during a 3-h PGx workshop. PGx knowledge was assessed by pre- and post-tests. Focus groups assessed the impact of the workshop on attitudes toward the clinical utility of PGx. RESULTS: Both student knowledge of PGx, and its perceived clinical utility, increased immediately following the workshop. However, exposure to PGx on clinical rotations following the workshop seemed to influence student attitudes toward PGx utility. CONCLUSION: Personal CYP2D6 testing improves both knowledge and comfort with PGx. Continued exposure to PGx concepts is important for transfer of learning.

Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae.

A broad spectrum of genetic damage results from exposure to hexavalent chromium. These lesions can result in DNA and RNA polymerase arrest, chromosomal aberrations, point mutations and deletions. Because of the complexity of Cr genotoxicity, the repair of Cr(VI)-induced DNA damage is poorly understood. Therefore, our aim was to investigate the sensitivities of DNA repair-deficient Saccharomyces cerevisiae strains to Cr(VI)-induced growth inhibition and lethality. Wild-type, translesion synthesis (rev3) and excision repair (apn1, ntg1, ntg2, rad1) mutants exhibited similar survival following Cr(VI) treatment (0-50mM) and underwent at least one population doubling within 2-4h post-treatment. The simultaneous loss of several excision repair genes (apn1 rad1 ntg1 ntg2) led to slower growth after Cr(VI) exposure (10mM) manifested as an initial delay in S phase progression. Higher concentrations of Cr(VI) (25mM) resulted in a prolonged transit through S phase in every strain tested. A G(2)/M arrest was evident within 1-2h after Cr(VI) treatment (10mM) in all strains and cells subsequently divided after this transient delay. In contrast to all other strains, only recombination-deficient (rad52, rad52 rev3) yeast were markedly hypersensitive towards Cr(VI) lethality. RAD52 mutant strains (rad52, rad52 rev3) also exhibited a significant delay (>6h) in the resumption of replication after Cr(VI) exposure which was related to the immediate and apparently terminal arrest of these yeast in G(2)/M after Cr(VI) treatment. These results, taken together with the recombinogenic effects of Cr(VI) in yeast containing a functional RAD52 gene, suggest that RAD52-mediated recombination is critical for the normal processing of lethal Cr-induced genetic lesions and exit from G(2) arrest. Furthermore, only the combined inactivation of multiple excision repair genes affects cell growth after Cr(VI) treatment.