Travis J. O'Brien

Ethnicity-specific pharmacogenetics: The case of warfarin in African Americans

Using a derivation cohort (N=349), we developed the first warfarin dosing algorithm that includes recently discovered polymorphisms in VKORC1 and CYP2C9 associated with warfarin dose requirement in African Americans (AAs). We tested our novel algorithm in an independent cohort of 129 AAs and compared the dose prediction to the International Warfarin Pharmacogenetics Consortium (IWPC) dosing algorithms. Our algorithm explains more of the phenotypic variation (R2=0.27) than the IWPC pharmacogenomics (R2=0.15) or clinical (R2=0.16) algorithms. Among high-dose patients, our algorithm predicted a higher proportion of patients within 20% of stable warfarin dose (45% vs 29% and 2% in the IWPC pharmacogenomics and clinical algorithms, respectively). In contrast to our novel algorithm, a significant inverse correlation between predicted dose and percent West African ancestry was observed for the IWPC pharmacogenomics algorithm among patients requiring ⩾60 mg per week (β=−2.04, P=0.02).

DNA Polymerase ζ 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.

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.

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.

First report of warfarin dose requirements in patients possessing the CYP2C9*12 allele

BACKGROUND Warfarin is the most frequently prescribed anticoagulant in North America and Europe. It is administered as a racemate, but S-warfarin is principally responsible for its anticoagulant activity. Cytochrome P450 (CYP) 2C9 is the enzyme primarily responsible for the metabolism of S-warfarin. Numerous variant alleles of CYP2C9 have been identified. The CYP2C9*12 (rs9332239) allele harbors a P489S substitution in CYP2C9 which has been shown to result in a 40% decline in catalytic activity in vitro. CASES Four Caucasian patients with a low mean weekly warfarin dose (MWWD) were genotyped for CYP2C9, VKORC1 and APOE variant alleles. None of the four patients carried the common CYP2C9 variant alleles (*2, *3, *5, *6, *7, *8, *9, *11, *13) despite a relatively low MWWD (23.4±7.94 mg) compared to 208 patients carrying the CYP29C9*1 genotype (32.2±12.65 mg). Given that CYP2C9*12 confers decreased in vitro activity to the enzyme, we investigated whether these patients carried this allele. All four patients were CYP2C9*12 CT heterozygotes. Individual comparisons with patients possessing the same VKORC1 and APOE genotypes also demonstrated lower dose requirements in the patients that possessed CYP2C9*12 allele. CONCLUSIONS There are no reports of the clinical impact of rs9332239 on CYP2C9 substrates. This is the first report of patients with the rare CYP2C9*12 genotype and lower warfarin dose requirements.

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.

Integrated analysis of genetic variation and gene expression reveals novel variant for increased warfarin dose requirement in African Americans.

Essentials Genetic variants controlling gene regulation have not been explored in pharmacogenomics. We tested liver expression quantitative trait loci for association with warfarin dose response. A novel predictor for increased warfarin dose response in African Americans was identified. Precision medicine must take into account population‐specific variation in gene regulation.

Abstract 2131: Plk1 decreases DNA double strand break (DSB) formation during replication- stalling genotoxic stress

Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. The aim of the present study was to determine the ability of Plk1 to affect the DNA damage response in the face of different forms of DSB-inducing genotoxic stress. We employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], the anticancer drug and topoisomerase II inhibitor, etoposide, as well as the oxidizer, H2O2. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by the respective genotoxins under the condition of Plk1 activation by using the comet assay and γH2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated both Cr(VI) and etoposide-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control-transfected cells. In sharp contrast, treatment with 150 μM H2O2 induced a similar level of DNA DSBs in both the c/a T210D mutant- and vector-transfected cells. These data highlight the ability of Plk1 to decrease DNA DSBs uniquely under conditions of replication-stalling genotoxic stress. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates decreased DNA DSBs after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to abrogate DNA DSB formation after either Cr(VI) or etoposide exposure may occur at the expense of genomic stability. Supported by NIH grants CA107972 and ES017334 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2131. doi:1538-7445.AM2012-2131

Abstract 3914: Plk1 enhances DNA double strand break (DSB) repair during acute genotoxin exposure

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression in cancer cells during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. In this study we employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], which is a well-documented genotoxin of occupational and environmental concern. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study we measured DNA DSB induction by Cr(VI) in HLFs under the condition of Plk1 activation by using the comet assay and gamma H2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We found that DNA DSBs increased > 2 fold 15 min after 3 μM Cr(VI) exposure, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated Cr(VI)-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control transfected cells. Since Cr(VI)-induced DNA DSBs were equally formed by 15 min in both vector- and Plk1 T210D-transfected cells, these data highlight the heretofore unreported ability of Plk1 to enhance DNA repair. Our previous report found that Plk1 expression increased survival and mutagenesis of wt S. cerevisiae treated with Cr(VI). Therefore, we also determined the ability of Plk1 to “rescue” repair deficient rad52 yeast from Cr(VI)-induced lethality. S. cerevisae transformed with Plk1-Gal-HA-EGFP plasmid grown under inducible conditions overexpress Plk1. We found that induction of Plk1 in rad52 yeast significantly protected them from Cr(VI) clonogenic lethality. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates DNA damage repair after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to provoke DNA DSB repair after Cr(VI) exposure occurs at the expense of genomic stability. Supported by NIH grants CA107972 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2011-3914

Abstract 2978: Nucleotide excision repair contributes to hexavalent chromium-induced double strand breaks and chromosomal damage

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Certain hexavalent chromium (Cr(VI)) compounds are known human lung carcinogens. Cr(VI) causes several types of DNA damage including bulky adducts and crosslinks. Cr(VI)-induced tumors exhibit chromosomal instability (CIN), however, very little is known about mechanisms of these effects and the role that DNA repair plays in this process. Nucleotide excision repair (NER) is known to be involved in the repair of bulky DNA damage. Unrepaired or misrepaired lesions can lead to chromosomal aberrations (CAs). We investigated the role of NER leading to CAs using Chinese hamster ovary (CHO) parental (AA8) and NER-deficient cells (UV-5). We found that UV-5 cells displayed attenuated Cr(VI)-induced chromosome damage. In general, DNA lesions need to be converted to DNA double strand breaks (DSBs) in order to give rise to CAs. Therefore, we used the neutral Comet assay to measure the induction and repair of DSBs after Cr(VI)-induced damage. We found that both parental and NER-deficient cell lines displayed low levels of DSBs immediately after Cr(VI) treatment. Interestingly, DSB formation peaked after a 24h recovery period, and NER-deficient cells exhibited lower overall levels of DSBs than repair-proficient cells. All damage in both cell lines was repaired by 48h following Cr(VI) exposure. Taken together, our results suggest that Cr(VI)-induced DSBs are not directly formed by Cr(VI) exposure. Instead, these lesions develop as a consequence of repair of, or replication past, Cr(VI)-induced lesions. Importantly, our data suggests that NER contributes to Cr(VI)-induced genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2978.