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Average-risk colorectal cancer (CRC) screening is broadly recommended, using one of several endorsed test options. However, CRC screening participation rates remain below national goals. To gain further insights regarding recent, population-based patterns in overall and test-specific CRC screening participation, we conducted a retrospective study of adults, ages 50–75 years, utilizing comprehensive data resources from the Rochester Epidemiology Project (REP). Among residents of Olmsted County, MN eligible and due for CRC screening, we identified 5818 residents across three annual cohorts who completed screening between 1/1/2016 and 12/31/2018. We summarized CRC screening rates as incidence per 1000 population and used Poisson regression to test for overall and mode-specific CRC trends. We also analyzed rates of follow-up colonoscopy within 6-months after a positive stool-based screening result. While no significant differences over time were observed in overall CRC screening incidence rates among those due for screening, we observed a statistically significant increase in mt-sDNA test and statistically significant decreases in screening colonoscopy and FIT/FOBT test completion rates; differences in screening overall and by modality were observed by age, sex, and race/ethnicity. The diagnostic colonoscopy follow-up rate within six months after a positive stool-based test was significantly higher following mt-sDNA (84.9%) compared to FIT/FOBT (42.6%). In this retrospective, population-based study, overall CRC screening incidence rates remained stable from 2016 to 2018, while test-specific rates for mt-sDNA significantly increased and decreased for colonoscopy and FIT/FOBT. Adherence with follow-up colonoscopy after a positive stool-based test was significantly higher among patients who underwent mt-sDNA screening compared to FIT/FOBT.

Incidence rates for endometrial cancer (EC) are rising, particularly in postmenopausal and obese women. Previously, we showed that the uterine and vaginal microbiome distinguishes patients with EC from those without. Here, we sought to examine the impact of patient factors (such as menopause status, body mass index, and vaginal pH) in the microbiome in the absence of EC and how these might contribute to the microbiome signature in EC. We find that each factor independently alters the microbiome and identified postmenopausal status as the main driver of a polymicrobial network associated with EC (ECbiome). We identified Porphyromas somerae presence as the most predictive microbial marker of EC and we confirm this using targeted qPCR, which could be of use in detecting EC in high-risk, asymptomatic women. Given the established pathogenic behavior of P. somerae and accompanying network in tissue infections and ulcers, future investigation into their role in EC is warranted.

Cytochrome (CYP) P450 enzymes have a primary role in antidepressant metabolism and variants in these polymorphic genes are targets for pharmacogenetic investigation. This is the first meta-analysis to investigate how CYP2C19 polymorphisms predict citalopram/escitalopram efficacy and side effects.CYP2C19 phenotypes comprise poor metabolizers (PM), intermediate and intermediate+ metabolizers (IM; IM+), extensive and extensive+ metabolizers (EM [wild type]; EM+) and ultra-rapid metabolizers (UM) defined by the two most common CYP2C19 functional polymorphisms (rs4244285 and rs12248560) in Caucasians. These polymorphisms were genotyped or imputed from genome-wide data in four samples treated with citalopram or escitalopram (GENDEP, STAR*D, GenPod, PGRN-AMPS). Treatment efficacy was percentage symptom improvement and remission. Side effect data were available at weeks 2–4, 6 and 9 in three of the investigated samples. A fixed-effects meta-analysis was performed using EM as the reference group.Analysis of 2558 patients for efficacy and 2037 patients for side effects showed that PMs had higher symptom improvement (SMD=0.43, CI=0.19–0.66) and higher remission rates (OR=1.55, CI=1.23–1.96) compared to EMs. At weeks 2–4, PMs showed higher risk of gastro-intestinal (OR=1.26, CI=1.08–1.47), neurological (OR=1.28, CI=1.07–1.53) and sexual side effects (OR=1.52, CI=1.23–1.87; week 6 values similar). No difference was seen at week 9 or in total side effect burden. PMs did not have higher risk of dropout at week 4 compared to EMs. Antidepressant dose was not different among CYP2C19 groups.CYP2C19 polymorphisms may provide helpful information for guiding citalopram/escitalopram treatment, despite PMs are relatively rare among Caucasians (~2%).

Background Neprilysin is a metalloprotease involved in proteolysis of numerous peptides, including natriuretic peptides, and is of prognostic and therapeutic importance in heart failure with reduced ejection fraction. No studies have investigated circulating neprilysin in the community, its clinical correlates, or its relationship to cardiovascular disease in the general population. Methods and Results Plasma neprilysin was measured in 1536 participants from Olmsted County, Minnesota, using a commercially available sandwich ELISA assay. Clinical and echocardiographic correlates and subsequent outcomes were determined. Soluble neprilysin is non‐normally distributed in the community (median: 3.9 ng/mL; interquartile range: 1.0–43.0 ng/mL). There was no relationship between plasma neprilysin and age (Spearman correlation: −0.04, P =0.16); body mass index (Spearman correlation: −0.04, P =0.16); glomerular filtration rate (Spearman correlation: −0.007, P =0.8); or A‐, B‐, or C‐type natriuretic peptides (Spearman correlation: 0.03, P =0.22; −0.001, P =0.96; 0.01, P =0.67, respectively). Among tertiles of neprilysin, the lowest tertile group had the highest prevalence of smokers ( P P =0.04), dyslipidemia ( P =0.03), and diastolic dysfunction ( P =0.02). Soluble neprilysin was not prospectively associated with death or heart failure over a median of 10.7 years. Conclusions In a large community‐based cohort, for the first time, we described the distribution of circulating neprilysin in the general community. We observed that neprilysin does not correlate with natriuretic peptide levels and is not independently associated with adverse outcomes. The novel associations observed between low soluble neprilysin levels and an adverse cardiometabolic and smoking profile requires further investigation.

ObjectiveObesity is a risk factor for colorectal cancer (CRC), accounting for more than 14% of CRC incidence. Microbial dysbiosis and chronic inflammation are common characteristics in both obesity and CRC. Human and murine studies, together, demonstrate the significant impact of the microbiome in governing energy metabolism and CRC development; yet, little is understood about the contribution of the microbiome to development of obesity-associated CRC as compared to individuals who are not obese.DesignIn this study, we conducted a meta-analysis using five publicly available stool and tissue-based 16S rRNA and whole genome sequencing (WGS) data sets of CRC microbiome studies. High-resolution analysis was employed for 16S rRNA data, which allowed us to achieve species-level information to compare with WGS. ResultsCharacterisation of the confounders between studies, 16S rRNA variable region and sequencing method did not reveal any significant effect on alpha diversity in CRC prediction. Both 16S rRNA and WGS were equally variable in their ability to predict CRC. Results from diversity analysis confirmed lower diversity in obese individuals without CRC; however, no universal differences were found in diversity between obese and non-obese individuals with CRC. When examining taxonomic differences, the probability of being classified as CRC did not change significantly in obese individuals for all taxa tested. However, random forest classification was able to distinguish CRC and non-CRC stool when body mass index was added to the model.ConclusionOverall, microbial dysbiosis was not a significant factor in explaining the higher risk of colon cancer among individuals with obesity.

Paul Y Takahashi,1 Gregory D Jenkins,2 Benjamin P Welkie,2 Shannon K McDonnell,2 Jared M Evans,2 James R Cerhan,2 Janet E Olson,2 Stephen N Thibodeau,3 Mine S Cicek,3 Euijung Ryu2 1Division of Primary Care Internal Medicine, 2Department of Health Sciences Research, 3Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Purpose: In aging adults, mitochondrial dysfunction may be an important contributor. We evaluated the association between mitochondrial DNA (mtDNA) copy number, which is a biomarker for mitochondrial function, and self-rated health status.Patients and methods: We conducted a cross-sectional study of patients enrolled within the Mayo Clinic Biobank. We utilized the questionnaire and sequence data from 944 patients. We examined the association between mtDNA copy number and self-rated health status with 3 collapsed categories for the latter variable (excellent/very good, good, and fair/poor). For analysis, we used proportional odds models after log-transforming mtDNA copy number, and we adjusted for age and sex. Results: We found the median age at enrollment was 61 years (25th–75th percentile: 51–71), and 64% reported excellent or very good health, 31% reported good health, and 6% reported fair/poor health. Overall, the median mtDNA copy number was 88.9 (25th–75th percentile: 77.6–101.1). Higher mtDNA copy number was found for subjects reporting better self-rated health status after adjusting for age, sex, and comorbidity burden (OR =2.3 [95% CI: 1.2–4.5] for having better self-rated health for a one-unit increase in log-transformed mtDNA copy number). Conclusion: We found that a higher mtDNA copy number is associated with better self-rated health status after adjustment for age, sex, and comorbidity burden. The current study implies that mtDNA copy number may serve as a biomarker for self-reported health. Further studies, potentially including cohort studies, may be required. Keywords: mitochondrial DNA copy number, self-rated health, personalized medicine

There are currently no reliable approaches for correctly identifying which patients with major depressive disorder (MDD) will respond well to antidepressant therapy. However, recent genetic advances suggest that Polygenic Risk Scores (PRS) could allow MDD patients to be stratified for antidepressant response. We used PRS for MDD and PRS for neuroticism as putative predictors of antidepressant response within three treatment cohorts: The Genome-based Therapeutic Drugs for Depression (GENDEP) cohort, and 2 sub-cohorts from the Pharmacogenomics Research Network Antidepressant Medication Pharmacogenomics Study PRGN-AMPS (total patient number = 760). Results across cohorts were combined via meta-analysis within a random effects model. Overall, PRS for MDD and neuroticism did not significantly predict antidepressant response but there was a consistent direction of effect, whereby greater genetic loading for both MDD (best MDD result, p < 5*10–5 MDD-PRS at 4 weeks, β = -0.019, S.E = 0.008, p = 0.01) and neuroticism (best neuroticism result, p < 0.1 neuroticism-PRS at 8 weeks, β = -0.017, S.E = 0.008, p = 0.03) were associated with less favourable response. We conclude that the PRS approach may offer some promise for treatment stratification in MDD and should now be assessed within larger clinical cohorts.

Intestinal barrier function and microbiota are integrally related and play critical roles in maintenance of host physiology. Sex is a key biologic variable for several disorders. Our aim was to determine sex-based differences in response to perturbation and subsequent recovery of intestinal barrier function and microbiota in healthy humans. Twenty-three volunteers underwent duodenal biopsies, mucosal impedance, and in vivo permeability measurement. Permeability testing was repeated after administration of indomethacin, then 4 to 6 wk after its discontinuation. Duodenal and fecal microbiota composition was determined using 16S rRNA amplicon sequencing. Healthy women had lower intestinal permeability and higher duodenal and fecal microbial diversity than healthy men. Intestinal permeability increases after indomethacin administration in both sexes. However, only women demonstrated decreased fecal microbial diversity, including an increase in Prevotella abundance, after indomethacin administration. Duodenal microbiota composition did not show sex-specific changes. The increase in permeability and microbiota changes normalized after discontinuation of indomethacin. In summary, women have lower intestinal permeability and higher microbial diversity. Intestinal permeability is sensitive to perturbation but recovers to baseline. Gut microbiota in women is sensitive to perturbation but appears to be more stable in men. Sex-based differences in intestinal barrier function and microbiome should be considered in future studies.-Edogawa, S., Peters, S. A., Jenkins, G. D., Gurunathan, S. V., Sundt, W. J., Johnson, S., Lennon, R. J., Dyer, R. B., Camilleri, M., Kashyap, P. C., Farrugia, G., Chen, J., Singh, R. J., Grover, M. Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

Background: Loss-of-function mutations in GRN cause frontotemporal lobar degeneration (FTLD). Patients with GRN mutations present with a uniform subtype of TAR DNA-binding protein 43 (TDP-43) pathology at autopsy (FTLD-TDP type A); however, age at onset and clinical presentation are variable, even within families. We aimed to identify potential genetic modifiers of disease onset and disease risk in GRN mutation carriers. Methods: The study was done in three stages: a discovery stage, a replication stage, and a meta-analysis of the discovery and replication data. In the discovery stage, genome-wide logistic and linear regression analyses were done to test the association of genetic variants with disease risk (case or control status) and age at onset in patients with a GRN mutation and controls free of neurodegenerative disorders. Suggestive loci (p−5) were genotyped in a replication cohort of patients and controls, followed by a meta-analysis. The effect of genome-wide significant variants at the GFRA2 locus on expression of GFRA2 was assessed using mRNA expression studies in cerebellar tissue samples from the Mayo Clinic brain bank. The effect of the GFRA2 locus on progranulin concentrations was studied using previously generated ELISA-based expression data. Co-immunoprecipitation experiments in HEK293T cells were done to test for a direct interaction between GFRA2 and progranulin. Findings: Individuals were enrolled in the current study between Sept 16, 2014, and Oct 5, 2017. After quality control measures, statistical analyses in the discovery stage included 382 unrelated symptomatic GRN mutation carriers and 1146 controls free of neurodegenerative disorders collected from 34 research centres located in the USA, Canada, Australia, and Europe. In the replication stage, 210 patients (67 symptomatic GRN mutation carriers and 143 patients with FTLD without GRN mutations pathologically confirmed as FTLD-TDP type A) and 1798 controls free of neurodegenerative diseases were recruited from 26 sites, 20 of which overlapped with the discovery stage. No genome-wide significant association with age at onset was identified in the discovery or replication stages, or in the meta-analysis. However, in the case-control analysis, we replicated the previously reported TMEM106B association (rs1990622 meta-analysis odds ratio [OR] 0·54, 95% CI 0·46–0·63; p=3·54 × 10−16), and identified a novel genome-wide significant locus at GFRA2 on chromosome 8p21.3 associated with disease risk (rs36196656 meta-analysis OR 1·49, 95% CI 1·30–1·71; p=1·58 × 10−8). Expression analyses showed that the risk-associated allele at rs36196656 decreased GFRA2 mRNA concentrations in cerebellar tissue (p=0·04). No effect of rs36196656 on plasma and CSF progranulin concentrations was detected by ELISA; however, co-immunoprecipitation experiments in HEK293T cells did suggest a direct binding of progranulin and GFRA2. Interpretation: TMEM106B-related and GFRA2-related pathways might be future targets for treatments for FTLD, but the biological interaction between progranulin and these potential disease modifiers requires further study. TMEM106B and GFRA2 might also provide opportunities to select and stratify patients for future clinical trials and, when more is known about their potential effects, to inform genetic counselling, especially for asymptomatic individuals. Funding: National Institute on Aging, National Institute of Neurological Disorders and Stroke, Canadian Institutes of Health Research, Italian Ministry of Health, UK National Institute for Health Research, National Health and Medical Research Council of Australia, and the French National Research Agency.

BACKGROUND Critically ill patients frequently receive plasma transfusion under the assumptions that abnormal coagulation test results confer increased risk of bleeding and that plasma transfusion will decrease this risk. However, the effect of prophylactic plasma transfusion remains poorly understood. The objective of this study was to determine the relationship between prophylactic plasma transfusion and bleeding complications in critically ill patients. METHODS This is a retrospective cohort study of adults admitted to the intensive care unit (ICU) at a single academic institution between January 1, 2009 and December 31, 2013. Inclusion criteria included age ≥18 years and an international normalized ratio measured during ICU admission. Multivariable propensity-matched analyses were used to evaluate associations between prophylactic plasma transfusion and outcomes of interest with a primary outcome of red blood cell transfusion in the ensuing 24 hours and secondary outcomes of hospital- and ICU-free days and mortality within 30 days of ICU discharge. RESULTS A total of 27,561 patients were included in the investigation with 2472 (9.0%) receiving plasma therapy and 1105 (44.7%) for which plasma transfusion was prophylactic in nature. In multivariable propensity-matched analyses, patients receiving plasma had higher rates of red blood cell transfusion (odds ratio: 4.3 [95% confidence interval: 3.3-5.7], P < .001) and fewer hospital-free days (estimated % increase: -11.0% [95% confidence interval: -11.4, -10.6%], P < .001). There were no significant differences in ICU-free days or mortality. These findings appeared robust, persisting in multiple predefined sensitivity analyses. CONCLUSIONS Prophylactic administration of plasma in the critically ill was not associated with improved clinical outcomes. Further investigation examining the utility of plasma transfusion in this population is warranted.

Paul Y Takahashi,1 Euijung Ryu,2 Jyotishman Pathak,2 Gregory D Jenkins,2 Anthony Batzler,2 Matthew A Hathcock,2 John Logan Black,3 Janet E Olson,2 James R Cerhan,2 Suzette J Bielinski2 1Division of Primary Care Internal Medicine, Department of Medicine, 2Department of Health Sciences Research, 3Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Background: Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism of clinically used drugs and other environmental exposures, but it is unclear whether the CYP2D6 phenotype is associated with adverse health outcomes. The aim was to determine the association of CYP2D6 phenotype with the risk of hospitalization or an emergency department (ED) visit among a group of primary care patients. Methods: In this study, 929 adult patients underwent CYP2D6 testing. The primary outcome was risk of hospitalization or an ED visit from January 2005 through September 2014. CYP2D6 genotypes were interpreted as 1 of 7 clinical phenotypes, from ultrarapid to poor metabolizer, and patients with the extensive metabolizer phenotype were used as the reference group. The hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated for finding the association of CYP2D6 phenotypes with the risk of hospitalization or an ED visit by using Cox proportional hazard models and adjusting for age and sex. Results: The median age was 49 years (interquartile range, 46–52 years); 74% of patients had 3 or fewer chronic conditions, 285 had at least 1 hospitalization, and 496 had at least 1 ED visit. The risk of hospitalization was higher among patients who were ultrarapid metabolizers compared to extensive metabolizers (47% vs 30%; HR, 1.69; 95% CI, 1.11–2.57), as was the risk of an ED visit (62% vs 49%; HR, 1.50; 95% CI, 1.05–2.14). For poor metabolizers compared to extensive metabolizers, there was no difference in the risk of hospitalization (HR, 0.95; 95% CI, 0.58–1.56), but there was an increase in the risk of an ED visit (HR, 1.38; 95% CI, 0.96–1.98) (the difference was not statistically significant). Conclusion: We found an increased risk of hospitalization or an ED visit among ultrarapid compared to extensive CYP2D6 metabolizers. Further research identifying the mechanisms of the association and ultimate clinical utility is warranted. Keywords: cohort study, pharmacogenomics, emergency department, hospitalization

Metformin is currently considered as a promising anticancer agent in addition to its anti-diabetic effect. To better individualize metformin therapy and explore novel molecular mechanisms in cancer treatment, we conducted a pharmacogenomic study using 266 lymphoblastoid cell lines (LCLs). Metformin cytotoxicity assay was performed using the MTS assay. Genome-wide association (GWA) analyses were performed in LCLs using 1.3 million SNPs, 485k DNA methylation probes, 54k mRNA expression probe sets, and metformin cytotoxicity (IC50s). Top candidate genes were functionally validated using siRNA screening, followed by MTS assay in breast cancer cell lines. Further study of one top candidate, STUB1, was performed to elucidate the mechanisms by which STUB1 might contribute to metformin action. GWA analyses in LCLs identified 198 mRNA expression probe sets, 12 SNP loci, and 5 DNA methylation loci associated with metformin IC50 with P-values

© 2016 the American Physiological Society. Prior to the initiation of menopausal hormone treatment (MHT), genetic variations in the innate immunity pathway were found to be associated with carotid artery intima-medial thickness (CIMT) and coronary arterial calcification (CAC) in women (n = 606) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS). Whether MHT might affect these associations is unknown. The association of treatment outcomes with variation in the same 764 candidate genes was evaluated in the same KEEPS participants 4 yr after randomization to either oral conjugated equine estrogens (0.45 mg/day), transdermal 17β-estradiol (50 μg/day), each with progesterone (200 mg/day) for 12 days each month, or placebo pills and patch. Twenty SNPs within the innate immunity pathway most related with CIMT after 4 yr were not among those associated with CIMT prior to MHT. In 403 women who completed the study in their assigned treatment group, single nucleotide polymorphisms (SNPs) within the innate immunity pathway were found to alter the treatment effect on 4 yr change in CIMT (i.e., significant interaction between treatment and genetic variation in the innate immunity pathway; P < 0.001). No SNPs by treatment effects were observed with changes of CAC >5 Agatston units after 4 yr. Results of this study suggest that hormonal status may interact with genetic variants to influence cardiovascular phenotypes, specifically, the pharmacogenomic effects within the innate immunity pathway for CIMT.

Background Triptolide is a therapeutic diterpenoid derived from the Chinese herb Tripterygium wilfordii Hook f. Triptolide has been shown to induce apoptosis by activation of pro-apoptotic proteins, inhibiting NFkB and c-KIT pathways, suppressing the Jak2 transcription, activating MAPK8/JNK signaling and modulating the heat shock responses. Results In the present study, we used lymphoblast cell lines (LCLs) derived from 55 unrelated Caucasian subjects to identify genetic markers predictive of cellular sensitivity to triptolide using genome wide association study. Our results identified SNPs on chromosome 2 associated with triptolide IC50 (p

The mammalian target of rapamycin (mTOR) inhibitors, a set of promising potential anti-cancer agents, has shown response variability among individuals. This study aimed to identify novel biomarkers and mechanisms that might influence the response to Rapamycin and Everolimus. Genome-wide association (GWA) analyses involving single nucleotide polymorphisms (SNPs), mRNA, and microRNAs microarray data were assessed for association with area under the cytotoxicity dose response curve (AUC) of two mTOR inhibitors in 272 human lymphoblastoid cell lines (LCLs). Integrated analysis among SNPs, expression data, microRNA data and AUC values were also performed to help select candidate genes for further functional characterization. Functional validation of candidate genes using siRNA screening in multiple cell lines followed by MTS assays for the two mTOR inhibitors were performed. We found that 16 expression probe sets (genes) that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors. One hundred and twenty seven and one hundred SNPs had P < 10−4, while 8 and 10 SNPs had P < 10−5 with Rapamycin and Everolimus AUC, respectively. Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line. Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs. In summary, this study identified genes and a microRNA that might contribute to response to mTOR inhibitors.

Purpose FKBP51, (FKBP5), is a negative regulator of Akt. Variability in FKBP5 expression level is a major factor contributing to variation in response to chemotherapeutic agents including gemcitabine, a first line treatment for pancreatic cancer. Genetic variation in FKBP5 could influence its function and, ultimately, treatment response of pancreatic cancer. Experimental Design We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing on response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired normal-tumor samples. Next, genotype-phenotype association studies were performed with overall survival as well as with FKBP5 gene expression in tumor using the same samples in which resequencing had been performed, followed by functional genomics studies. Results In-depth resequencing identified 404 FKBP5 single nucleotide polymorphisms (SNPs) in normal and tumor DNA. SNPs with the strongest associations with survival or FKBP5 expression were subjected to functional genomic study. Electromobility shift assay showed that the rs73748206 “A(T)” SNP altered DNA-protein binding patterns, consistent with significantly increased reporter gene activity, possibly through its increased binding to Glucocorticoid Receptor (GR). The effect of rs73748206 was confirmed on the basis of its association with FKBP5 expression by affecting the binding to GR in lymphoblastoid cell lines derived from the same patients for whom DNA was used for resequencing. Conclusion This comprehensive FKBP5 resequencing study provides insights into the role of genetic variation in variation of gemcitabine response.

The selective estrogen receptor modulators (SERM) tamoxifen and raloxifene can reduce the occurrence of breast cancer in high-risk women by 50%, but this U.S. Food and Drug Administration-approved prevention therapy is not often used. We attempted to identify genetic factors that contribute to variation in SERM breast cancer prevention, using DNA from the NSABP P-1 and P-2 breast cancer prevention trials. An initial discovery genome-wide association study identified common single-nucleotide polymorphisms (SNP) in or near the ZNF423 and CTSO genes that were associated with breast cancer risk during SERM therapy. We then showed that both ZNF423 and CTSO participated in the estrogen-dependent induction of BRCA1 expression, in both cases with SNP-dependent variation in induction. ZNF423 appeared to be an estrogen-inducible BRCA1 transcription factor. The OR for differences in breast cancer risk during SERM therapy for subjects homozygous for both protective or both risk alleles for ZNF423 and CTSO was 5.71. Significance: This study identified novel, functionally polymorphic genes involved in the estrogen-dependent regulation of BRCA1 expression, as well as a novel mechanism for genetic variation in SERM therapeutic effect. These observations, and definition of their underlying mechanisms, represent steps toward pharmacogenomically individualized SERM breast cancer prevention. Cancer Discov; 3(7); 812–25. ©2013 AACR. See related commentary by Machiela and Chanock, p. 728 This article is highlighted in the In This Issue feature, p. 705

We performed a discovery genome-wide association study to identify genetic factors associated with variation in plasma estradiol (E2) concentrations using DNA from 772 postmenopausal women with estrogen receptor (ER)-positive breast cancer prior to the initiation of aromatase inhibitor therapy. Association analyses showed that the single nucleotide polymorphisms (SNP) (rs1864729) with the lowest P value (P = 3.49E-08), mapped to chromosome 8 near TSPYL5. We also identified 17 imputed SNPs in or near TSPYL5 with P values < 5E-08, one of which, rs2583506, created a functional estrogen response element. We then used a panel of lymphoblastoid cell lines (LCLs) stably transfected with ERα with known genome-wide SNP genotypes to demonstrate that TSPYL5 expression increased after E2 exposure of cells heterozygous for variant TSPYL5 SNP genotypes, but not in those homozygous for wild-type alleles. TSPYL5 knockdown decreased, and overexpression increased aromatase (CYP19A1) expression in MCF-7 cells, LCLs, and adipocytes through the skin/adipose (I.4) promoter. Chromatin immunoprecipitation assay showed that TSPYL5 bound to the CYP19A1 I.4 promoter. A putative TSPYL5 binding motif was identified in 43 genes, and TSPYL5 appeared to function as a transcription factor for most of those genes. In summary, genome-wide significant SNPs in TSPYL5 were associated with elevated plasma E2 in postmenopausal breast cancer patients. SNP rs2583506 created a functional estrogen response element, and LCLs with variant SNP genotypes displayed increased E2-dependent TSPYL5 expression. TSPYL5 induced CYP19A1 expression and that of many other genes. These studies have revealed a novel mechanism for regulating aromatase expression and plasma E2 concentrations in postmenopausal women with ER(+) breast cancer.

It is believed that multiple genetic variants with small individual effects contribute to the risk of alcohol dependence. Such polygenic effects are difficult to detect in genome-wide association studies that test for association of the phenotype with each single nucleotide polymorphism (SNP) individually. To overcome this challenge, gene-set analysis (GSA) methods that jointly test for the effects of pre-defined groups of genes have been proposed. Rather than testing for association between the phenotype and individual SNPs, these analyses evaluate the global evidence of association with a set of related genes enabling the identification of cellular or molecular pathways or biological processes that play a role in development of the disease. It is hoped that by aggregating the evidence of association for all available SNPs in a group of related genes, these approaches will have enhanced power to detect genetic associations with complex traits. We performed GSA using data from a genome-wide study of 1165 alcohol-dependent cases and 1379 controls from the Study of Addiction: Genetics and Environment (SAGE), for all 200 pathways listed in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results demonstrated a potential role of the ‘synthesis and degradation of ketone bodies’ pathway. Our results also support the potential involvement of the ‘neuroactive ligand–receptor interaction’ pathway, which has previously been implicated in addictive disorders. These findings demonstrate the utility of GSA in the study of complex disease, and suggest specific directions for further research into the genetic architecture of alcohol dependence.

Background/Aims: Tests for whether observed genotype proportions fit Hardy Weinberg Equilibrium (HWE) are widely used in population genetics analyses, as well as to evaluate quality of genotype data. To date, all methods testing for HWE require subjects to be classified into discrete categories, yet it is becoming clear that the distribution of allele frequencies tends to be smooth over geographic regions. Methods: To evaluate the HWE assumption, we develop new approaches to model allele frequencies as functions of covariates, and use these models to test whether there is residual correlation between the two alleles of subjects; lack of residual correlation supports the null hypothesis of HWE, but conditional on how the covariates influence the allele frequencies. Results: By simulations, we illustrate that a simple statistical test of residual correlation of alleles adequately controls the type I error rate, while maintaining power that is comparable to standard tests for HWE. Conclusion: Our approach can be implemented in standard software, enabling more flexible and powerful ways to evaluate the association of covariates with allele frequencies and whether these associations ‘explain' departures from HWE when the covariates are ignored, opening new strategies to evaluate the quality of genotype data generated by next-generation sequencing assays.

Mycophenolic acid (MPA) is commonly used to treat patients with solid organ transplants during maintenance immunosuppressive therapy. Response to MPA varies widely, both for efficacy and drug-induced toxicity. A portion of this variation can be explained by pharmacokinetic and pharmacodynamic factors, including genetic variation in MPA-metabolizing UDP-glucuronyltransferase isoforms and the MPA targets, inosine monophosphate dehydrogenase 1 and 2. However, much of the variation in MPA response presently remains unexplained. We set out to determine whether there might be additional genes that modify response to MPA by performing a genome-wide association study between basal gene mRNA expression profiles and an MPA cytotoxicity phenotype using a 271 human lymphoblastoid cell line model system to identify and functionally validate genes that might contribute to variation in MPA response. Our association study identified 41 gene expression probe sets, corresponding to 35 genes, that were associated with MPA cytotoxicity as a drug response phenotype (p < 1 × 10−6). Follow-up siRNA-mediated knockdown-based functional validation identified four of these candidate genes, C17orf108, CYBRD1, NASP, and RRM2, whose knockdown shifted the MPA cytotoxicity curves in the direction predicted by the association analysis. These studies have identified novel candidate genes that may contribute to variation in response to MPA therapy and, as a result, may help make it possible to move toward more highly individualized MPA-based immunosuppressive therapy.

Radiation therapy is used to treat half of all cancer patients. Response to radiation therapy varies widely among patients. Therefore, we performed a genome-wide association study (GWAS) to identify biomarkers to help predict radiation response using 277 ethnically defined human lymphoblastoid cell lines (LCLs). Basal gene expression levels and 1.3 million genome-wide single nucleotide polymorphism (SNP) markers from both Affymetrix and Illumina platforms were assayed for all 277 human LCLs. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assays for radiation cytotoxicity were also performed to obtain area under the curve (AUC) as a radiation response phenotype for use in the association studies. Functional validation of candidate genes, selected from an integrated analysis that used SNP, expression, and AUC data, was performed with multiple cancer cell lines using specific siRNA knockdown, followed by MTS and colony-forming assays. A total of 27 loci, each containing at least two SNPs within 50 kb with P-values less than 10−4 were associated with radiation AUC. A total of 270 expression probe sets were associated with radiation AUC with P < 10−3. The integrated analysis identified 50 SNPs in 14 of the 27 loci that were associated with both AUC and the expression of 39 genes, which were also associated with radiation AUC (P < 10−3). Functional validation using siRNA knockdown in multiple tumor cell lines showed that C13orf34, MAD2L1, PLK4, TPD52, and DEPDC1B each significantly altered radiation sensitivity in at least two cancer cell lines. Studies performed with LCLs can help to identify novel biomarkers that might contribute to variation in response to radiation therapy and enhance our understanding of mechanisms underlying that variation.

Gene set methods aim to assess the overall evidence of association of a set of genes with a phenotype, such as disease or a quantitative trait. Multiple approaches for gene set analysis of expression data have been proposed. They can be divided into two types: competitive and self-contained. Benefits of self-contained methods include that they can be used for genome-wide, candidate gene, or pathway studies, and have been reported to be more powerful than competitive methods. We therefore investigated ten self-contained methods that can be used for continuous, discrete and time-to-event phenotypes. To assess the power and type I error rate for the various previously proposed and novel approaches, an extensive simulation study was completed in which the scenarios varied according to: number of genes in a gene set, number of genes associated with the phenotype, effect sizes, correlation between expression of genes within a gene set, and the sample size. In addition to the simulated data, the various methods were applied to a pharmacogenomic study of the drug gemcitabine. Simulation results demonstrated that overall Fisher's method and the global model with random effects have the highest power for a wide range of scenarios, while the analysis based on the first principal component and Kolmogorov-Smirnov test tended to have lowest power. The methods investigated here are likely to play an important role in identifying pathways that contribute to complex traits.

Background In recent years, capabilities for genotyping large sets of single nucleotide polymorphisms (SNPs) has increased considerably with the ability to genotype over 1 million SNP markers across the genome. This advancement in technology has led to an increase in the number of genome-wide association studies (GWAS) for various complex traits. These GWAS have resulted in the implication of over 1500 SNPs associated with disease traits. However, the SNPs identified from these GWAS are not necessarily the functional variants. Therefore, the next phase in GWAS will involve the refining of these putative loci. Methodology A next step for GWAS would be to catalog all variants, especially rarer variants, within the detected loci, followed by the association analysis of the detected variants with the disease trait. However, sequencing a locus in a large number of subjects is still relatively expensive. A more cost effective approach would be to sequence a portion of the individuals, followed by the application of genotype imputation methods for imputing markers in the remaining individuals. A potentially attractive alternative option would be to impute based on the 1000 Genomes Project; however, this has the drawbacks of using a reference population that does not necessarily match the disease status and LD pattern of the study population. We explored a variety of approaches for carrying out the imputation using a reference panel consisting of sequence data for a fraction of the study participants using data from both a candidate gene sequencing study and the 1000 Genomes Project. Conclusions Imputation of genetic variation based on a proportion of sequenced samples is feasible. Our results indicate the following sequencing study design guidelines which take advantage of the recent advances in genotype imputation methodology: Select the largest and most diverse reference panel for sequencing and genotype as many “anchor” markers as possible.

Aromatase (CYP19) is a critical enzyme in estrogen biosynthesis and aromatase inhibitors (AI) are employed widely for endocrine therapy in postmenopausal women with breast cancer. We hypothesized that single nucleotide polymorphisms (SNPs) in the CYP19 gene may alter the effectiveness of AI therapy in the neoadjuvant setting. Genomic DNA was obtained for sequencing from 52 women pre-AI and post-AI treatment in this setting. Additionally, genomic DNA obtained from 82 samples of breast cancer and 19 samples of normal breast tissue was subjected to resequencing. No differences in CYP19 sequence were observed between tumor and germ-line DNA in the same patient. A total of 48 SNPs were identified including 4 novel SNPs when compared with previous resequencing data. For genotype-phenotype association studies, we determined the levels of aromatase activity, estrone, estradiol, and tumor size in patients pre-AI and post-AI treatment. We defined two tightly linked SNPs (rs6493497 and rs7176005 in the 5'-flanking region of CYP19 exon 1.1) that were significantly associated with a greater change in aromatase activity after AI treatment. In a follow-up study of 200 women with early-stage breast cancer who were treated with adjuvant anastrozole, these same two SNPs were also associated with higher plasma estradiol levels in patients pre-AI and post-AI treatment. Electrophoretic mobility shift and reporter gene assays confirmed likely functional effects of these two SNPs on transcription of CYP19. Our findings indicate that two common genetic polymorphisms in the aromatase gene CYP19 vary the response of breast cancer patients to aromatase inhibitors. Cancer Res; 70(1); 319–28

Cancer patients show large individual variation in their response to chemotherapeutic agents. Gemcitabine (dFdC) and AraC, two cytidine analogues, have shown significant activity against a variety of tumors. We previously used expression data from a lymphoblastoid cell line-based model system to identify genes that might be important for the two drug cytotoxicity. In the present study, we used that same model system to perform a genome-wide association (GWA) study to test the hypothesis that common genetic variation might influence both gene expression and response to the two drugs. Specifically, genome-wide single nucleotide polymorphisms (SNPs) and mRNA expression data were obtained using the Illumina 550K(R) HumanHap550 SNP Chip and Affymetrix U133 Plus 2.0 GeneChip, respectively, for 174 ethnically-defined "Human Variation Panel" lymphoblastoid cell lines. Gemcitabine and AraC cytotoxicity assays were performed to obtain IC(50) values for the cell lines. We then performed GWA studies with SNPs, gene expression and IC(50) of these two drugs. This approach identified SNPs that were associated with gemcitabine or AraC IC(50) values and with the expression regulation for 29 genes or 30 genes, respectively. One SNP in IQGAP2 (rs3797418) was significantly associated with variation in both the expression of multiple genes and gemcitabine and AraC IC(50). A second SNP in TGM3 (rs6082527) was also significantly associated with multiple gene expression and gemcitabine IC50. To confirm the association results, we performed siRNA knock down of selected genes with expression that was associated with rs3797418 and rs6082527 in tumor cell and the knock down altered gemcitabine or AraC sensitivity, confirming our association study results. These results suggest that the application of GWA approaches using cell-based model systems, when combined with complementary functional validation, can provide insights into mechanisms responsible for variation in cytidine analogue response.

Catechol O-methyltransferase (COMT) is expressed as both soluble (S) and membrane-bound (MB) isoforms, with S-COMT predominantly expressed in the liver. A common nonsynonymous single nucleotide polymorphism (SNP), 472GA (108/158ValMet, S/MB), has been associated with variation in levels of COMT enzyme activity and thermal stability. We set out to test the hypothesis that additional COMT polymorphisms might also be associated with phenotypic variation.We phenotyped 268 liver biopsy samples for S-COMT activity and thermal stability, resequenced a portion of the gene that had not been resequenced earlier, and genotyped DNA from these same samples for 16 COMT polymorphisms.There was a significant association between the two COMT phenotypes and genotype at the codon 108 SNP. A haplotype-based approach was then used to assess the possible association of other polymorphisms with phenotype. Specifically, the codon 108 SNP explained 20.4% of variance in enzyme activity (P10), and 59% of variance in thermal stability (P10). Haplotypes that included SNPs at cDNA nucleotides 408 and 472 explained additional variance in enzyme activity (up to 24.4%), and the addition to the haplotype of a SNP at intron 2 (51) explained a total of 27.5% of the variance. However, no SNPs beyond that at the nucleotide 472GA polymorphism were associated with variation in thermal stability. We also observed a three-fold variation in the ability of reporter gene constructs for 'proximal promoter' haplotypes to drive transcription.The common COMT 108ValMet polymorphism is associated with human liver S-COMT activity and thermal stability, but additional COMT SNPs also contribute to variation in activity.

Background: Taxane is one of the first line treatments of lung cancer. In order to identify novel single nucleotide polymorphisms (SNPs) that might contribute to taxane response, we performed a genome-wide association study (GWAS) for two taxanes, paclitaxel and docetaxel, using 276 lymphoblastoid cell lines (LCLs), followed by genotyping of top candidate SNPs in 874 lung cancer patient samples treated with paclitaxel. Methods: GWAS was performed using 1.3 million SNPs and taxane cytotoxicity IC50 values for 276 LCLs. The association of selected SNPs with overall survival in 76 small or 798 non-small cell lung cancer (SCLC, NSCLC) patients were analyzed by Cox regression model, followed by integrated SNP-microRNA-expression association analysis in LCLs and siRNA screening of candidate genes in SCLC (H196) and NSCLC (A549) cell lines. Results: 147 and 180 SNPs were associated with paclitaxel or docetaxel IC50s with p-values, National Institutes of Health (U.S.) ( NIH grant K22 CA130828), National Institutes of Health (U.S.) (NIH grant R01 CA138461), National Institutes of Health (U.S.) (NIH grant U19 GM61388), National Institutes of Health (U.S.) (Pharmacogenomics Research Network (R01 CA80127), National Institutes of Health (U.S.) (Pharmacogenomics Research Network (R01 CA84354)), National Institutes of Health (U.S.) (Pharmacogenomics Research Network (R01 CA105857))

SummaryAkt is a central regulator of cell growth. Its activity can be negatively regulated by the phosphatase PHLPP that specifically dephosphorylates the hydrophobic motif of Akt (Ser473 in Akt1). However, how PHLPP is targeted to Akt is not clear. Here we show that FKBP51 (FK506-binding protein 51) acts as a scaffolding protein for Akt and PHLPP and promotes dephosphorylation of Akt. Furthermore, FKBP51 is downregulated in pancreatic cancer tissue samples and several cancer cell lines. Decreased FKBP51 expression in cancer cells results in hyperphosphorylation of Akt and decreased cell death following genotoxic stress. Overall, our findings identify FKBP51 as a negative regulator of the Akt pathway, with potentially important implications for cancer etiology and response to chemotherapy.

Objective: The objectives of this study were the comparison of patients who needed mesh closure of the abdomen with patients who underwent standard abdominal closure after ruptured abdominal aortic aneurysm repair and the determination of the impact of timing of mesh closure on multiple organ failure (MOF) and mortality. Methods: We performed a case-control study of patients who needed mesh-based abdominal closure (n = 45) as compared with patients who underwent primary closure (n = 90) after ruptured abdominal aortic aneurysm repair. Results: Before surgery, the patients who needed mesh abdominal closure had more blood loss (8 g versus 12 g of hemoglobin; P

Background Two cytidine analogues, gemcitabine and cytosine arabinoside (AraC), are widely used in the treatment of a variety of cancers with a large individual variation in response. To identify potential genetic biomarkers associated with response to these two drugs, we used a human lymphoblastoid cell line (LCL) model system with extensive genomic data, including 1.3 million SNPs and 54,000 basal expression probesets to perform genome-wide association studies (GWAS) with gemcitabine and AraC IC50 values. Results We identified 11 and 27 SNP loci significantly associated with gemcitabine and AraC IC50 values, respectively. Eleven candidate genes were functionally validated using siRNA knockdown approach in multiple cancer cell lines. We also characterized the potential mechanisms of genes by determining their influence on the activity of 10 cancer-related signaling pathways using reporter gene assays. Most SNPs regulated gene expression in a trans manner, except 7 SNPs in the PIGB gene that were significantly associated with both the expression of PIGB and gemcitabine cytotoxicity. Conclusion These results suggest that genetic variation might contribute to drug response via either cis- or trans- regulation of gene expression. GWAS analysis followed by functional pharmacogenomics studies might help identify novel biomarkers contributing to variation in response to these two drugs and enhance our understanding of underlying mechanisms of drug action.