Genome-wide Association Approach Research Articles

Genes of the Unfolded Protein Response Pathway Harbor Risk Alleles for Primary Open Angle Glaucoma

The statistical power of genome-wide association (GWA) studies to detect risk alleles for human diseases is limited by the unfavorable ratio of SNPs to study subjects. This multiple testing problem can be surmounted with very large population sizes when common alleles of large effects give rise to disease status. However, GWA approaches fall short when many rare alleles may give rise to a common disease, or when the number of subjects that can be recruited is limited. Here, we demonstrate that this multiple testing problem can be overcome by a comparative genomics approach in which an initial genome-wide screen in a genetically amenable model organism is used to identify human orthologues that may harbor risk alleles for adult-onset primary open angle glaucoma (POAG). Glaucoma is a major cause of blindness, which affects over 60 million people worldwide. Several genes have been associated with juvenile onset glaucoma, but genetic factors that predispose to adult onset primary open angle glaucoma (POAG) remain largely unknown. Previous genome-wide analysis in a Drosophila ocular hypertension model identified transcripts with altered regulation and showed induction of the unfolded protein response (UPR) upon overexpression of transgenic human glaucoma-associated myocilin (MYOC). We selected 16 orthologous genes with 62 polymorphic markers and identified in two independent human populations two genes of the UPR that harbor POAG risk alleles, BIRC6 and PDIA5. Thus, effectiveness of the UPR in response to accumulation of misfolded or aggregated proteins may contribute to the pathogenesis of POAG and provide targets for early therapeutic intervention.

At the End of the Beginning

At the End of the Beginning

Abstract 2660: Downregulation of Bora gene expression in human tumor cells results in radioresistance by promoting repair of double strand breaks

Abstract DNA double-strand breaks (DSBs) are highly cytotoxic lesions caused by genotoxin such as ionizing radiation (IR). Following DSB, cells activate the DNA-damage response pathway by activating protein kinases such as ATM, ATR and DNA-PK, which then trigger histone H2AX phosphorylation and the accumulation of proteins such as Mediator of DNA damage checkpoint protein 1 (MDC1), 53BP1, and BRCA1 into IR-induced foci. This process initiates the repair of DSB. Previously we have identified a novel biomarker for radiation response, Bora, by using a genome-wide association approach using data generated with 300 human lymphoblastoid cell liens. Bora is known as a cofactor of Aurora-A, which is involved in mitosis regulation. During G2, Aurora-A, in complex with Bora, phosphorylates Plk1 at Thr-210. Activated Plk1 phosphorylates Cdc25C and Wee1, which induces activation of cyclin B-cdk1 complexes and promotes mitotic entry. In this study, we want to determine the underlying mechanisms by which Bora regulates response to IR. We found that knockdown of Bora in human cancer cell lines including HupT3, MIAPaCa-2 and Hela (siBora) exhibit increased clonogenic survival rate in response to IR, an observation that is opposite from the effect of Plk1 knockdown, suggesting a different mechanism involved. Knockdown of Bora also desensitize cancer cells to chemotherapeutic agents including gemcitabine and oxiplatin. This enhanced resistance to genotoxin is primarily due to increased DNA DSB repair. More than 90% of DSBs were repaired in siBora cells compared with 60% in control cells 8 hours after cell exposure to 2 Gy IR. Upon IR, more cells with Bora knockdown retained in G2-M phase compared with control. Furthermore, Bora directly interacts with BRCT domain of MDC1 via its C terminus, and over expression of Bora can abolish MDC1 foci formation induced by IR. All of these observations suggested a role of Bora in DNA damage response pathway and in turn, in chemo-radio resistance. Indeed, overexpression of the full length or C-terminus of Bora sensitized cancer cells to IR treatment, while the N-terminal Bora did not have significant effect on IR response. In summary, we have identified a novel function of Bora involved in DNA damage and repair pathway, which indicates a broad role of Bora in both tumorgenesis and chemo-radio resistance. 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 2660. doi:10.1158/1538-7445.AM2011-2660

Abstract 5452: Functional genome-wide approach identifies genes associated with chemotherapeutic susceptibility in individuals of African descent

Abstract Chemotherapeutic agents are used in the treatment of many cancers, yet variable resistance and toxicities among individuals limit successful outcomes. Several studies have indicated outcome differences associated with ancestry among patients with various cancer types. We performed genome-wide association studies (GWAS) in lymphoblastoid cell lines derived from Yoruba (YRI; n = 176) and African American (ASW; n = 83) populations and identified SNP predictors of cellular susceptibility to cisplatin, carboplatin, capecitabine and ara-C. To account for population structure in African American population, we incorporated local ancestry information into our association model. We found that chemotherapeutic susceptibility SNPs in the YRI are enriched for expression quantitative trait loci (eQTLs, p<10-3). Most of these are trans eQTLs and thus simply examining the region directly surrounding a SNP that associates with a phenotype of interest for possible functional genes is inadequate. Traditional GWAS that test single markers are powered to detect SNP associations with relatively strong effects, but are unable to detect multiple smaller effects working together. Using a novel functional gene-based genome-wide association approach, we identified 10, 13, and 1 suggestive candidate genes for association with cytarabine-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10-4) in ASW. Functional GWAS in the YRI resulted in the genome-wide significant association of 234 genes with cisplatin IC50, 81 genes with 5’-deoxyfluorouridine AUC, 64 genes with carboplatin IC50 and 13 genes with cytarabine AUC (p<10-6). Our genome-wide SNP- and gene-based approaches that incorporate local ancestry information can be used to elucidate genes functionally contributing to a wide range of phenotypes in African American populations. 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 5452. doi:10.1158/1538-7445.AM2011-5452

Data Integration Workflow for Search of Disease Driving Genes and Genetic Variants

Comprehensive characterization of a gene's impact on phenotypes requires knowledge of the context of the gene. To address this issue we introduce a systematic data integration method Candidate Genes and SNPs (CANGES) that links SNP and linkage disequilibrium data to pathway- and protein-protein interaction information. It can be used as a knowledge discovery tool for the search of disease associated causative variants from genome-wide studies as well as to generate new hypotheses on synergistically functioning genes. We demonstrate the utility of CANGES by integrating pathway and protein-protein interaction data to identify putative functional variants for (i) the p53 gene and (ii) three glioblastoma multiforme (GBM) associated risk genes. For the GBM case, we further integrate the CANGES results with clinical and genome-wide data for 209 GBM patients and identify genes having effects on GBM patient survival. Our results show that selecting a focused set of genes can result in information beyond the traditional genome-wide association approaches. Taken together, holistic approach to identify possible interacting genes and SNPs with CANGES provides a means to rapidly identify networks for any set of genes and generate novel hypotheses. CANGES is available in http://csbi.ltdk.helsinki.fi/CANGES/

Genetics of cortisol secretion and depressive symptoms: A candidate gene and genome wide association approach

Depressive patients often have altered cortisol secretion, but few studies have investigated genetic variants in relation to both cortisol secretion and depression. To identify genes related to both these conditions, we: (1) tested the association of single nucleotide polymorphisms (SNPs) in hypothalamic-pituitary-adrenal-axis (HPA-axis) candidate genes with a summary measure of total cortisol secretion during the day (cortisol(AUC)), (2) performed a genome wide association study (GWAS) of cortisol(AUC), and (3) tested the association of identified cortisol-related SNPs with depressive symptoms. We analyzed data on candidate SNPs for the HPA-axis, genome-wide scans, cortisol secretion (n=1711) and depressive symptoms (the Centre for Epidemiology Studies Depression Scale, CES-D) (n=2928) in elderly persons of the Rotterdam Study. We used data from the Whitehall II study (n=2836) to replicate the GWAS findings. Of the 1456 SNPs in 33 candidate genes, minor alleles of 4 SNPs (rs9470080, rs9394309, rs7748266 and rs1360780) in the FKBP5 gene were associated with a decreased cortisol(AUC) (p<1×10(-4) after correction for multiple testing using permutations). These SNPs were also associated with an increased risk of depressive symptoms (rs9470080: OR 1.19 (95%CI 1.0; 1.4)). The GWAS for cortisol yielded 2 SNPs with p-values of 1×10(-06) (rs8062512, rs2252459), but these associations could not be replicated. These results suggest that variation in the FKBP5 gene is associated with both cortisol(AUC) and the likelihood of depressive symptoms.

Rheumatoid Arthritis: An Orchestra of Genetic, Autoimmune and Environmental Factors

Clinical presentation of rheumatoid arthritis (RA) may be the results of a combination of genetic and environmental risk factors resulting in a prominent autoimmune component. Evidence from case-control studies, either using the candidate gene or genome-wide association approaches, have revealed more than 30 loci that are associated with RA susceptibility. Many RA-associated gene variants are involved in pathways of T-cell, B-cell and NF-κB signaling. HLA-DRB1 shared epitope is a major determinant of genetic predisposition to RA development in different ethnic groups, which is involved in T-cell antigen presentation and the production of anti-cyclic citrullinated peptides antibodies (ACPA). The presence or absence of ACPA appears to stratify RA patients into two distinct subsets with different genetic profiles, clinical courses and histological findings. In addition to the shared epitope, a growing number of gene variants is associated with RA in multiple ethnic groups, including STAT4, AFF3, CCR6, CCL21, and BLK. Some of the RA-associated gene variants may be particularly important in a specific ethnic group; for example, PTPN22 in populations of European ancestry and PADI4 in Asians. Emerging evidence has shown that many disease-associated loci are shared among multiple autoimmune diseases, including type 1 diabetes, systemic lupus erythematosus, inflammatory bowel disease and multiple sclerosis, suggesting the presence of common pathways in the pathophysiology of these diseases. In addition to genetic risk factors, recent data have implicated cigarette smoking and infection of P. ginivitis as environmental risk factors that may potentiate disease risk in genetically susceptible individuals. Frequent and long-term exposure to insecticides also may increase risks for RA development. Further studies to understand functional consequences of disease-associated gene variants and gene-environment interactions that impact on the immune system are likely to lead to the development of novel therapies and/or prevention strategies for RA.

Genetics of Common Polygenic Ischaemic Stroke: Current Understanding and Future Challenges

Stroke is the third commonest cause of death and the major cause of adult neurological disability worldwide. While much is known about conventional risk factors such as hypertension, diabetes and incidence of smoking, these environmental factors only account for a proportion of stroke risk. Up to 50% of stroke risk can be attributed to genetic risk factors, although to date no single risk allele has been convincingly identified as contributing to this risk. Advances in the field of genetics, most notably genome wide association studies (GWAS), have revealed genetic risks in other cardiovascular disease and these techniques are now being applied to ischaemic stroke. This paper covers previous genetic studies in stroke including candidate gene studies, discusses the genome wide association approach, and future techniques such as next generation sequencing and the post-GWAS era. The review also considers the overlap from other cardiovascular diseases and whether findings from these may also be informative in ischaemic stroke.

Sorting Out Cholesterol and Coronary Artery Disease

A genomewide association study published in the Journal 3 years ago reported the identification of a DNA variant on chromosome 1p13 that increased the relative risk of coronary artery disease by 29% per allele.1 A few months later, the same genetic variant was also found — again through a genomewide association approach — to increase serum levels of low-density lipoprotein (LDL) cholesterol.2 At that time, little was known about the genes at the 1p13 locus, so the functional link between the DNA variant and the increase in LDL cholesterol and coronary risk was puzzling. Basic research carried out by Musunuru . . .

A genome-wide approach identifies that the aspartate metabolism pathway contributes to asparaginase sensitivity

Asparaginase is an important component for treatment of childhood acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in the cytotoxicity of asparaginase, we used a genome-wide association approach using the HapMap lymphoblastoid cell lines (87 CEU trio members) and 54 primary ALL leukemic blast samples at diagnosis. Asparaginase sensitivity was assessed as the drug concentration necessary to inhibit 50% of growth (inhibitory concentration (IC)(50)). In CEU lines, we tested 2,390,203 single-nucleotide polymorphism (SNP) genotypes at the individual SNP (P<0.001) and gene level (P<0.05), and identified 329 SNPs representing 94 genes that were associated with asparaginase IC(50). The aspartate metabolism pathway was the most overrepresented among 199 pathways evaluated (P=8.1 × 10(-3)), with primary involvement of adenylosuccinate lyase and aspartyl-tRNA synthetase genes. We validated that SNPs in the aspartate metabolism pathway were also associated with asparaginase sensitivity in primary ALL leukemic blast samples (P=5.5 × 10(-5)). Our genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited genomic interindividual variation in a plausible candidate pathway can contribute to asparaginase sensitivity.

Radiation pharmacogenomics: A genome-wide association approach to identify radiation response biomarkers using human lymphoblastoid cell lines

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.

Quantitative assessment of the influence of hematopoietically expressed homeobox variant (rs1111875) on type 2 diabetes risk

Hematopoietically expressed homeobox ( HHEX) gene encodes for a transcription factor involved in Wnt/β-catenin signaling pathway which has attracted considerable attention as a candidate gene for type 2 diabetes (T2D) since it was first identified through genome wide association approach. The relationship between HHEX and T2D has been reported in various ethnic groups; however, these studies have yielded contradictory results. To investigate this inconsistency, we performed a meta-analysis of 26 studies involving a total of 110,875 subjects for rs1111875 of the HHEX gene to evaluate the effect of HHEX on genetic susceptibility for T2D. An overall random effects odds ratio of 1.16 (95% CI: 1.13–1.20) was found for C allele versus T allele. Significant results were also observed using dominant (OR = 1.21, 95% CI: 1.16–1.25) or recessive genetic model (OR = 1.24, 95% CI: 1.18–1.30). There was strong evidence of heterogeneity ( P < 0.001), which largely disappeared after stratification by ethnicity. In the subgroup analysis by sample size, source of controls and diagnostic criterion, significantly increased risks were found for the polymorphism in all genetic models. This meta-analysis demonstrated that the C allele of rs1111875 of HHEX is a risk factor associated with increased T2D susceptibility, but these associations vary in different ethnic populations.

Compilation of a Comprehensive Gene Panel for Systematic Assessment of Genes that Govern an Individual’s Drug Responses

Polymorphisms of genes involved in the pharmacokinetic and pharmacodynamic processes underlie the divergent drug responses among individuals. Despite some successes in identifying these polymorphisms, the candidate gene approach suffers from insufficient gene coverage whereas the genome-wide association approach is limited by less than ideal coverage of SNPs in some important genes. To expand the potential of the candidate approach, we aim to delineate a comprehensive network of drug-response genes for in-depth genetic studies. Pharmacologically important genes were extracted from various sources including literatures and web resources. These genes, along with their homologs and regulatory miRNAs, were organized based on their pharmacological functions and weighted by literature evidence and confidence levels. Their coverage was evaluated by analyzing three commercial SNP chips commonly used for genome-wide association studies: Affymetrix SNP array 6.0, Illumina HumanHap1M and Illumina Omni. A panel of drug-response genes was constructed, which contains 923 pharmacokinetic genes, 703 pharmacodynamic genes and 720 miRNAs. There are only 16.7% of these genes whose all known SNPs can be directly or indirectly (r(2) > 0.8) captured by the SNP chips with coverage of more than 80%. This is possibly because these SNPs chips have notably poor performance over rare SNPs and miRNA genes. We have compiled a panel of candidate genes that may be pharmacologically important. Using this knowledgebase, we are able to systematically evaluate genes and their variants that govern an individual's response to a given pharmaceutical therapy. This approach can serve as a necessary complement to genome-wide associations.

Germline Genetic Markers for Urinary Bladder Cancer Risk, Prognosis and Treatment Response

After decades of research using a candidate gene approach, only NAT2 and GSTM1 have consistently been demonstrated to be germline genetic susceptibility markers for urinary bladder cancer (UBC). The recent shift to an agnostic genome-wide association approach led to the identification of several UBC susceptibility loci, and provided valuable leads for new mechanistic insights into UBC carcinogenesis. The markers do not have sufficient discriminatory ability yet to be applied for risk assessment in the population and the question is whether they ever will. Prognostic and predictive studies in UBC are still in their infancy compared with etiologic studies. In the future, focus on a genome-wide association approach possibly using whole-genome sequence data, consortia formation and meta-analyses, and blood and tumor tissue collection, preferably in the context of randomized controlled trials will stimulate well designed and sufficiently powered studies, and thereby enhance the elucidation of genetic prognostic and predictive markers.

Heritability in the Efficiency of Nonsense-Mediated mRNA Decay in Humans

BackgroundIn eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences.Principal FindingsUsing previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD.ConclusionsWhile we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

Heritability of the human infectious reservoir of malaria parasites.

BackgroundStudies on human genetic factors associated with malaria have hitherto concentrated on their role in susceptibility to and protection from disease. In contrast, virtually no attention has been paid to the role of human genetics in eliciting the production of parasite transmission stages, the gametocytes, and thus enhancing the spread of disease.Methods and FindingsWe analysed four longitudinal family-based cohort studies from Senegal and Thailand followed for 2–8 years and evaluated the relative impact of the human genetic and non-genetic factors on gametocyte production in infections of Plasmodium falciparum or P. vivax. Prevalence and density of gametocyte carriage were evaluated in asymptomatic and symptomatic infections by examination of Giemsa-stained blood smears and/or RT-PCR (for falciparum in one site). A significant human genetic contribution was found to be associated with gametocyte prevalence in asymptomatic P. falciparum infections. By contrast, there was no heritability associated with the production of gametocytes for P. falciparum or P. vivax symptomatic infections. Sickle cell mutation, HbS, was associated with increased gametocyte prevalence but its contribution was small.ConclusionsThe existence of a significant human genetic contribution to gametocyte prevalence in asymptomatic infections suggests that candidate gene and genome wide association approaches may be usefully applied to explore the underlying human genetics. Prospective epidemiological studies will provide an opportunity to generate novel and perhaps more epidemiologically pertinent gametocyte data with which similar analyses can be performed and the role of human genetics in parasite transmission ascertained.

A Compendium of Genome-Wide Associations for Cancer: Critical Synopsis and Reappraisal

Since 2007, genome-wide association (GWA) studies have identified numerous well-supported, novel genetic risk loci for common cancers; however, there are concerns that this technology is reaching its limits. We provide an overview of GWA-identified genetic associations with solid tumors. We simulated the distribution of population risk alleles for colorectal, prostate, testicular, and thyroid cancers based on genetic variants identified in GWA studies. We also evaluated whether statistical power to detect typical genetic effects could be improved with studies performing GWA analyses of all available samples rather than multistage designs. Fifty-six eligible articles yielded 92 eligible associations between cancer phenotypes and genetic variants with a median per-allele odds ratio (OR) of 1.22 (interquartile range = 1.15-1.36). Half of the associations pertained to prostate, colorectal, or breast cancer. Individuals at the upper quartile of simulated risk had only 2.1- to 4.2-fold higher relative risk than those in the lower quartile. Comprehensive evaluation of currently available samples with GWA platforms would yield few additional variants with per-allele OR = 1.4, but many more variants with OR = 1.2 could be detected; statistical power to detect weak associations (OR = 1.07) would still be negligible. The GWA approach is effective in identifying common genetic variants with moderate effect; however, identifying loci with very small effects and rare variants will require major new efforts. At present, the utility of GWA-identified risk loci in risk stratification for cancer is limited.

A combined genome-wide linkage and association approach to find susceptibility loci for platelet function phenotypes in European American and African American families with coronary artery disease

BackgroundThe inability of aspirin (ASA) to adequately suppress platelet aggregation is associated with future risk of coronary artery disease (CAD). Heritability studies of agonist-induced platelet function phenotypes suggest that genetic variation may be responsible for ASA responsiveness. In this study, we leverage independent information from genome-wide linkage and association data to determine loci controlling platelet phenotypes before and after treatment with ASA.MethodsClinical data on 37 agonist-induced platelet function phenotypes were evaluated before and after a 2-week trial of ASA (81 mg/day) in 1231 European American and 846 African American healthy subjects with a family history of premature CAD. Principal component analysis was performed to minimize the number of independent factors underlying the covariance of these various phenotypes. Multi-point sib-pair based linkage analysis was performed using a microsatellite marker set, and single-SNP association tests were performed using markers from the Illumina 1 M genotyping chip from deCODE Genetics, Inc. All analyses were performed separately within each ethnic group.ResultsSeveral genomic regions appear to be linked to ASA response factors: a 10 cM region in African Americans on chromosome 5q11.2 had several STRs with suggestive (p-value < 7 × 10-4) and significant (p-value < 2 × 10-5) linkage to post aspirin platelet response to ADP, and ten additional factors had suggestive evidence for linkage (p-value < 7 × 10-4) to thirteen genomic regions. All but one of these factors were aspirin response variables. While the strength of genome-wide SNP association signals for factors showing evidence for linkage is limited, especially at the strict thresholds of genome-wide criteria (N = 9 SNPs for 11 factors), more signals were considered significant when the association signal was weighted by evidence for linkage (N = 30 SNPs).ConclusionsOur study supports the hypothesis that platelet phenotypes in response to ASA likely have genetic control and the combined approach of linkage and association offers an alternative approach to prioritizing regions of interest for subsequent follow-up.

Copy number variation and cytidine analogue cytotoxicity: A genome-wide association approach

BackgroundThe human genome displays extensive copy-number variation (CNV). Recent discoveries have shown that large segments of DNA, ranging in size from hundreds to thousands of nucleotides, are either deleted or duplicated. This CNV may encompass genes, leading to a change in phenotype, including drug response phenotypes. Gemcitabine and 1-β-D-arabinofuranosylcytosine (AraC) are cytidine analogues used to treat a variety of cancers. Previous studies have shown that genetic variation may influence response to these drugs. In the present study, we set out to test the hypothesis that variation in copy number might contribute to variation in cytidine analogue response phenotypes.ResultsWe used a cell-based model system consisting of 197 ethnically-defined lymphoblastoid cell lines for which genome-wide SNP data were obtained using Illumina 550 and 650 K SNP arrays to study cytidine analogue cytotoxicity. 775 CNVs with allele frequencies > 1% were identified in 102 regions across the genome. 87/102 of these loci overlapped with previously identified regions of CNV. Association of CNVs with gemcitabine and AraC IC50 values identified 11 regions with permutation p-values < 0.05. Multiplex ligation-dependent probe amplification assays were performed to verify the 11 CNV regions that were associated with this phenotype; with false positive and false negative rates for the in-silico findings of 1.3% and 0.04%, respectively. We also had basal mRNA expression array data for these same 197 cell lines, which allowed us to quantify mRNA expression for 41 probesets in or near the CNV regions identified. We found that 7 of those 41 genes were highly expressed in our lymphoblastoid cell lines, and one of the seven genes (SMYD3) that was significant in the CNV association study was selected for further functional experiments. Those studies showed that knockdown of SMYD3, in pancreatic cancer cell lines increased gemcitabine and AraC resistance during cytotoxicity assay, consistent with the results of the association analysis.ConclusionsThese results suggest that CNVs may play a role in variation in cytidine analogue effect. Therefore, association studies of CNVs with drug response phenotypes in cell-based model systems, when paired with functional characterization, might help to identify CNV that contributes to variation in drug response.

Abstract 2599: Taxane pharmacogenomics: Identifying genetic variation associated with drug cytotoxicity using a genome-wide association approach and a cell line model system

Abstract Background Taxane, a class of common anticancer agent, is widely used to treat many cancers. Among the taxanes, paclitaxel and docetaxel are the most commonly used agents. The mechanism of action for taxane drugs is through stabilizing microtubules by enhancing tubulin assembly and against depolymerization, thereby disrupting the dynamics of the microtubule. Large inter-individual variation has been observed in response to taxane treatment. Therefore, in the present study, we conducted a genome-wide association (GWA) study to identify genetic polymorphisms that might contribute to variation in response to taxanes drugs using the “Human Variation Panel” lymphoblastoid cell line (LCL) model system that consists of 287 ethnically defined cell lines. Method Genome-wide SNP and basal gene expression data were obtained for 287 LCLs using Illumina HumanHap 550K, 510S BeadChips, Affymetix SNP Array 6.0 and Affymetrix U133 plus 2.0 GeneChip. Cytotoxicity assay was also performed in the same cell lines for both paclitaxel and docetaxel. Candidate genes were selected based on an integrated analysis of genome-wide SNPs, expression array data and IC50 values (a cytotoxicity phenotype indicating the drug concentration required for 50% inhibition of maximal cell growth) for paclitaxel and docetaxel. SiRNA screening was performed to functionally validate those top candidates, followed by MTS assay in a breast cancer cell line, MDA-MB231. Results Of the 54,000 probe sets considered, 390 probe sets, representing 272 unique annotated genes, were found to be associated with docetaxel cytotoxicity (IC50 value) with p value &amp;lt;10−3, while 176 probe sets, representing 124 unique genes, were associated with paclitaxel IC50. Of these probe sets, 50 probe sets were associated with IC50 for both drugs. Analysis of 1.3 million SNPs identified 11 loci, defined as a region containing at least 1 SNP of p value &amp;lt;10−5 and 1 SNP of p value &amp;lt;10−4 within 50kb, associated with docetaxel IC50, while 7 loci were associated with paclitaxel IC50. One locus was found to be associated with both drugs. Previous studies suggested that gene expression is regulated in a cell-type specific manner. Therefore, based on our analysis and biological implication, we selected 16 top candidate genes that were expressed in our LCLs to perform functional validation studies using siRNA, followed by MTS assay in a breast cancer cell line, MDA-MB231. Functional studies showed that knockdown of SLC44A1 and HSPA5 significantly desensitized the cancer cell to both taxanes treatment. Conclusion GWAS using LCL model system could provide potential pharmacogenomic candidates for future clinical translational studies. Functional validation in tumor cell lines may help determine the genomic mechanism contributing to the inter-individual variation of taxane response. 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 2599.