Role of KCNMA1 in Breast Cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca²⁺-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3–7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.     

Batch effect correction for genome-wide methylation data with Illumina Infinium platform

Background

Paclitaxel is a microtubule-stabilizing drug that has been commonly used in treating cancer. Due to genetic heterogeneity within patient populations, therapeutic response rates often vary. Here we used the NCI60 panel to identify SNPs associated with paclitaxel sensitivity. Using the panel's GI50 response data available from Developmental Therapeutics Program, cell lines were categorized as either sensitive or resistant. PLINK software was used to perform a genome-wide association analysis of the cellular response to paclitaxel with the panel's SNP-genotype data on the Affymetrix 125 k SNP array. FastSNP software helped predict each SNP's potential impact on their gene product. mRNA expression differences between sensitive and resistant cell lines was examined using data from BioGPS. Using Haploview software, we investigated for haplotypes that were more strongly associated with the cellular response to paclitaxel. Ingenuity Pathway Analysis software helped us understand how our identified genes may alter the cellular response to paclitaxel.

Results

43 SNPs were found significantly associated (FDR < 0.005) with paclitaxel response, with 10 belonging to protein-coding genes (CFTR, ROBO1, PTPRD, BTBD12, DCT, SNTG1, SGCD, LPHN2, GRIK1, ZNF607). SNPs in GRIK1, DCT, SGCD and CFTR were predicted to be intronic enhancers, altering gene expression, while SNPs in ZNF607 and BTBD12 cause conservative missense mutations. mRNA expression analysis supported these findings as GRIK1, DCT, SNTG1, SGCD and CFTR showed significantly (p < 0.05) increased expression among sensitive cell lines. Haplotypes found in GRIK1, SGCD, ROBO1, LPHN2, and PTPRD were more strongly associated with response than their individual SNPs.

Conclusions

Our study has taken advantage of available genotypic data and its integration with drug response data obtained from the NCI60 panel. We identified 10 SNPs located within protein-coding genes that were not previously shown to be associated with paclitaxel response. As only five genes showed differential mRNA expression, the remainder would not have been detected solely based on expression data. The identified haplotypes highlight the role of utilizing SNP combinations within genomic loci of interest to improve the risk determination associated with drug response. These genetic variants represent promising biomarkers for predicting paclitaxel response and may play a significant role in the cellular response to paclitaxel.     

NCI60 cancer cell line panel data and RNAi analysis help identify EAF2 as a modulator of simvastatin and lovastatin response in HCT-116 cells

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the pharmacological data available for all the NCI60 cell lines to classify simvastatin or lovastatin resistant and sensitive cell lines, respectively. Next, we performed whole-genome single marker case-control association tests for the lovastatin and simvastatin resistant and sensitive cells using their publicly available Affymetrix 125K SNP genomic data. The results were then evaluated using RNAi methodology. After correction of the p-values for multiple testing using False Discovery Rate, our results identified three genes (NRP1, COL13A1, MRPS31) and six genes (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) associated with resistance to simvastatin and lovastatin, respectively. Functional validation using RNAi confirmed that silencing of EAF2 expression modulated the response of HCT-116 colon cancer cells to both statins. In summary, we have successfully utilized the publicly available data on the NCI60 cell lines to perform whole-genome association studies for simvastatin and lovastatin. Our results indicated genes involved in the cellular response to these statins and siRNA studies confirmed the role of the EAF2 in response to these drugs in HCT-116 colon cancer cells.     

Multiple Functional Risk Variants in a SMAD7 Enhancer Implicate a Colorectal Cancer Risk Haplotype

Genome-wide association studies (GWAS) of colorectal cancer (CRC) have led to the identification of a number of common variants associated with modest risk. Several risk variants map within the vicinity of TGFβ/BMP signaling pathway genes, including rs4939827 within an intron of SMAD7 at 18q21.1. A previous study implicated a novel SNP (novel 1 or rs58920878) as a functional variant within an enhancer element in SMAD7 intron 4. In this study, we show that four SNPs including novel 1 (rs6507874, rs6507875, rs8085824, and rs58920878) in linkage disequilibrium (LD) with the index SNP rs4939827 demonstrate allele-specific enhancer effects in a large, multi-component enhancer of SMAD7. All four SNPs demonstrate allele-specific protein binding to nuclear extracts of CRC cell lines. Furthermore, some of the risk-associated alleles correlate with increased expression of SMAD7 in normal colon tissues. Finally, we show that the enhancer is responsive to BMP4 stimulation. Taken together, we propose that the associated CRC risk at 18q21.1 is due to four functional variants that regulate SMAD7 expression and potentially perturb a BMP negative feedback loop in TGFβ/BMP signaling pathways.     

A genetic variant in the gene encoding the stress70 protein chaperone family member STCH is associated with gastric cancer in the Japanese population

Association analysis, based on linkage disequilibrium between specific alleles in the candidate loci and nearby genetic markers, has been proposed to identify genes conferring susceptibility to multifactorial diseases. Using the affected sib-pair method, we previously mapped four candidate chromosomal regions, 1p32, 2q33-q35, 11p13-p14, and 21q21, for gastric cancer by linkage analysis. To identify genes involved in the disease, we performed a gene-based association analysis of 66 genes, located on 21p11-21q22, using 126 single nucleotide polymorphisms (SNPs) as genetic markers in 373 patients with 250 controls. We found a significant association of five SNPs in the stress70 protein chaperon family member STCH gene with gastric cancer, especially with the non-cardia localization subgroup (P = 0.0005-0.02, odds ratio = 1.44-1.72). Comparisons of haplotype frequency showed significant association between TTGGC haplotype and gastric cancer (P = 0.0001, odds ratio = 1.59). These results suggest that, in the Japanese population, STCH might be a new candidate for conferring susceptibility to this disease.     

Refining Susceptibility Loci of Chronic Obstructive Pulmonary Disease with Lung eqtls

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide. Recent genome-wide association studies (GWAS) have identified robust susceptibility loci associated with COPD. However, the mechanisms mediating the risk conferred by these loci remain to be found. The goal of this study was to identify causal genes/variants within susceptibility loci associated with COPD. In the discovery cohort, genome-wide gene expression profiles of 500 non-tumor lung specimens were obtained from patients undergoing lung surgery. Blood-DNA from the same patients were genotyped for 1,2 million SNPs. Following genotyping and gene expression quality control filters, 409 samples were analyzed. Lung expression quantitative trait loci (eQTLs) were identified and overlaid onto three COPD susceptibility loci derived from GWAS; 4q31 (HHIP), 4q22 (FAM13A), and 19q13 (RAB4B, EGLN2, MIA, CYP2A6). Significant eQTLs were replicated in two independent datasets (n = 363 and 339). SNPs previously associated with COPD and lung function on 4q31 (rs1828591, rs13118928) were associated with the mRNA expression of HHIP. An association between mRNA expression level of FAM13A and SNP rs2045517 was detected at 4q22, but did not reach statistical significance. At 19q13, significant eQTLs were detected with EGLN2. In summary, this study supports HHIP, FAM13A, and EGLN2 as the most likely causal COPD genes on 4q31, 4q22, and 19q13, respectively. Strong lung eQTL SNPs identified in this study will need to be tested for association with COPD in case-control studies. Further functional studies will also be needed to understand the role of genes regulated by disease-related variants in COPD.     

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.     

Genome-wide associations of gene expression variation in humans

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12–13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.     

A Genome-Wide Association Study of the Maize Hypersensitive Defense Response Identifies Genes That Cluster in Related Pathways

Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (Rp1-D21), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of Rp1-D21. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants.     

Identification of candidate genes for dyslexia susceptibility on chromosome 18

Background

Six independent studies have identified linkage to chromosome 18 for developmental dyslexia or general reading ability. Until now, no candidate genes have been identified to explain this linkage. Here, we set out to identify the gene(s) conferring susceptibility by a two stage strategy of linkage and association analysis.

Methodology/Principal Findings

Linkage analysis: 264 UK families and 155 US families each containing at least one child diagnosed with dyslexia were genotyped with a dense set of microsatellite markers on chromosome 18. Association analysis: Using a discovery sample of 187 UK families, nearly 3000 SNPs were genotyped across the chromosome 18 dyslexia susceptibility candidate region. Following association analysis, the top ranking SNPs were then genotyped in the remaining samples. The linkage analysis revealed a broad signal that spans approximately 40 Mb from 18p11.2 to 18q12.2. Following the association analysis and subsequent replication attempts, we observed consistent association with the same SNPs in three genes; melanocortin 5 receptor (MC5R), dymeclin (DYM) and neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L).

Conclusions

Along with already published biological evidence, MC5R, DYM and NEDD4L make attractive candidates for dyslexia susceptibility genes. However, further replication and functional studies are still required.     

Association Study of Polymorphisms in Selenoprotein Genes and Kashin–Beck Disease and Serum Selenium/Iodine Concentration in a Tibetan Population

Background

Kashin-Beck disease is a kind of degenerative osteoarthropathy. Genetic factors may play an important role in the pathogenesis of KBD.

Objective

To investigate the association of the selenoprotein genes GPX1 (rs1050450, rs1800668, and rs3811699), TrxR2 (rs5748469), and DIO2 (rs225014) with Kashin-Beck disease (KBD) in a Tibetan population and to investigate the association of these SNPs with the serum iodine/selenium concentration in the Tibetan population.

Design

Five SNPs including rs1050450, rs1800668, and rs3811699 in the GPX1 gene, rs5748469 in the TrxR2 gene, and rs225014 in the DIO2 gene were analyzed in Tibetan KBD patients and controls using the SNaPshot method. P trend values of the SNPs were calculated using an additive model.

Results

None of the five SNPs in the three genes showed a significant association with KBD. Haplotypes TCC, TTC and TTT of rs1050450, rs1800668 and rs3811699 in GPX1 showed a significant association with KBD and controls with P value of 0.0421, 5.0E-4 and 0.0066, respectively. The GPX1 gene (rs1050450) showed a potential significant association with the iodine concentration in the Tibetan study population (P = 0.02726). However, no such association was detected with the selenium concentration (P = 0.2849).

Conclusion(s)

In this study, we showed that single SNPs in the genes GPX1 (rs1050450, rs1800668 and rs3811699), TrxR2 (rs5748469), and DIO2 (rs225014) may not be significantly associated with KBD in a Tibetan population. However, haplotype analysis of SNPs rs1050450, rs1800668 and rs3811699 in GPX1 gene showed a significant association with KBD. The results suggested that GPX1 gene play a protective role in the susceptivity of KBD in Tibetans. Furthermore, the GPX1 gene (rs1050450) may be significantly associated with the serum iodine concentration in Tibetans.     

Genome-wide linkage scan and association study of PARL to the expression of LHON families in Thailand

Leber hereditary optic neuropathy (LHON) is the most common mitochondrially inherited disease causing blindness, preferentially in young adult males. Most of the patients carry the G11778A mitochondrial DNA (mtDNA) mutation. However, the marked incomplete penetrance and the gender bias indicate some additional genetic and/or environmental factors to disease expression. Herein, we first conducted a genome-wide linkage scan with 400 microsatellite markers in 9 large Thai LHON G11778A pedigrees. Using an affecteds-only nonparametric linkage analysis, 4 regions on chromosomes 3, 12, 13 and 18 showed Zlr scores greater than 2 (P < 0.025), which is consistently significant across several linkage statistics. The most suggestive marker D3S1565 (Zlr > 2 in 10 of 16 allele sharing models tested) was then expanded to include the region 3q26.2–3q28 covering SLC7A14 (3q26.2), MFN1 (3q26.32), MRPL47 (3q26.33), MCCC1 (3q27.1), PARL (3q27.1) and OPA1 (3q28–q29). All of these candidate genes were selected from the Maestro database and had known to be localized in mitochondria. Sixty tag SNPs were genotyped in 86 cases, 211 of their relatives and 32 unrelated Thai controls, by multiplex-PCR-based Invader assay. Analyses using a powerful association testing tool that adjusts for relatedness (the M_QLS statistic) showed the most evidence of association between two SNPs, rs3749446 and rs1402000 (located in PARL presenilins-associated rhomboid-like) and LHON expression (both P = 8.8 × 10^?5). The mitochondrial PARL protease has been recently known to play a role with a dynamin-related OPA1 protein in preventing apoptotic events by slowing down the release of cytochrome c out of mitochondrial cristae junctions. Moreover, PARL is required to activate the intramembranous proteolyses resulting in the degradation of an accumulated pro-apoptotic protein in the outer mitochondrial membrane. Under these circumstances, variants of PARL are suggested to influence cell death by apoptosis which has long been believed to intrigue the neurodegeneration of LHON.     

Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility

Introduction

Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region.

Methods

To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry.

Results

Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region.

Conclusions

Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.     

Family-Based Association Study of Pulmonary Function in a Population in Northeast Asia

The spirometric measurement of pulmonary function by measuring the forced expiratory volume in one second (FEV₁) is a heritable trait that reflects the physiological condition of the lung and airways. Genome-wide linkage and association studies have identified a number of genes and genetic loci associated with pulmonary function. However, limited numbers of studies have been reported for Asian populations. In this study, we aimed to investigate genetic evidence of pulmonary function in a population in northeast Asia. We conducted a family-based association test with 706 GENDISCAN study participants from 72 Mongolian families to determine candidate genetic determinants of pulmonary function. For the replication, we chose seven candidate single nucleotide polymorphisms (SNPs) from the 5 loci, and tested 1062 SNPs for association with FEV₁ from 2,729 subjects of the Korea Healthy Twin study. We identified TMEM132C as a potential candidate gene at 12q24.3, which is a previously reported locus of asthma and spirometric indices. We also found two adjacent candidate genes (UNC93A and TTLL2) in the 6q27 region, which has been previously identified as a pulmonary function locus in the Framingham cohort study. Our findings suggest that novel candidate genes (TMEM132C, UNC93A and TTLL2) in two different regions are associated with pulmonary function in a population in northeast Asia.     

Methylseleninic acid activates Keap1/Nrf2 pathway via up-regulating miR-200a in human oesophageal squamous cell carcinoma cells

Oesophageal squamous cell carcinoma (ESCC) occurs at a very high rates in certain regions of China. There are increasing evidences demonstrating that selenium could act as a potential anti-oesophageal cancer agent, but the precise mechanisms involved are still not completely understood. Methylseleninic acid (MSA), as a potent second-generation selenium compound, is a promising chemopreventive agent. Previous studies demonstrated that the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2) system plays a critical role in cancer prevention, but little is known about its association with MSA in ESCC cells. In the present study, we observed that MSA treatment significantly down-regulated Keap1, induced nuclear accumulation of Nrf2 and enhance the antioxidant response element (ARE) promoter activity in ESCC cells. MSA could also significantly induce miR-200a expression and inhibit Keap1 directly. Antagomir-200a could attenuate MSA treatment-induced Keap1 down-regulation in ESCC cells. Moreover, MSA-induced miR-200a expression was dependent on the mediation of Krüpple-like factor 4 (KLF4). These results reaffirm the potential role of MSA as a chemopreventive agent via the regulation of KLF4/miR-200a/Keap1/Nrf2 axis in ESCC cells.     

Gemcitabine and Arabinosylcytosin Pharmacogenomics: Genome-Wide Association and Drug Response Biomarkers

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® 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 IC₅₀ values for the cell lines. We then performed GWA studies with SNPs, gene expression and IC₅₀ of these two drugs. This approach identified SNPs that were associated with gemcitabine or AraC IC₅₀ 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 IC₅₀. 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.