Breast cancer is the most common malignancy among women in worldwide including Japan. Several studies have identified common genetic variants to be associated with the risk of breast cancer. Due to the complex linkage disequilibrium structure and various environmental exposures in different populations, it is essential to identify variants associated with breast cancer in each population, which subsequently facilitate the better understanding of mammary carcinogenesis. In this study, we conducted a genome-wide association study (GWAS) as well as whole-genome imputation with 2,642 cases and 2,099 unaffected female controls. We further examined 13 suggestive loci (P<1.0×10−5) using an independent sample set of 2,885 cases and 3,395 controls and successfully validated two previously-reported loci, rs2981578 (combined P-value of 1.31×10−12, OR = 1.23; 95% CI = 1.16–.30) on chromosome 10q26 (FGFR2), rs3803662 (combined P-value of 2.79×10−11, OR = 1.21; 95% CI = 1.15–.28) and rs12922061 (combined P-value of 3.97×10−10, OR = 1.23; 95% CI = 1.15–.31) on chromosome 16q12 (TOX3-LOC643714). Weighted genetic risk score on the basis of three significantly associated variants and two previously reported breast cancer associated loci in East Asian population revealed that individuals who carry the most risk alleles in category 5 have 2.2 times higher risk of developing breast cancer in the Japanese population than those who carry the least risk alleles in reference category 1. Although we could not identify additional loci associated with breast cancer, our study utilized one of the largest sample sizes reported to date, and provided genetic status that represent the Japanese population. Further local and international collaborative study is essential to identify additional genetic variants that could lead to a better, accurate prediction for breast cancer.
Sensory peripheral neuropathy is a common and sometimes debilitating toxicity associated with paclitaxel therapy. This study aims to identify genetic risk factors for development of this toxicity.
A prospective pharmacogenetic analysis of primary breast cancer patients randomized to the paclitaxel arm of CALGB 40101 was used to identify genetic predictors of the onset and severity of sensory peripheral neuropathy. A genome-wide association study in 855 subjects of European ancestry was performed and findings were replicated in additional European (n = 154) and African American (n = 117) subjects.
A single nucleotide polymorphism in FGD4 was associated with the onset of sensory peripheral neuropathy in the discovery cohort (rs10771973; HR, 1.57; 95% CI, 1.30–1.91; P = 2.6 × 10−6) and in a European (HR, 1.72; 95% CI, 1.06–2.80; P = 0.013) and African American (HR, 1.93; 95% CI, 1.13-3.28; P = 6.7 × 10−3) replication cohort. There is also evidence that markers in additional genes, including EPHA5 (rs7349683) and FZD3 (rs10771973), were associated with the onset or severity of paclitaxel-induced sensory peripheral neuropathy.
A genome-wide association study has identified novel genetic markers of paclitaxel-induced sensory peripheral neuropathy, including a common polymorphism in FGD4, a congenital peripheral neuropathy gene. These findings suggest that genetic variation may contribute to variation in development of this toxicity. Validation of these findings may allow for the identification of patients at increased risk of peripheral neuropathy and inform the use of an alternative to paclitaxel and/or the clinical management of this toxicity.
paclitaxel; peripheral neuropathy; breast cancer; pharmacogenetics; genome-wide association study
Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity, affecting around 2% of adolescents worldwide. Genetic factors play an important role in its etiology. Using a genome-wide association study (GWAS), we recently identified novel AIS susceptibility loci on chromosomes 10q24.31 and 6q24.1. To identify more AIS susceptibility loci relating to its severity and progression, we performed GWAS by limiting the case subjects to those with severe AIS. Through a two-stage association study using a total of ∼12,000 Japanese subjects, we identified a common variant, rs12946942 that showed a significant association with severe AIS in the recessive model (P = 4.00×10−8, odds ratio [OR] = 2.05). Its association was replicated in a Chinese population (combined P = 6.43×10−12, OR = 2.21). rs12946942 is on chromosome 17q24.3 near the genes SOX9 and KCNJ2, which when mutated cause scoliosis phenotypes. Our findings will offer new insight into the etiology and progression of AIS.
VKORC1 and CYP2C9 are important contributors to warfarin dose variability, but explain less variability for individuals of African descent than for those of European or Asian descent. We aimed to identify additional variants contributing to warfarin dose requirements in African Americans.
We did a genome-wide association study of discovery and replication cohorts. Samples from African-American adults (aged ≥18 years) who were taking a stable maintenance dose of warfarin were obtained at International Warfarin Pharmacogenetics Consortium (IWPC) sites and the University of Alabama at Birmingham (Birmingham, AL, USA). Patients enrolled at IWPC sites but who were not used for discovery made up the independent replication cohort. All participants were genotyped. We did a stepwise conditional analysis, conditioning first for VKORC1 −1639G→A, followed by the composite genotype of CYP2C9*2 and CYP2C9*3. We prespecified a genome-wide significance threshold of p<5×10−8 in the discovery cohort and p<0·0038 in the replication cohort.
The discovery cohort contained 533 participants and the replication cohort 432 participants. After the prespecified conditioning in the discovery cohort, we identified an association between a novel single nucleotide polymorphism in the CYP2C cluster on chromosome 10 (rs12777823) and warfarin dose requirement that reached genome-wide significance (p=1·51×10−8). This association was confirmed in the replication cohort (p=5·04×10−5); analysis of the two cohorts together produced a p value of 4·5×10−12. Individuals heterozygous for the rs12777823 A allele need a dose reduction of 6·92 mg/week and those homozygous 9·34 mg/week. Regression analysis showed that the inclusion of rs12777823 significantly improves warfarin dose variability explained by the IWPC dosing algorithm (21% relative improvement).
A novel CYP2C single nucleotide polymorphism exerts a clinically relevant effect on warfarin dose in African Americans, independent of CYP2C9*2 and CYP2C9*3. Incorporation of this variant into pharmacogenetic dosing algorithms could improve warfarin dose prediction in this population.
National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.
The recent development of massively parallel sequencing technology has allowed the creation of comprehensive catalogs of genetic variation. However, due to the relatively high sequencing error rate for short read sequence data, sophisticated analysis methods are required to obtain high-quality variant calls. Here, we developed a probabilistic multinomial method for the detection of single nucleotide variants (SNVs) as well as short insertions and deletions (indels) in whole genome sequencing (WGS) and whole exome sequencing (WES) data for single sample calling. Evaluation with DNA genotyping arrays revealed a concordance rate of 99.98% for WGS calls and 99.99% for WES calls. Sanger sequencing of the discordant calls determined the false positive and false negative rates for the WGS (0.0068% and 0.17%) and WES (0.0036% and 0.0084%) datasets. Furthermore, short indels were identified with high accuracy (WGS: 94.7%, WES: 97.3%). We believe our method can contribute to the greater understanding of human diseases.
Chronic kidney disease (CKD) is well known as a strong risk factor for both of end-stage renal disease and cardiovascular disease. To clarify the association of polymorphisms in the genes encoding antioxidant enzymes (SOD2, CAT, GPx, TXNRD, SEPP1, SEP15 and SELS) with the risk of CKD in Japanese, we examined this association using the cross-sectional data of Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. The subjects were 3,285 men and women, aged 35–69 years, selected from J-MICC Study participants for whom genotyping were conducted by multiplex polymerase chain reaction-based Invader assay. The prevalence of CKD was determined for CKD stages 3–5 (eGFR <60 ml/min/1.73 m2). When those with CAT C-262T C/C were defined as reference, those with CAT C-262T C/T demonstrated the OR for CKD of 0.67 (95% CI 0.43–1.06) with the marginally significant trend for decreased odds ratio with increasing numbers of T allele (p = 0.070). There were no significant associations between the other polymorphisms with CKD risk. The present study found a marginally significant trend of the decreased risk of CKD with increasing numbers of T allele of CAT, which may suggest the possibility of personalized risk estimation of this life-limiting disease in the near future.
antioxidant enzymes; genetic predisposition to disease; single nucleotide polymorphisms; chronic kidney disease
Peptic ulcer is one of the most common gastrointestinal disorders with complex etiology. Recently we conducted the genome wide association study for duodenal ulcer and identified disease susceptibility variations at two genetic loci corresponding to the Prostate stem cell antigen (PSCA) gene and the ABO blood group (ABO) gene. Here we investigated the association of these variations with gastric ulcer in two Japanese case-control sample sets, a total of 4,291 gastric ulcer cases and 22,665 controls. As a result, a C-allele of rs2294008 at PSCA increased the risk of gastric ulcer with odds ratio (OR) of 1.13 (P value of 5.85×10−7) in an additive model. On the other hand, SNP rs505922 on ABO exhibited inconsistent result between two cohorts. Our finding implies presence of the common genetic variant in the pathogenesis of gastric and duodenal ulcers.
Age at menarche (AAM) is a complex trait involving both genetic and environmental factors. To identify the genetic factors associated with AAM, we conducted a large-scale meta-analysis of genome-wide association studies using more than 15,000 Japanese female samples. Here, we identified an association between SNP (single nucleotide polymorphism) rs364663 at the LIN28B locus and AAM, with a P-value of 5.49×10−7 and an effect size of 0.089 (year). We also evaluated 33 SNPs that were previously reported to be associated with AAM in women of European ancestry. Among them, two SNPs rs4452860 and rs7028916 in TMEM38B indicated significant association with AAM in the same directions as reported in previous studies (P = 0.0013 with an effect size of 0.051) even after Bonferroni correction for the 33 SNPs. In addition, six loci in or near CCDC85A, LOC100421670, CA10, ZNF483, ARNTL, and RXRG exhibited suggestive association with AAM (P<0.05). Our findings elucidated the impact of genetic variations on AAM in the Japanese population.
There have been few genome-wide association studies (GWAS) of prostate cancer among diverse populations. To search for novel prostate cancer risk variants, we conducted GWAS of prostate cancer in Japanese and Latinos. In addition, we tested prostate cancer risk variants and developed genetic risk models of prostate cancer for Japanese and Latinos.
Our first stage GWAS of prostate cancer included Japanese (cases/controls=1,033/1,042) and Latino (cases/controls=1,043/1,057) from the Multiethnic Cohort. Significant associations from stage 1 (P < 1.0×10−4) were examined in silico in GWAS of prostate cancer (stage 2) in Japanese (cases/controls=1,583/3,386) and Europeans (cases/controls=1,854/1,894).
No novel stage 1 SNPs outside of known risk regions reached genome-wide significance. For Japanese, in stage 1, the most notable putative novel association was seen with 10 SNPs (P<8.0. x10−6) at chromosome 2q33; however, this was not replicated in stage 2. For Latinos, the most significant association was observed with rs17023900 at the known 3p12 risk locus (stage 1: OR=1.45; P=7.01×10−5 and stage 2: OR=1.58; P =3.05×10−7). The majority of the established risk variants for prostate cancer, 79% and 88%, were positively associated with prostate cancer in Japanese and Latinos (stage I), respectively. The cumulative effects of these variants significantly influence prostate cancer risk (OR per allele=1.10; P = 2.71×10−25 and OR=1.07; P = 1.02×10−16 for Japanese and Latinos, respectively).
Conclusion and Impact
Our GWAS of prostate cancer did not identify novel genome-wide significant variants. However, our findings demonstrate that established risk variants for prostate cancer significantly contribute to risk among Japanese and Latinos.
Hepatitis C virus (HCV) infection is the major cause of hepatocellular carcinoma (HCC) in Japan. We previously identified the association of SNP rs2596542 in the 5' flanking region of the MHC class I polypeptide-related sequence A (MICA) gene with the risk of HCV-induced HCC. In the current study, we performed detailed functional analysis of 12 candidate SNPs in the promoter region and found that a SNP rs2596538 located at 2.8 kb upstream of the MICA gene affected the binding of a nuclear protein(s) to the genomic segment including this SNP. By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, we identified that transcription factor Specificity Protein 1 (SP1) can bind to the protective G allele, but not to the risk A allele. In addition, reporter construct containing the G allele was found to exhibit higher transcriptional activity than that containing the A allele. Moreover, SNP rs2596538 showed stronger association with HCV-induced HCC (P = 1.82×10−5 and OR = 1.34) than the previously identified SNP rs2596542. We also found significantly higher serum level of soluble MICA (sMICA) in HCV-induced HCC patients carrying the G allele than those carrying the A allele (P = 0.00616). In summary, we have identified a functional SNP that is associated with the expression of MICA and the risk for HCV-induced HCC.
We set out to test the hypothesis that pharmacometabolomic data could be efficiently merged with pharmacogenomic data by SNP imputation of metabolomic-derived pathway data on a “scaffolding” of genome-wide association (GWA) SNP data to broaden and accelerate “pharmacometabolomics-informed pharmacogenomic” studies by eliminating the need for initial genotyping and by making broader SNP association testing possible.
We previously genotyped 131 tag SNPs for six genes encoding enzymes in the glycine synthesis and degradation pathway using DNA from 529 depressed patients treated with citalopram/escitalopram to pursue a glycine metabolomics “signal” associated with selective serotonine reuptake inhibitor response. We identified a significant SNP in the glycine dehydrogenase gene. Subsequently, GWAS SNP data were generated for the same patients. In this study, we compared SNP imputation within 200 kb of these same six genes with results of the previous tag SNP strategy as a rapid strategy for merging pharmacometabolomic and pharmacogenomic data.
Imputed genotype data provided greater coverage and higher resolution than did tag SNP genotyping, with a higher average genotype concordance between genotyped and imputed SNP data for “1000 Genomes” (96.4%) than HapMap 2 (93.2%) imputation. Many low p-value SNPs with novel locations within genes were observed for imputed compared with tag SNPs, thus altering the focus for subsequent functional genomic studies.
These results indicate that the use of GWAS data to impute SNPs for genes in pathways identified by other “omics” approaches makes it possible to rapidly and economically identify SNP markers to “broaden” and accelerate pharmacogenomic studies.
Pharmacometabolomics; pharmacogenomics; imputation; tag SNPs; 1000 Genomes; HapMap; selective serotonin reuptake inhibitors; SSRIs; major depressive disorder; MDD
Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness (AHR). The genetics of asthma have been widely studied in mouse and human, and homologous genomic regions have been associated with mouse AHR and human asthma-related phenotypes. Our goal was to identify asthma-related genes by integrating AHR associations in mouse with human genome-wide association study (GWAS) data. We used Efficient Mixed Model Association (EMMA) analysis to conduct a GWAS of baseline AHR measures from males and females of 31 mouse strains. Genes near or containing SNPs with EMMA p-values <0.001 were selected for further study in human GWAS. The results of the previously reported EVE consortium asthma GWAS meta-analysis consisting of 12,958 diverse North American subjects from 9 study centers were used to select a subset of homologous genes with evidence of association with asthma in humans. Following validation attempts in three human asthma GWAS (i.e., Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG) and two human AHR GWAS (i.e., SHARP, DAG), the Kv channel interacting protein 4 (KCNIP4) gene was identified as nominally associated with both asthma and AHR at a gene- and SNP-level. In EVE, the smallest KCNIP4 association was at rs6833065 (P-value 2.9e-04), while the strongest associations for Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG were 1.5e-03, 1.0e-03, 3.1e-03 at rs7664617, rs4697177, rs4696975, respectively. At a SNP level, the strongest association across all asthma GWAS was at rs4697177 (P-value 1.1e-04). The smallest P-values for association with AHR were 2.3e-03 at rs11947661 in SHARP and 2.1e-03 at rs402802 in DAG. Functional studies are required to validate the potential involvement of KCNIP4 in modulating asthma susceptibility and/or AHR. Our results suggest that a useful approach to identify genes associated with human asthma is to leverage mouse AHR association data.
Background and Aims
CALGB 80303 was a randomized, phase III study in advanced pancreatic cancer patients treated with gemcitabine plus either bevacizumab or placebo. We prospectively collected germline DNA and conducted a genome-wide association study (GWAS) using overall survival (OS) as the endpoint.
DNA from 351 patients was genotyped for >550,000 single nucleotide polymorphisms (SNPs). Associations between OS and SNPs were investigated using the log-linear two-way multiplicative Cox proportional-hazards model. The subset of 294 genetically European patients was used for the primary analysis.
A nonsynonymous SNP in IL17F (rs763780, H161R) and an intronic SNP in strong linkage disequilibrium (rs7771466) were associated with OS using genome-wide criteria (p≤10−7). Median OS was significantly shorter (p 2.61×10−8) for the rs763780 heterozygotes (3.1 months, 95% CI 2.3–4.3) as compared to the patients without this variant (6.8 months, 5.8–7.3). After adjustment by stratification factors, the p value for the association was 9.51×10−7.
The variant 161R form of interleukin-17F is a natural antagonist of the anti-angiogenic effects of wild-type 161H interleukin-17F, and angiogenesis may play an important role in the metastatic spread of pancreatic cancer. In this preliminary study, we hypothesize that the angiogenesis potential of pancreatic cancers in patients with variant interleukin-17F is higher than that of tumors in patients with wild-type interleukin-17F, conferring worse prognosis. This exploratory GWAS may provide the foundation for testing the biology and clinical effects of novel genes and their heritable variants through mechanistic and confirmatory studies in pancreatic cancer.
GWAS; pharmacogenetics; bevacizumab; pancreatic cancer; gemcitabine
Gene-set analyses have been widely used in gene expression studies, and some of the developed methods have been extended to genome wide association studies (GWAS). Yet, complications due to linkage disequilibrium (LD) among single nucleotide polymorphisms (SNPs), and variable numbers of SNPs per gene and genes per gene-set, have plagued current approaches, often leading to ad hoc “fixes”. To overcome some of the current limitations, we developed a general approach to scan GWAS SNP data for both gene-level and gene-set analyses, building on score statistics for generalized linear models, and taking advantage of the directed acyclic graph structure of the gene ontology when creating gene-sets. However, other types of gene-set structures can be used, such as the popular Kyoto Encyclopedia of Genes and Genomes (KEGG). Our approach combines SNPs into genes, and genes into gene-sets, but assures that positive and negative effects of genes on a trait do not cancel. To control for multiple testing of many gene-sets, we use an efficient computational strategy that accounts for LD and provides accurate step-down adjusted p-values for each gene-set. Application of our methods to two different GWAS provide guidance on the potential strengths and weaknesses of our proposed gene-set analyses.
gene-sets; genome wide association; pathways; score statistics
Nitric oxide is a key molecule not only in the cardiovascular system, but also in the metabolic-endocrine system. The purpose of this study was to examine possible associations of the NOS3 T-786C polymorphism (rs2070744) with serum lipid levels on the basis of lifestyle factors for tailoring prevention of dyslipidemia.
For this cross-sectional study, a total of 2226 subjects aged 35 to 69 years (1084 men and 1142 women) were selected from Japanese participants in the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. They were recruited in eight areas throughout Japan between February 2004 and November 2008.
In a stratified analysis by leisure-time physical activity, the likelihood of hypertriglyceridemia (serum triglyceride levels ≥ 150 mg/dL) among subjects with the C allele was significantly lower than those without it in the active group (OR = 0.43, 95% CI = 0.22-0.84 in the fasting group), but not in the sedentary group. A gene-environment interaction between the T-786C polymorphism and leisure-time physical activity for hypertriglyceridemia was significant (P = 0.007 in the fasting group). Additionally, serum triglyceride levels (mean ± SD) across leisure-time physical activity classes decreased significantly only in the TC + CC genotype group (111 ± 60 mg/dL for sedentary, 95 ± 48 mg/dL for moderately active, 88 ± 44 mg/dL for very active, P for trend = 0.008 in the fasting group), but not in the TT genotype group. Total cholesterol, high-density lipoprotein (HDL) cholesterol, and non-HDL cholesterol levels had no significant association with the polymorphism.
This study suggests that the NOS3 T-786C polymorphism modifies the effect of leisure-time physical activity on serum triglyceride levels.
Cross-sectional study; Gene-environment interaction; Hypertriglyceridemia; Lifestyle-related disease; NOS3; Tailoring prevention
Controversy exists regarding the association between CYP2D6 enzyme activity and tamoxifen effectiveness in the adjuvant treatment of invasive breast cancer; however this association in the primary prevention of breast cancer is unknown.
We performed a nested case-control study in the context of the NSABP P-1 and P-2 prevention trials to determine the impact of CYP2D6 genotype, CYP2D6 inhibitor use, as well as metabolizer status (CYP2D6 genotype combined with CYP2D6 inhibitor use), on breast cancer events. Women who developed breast cancer (both non-invasive and invasive) while on five years of SERM therapy (cases) were matched to controls free of breast cancer. Comprehensive CYP2D6 genotyping was performed for alleles associated with absent (*3, *4, *5, *6), reduced (*10, *17, *41), and increased (*1XN and *2XN) enzyme activity. Information regarding the use of CYP2D6 inhibitors was recorded.
591 cases were matched to 1126 controls and DNA was genotyped in >97%. In patients treated with tamoxifen, there was no association of CYP2D6 genotype [OR(extensive/poor metabolizer): 0.90; 95% CI 0.46-1.74, p=0.74), use of a potent CYP2D6 inhibitor (OR 0.92; 95% CI 0.575-1.486), or CYP2D6 metabolizer status (OR 1.03; 95% CI 0.669-1.607) with breast cancer occurrence. Likewise, there was no association between any CYP2D6 metabolism parameter with breast cancer events in raloxifene treated patients.
In the NSABP P1 and P2 clinical trials, alterations in CYP2D6 metabolism are not associated with either tamoxifen or raloxifene efficacy.
tamoxifen; breast cancer; prevention; CYP2D6; polymorphism
Prostate specific antigen (PSA) is widely used as a diagnostic biomarker for prostate cancer (PC). However, due to its low predictive performance, many patients without PC suffer from the harms of unnecessary prostate needle biopsies. The present study aims to evaluate the reproducibility and performance of a genetic risk prediction model in Japanese and estimate its utility as a diagnostic biomarker in a clinical scenario. We created a logistic regression model incorporating 16 SNPs that were significantly associated with PC in a genome-wide association study of Japanese population using 689 cases and 749 male controls. The model was validated by two independent sets of Japanese samples comprising 3,294 cases and 6,281 male controls. The areas under curve (AUC) of the model were 0.679, 0.655, and 0.661 for the samples used to create the model and those used for validation. The AUCs were not significantly altered in samples with PSA 1–10 ng/ml. 24.2% and 9.7% of the patients had odds ratio <0.5 (low risk) or >2 (high risk) in the model. Assuming the overall positive rate of prostate needle biopsies to be 20%, the positive biopsy rates were 10.7% and 42.4% for the low and high genetic risk groups respectively. Our genetic risk prediction model for PC was highly reproducible, and its predictive performance was not influenced by PSA. The model could have a potential to affect clinical decision when it is applied to patients with gray-zone PSA, which should be confirmed in future clinical studies.
Smoking is a major public health problem, but the genetic factors associated with smoking behaviors are not fully elucidated. Here, we have conducted an integrated genome-wide association study to identify common copy number polymorphisms (CNPs) and single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) in Japanese smokers ( = 17,158). Our analysis identified a common CNP with a strong effect on CPD (rs8102683; ) in the 19q13 region, encompassing the CYP2A6 locus. After adjustment for the associated CNP, we found an additional associated SNP (rs11878604; ) located 30 kb downstream of the CYP2A6 gene. Imputation of the CYP2A6 locus revealed that haplotypes underlying the CNP and the SNP corresponded to classical, functional alleles of CYP2A6 gene that regulate nicotine metabolism and explained 2% of the phenotypic variance of CPD (ANOVA -test ). These haplotypes were also associated with smoking-related diseases, including lung cancer, chronic obstructive pulmonary disease and arteriosclerosis obliterans.
MHC class I polypeptide-related chain A (MICA) molecule is induced in response to viral infection and various types of stress. We recently reported that a single nucleotide polymorphism (SNP) rs2596542 located in the MICA promoter region was significantly associated with the risk for hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) and also with serum levels of soluble MICA (sMICA). In this study, we focused on the possible involvement of MICA in liver carcinogenesis related to hepatitis B virus (HBV) infection and examined correlation between the MICA polymorphism and the serum sMICA levels in HBV-induced HCC patients. The genetic association analysis revealed a nominal association with an SNP rs2596542; a G allele was considered to increase the risk of HBV-induced HCC (P = 0.029 with odds ratio of 1.19). We also found a significant elevation of sMICA in HBV-induced HCC cases. Moreover, a G allele of SNP rs2596542 was significantly associated with increased sMICA levels (P = 0.009). Interestingly, HCC patients with the high serum level of sMICA (>5 pg/ml) exhibited poorer prognosis than those with the low serum level of sMICA (≤5 pg/ml) (P = 0.008). Thus, our results highlight the importance of MICA genetic variations and the significance of sMICA as a predictive biomarker for HBV-induced HCC.
Rheumatoid arthritis is an autoimmune disease with a complex etiology, leading to inflammation of synovial tissue and joint destruction. Through a genome-wide association study (GWAS) and two replication studies in the Japanese population (7,907 cases and 35,362 controls), we identified two gene loci associated with rheumatoid arthritis susceptibility (NFKBIE at 6p21.1, rs2233434, odds ratio (OR) = 1.20, P = 1.3×10−15; RTKN2 at 10q21.2, rs3125734, OR = 1.20, P = 4.6×10−9). In addition to two functional non-synonymous SNPs in NFKBIE, we identified candidate causal SNPs with regulatory potential in NFKBIE and RTKN2 gene regions by integrating in silico analysis using public genome databases and subsequent in vitro analysis. Both of these genes are known to regulate the NF-κB pathway, and the risk alleles of the genes were implicated in the enhancement of NF-κB activity in our analyses. These results suggest that the NF-κB pathway plays a role in pathogenesis and would be a rational target for treatment of rheumatoid arthritis.
Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting approximately 1% of the general adult population. More than 30 susceptibility loci for RA have been identified through genome-wide association studies (GWAS), but the disease-causal variants at most loci remain unknown. Here, we performed replication studies of the candidate loci of our previous GWAS using Japanese cohorts and identified variants in NFKBIE and RTKN2 gene loci that were associated with RA. To search for causal variants in both gene regions, we first examined non-synonymous (ns)SNPs that alter amino-acid sequences. As NFKBIE and RTKN2 are known to be involved in the NF-κB pathway, we evaluated the effects of nsSNPs on NF-κB activity. Next, we screened in silico variants that may regulate gene transcription using publicly available epigenetic databases and subsequently evaluated their regulatory potential using in vitro assays. As a result, we identified multiple candidate causal variants in NFKBIE (2 nsSNPs and 1 regulatory SNP) and RTKN2 (2 regulatory SNPs), indicating that our integrated in silico and in vitro approach is useful for the identification of causal variants in the post–GWAS era.
Multiple genetic loci associated with obesity or body mass index (BMI) have been identified through genome-wide association studies conducted predominantly in populations of European ancestry. We conducted a meta-analysis of associations between BMI and approximately 2.4 million SNPs in 27,715 East Asians, followed by in silico and de novo replication in 37,691 and 17,642 additional East Asians, respectively. We identified ten BMI-associated loci at the genome-wide significance level (P<5.0×10−8), including seven previously identified loci (FTO, SEC16B, MC4R, GIPR/QPCTL, ADCY3/RBJ, BDNF, and MAP2K5) and three novel loci in or near the CDKAL1,PCSK1, and GP2 genes. Three additional loci nearly reached the genome-wide significance threshold, including two previously identified loci in the GNPDA2 and TFAP2B genes and a new locus near PAX6, which all had P<5.0×10−7. Findings from this study may shed light on new pathways involved in obesity and demonstrate the value of conducting genetic studies in non-European populations.
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV1) before and after the administration of a short-acting β2-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of β2-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV1 after administration of a β2-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased β2-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of β2-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to β2-agonists through GWAS.
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function before and after the administration of short-acting β2-agonists, common medications used for asthma treatment. We performed a genome-wide association study of BDR with 1,644 white asthmatic subjects from six drug clinical trials and attempted to replicate these findings in 1,051 white subjects from two independent cohorts. The most significant associated variant was near the SPATS2L gene. We knocked down SPATS2L mRNA in human airway smooth muscle cells and found that β2-adrenergic receptor levels increased, suggesting that SPATS2L may be a regulator of BDR. Our results highlight the promise of pursuing GWAS results that do not necessarily reach genome-wide significance and are an example of how results from pharmacogenetic GWAS can be studied functionally.
We performed a genome-wide association study (GWAS) on levels of serum total protein (TP), albumin (ALB), and non-albumin protein (NAP). We analyzed SNPs on autosomal chromosomes using data from 9,103 Japanese individuals, followed by a replication study of 1,600 additional individuals. We confirmed the previously- reported association of GCKR on chromosome 2p23.3 with serum ALB (rs1260326, Pmeta = 3.1×10−9), and additionally identified the significant genome-wide association of rs4985726 in TNFRSF13B on 17p11.2 with both TP and NAP (Pmeta = 1.2×10−14 and 7.1×10−24, respectively). For NAP, rs3803800 and rs11552708 in TNFSF13 on 17p13.1 (Pmeta = 7.2×10−15 and 7.5×10−10, respectively) as well as rs10007186 on 4q21.2 near ANXA3 (Pmeta = 1.3×10−9) also indicated significant associations. Interestingly, TNFRSF13B and TNFSF13 encode a tumor necrosis factor (TNF) receptor and its ligand, which together constitute an important receptor-ligand axis for B-cell homeostasis and immunoglobulin production. Furthermore, three SNPs, rs4985726, rs3803800, and rs11552708 in TNFRSF13B and TNFSF13, were indicated to be associated with serum levels of IgG (P<2.3×10−3) and IgM (P<0.018), while rs3803800 and rs11552708 were associated with IgA (P<0.013). Rs10007186 in 4q21.2 was associated with serum levels of IgA (P = 0.036), IgM (P = 0.019), and IgE (P = 4.9×10−4). Our results should add interesting knowledge about the regulation of major serum components.
Arthralgias and myalgias are major side effects associated with aromatase inhibitor (AI) therapy of breast cancer. In a recent genome-wide association study, we identified SNPs - including one that created an estrogen response element near the 3' end of the T-cell leukemia 1A (TCL1A) gene - that were associated with musculoskeletal pain in women on adjuvant AI therapy for breast cancer. We also showed estrogen-dependent, SNP-modulated variation in TCL1A expression and, in preliminary experiments, showed that TCL1A could induce IL-17RA expression. In the present study, we set out to determine whether these SNPs might influence cytokine expression and effect more widely, and, if so, to explore the mechanism of TCL1A-related AI-induced side effects.
The functional genomic experiments performed included determinations of TCL1A, cytokine and cytokine receptor expression in response to estrogen treatment of U2OS cells and lymphoblastoid cell lines that had been stably transfected with estrogen receptor alpha. Changes in mRNA and protein expression after gene knockdown and overexpression were also determined, as was NF-κB transcriptional activity.
Estradiol (E2) increased TCL1A expression and, in a TCL1A SNP-dependent fashion, also altered the expression of IL-17, IL-17RA, IL-12, IL-12RB2 and IL-1R2. TCL1A expression was higher in E2-treated lymphoblastoid cell lines with variant SNP genotypes, and induction of the expression of cytokine and cytokine receptor genes was mediated by TCL1A. Finally, estrogen receptor alpha blockade with ICI-182,780 in the presence of E2 resulted in greatly increased NF-κB transcriptional activity, but only in cells that carried variant SNP genotypes. These results linked variant TCL1A SNP sequences that are associated with AI-dependent musculoskeletal pain with increased E2-dependent TCL1A expression and with downstream alterations in cytokine and cytokine receptor expression as well as NF-κB transcriptional activity.
SNPs near the 3' terminus of TCL1A were associated with AI-dependent musculoskeletal pain. E2 induced SNP-dependent TCL1A expression, which in turn altered IL-17, IL-17RA, IL-12, IL-12RB2, and IL-1R2 expression as well as NF-κB transcriptional activity. These results provide a pharmacogenomic explanation for a clinically important adverse drug reaction as well as insights into a novel estrogen-dependent mechanism for the modulation of cytokine and cytokine receptor expression.