Genome Association Scanning Research Articles

In Silico Whole Genome Association Scan for Murine Prepulse Inhibition

BackgroundThe complex trait of prepulse inhibition (PPI) is a sensory gating measure related to schizophrenia and can be measured in mice. Large-scale public repositories of inbred mouse strain genotypes and phenotypes such as PPI can be used to detect Quantitative Trait Loci (QTLs) in silico. However, the method has been criticized for issues including insufficient number of strains, not controlling for false discoveries, the complex haplotype structure of inbred mice, and failing to account for genotypic and phenotypic subgroups.Methodology/Principal FindingsWe have implemented a method that addresses these issues by incorporating phylogenetic analyses, multilevel regression with mixed effects, and false discovery rate (FDR) control. A genome-wide scan for PPI was conducted using over 17,000 single nucleotide polymorphisms (SNPs) in 37 strains phenotyped. Eighty-nine SNPs were significant at a false discovery rate (FDR) of 5%. After accounting for long-range linkage disequilibrium, we found 3 independent QTLs located on murine chromosomes 1 and 13. One of the PPI positives corresponds to a region of human chromosome 6p which includes DTNBP1, a gene implicated in schizophrenia. Another region includes the gene Tsn which alters PPI when knocked out. These genes also appear to have correlated expression with PPI.Conclusions/SignificanceThese results support the usefulness of using an improved in silico mapping method to identify QTLs for complex traits such as PPI which can be then be used for to help identify loci influencing schizophrenia in humans.

The role of the CD58 locus in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system associated with demyelination and axonal loss. A whole genome association scan suggested that allelic variants in the CD58 gene region, encoding the costimulatory molecule LFA-3, are associated with risk of developing MS. We now report additional genetic evidence, as well as resequencing and fine mapping of the CD58 locus in patients with MS and control subjects. These efforts identify a CD58 variant that provides further evidence of association with MS (P = 1.1 x 10(-6), OR 0.82) and the single protective effect within the CD58 locus is captured by the rs2300747(G) allele. This protective rs2300747(G) allele is associated with a dose-dependent increase in CD58 mRNA expression in lymphoblastic cell lines (P = 1.1 x 10(-10)) and in peripheral blood mononuclear cells from MS subjects (P = 0.0037). This protective effect of enhanced CD58 expression on circulating mononuclear cells in patients with MS is supported by finding that CD58 mRNA expression is higher in MS subjects during clinical remission. Functional investigations suggest a potential mechanism whereby increases in CD58 expression, mediated by the protective allele, up-regulate the expression of transcription factor FoxP3 through engagement of the CD58 receptor, CD2, leading to the enhanced function of CD4(+)CD25(high) regulatory T cells that are defective in subjects with MS.

The emergence of whole genome association scans in barley

Barley geneticists are currently using association genetics to identify and fine map traits directly in elite plant breeding material. This has been made possible by the development of a highly parallel SNP assay platform that provides sufficient marker density for genome-wide scans and linkage disequilibrium-led gene identification. By leveraging the combined resources of the barley research and breeding sectors, marker-trait associations are being identified and a renewed interest has emerged in novel strategies for barley improvement. New database and visualization tools have been developed and statistical methods adapted from human genetics to account for complexities in the datasets. Exciting early results suggest that association genetics will assume a central role in establishing genotype-to-phenotype relationships.

Exploration of the Genetic Basis of GVHD by Genetic Association Studies

Graft-versus-host disease (GVHD), as well as graft-versus-leukemia effect (GVL), are essentially allo-immune reactions, which are induced by the engrafted donor T cells that recognize the host-derived allo-antigens presented on their targets (Figure 1). In HLA-matched transplantation, these antigens are called minor histocompatibility antigens (mHags), and are typically defined by the host single nucleotide polymorphisms (SNPs) that are not shared by the donor and therefore considered to be genetically mismatched between the donor and the recipient [1–3]. Thus, the development of both allo-reactions absolutely depends on the presence of 1 or more mismatched mHags, although these reactions could be further modified by other genetic as well as environmental factors, including, cytokine polymorphisms and GVHD prophylaxis. So, in view of better preventing GVHD and specifically targeting allo-immunity to the tumor component, central questions are what mHAgs are responsible for the development of GVHD or GVL and what genetic factors can influence the overall reactions, which are the plausible targets of genome-wide association studies (GWAS) [4-8]. To identify the genetic basis of GVHD, we conducted GWAS by genotyping more than 500,000

Genome-Wide Association Studies of Genetic Incompatibility That Is Relevant to the Development of GvHD in Unrelated Bone Marrow Transplantation

Allogeneic stem cell transplantation (allo-SCT) is one of the most effective therapeutic options for blood cell cancers. While its major anti-leukemic benefits are obtained from allo-immune reactions against leukemic cells, or GVL, the same kind of allo-reactions could be also directed to normal host tissues, giving rise to a severe complication, know as graft versus host disease (GvHD). In HLA-matched transplantation, the development of both reactions absolutely depends on the presence of one or more mismatched minor histocompatibility antigens (mHAgs) and could be further modified by other genetic as well as environmental factors, including for example, cytokine polymorphisms and GvHD prophylaxis. Thus, in view of better preventing GvHD and specifically targeting allo-immunity to the tumor component, it is critical to understand what mHAgs are mismatched and responsible for the development of GVHD or GVL and what genetic factors can influence the overall reactions. To address these questions, we conducted whole genome association studies by genotyping more than 500,000 SNPs in donors and recipients of 1598 unrelated transplants from Japan Marrow Donation Program (JMDP). All transplants were matched for HLA-A, B, C, DRB1 and DQB1, while 1033 (63%) transplants were mismatched for HLA-DPB1. 656 (41.7%) and 245 (14.9%) of transplants had developed grade II–IV and III–IV of acute GvHD (aGvHD), respectively. Overall call rates exceeded 98% both in donors and in recipients. Unobserved HapMap PhaseII SNPs were rigorously imputed using genotyped SNPs. After excluding those SNPs showing <95% call rate, deviation from Hardy-Weinberg equilibrium, or <5% minor allele frequency, 1,276,699 SNPs were tested for association with development of acute and chronic GvHD, relapse, and overall survival, by calculating LogRank statistics for each SNP according to single genotypes in donors and recipients or based on mismatch in genotypes between donor and recipient. Statistical thresholds for genome-wide-P value of 0.05 were determined empirically by doing 1,000 permutations for each analysis. In the analysis of mismatched genotypes, SNPs around the HLA-DPB1 locus uniquely showed a strong association with the development of >grade II aGvHD with the maximum P-value of 1.81 × 10−9 at rs6937034, and thus, successfully captured the association of DPB1 allele mismatch as directly defined by HLA typing (HR = 1.91, P= 2.88 × 10−13). To facilitate the identification of target mHAgs for aGvHD, we performed subgroup analysis, where association tests were confined to those transplants sharing particular HLA types based on the fact that recognition of mHAgs is restricted to particular HLA contexts (HLA restriction). Six loci was identified as candidate mHAg loci whose mismatch may confer increased risk for development of aGvHD. These included rs17473423 on chr12 associated with an A*2402/B*5201/Cw*1202/DRB1*1501/DQB1*0601 allele set shared in ~40% of unrelated transplants in Japanese (grade III–IV aGvHD with maximum P=3.99 × 10−13), rs9657655 on chr9 associated with another common allele in Japanese, A*3303/B*4403/Cw*1403 (grade III–IV aGvHD with maximum P=8.56 × 10−10), and other four loci associated with DQB1*0501, Cw*0102, B*5201, and Cw*1202. Two SNPs in patients were also found to be associated with aGvHD, rs5998746 on chr22 (P=3.41 × 10−8) and rs11873016 on chr18 (P=1.26 × 10−8), although no donor SNPs showed significant associations). Similarly, we identified four candidate SNPs associated with the development of severe cGvHD or relapse. Current study provided a unique opportunity in that combination of two different genotypes, not merely genotypes of single individuals, that is associated with particular disease phenotypes, is explored by whole genome association scans. Although further replication studies and biological confirmation are required, our results suggest that whole genome association studies of allo-SCT could provide a novel clue to understanding the genetic basis of allo-SCT.

Impact of Highly Conserved HLA Haplotype on Acute Graft-Versus-Host Disease in Unrelated Bone Marrow Transplantation

[INTRODUCTION] Although the effect of HLA locus and/or HLA antigen matching on clinical outcome of unrelated bone marrow transplantation (UR-BMT) has been well characterized, the effect of HLA haplotype (HP) itself is little known. The large scale genotyping of HLA-A, B, C, DRB1, DQB1, DPB1 alleles and SNPs among HLA region by whole genome association scans (WGAS) made it possible to ellucidate conserved common HPs and their impact to acute GVHD in HLA allele matched Japanese patient-donor pairs through Japan Marrow Donor Program.[PATIENTS AND METHODS] 5210 patients who received T cell replete marrow were retrospectively typed for HLA-A, B, C, DRB1, DQB1 and DPB1 allele, and 1810 pairs performed WGAS using Affimetrics 500k array. HLA allele fullmatch was found in 712 pairs, of whom 451 pairs were genotyped 912 SNPs between HLA-A and HLA-DPB2 (from nucleotide 29989443 to 33205942, 3.2Mb). 45 common HLA-A, B, C, DRB1, DQB1, DPB1 HPs among Japanese (designated as HP-P1 to HP-P45) was determined according to the combination of HLA alleles. The probabilities of acute GVHD (grade 2–4) were estimated using the cumulative incidence function, where death without acute GVHD was regarded as a competing risk. Hazard risk (HR) of grade 2–4 acute GVHD was assesed by multivariable Cox regression analysis adjusting with 8 clincal factors.[RESULTS] 1) 220 pairs determined both HPs, 354 one HP, 64 no HP and 74 HP with ambiguility of HP. There was no difference of acute GVHD between these 4 groups (26.7%, 33.2%, 28.6%, 35.8%, respectively). And that, donor and recipient mismatching rate of SNPs was not associated with acute GVHD; less than 5 mismatch SNPs (n=126) showed 32.2%, 6–50 (n=105) 32.4%, 51–100 (n=102) 27.5%, and more than 101 (n=118) 31.4%. 2) Most frequent HP (HP-P1: HLA- A*2402- Cw*1202- B*5201- DRB1*1502- DQB1*0601- DPB1*0901) was found in 331 pairs, and the second HP (HP-P2: HLA-A*3303- Cw*1403- B*4403- DRB1*1302- DQB1* 0604- DPB1*0401) in 111 pairs. 68 out of 72 homozygous HP-P1/P1 individuals had identical homozygous alleles for more than 98% of 912 consecutive SNPs. Of 10 HP-P2/P2 homozygous individuals, all individuals were identical homozygous alleles for 99% of 1310 consecutive SNPs extending from nucleotide 29001906 to 33987897 (4.9 Mb). Further more, 32 out of 34 heterozygous HP-P1/P2 individuals had more than 99% identical consecutive 912 alleles. 3) Considerable number of pairs with HP-P1 made it possible to compare another HPs for the risk of acute GVHD. Notably, 16.2% of acute GVHD in homozygous HP-P1/P1 pair (n=33) and 12.0% of acute GVHD in HP-P1/P2 pair (n=25) were significaitly lower than 39.1% in pair with HP-P1 and HP-P3-45 (n=246) (p=0.01), and HR was 0.41 (p=0.04) and 0.30 (p=0.04) respectively. 4) When analyzed an individual HP, patients with HP-P2 showed significant lower incidence of acute GVHD (22.3% n=111) than HP-P2 negative one (32.7% n=601) (p=0.031), and HR was 0.63 (p=0.032), and the other HPs did no significant difference.[CONCLUSIONS]Donor and recipient mismatching rate of SNPs in HLA region had no correlation with acute GVHD in HLA full matched UR-BMT in JMDP.Common HP-P1 and HP-P2 were highly conserved through HLA-A to HLA-DPB2.Patients with HP-P1/P1 and those with HP-P1/P2 showed lower incidence of acute GVHD, which is compatible with BMT from HLA identical sibling among Japanese.Specific HP (HP-P2)itself reduced the incidence of acute GVHD.These results contribute to favorable outcome of UR-BMT and to ellucidate the mechanism of acute GVHD.

Identification of human minor histocompatibility antigens based on genetic association with highly parallel genotyping of pooled DNA

AbstractMinor histocompatibility (H) antigens are the molecular targets of allo-immunity responsible both for the development of antitumor effects and for graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, despite their potential clinical use, our knowledge of human minor H antigens is largely limited by the lack of efficient methods of their characterization. Here we report a robust and efficient method of minor H gene discovery that combines whole genome association scans (WGASs) with cytotoxic T-lymphocyte (CTL) assays, in which the genetic loci of minor H genes recognized by the CTL clones are precisely identified using pooled-DNA analysis of immortalized lymphoblastoid cell lines with/without susceptibility to those CTLs. Using this method, we have successfully mapped 2 loci: one previously characterized (HMSD encoding ACC-6), and one novel. The novel minor H antigen encoded by BCL2A1 was identified within a 26 kb linkage disequilibrium block on chromosome 15q25, which had been directly mapped by WGAS. The pool size required to identify these regions was no more than 100 individuals. Thus, once CTL clones are generated, this method should substantially facilitate discovery of minor H antigens applicable to targeted allo-immune therapies and also contribute to our understanding of human allo-immunity.

Use of expression data and the CGEMS genome-wide breast cancer association study to identify genes that may modify risk in BRCA1/2 mutation carriers

Germline mutations in BRCA1 or BRCA2 confer an increased lifetime risk of developing breast or ovarian cancer, but variable penetrance suggests that cancer susceptibility is influenced in part by modifier genes. Microarray expression profiling was conducted for 69 irradiated lymphoblastoid cell lines derived from healthy controls, or from cancer-affected women with a strong family history of breast and ovarian cancer carrying pathogenic mutations in BRCA1 or BRCA2, or with no BRCA1/2 mutations (BRCAX). Genes discriminating between BRCA1, BRCA2 or BRCAX and controls were stratified based on irradiation response and/or cell cycle involvement. Gene lists were aligned against genes tagged with single nucleotide polymorphisms (SNPs) determined by the Cancer Genetic Markers of Susceptibility (CGEMS) Breast Cancer Whole Genome Association Scan to be nominally associated with breast cancer risk. Irradiation responsive genes whose expression correlated with BRCA1 and/or BRCA2 mutation status were more likely to be tagged by risk-associated SNPs in the CGEMS dataset (BRCA1, P = 0.0005; BRCA2, P = 0.01). In contrast, irradiation responsive genes correlating with BRCAX status were not enriched in the CGEMS dataset. Classification of expression data by involvement in cell cycle processes did not enrich for genes tagged by risk-associated SNPs, for BRCA1, BRCA2 or BRCAX groups. Using a novel combinatorial approach, we have identified a subset of irradiation responsive genes as high priority candidate BRCA1/2 modifier genes. Similar approaches may be used to identify genes and underlying genetic risk factors that interact with exogenous stimulants to cause or modify any disease, without a priori knowledge of the pathways involved.

Simulating association studies: a data-based resampling method for candidate regions or whole genome scans

Reductions in genotyping costs have heightened interest in performing whole genome association scans and in the fine mapping of candidate regions. Improvements in study design and analytic techniques will require the simulation of datasets with realistic patterns of linkage disequilibrium and allele frequencies for typed SNPs. We describe a general approach to simulate genotyped datasets for standard case-control or affected child trio data, by resampling from existing phased datasets. The approach allows for considerable flexibility in disease models, potentially involving a large number of interacting loci. The method is most applicable for diseases caused by common variants that have not been under strong selection, a class specifically targeted by the International HapMap project. Using the three population Phase I/II HapMap data as a testbed for our approach, we have implemented the approach in HAP-SAMPLE, a web-based simulation tool.

Familial correlations and gene‐environment interactions for myopia

Abstract Purpose: To investigate the combined contributions of environmental and genetic risk factors for myopia in children. Methods: Children aged 7 to 9 years from 3 schools were recruited and the children have been followed annually since 1999. Cycloplegic autorefraction and biometry measures were conducted annually in the schools, and buccal specimens were collected. TheWhole Genome Association (WGA) scans of buccal DNA using the Illumina Sentrix HumanHap 550 Genotyping BeadChip assay will be performed. We propose to compare the top ranked genes of 1250 children with greater change in refraction with children with the smallest changes in refraction. Results: There were 1478 Chinese, 349 Malays and 152 children who were Indian and other races. Using a Cox Proportional Hazards model, children with 2 myopic parents had 3.89 times higher risks of myopia, compared with children with 0 myopic parents. Parental history of myopia interacted with reading in books per week to increase the risks of myopia, suggesting gene‐environment interaction. There were 159 sibling pairs in this cohort. After adjusting for age and sex, the between‐sibling correlation in refractive error was 0.447 (95% confidence interval 0.314, 0.564) and between‐sibling 1‐year change in refractive error was high at 0.420 (95% confidence interval 0.282, 0.543). The pilot WGA study of matched sets of 5 unamplified buccal DNA samples, 5 amplified buccal DNA samples, and 5 saliva DNA samples using the Oragene saliva kit revealed mean call rates of 0.9986, 0.9881, and 0.9929, respectively. Conclusions: Thus, both the contributions of environment and genes will be documented in detail in this cohort.

Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits.

The obesity epidemic is responsible for a substantial economic burden in developed countries and is a major risk factor for type 2 diabetes and cardiovascular disease. The disease is the result not only of several environmental risk factors, but also of genetic predisposition. To take advantage of recent advances in gene-mapping technology, we executed a genome-wide association scan to identify genetic variants associated with obesity-related quantitative traits in the genetically isolated population of Sardinia. Initial analysis suggested that several SNPs in the FTO and PFKP genes were associated with increased BMI, hip circumference, and weight. Within the FTO gene, rs9930506 showed the strongest association with BMI (p = 8.6 ×10− 7), hip circumference (p = 3.4 × 10− 8), and weight (p = 9.1 × 10− 7). In Sardinia, homozygotes for the rare “G” allele of this SNP (minor allele frequency = 0.46) were 1.3 BMI units heavier than homozygotes for the common “A” allele. Within the PFKP gene, rs6602024 showed very strong association with BMI (p = 4.9 × 10− 6). Homozygotes for the rare “A” allele of this SNP (minor allele frequency = 0.12) were 1.8 BMI units heavier than homozygotes for the common “G” allele. To replicate our findings, we genotyped these two SNPs in the GenNet study. In European Americans (N = 1,496) and in Hispanic Americans (N = 839), we replicated significant association between rs9930506 in the FTO gene and BMI (p-value for meta-analysis of European American and Hispanic American follow-up samples, p = 0.001), weight (p = 0.001), and hip circumference (p = 0.0005). We did not replicate association between rs6602024 and obesity-related traits in the GenNet sample, although we found that in European Americans, Hispanic Americans, and African Americans, homozygotes for the rare “A” allele were, on average, 1.0–3.0 BMI units heavier than homozygotes for the more common “G” allele. In summary, we have completed a whole genome–association scan for three obesity-related quantitative traits and report that common genetic variants in the FTO gene are associated with substantial changes in BMI, hip circumference, and body weight. These changes could have a significant impact on the risk of obesity-related morbidity in the general population.

Gene discovery in diabetic nephropathy

Diabetic nephropathy affects approximately one third of diabetic patients and is the leading cause of end-stage renal disease. Identification of genetic susceptibility factors contributing to the development of diabetic nephropathy could facilitate prediction, development of improved treatments, and prevention of this devastating complication of diabetes. Although investigations to identify the causal genetic variants associated with diabetic nephropathy have been inconclusive, new approaches, including whole genome association scanning, offer hope for the future.

SNPs, Haplotypes, and Cancer: Applications in Molecular Epidemiology

SNPs, Haplotypes, and Cancer: Applications in Molecular Epidemiology

Linkage disequilibrium mapping identifies a 390 kb region associated with CYP2D6 poor drug metabolising activity.

The cytochrome p450 enzyme, CYP2D6, metabolises approximately 20% of marketed drugs. CYP2D6 multiple variants are associated with altered enzyme activities. Genotyping 1018 Caucasians for CYP2D6 polymorphisms (G1846A, delT1707, delA2549 and A2935C), known to result in the recessive CYP2D6 poor drug metaboliser (PM) phenotype, identified 41 individuals with predicted PM phenotype. These 41 individuals were classified as 'cases'. Single nucleotide polymorphisms (SNPs) mapping within an 880 kb region flanking CYP2D6, were identified to evaluate potential association between genetic variation and the CYP2D6 PM phenotype. The 41 PM cases and 977 controls were genotyped and analysed for 27 SNPs. Associations were observed across a 390 kb region between 14 SNPs and the PM phenotype (P values from 6.20 x 10(-4) to 4.54 x 10(-35)). Haplotype analysis revealed more significant levels of association (P = 3.54 x 10(-56)). Strong (D' > 0.7) linkage disequilibrium (LD) between SNPs was observed across the same 390 kb region associated with the CYP2D6 phenotype. The observed phenotype:genotype association reached genome-wide levels of significance, and supports the strategy for potential application of LD mapping and whole genome association scans to pharmacogenetic studies.