Will the real disease gene please stand up?

A common dilemma arising in linkage studies of complex genetic diseases is the selection of positive signals, their follow-up with association studies and discrimination between true and false positive results. Several strategies for overcoming these issues have been devised. Using the Genetic Analysis Workshop 14 simulated dataset, we aimed to apply different analytical approaches and evaluate their performance in discerning real associations. We considered a) haplotype analyses, b) different methods adjusting for multiple testing, c) replication in a second dataset, and d) exhaustive genotyping of all markers in a sufficiently powered, large sample group. We found that haplotype-based analyses did not substantially improve over single-point analysis, although this may reflect the low levels of linkage disequilibrium simulated in the datasets provided. Multiple testing correction methods were in general found to be over-conservative. Replication of nominally positive results in a second dataset appears to be less stringent, resulting in the follow-up of false positives. Performing a comprehensive assay of all markers in a large, well-powered dataset appears to be the most effective strategy for complex disease gene identification

Two-Stage Two-Locus Models in Genome-Wide Association

Studies in model organisms suggest that epistasis may play an important role in the etiology of complex diseases and traits in humans. With the era of large-scale genome-wide association studies fast approaching, it is important to quantify whether it will be possible to detect interacting loci using realistic sample sizes in humans and to what extent undetected epistasis will adversely affect power to detect association when single-locus approaches are employed. We therefore investigated the power to detect association for an extensive range of two-locus quantitative trait models that incorporated varying degrees of epistasis. We compared the power to detect association using a single-locus model that ignored interaction effects, a full two-locus model that allowed for interactions, and, most important, two two-stage strategies whereby a subset of loci initially identified using single-locus tests were analyzed using the full two-locus model. Despite the penalty introduced by multiple testing, fitting the full two-locus model performed better than single-locus tests for many of the situations considered, particularly when compared with attempts to detect both individual loci. Using a two-stage strategy reduced the computational burden associated with performing an exhaustive two-locus search across the genome but was not as powerful as the exhaustive search when loci interacted. Two-stage approaches also increased the risk of missing interacting loci that contributed little effect at the margins. Based on our extensive simulations, our results suggest that an exhaustive search involving all pairwise combinations of markers across the genome might provide a useful complement to single-locus scans in identifying interacting loci that contribute to moderate proportions of the phenotypic variance

GLIDERS - A web-based search engine for genome-wide linkage disequilibrium between HapMap SNPs

<p>Abstract</p> <p>Background</p> <p>A number of tools for the examination of linkage disequilibrium (LD) patterns between nearby alleles exist, but none are available for quickly and easily investigating LD at longer ranges (>500 kb). We have developed a web-based query tool (GLIDERS: Genome-wide LInkage DisEquilibrium Repository and Search engine) that enables the retrieval of pairwise associations with r²≥ 0.3 across the human genome for any SNP genotyped within HapMap phase 2 and 3, regardless of distance between the markers.</p> <p>Description</p> <p>GLIDERS is an easy to use web tool that only requires the user to enter rs numbers of SNPs they want to retrieve genome-wide LD for (both nearby and long-range). The intuitive web interface handles both manual entry of SNP IDs as well as allowing users to upload files of SNP IDs. The user can limit the resulting inter SNP associations with easy to use menu options. These include MAF limit (5-45%), distance limits between SNPs (minimum and maximum), r²(0.3 to 1), HapMap population sample (CEU, YRI and JPT+CHB combined) and HapMap build/release. All resulting genome-wide inter-SNP associations are displayed on a single output page, which has a link to a downloadable tab delimited text file.</p> <p>Conclusion</p> <p>GLIDERS is a quick and easy way to retrieve genome-wide inter-SNP associations and to explore LD patterns for any number of SNPs of interest. GLIDERS can be useful in identifying SNPs with long-range LD. This can highlight mis-mapping or other potential association signal localisation problems.</p

Linkage Disequilibrium Mapping via Cladistic Analysis of Single-Nucleotide Polymorphism Haplotypes

We present a novel approach to disease-gene mapping via cladistic analysis of single-nucleotide polymorphism (SNP) haplotypes obtained from large-scale, population-based association studies, applicable to whole-genome screens, candidate-gene studies, or fine-scale mapping. Clades of haplotypes are tested for association with disease, exploiting the expected similarity of chromosomes with recent shared ancestry in the region flanking the disease gene. The method is developed in a logistic-regression framework and can easily incorporate covariates such as environmental risk factors or additional unlinked loci to allow for population structure. To evaluate the power of this approach to detect disease-marker association, we have developed a simulation algorithm to generate high-density SNP data with short-range linkage disequilibrium based on empirical patterns of haplotype diversity. The results of the simulation study highlight substantial gains in power over single-locus tests for a wide range of disease models, despite overcorrection for multiple testing

An Evaluation of the Performance of Tag SNPs Derived from HapMap in a Caucasian Population

The Haplotype Map (HapMap) project recently generated genotype data for more than 1 million single-nucleotide polymorphisms (SNPs) in four population samples. The main application of the data is in the selection of tag single-nucleotide polymorphisms (tSNPs) to use in association studies. The usefulness of this selection process needs to be verified in populations outside those used for the HapMap project. In addition, it is not known how well the data represent the general population, as only 90–120 chromosomes were used for each population and since the genotyped SNPs were selected so as to have high frequencies. In this study, we analyzed more than 1,000 individuals from Estonia. The population of this northern European country has been influenced by many different waves of migrations from Europe and Russia. We genotyped 1,536 randomly selected SNPs from two 500-kbp ENCODE regions on Chromosome 2. We observed that the tSNPs selected from the CEPH (Centre d'Etude du Polymorphisme Humain) from Utah (CEU) HapMap samples (derived from US residents with northern and western European ancestry) captured most of the variation in the Estonia sample. (Between 90% and 95% of the SNPs with a minor allele frequency of more than 5% have an r (2) of at least 0.8 with one of the CEU tSNPs.) Using the reverse approach, tags selected from the Estonia sample could almost equally well describe the CEU sample. Finally, we observed that the sample size, the allelic frequency, and the SNP density in the dataset used to select the tags each have important effects on the tagging performance. Overall, our study supports the use of HapMap data in other Caucasian populations, but the SNP density and the bias towards high-frequency SNPs have to be taken into account when designing association studies

Evaluation and monitoring of terrestrial and aquatic insect biodiversity in forested and cleared watersheds at Camp Atterbury, Indiana.

Executive Summary Camp Atterbury is a 33,132 ha military installation near Edinburgh, Indiana. Construction of a 80 ha (4,550 ha with safety fan) Multi-Purpose Training Range (MPTR) began in 1998, and supports training for military vehicles and dismounted infantry, with a variety of stationary and moving targets. This study provides a baseline for long term monitoring and evaluation of natural communities to assess the impacts of construction of, and training in, the MPTR. We assessed both aquatic macroinvertebrate and terrestrial insect community diversity, abundance, and richness and similarity at a series of study plots using quantifiable, repeatable and replicated methods. These data provide baseline data facilitating long-term monitoring and assessment as a measure of ecosystem health, and allow evaluation of relationships between community composition and habitat metrics. Methods Eight terrestrial study sites, each comprised of a 30 m square plot, were randomly selected, with four of these placed in the cleared portions of the MPTR and four placed in adjacent upland forest. We used several sampling methods, with focus on three groups of taxa (all insect taxa, ants, and leafhoppers and kin) and compared the efficacy of both the methods and the groups as monitoring tools. Sampling methods included: 1) a Malaise trap (mesh tent-like device that captures flying insects) at each site; 2) four sweep sample transects at each site; 3) four leaf litter samples from each site, with invertebrates extracted using the Winkler method; and 4) Nine pitfall traps at each site. Samples were collect during Summer and Fall study periods, and this report gives results from the Summer sample period. Several habitat parameters were recorded, including a vegetation index, canopy cover, ground cover, and leaf litter depth. Dominant plant taxa were collected, and data loggers recorded soil and air temperature during the study. We sampled aquatic macroinvertebrates at three stream sites draining the MPTR. Invertebrates were collected in replicate samples with a dipnet and these were sorted and subsampled in the laboratory. Canopy cover and basic water chemistry data were collected, and data loggers recorded changes in terrestrial and aquatic temperature. An index of biotic integrity and taxon richness were used to evaluate the aquatic communities. Results and Discussion At least 409 taxa and 3776 specimens were collected at terrestrial sample sites during the Summer sampling period. In general, there were some differences among sites, among sampling methods, and among treatments (cleared MPTR versus forested) when we examined taxon richness and species diversity, but these differences could not always be fully resolved. While taxon richness and species diversity differed among treatments, and, in general, plots in the two treatments harbored different insect communities. Species accumulation curves and various estimators of taxon richness were used to evaluate the four sampling methods and the three groups of taxa (all taxa, ants, leafhoppers). Based on the performance of the different taxa (all, ants, leafhoppers) compared across the different methods (malaise sampling, Winkler extracted leaf litter samples, pitfall traps, and sweep samples), the single most effective taxon for monitoring was found to be the ants (Formicidae), and the single best method for monitoring was found to be pitfall trapping. We collected 818 specimens, primarily aquatic macroinvertebrates, from the three stream sites during Summer sampling. All three streams were dry during the fall sample period, and thus no aquatic macroinvertebrates were collected. Using Hilsenhoff’s (1988) family-level index of biotic integrity, water quality was classified as “good” at one site, and “fair” at the other two, although taxon richness was lowest at the site classified as good. In addition to invertebrates, numerous salamanders (Eurycea cirrigera, the Two-lined Salamander) were observed in the streams. 3 For aquatic invertebrates, we found that the small upstream portions that directly drained the MPTR only held water seasonally, and thus were not effective sites for monitoring of stream macroinvertebrates. There was insufficient separation between MPTR-influenced stream sites and control sites, and a lack of replication (few streams flowing away from the MPTR) precluded robust statistical analysis of the data we did obtain. The community of aquatic macroinvertebrates collected during this study appeared similar to the communities reported by Robinson (2004) elsewhere at Camp Atterbury in larger streams, and includes taxa typical of rocky bottom Midwestern forest streams. Fish were largely absent due to the intermittent nature of the streams. Salamanders were abundant in the streams, and because they are top predators in this seasonal habitat, they may be suitable subjects for studies of potential bioaccumulation of toxins. This study provides a snapshot of insect biodiversity at a point in time, thus providing baseline for any possible future monitoring of insect biodiversity. Sampling methods and analyses developed in this study could easily be implemented at a wide variety of other military installations to facilitate inventory and/or monitoring of insect biodiversity.Ope

Clustered Environments and Randomized Genes: A Fundamental Distinction between Conventional and Genetic Epidemiology

In a cross-sectional study Davey Smith and colleagues show why observational studies can produce misleading claims regarding potential causal factors for disease, and illustrate the use of mendelian randomization to study environmentally modifiable risk factors

Interleukin-18 as a drug repositioning opportunity for inflammatory bowel disease: A Mendelian randomization study

Abstract: Support from human genetics increases the probability of success in drug development. However, few examples exist of successful genomically-driven drug repositioning. Given that a Mendelian form of severe enterocolitis is due to up-regulation of the interleukin-18 (IL18) signaling pathway, and pharmacologic inhibition of IL18 has been shown to reverse this enterocolitis, we undertook a Mendelian randomization study to test the causal effect of elevated IL18 levels on inflammatory bowel disease susceptibility (IBD) in 12,882 cases and 21,770 controls. Mendelian randomization is an established method to assess the role of biomarkers in disease etiology in a manner that minimizes confounding and prevents reverse causation. Using three SNPs that explained almost 7% of the variance in IL18 level, we found that each genetically predicted standard deviation increase in IL18 was associated with an increase in IBD susceptibility (odds ratio = 1.22, 95% CI = 1.11–1.34, P-value = 6 × 10−5). This association was further validated in 25,042 IBD cases and 34,915 controls (odds ratio = 1.13, 95% CI = 1.05–1.20). Recently, an anti-IL18 monoclonal antibody, which decreased free IL18 levels, was found to be safe, yet ineffective in a phase II trial for type 2 diabetes. Taken together, these genomic findings implicated IBD as an alternative indication for anti-IL18 therapy, which should be tested in randomized controlled trials

Lymphotoxin-α Gene and Risk of Myocardial Infarction in 6,928 Cases and 2,712 Controls in the ISIS Case-Control Study

Lymphotoxin-α (LTA) is a pro-inflammatory cytokine that plays an important role in the immune system and local inflammatory response. LTA is expressed in atherosclerotic plaques and has been implicated in the pathogenesis of atherosclerosis and coronary heart disease (CHD). Polymorphisms in the gene encoding lymphotoxin-α (LTA) on Chromosome 6p21 have been associated with susceptibility to CHD, but results in different studies appear to be conflicting. We examined the association of seven single nucleotide polymorphisms (SNPs) across the LTA gene, and their related haplotypes, with risk of myocardial infarction (MI) in the International Study of Infarct Survival (ISIS) case-control study involving 6,928 non-fatal MI cases and 2,712 unrelated controls. The seven SNPs (including the rs909253 and rs1041981 SNPs previously implicated in the risk of CHD) were in strong linkage disequilibrium with each other and contributed to six common haplotypes. Some of the haplotypes for LTA were associated with higher plasma concentrations of C-reactive protein (p = 0.004) and lower concentrations of albumin (p = 0.023). However, none of the SNPs or related haplotypes were significantly associated with risk of MI. The results of the ISIS study were considered in the context of six previously published studies that had assessed this association, and this meta-analysis found no significant association with CHD risk using a recessive model and only a modest association using a dominant model (with narrow confidence intervals around these risk estimates). Overall, these studies provide reliable evidence that these common polymorphisms for the LTA gene are not strongly associated with susceptibility to coronary disease