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The human proteome is a major source of therapeutic targets. Recent genetic association analyses of the plasma proteome enable systematic evaluation of the causal consequences of variation in plasma protein levels. Here, we estimated the effects of 1002 proteins on 225 phenotypes using two-sample Mendelian randomization (MR) and colocalization. Of 413 associations supported by evidence from MR, 130 (31.5%) were not supported by results of colocalization analyses, suggesting that genetic confounding due to linkage disequilibrium (LD) is widespread in naive phenome-wide association studies of proteins. Combining MR and colocalization evidence in cis-only analyses, we identified 111 putatively causal effects between 65 proteins and 52 disease-related phenotypes (www.epigraphdb.org/pqtl/). Evaluation of data from historic drug development programmes showed that target-indication pairs with MR and colocalization support were more likely to be approved, evidencing the value of our approach in identifying and prioritising potential therapeutic targets.

RORγt is an isoform of RORC, preferentially expressed in Th17 cells, that functions as a critical regulator of type 3 immunity. As murine Th17-driven inflammatory disease models were greatly diminished in RORC knock-out mice, this receptor was prioritised as an attractive therapeutic target for the treatment of several autoimmune diseases. Human genetic studies indicate a significant contributory role for RORC in several human disease conditions. Furthermore, genome-wide association studies (GWAS) report a significant association between inflammatory bowel disease (IBD) and the RORC regulatory variant rs4845604. To investigate if the rs4845604 variant may affect CD4+ T cell differentiation events, naïve CD4+ T cells were isolated from eighteen healthy subjects homozygous for the rs4845604 minor (A) or major (G) allele). Isolated cells from each subject were differentiated into distinct T cell lineages by culturing in either T cell maintenance medium or Th17 driving medium conditions for six days in the presence of an RORC inverse agonist (to prevent constitutive receptor activity) or an inactive diastereomer (control). Our proof of concept study indicated that genotype had no significant effect on the mean number of naïve CD4 T cells isolated, nor the frequency of Th1-like and Th17-like cells following six days of culture in any of the four culture conditions. Analysis of the derived RNA-seq count data identified genotype-driven transcriptional effects in each of the four culture conditions. Subsequent pathway enrichment analysis of these profiles reported perturbation of metabolic signalling networks, with the potential to affect the cellular detoxification response. This investigation reveals that rs4845604 genotype is associated with transcriptional effects in CD4+ T cells that may perturb immune and metabolic pathways. Most significantly, the rs4845604 GG, IBD risk associated, genotype may be associated with a differential detoxification response. This observation justifies further investigation in a larger cohort of both healthy and IBD-affected individuals.

Osteoarthritis is the most common musculoskeletal disease and the leading cause of disability globally. Here, we performed a genome-wide association study for osteoarthritis (77,052 cases and 378,169 controls), analyzing four phenotypes: knee osteoarthritis, hip osteoarthritis, knee and/or hip osteoarthritis, and any osteoarthritis. We discovered 64 signals, 52 of them novel, more than doubling the number of established disease loci. Six signals fine-mapped to a single variant. We identified putative effector genes by integrating expression quantitative trait loci (eQTL) colocalization, fine-mapping, and human rare-disease, animal-model, and osteoarthritis tissue expression data. We found enrichment for genes underlying monogenic forms of bone development diseases, and for the collagen formation and extracellular matrix organization biological pathways. Ten of the likely effector genes, including TGFB1 (transforming growth factor beta 1), FGF18 (fibroblast growth factor 18), CTSK (cathepsin K), and IL11 (interleukin 11), have therapeutics approved or in clinical trials, with mechanisms of action supportive of evaluation for efficacy in osteoarthritis.

A phenome-wide association study of variants in genes in the Th17 and IL-17 pathway was performed using self-reported phenotypes and genetic data from 521,000 research participants of 23andMe. Results replicated known associations with similar effect sizes for autoimmune traits illustrating self-reported traits can be a surrogate for clinically assessed conditions. Novel associations controlling for a false discovery rate of 5% included the association of the variant encoding p.Ile684Ser in TYK2 with increased risk of tonsillectomy, strep throat occurrences and teen acne, the variant encoding p.Arg381Gln in IL23R with a decrease in dandruff frequency, the variant encoding p.Asp10Asn in TRAF3IP2 with risk of male-pattern balding, and the RORC regulatory variant (rs4845604) with protection from allergies. This approach enabled rapid assessment of association with a wide variety of traits and investigation of traits with limited reported associations to overlay meaningful phenotypic context on the range of conditions being considered for drugs targeting this pathway.

The genome of the fission yeast, Schizosaccharomyces pombe, consists of some 14 million base pairs of DNA contained in three chromosomes. On account of its excellent genetics we used it as a test system for a strategy designed to map mammalian chromosomes and genomes. Data obtained from hybridization fingerprinting established an ordered library of 1,248 yeast artificial chromosome clones with an average size of 535 kilobases. The clones fall into three contigs completely representing the three chromosomes of the organism. This work provides a high resolution physical and clone map of the genome, which has been related to available genetic and physical map information.

Gridded on high density filters, a P1 genomic library of 17-fold coverage and a cosmid library of 8 genome equivalents, both made from S. pombe strain 972h-, were ordered by hybridizing genetic markers and individual clones from the two libraries. Yeast artificial chromosome (YAC) clones covering the entire genome were used to subdivide the libraries, and hybridization of short oligonucleotides and DNA pools made from randomly selected cosmids provided further mapping information. Restriction digests were generated as an independent confirmation of the clone order. The high resolution clone map was aligned to the genetic map and the physical Notl and YAC maps. The usefulness of the various mapping techniques and cloning procedures could be assessed upon the different data sets.

Migraine is a prevalent neurovascular disease with a significant genetic component. Linkage studies have so far identified migraine susceptibility loci on chromosomes 1, 4, 6, 11, 14, 19 and X. We performed a genome-wide scan of 92 Australian pedigrees phenotyped for migraine with and without aura and for a more heritable form of "severe" migraine. Multipoint non-parametric linkage analysis revealed suggestive linkage on chromosome 18p11 for the severe migraine phenotype (LOD*=2.32, P=0.0006) and chromosome 3q (LOD*=2.28, P=0.0006). Excess allele sharing was also observed at multiple different chromosomal regions, some of which overlap with, or are directly adjacent to, previously implicated migraine susceptibility regions. We have provided evidence for two loci involved in severe migraine susceptibility and conclude that dissection of the "migraine" phenotype may be helpful for identifying susceptibility genes that influence the more heritable clinical (symptom) profiles in affected pedigrees. Also, we concluded that the genetic aetiology of the common (International Headache Society) forms of the disease is probably comprised of a number of low to moderate effect susceptibility genes, perhaps acting synergistically, and this effect is not easily detected by traditional single-locus linkage analyses of large samples of affected pedigrees.

Linkage disequilibrium (LD) provides information about positional cloning, linkage, and evolution that cannot be inferred from other evidence, even when a correct sequence and a linkage map based on more than a handful of families become available. We present theory to construct an LD map for which distances are additive and population-specific maps are expected to be approximately proportional. For this purpose, there is only a modest difference in relative efficiency of haplotypes and diplotypes: resolving the latter into 2-locus haplotypes has significant cost or error and increases information by about 50%. LD maps for a cold spot in 19p13.3 and a more typical region in 3q21 are optimized by interval estimates. For a random sample and trustworthy map the value of LD at large distance can be predicted reliably from information over a small distance and does not depend on the evolutionary variance unless the sample size approaches the population size. Values of the association probability that can be distinguished from the value at large distance are determined not by population size but by time since a critical bottleneck. In these examples, omission of markers with significant Hardy–Weinberg disequilibrium does not improve the map, and widely discrepant draft sequences have similar estimates of the genetic parameters. The LD cold spot in 19p13.3 gives an unusually high estimate of time, supporting an argument that this relationship is general. As predicted for a region with ancient haplotypes or uniformly high recombination, there is no clear evidence of LD clustering. On the contrary, the 3q21 region is resolved into alternating blocks of stable and decreasing LD, as expected from crossover clustering. Construction of a genomewide LD map requires data not yet available, which may be complemented but not replaced by a catalog of haplotypes.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

982 progeny produced by a mouse Interspecific backcross between C57BL/6 and Mus spretus have been scored for at least 3 markers on each chromosome, completing an anchor map of 78 loci across the mouse genome. The anchor mapping identifies all the available recomblnants in each interanchor Interval allowing access to panels of mice that can be used for the high resolution mapping of any chromosome region. The large number of progeny recovered and scored from the Interspecific backcross allows us to resolve genetically markers that lie on average 200 kb apart on mouse chromosomes and within the cloning capacity of currently available YAC libraries. EUCIB provides the first genetic mapping resource specifically designed for the high resolution mapping of all regions of the mouse genome and will underpin the global physical mapping of the mouse genome. In addition, with the use of conserved sequences the facility is applicable to the high resolution comparative mapping of the mouse and human genomes. A new database has been implemented to support the computation of high resolution and ordered genetic maps. © 1994 Oxford University Press.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists are highly effective in the treatment of type 2 diabetes. In some patients, PPARgamma ligands are associated with fluid retention/oedema, for which the mechanism is not fully understood. A pharmacogenetic study was undertaken to investigate effects of variations in 21 candidate genes related to epithelial sodium channel (ENaC) pathways on oedema. This study used DNA samples collected from type 2 diabetes phase III clinical trials of the PPARgamma agonist farglitazar (administered alone or in combination with insulin or glyburide) and investigated oedema reported as an adverse event as phenotype. Initial case-control analysis of oedema identified candidate gene single nucleotide polymorphisms with significant associations. These included three polymorphisms in ENaCbeta subunit (SCNN1B) that showed significant associations (P0.05) with the two combination treatments in discrete regions of the gene, but not farglitazar treatment alone. Sequencing of SCNN1B in 207 Caucasian participants receiving farglitazar plus insulin or glyburide combination therapies, identified additional polymorphisms that were also significantly associated with oedema (P0.0005) and maintained the treatment-regional associations. Further covariate analysis accounting for clinical factors influencing oedema supported these observations. One of the SCNN1B polymorphisms, at position -405 of the 5' flanking region (rs34241435), was predicted to modify transcriptional interactions and in a transfected COS cell luciferase reporter gene assay exhibited higher promoter activity. These exploratory studies provide clinical pharmacogenetic and functional genomic evidence to support a pivotal role for ENaC regulation in PPARgamma-induced oedema and provide insight into mechanisms and possible management of this side effect.

BACKGROUND GW320659, a highly selective neuronal norepinephrine and dopamine re-uptake inhibitor, has been evaluated for the treatment of obesity. Scrutiny of the weight loss data from a phase II study (GlaxoSmithKline study OBS20001) showed a wide variation in weight loss response following GW320659 treatment and the possibility that the study population might include subgroups with enhanced weight loss response. METHODS Pharmacogenetic analysis was performed in 191 subjects prospectively ascertained from a Phase II dose ranging study to evaluate the influence of genotype on weight loss efficacy and safety of GW320659 in obese subjects. RESULTS Common genetic polymorphisms in the drug target (norepinephrine transporter protein 1, SLC6A2) and mechanism pathway (NMDA receptor channel NR1 subunit, GRIN1) were associated with increased weight loss following GW320659 treatment in a proportion (36%) of the study population. In the patient subgroup selected for these genotypes, GW320659 (15 mg/day) produced a significant difference in mean weight loss of 7.84 kg (SD 5.23, n = 14), compared to 2.53 kg (SD 5.17, n = 24) in the subgroup that did not possess the genotypes (P = 0.006). This subgroup also showed a highly significant weight loss response for GW320659 compared to placebo (+0.31 kg, SD 3.32, n = 16) with the same genotypes (P < 0.0001). In addition, there was no difference in placebo response between either subgroup. CONCLUSIONS Polymorphisms in SLC6A2 and GRIN1 could be used to maximize effective obesity pharmacotherapy by norepinephrine/dopamine transporter inhibitors by identifying patients that may be predisposed to particularly good treatment weight loss response.

A practical limitation to the identification of genetic profiles predictive of drug-induced adverse events is the number of patients with the adverse event that can be tolerated before the drug is withdrawn. Whole genome screening for regions of linkage disequilibrium (LD) associated with a particular phenotype may provide the mechanism to rapidly discover specific and sensitive profiles. We have used data from a large phase III clinical trial of tranilast and typed 76 SNPs over a 2.7 megabase region flanking the uridine diphosphate glucuronosyltranserferase 1A1 gene. Three SNPs within one LD block showed strong association with tranilast-induced hyperbilirubinemia (P

Genotyping data sets may contain errors that, in some instances, lead to false conclusions. Deviation from Hardy-Weinberg equilibrium (HWE) in random samples may be indicative of problematic assays. This study has analysed 107,000 genotypes generated by TaqMan, RFLP, sequencing or mass spectrometric methods from 443 single-nucleotide polymorphisms (SNPs). These SNPs are distributed both within genes and in intergenic regions. Genotype distributions for 36 out of 313 assays (11.5%) whose minor allele frequencies were >0.05 deviated from HWE (P

In recent years there has been increasing interest in mapping thehorse genome, particularly to identify disease and performance-enhancing genes. Although a number of horse mapping tools havebeen developed and have proved very useful (genetic maps, so-matic cell hybrid panels, and a BAC library), the benefits ofwhole-genome–radiation hybrid (WG-RH) mapping have not beenavailable. Its advantages over genetic linkage mapping are: (i) theresolving power is not lost in regions of the genome with a lowrecombination rate, because it does not rely on meiotic recombi-nation events; (ii) it is especially useful in animals such as thehorse with relatively long generation times and single births; and(iii) genetic and physical maps can be integrated, as both poly-morphic and non-polymorphic markers can be placed on the samemap. The WG-RH panel constructed in this study is the first suchpanel to be reported for the horse, and its preliminary character-ization demonstrates its usefulness for horse genome mapping.The panel was constructed by the fusion of horse embryonicendothelial primary lung cells (male) to the established hamsterfibroblast cell line A23 (Westerveld et al. 1971) by using themethod described in McCarthy et al. (1997). A series of fusions,involving irradiation (3000 rads) of donor cells prior to fusion withequal numbers of recipient cells, generated ∼160 hybrids in total.From these 160 hybrids, 94 were selected at random and screenedby using 20 widely distributed markers (representing 17 chromo-somes). Any hybrids that did not produce an amplification productwith these markers were screened by FISH to determine whetherthey contained horse DNA from other chromosomes or regions ofchromosomes (not represented by the 20 markers); hybrids foundto be negative by the FISH screen were replaced at random fromthe remaining unused hybrids, and the same PCR and FISH screen-ing procedure was repeated until 94 hybrids were assembled.These 94 hybrids were used for preliminary characterization as amapping panel. It is expected that the majority of the hybrids(>95%) characterized here will be represented in the TM99 panelof 94 hybrids that is being grown on a large scale by ResearchGenetics Inc. (Huntsville, Ala. 35801).The amount of horse DNA retained by the hybrids in the panelwas evaluated by examining the retention of the 20 widely dis-tributed horse markers. On average each marker was retained in27.8% of the hybrids (ranging from 10.6% to 71.3%; Table 1),implying that the panel as a whole retains the equivalent of ap-proximately 26 horse genomes. These retention frequencies com-pare well with those found for the human and mouse RH panels,which have been used successfully for creating whole-genomemaps (Gyapay et al. 1996; McCarthy et al. 1997).The mapping ability of the panel was assessed by producingRH maps for the two horse chromosomes with the most markersavailable, and comparing these with the latest genetic maps (Swin-burne et al. 2000a). In total, 39 markers on Chromosome (Chr) 1and 15 markers on Chr 10 were analyzed (Table 1). The averageretention of markers was 15.4% (ranging from 5.3% to 29.8%) onChr 1, and was higher, 25.4% (ranging from 16.0% to 44.7%) onChr 10. An increase in the retention frequency on smaller chro-mosomes was also noted in human and mouse RH panels (Gyapayet al. 1996; McCarthy et al. 1997). For each chromosome, linkagegroups with at least 4-LOD units support were identified; four suchgroups were found on Chr 1 and two were detected on Chr 10(Figs. 1 and 2). Within each of these linkage groups, frameworkmarkers were ordered with 3-LOD units support, and most of thenon-framework markers were ordered with 2-LOD units support.Some non-framework markers had lower statistical support fortheir order (shown in italics, Figs. 1 and 2). Similarly, the relativeorder of some linkage groups was suggested by linkage analysisbut had low statistical support [Chr 1 (groups B and C) and Chr 10(groups A and B)]. The relative order of the other RH linkagegroups was determined by comparison with the genetic map andFISH localizations.The genetic map can give a misleading impression of markerdensity because genetic distances may be small owing to regionshaving a low meiotic recombination rate. In such regions the mark-ers may actually be physically far apart and, therefore, at a lowerdensity than predicted by the genetic map. Since RH panels requirea high density of markers, this might account for the low statisticalsupport for the ordering of some markers and linkage groups. Alow density of available markers could also explain why two mark-ers (HLM5, ICA22) could not be placed on the RH map. HLM5 hasalready been shown to be 24 cM from its nearest neighboringmarker on the genetic map. Three other markers could not beplaced on the RH map (UM004, HTG12, and HMS15), but wereable to be placed on the genetic map in a region equivalent to RHlinkage group D; this discrepancy between the two maps should beresolved as both maps are characterized further.This study has demonstrated the ability of the horse WG-RHpanel to produce an accurate genome map as there is good agree-ment of the genetic and physical maps with the RH maps for Chrs1 and 10 (Figs. 1 and 2). Only a few differences between the mapswere observed, and experiences with human mapping suggest thatsuch differences are common and are resolved as more markers areincorporated into the maps (Walter et al. 1994). The WG-RH panelwas able to order some markers that co-segregate on the geneticlinkage map, i.e., Chr 1 markers LEX39 and ICA18, and ICA41 andICA32, and Chr 10 markers LEX8 and COR015, and NVHEQ18

Radiation hybrid panels are already available for genome mapping in human and mouse. In this study we have used two model organisms (chicken and zebrafish) to show that hybrid panels that contain a full complement of the donor genome can be generated by fusion to hamster cells. The quality of the resulting hybrids has been assessed using PCR and FISH. We confirmed the utility of our panels by establishing the percentage of donor DNA present in the hybrids. Our hybrid resources will allow inexpensive gene mapping and we expect that this technology can be transferred to many other species. Such successes are providing the basis for a new era of mapping tools, in the form of whole genome radiation hybrid panels, and are opening new possibilities for systematic genome analysis in the animal genetics community.

Radiation hybrid (RH) mapping is the most efficient method for generating long-range genomic maps using both polymorphic and nonpolymorphic markers. The basic principle of RH mapping is that the closer two loci are on a chromosome, the less likely they are to be separated by a radiation-induced break. Therefore, markers that are closely linked show correlated coretention patterns across the hybrid panel, where markers located at a large distance from one another are retained almost independently. There are a number of advantages to radiation hybrid mapping. RHs offer high map resolution for relatively small numbers of hybrids. The additional strength of this technique lies in allowing the efficient integration of physical and genetic maps, by facilitating the resolution of cosegregating genetic markers lying in recombination cool-spots on the meiotic map, and anchoring Yeast Artificial Chromosome (YAC) contigs to the genome map. By using a whole genome radiation hybrid (WG-RH) panel, anonymous markers can be both assigned to a chromosome and localized on that chromosome. The two most extensively used RH mapping programs are RHMAP and RHMAPPER. This chapter demonstrates the analysis of two WG-RH datasets using these programs, discussing the merits and limitations of both the packages.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.