Multi-site study of additive genetic effects on fractional anisotropy of cerebral white matter: Comparing meta and megaanalytical approaches for data pooling

Combining datasets across independent studies can boost statistical power by increasing the numbers of observations and can achieve more accurate estimates of effect sizes. This is especially important for genetic studies where a large number of observations are required to obtain sufficient power to detect and replicate genetic effects. There is a need to develop and evaluate methods for joint-analytical analyses of rich datasets collected in imaging genetics studies. The ENIGMA-DTI consortium is developing and evaluating approaches for obtaining pooled estimates of heritability through meta-and mega-genetic analytical approaches, to estimate the general additive genetic contributions to the intersubject variance in fractional anisotropy (FA) measured from diffusion tensor imaging (DTI). We used the ENIGMA-DTI data harmonization protocol for uniform processing of DTI data from multiple sites. We evaluated this protocol in five family-based cohorts providing data from a total of 2248 children and adults (ages: 9-85) collected with various imaging protocols. We used the imaging genetics analysis tool, SOLAR-Eclipse, to combine twin and family data from Dutch, Australian and Mexican-American cohorts into one large "mega-family". We showed that heritability estimates may vary from one cohort to another. We used two meta-analytical (the sample-size and standard-error weighted) approaches and a mega-genetic analysis to calculate heritability estimates across-population. We performed leave-one-out analysis of the joint estimates of heritability, removing a different cohort each time to understand the estimate variability. Overall, meta- and mega-genetic analyses of heritability produced robust estimates of heritability.

EVERSUN: A phase 2 trial of alternating sunitinib and everolimus as first-line therapy for advanced renal cell carcinoma

Background We hypothesised that alternating inhibitors of the vascular endothelial growth factor receptor (VEGFR) and mammalian target of rapamycin pathways would delay the development of resistance in advanced renal cell carcinoma (aRCC). Patients and methods A single-arm, two-stage, multicentre, phase 2 trial to determine the activity, feasibility, and safety of 12-week cycles of sunitinib 50 mg daily 4 weeks on / 2 weeks off, alternating with everolimus 10 mg daily for 5 weeks on / 1 week off, until disease progression or prohibitive toxicity in favourable or intermediate-risk aRCC. The primary end point was proportion alive and progression-free at 6 months (PFS6m). The secondary end points were feasibility, tumour response, overall survival (OS), and adverse events (AEs). The correlative objective was to assess biomarkers and correlate with clinical outcome. Results We recruited 55 eligible participants from September 2010 to August 2012. Demographics: mean age 61, 71% male, favourable risk 16%, intermediate risk 84%. Cycle 2 commenced within 14 weeks for 80% of participants; 64% received ≥22 weeks of alternating therapy; 78% received ≥22 weeks of any treatment. PFS6m was 29/55 (53%; 95% confidence interval [CI] 40% to 66%). Tumour response rate was 7/55 (13%; 95% CI 4% to 22%, all partial responses). After median follow-up of 20 months, 47 of 55 (86%) had progressed with a median progression-free survival of 8 months (95% CI 5–10), and 30 of 55 (55%) had died with a median OS of 17 months (95% CI 12–undefined). AEs were consistent with those expected for each single agent. No convincing prognostic biomarkers were identified. Conclusions The EVERSUN regimen was feasible and safe, but its activity did not meet pre-specified values to warrant further research. This supports the current approach of continuing anti-VEGF therapy until progression or prohibitive toxicity before changing treatment.

An immunodiagnostic assay for quantitation of specific ige to the major pollen allergen component, pas n 1, of the subtropical bahia grass

Background Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Methods Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Results: Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-Atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-Allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-Assay coefficient of variation for the BaGP-Allergic serum pool was 6.92%. Conclusions Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions.

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: Implications for the tertiary structure of the receptor and for an n-terminal binding site for HIV-1 gp120

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9/1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7/1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9/1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7/1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Effects of Tamoxifen and oestrogen on histology and radiographic density in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers

Mammographic density (MD) is a strong risk factor for breast cancer. It is altered by exogenous endocrine treatments, including hormone replacement therapy and Tamoxifen. Such agents also modify breast cancer (BC) risk. However, the biomolecular basis of how systemic endocrine therapy modifies MD and MD-associated BC risk is poorly understood. This study aims to determine whether our xenograft biochamber model can be used to study the effectiveness of therapies aimed at modulating MD, by examine the effects of Tamoxifen and oestrogen on histologic and radiographic changes in high and low MD tissues maintained within the biochamber model. High and low MD human tissues were precisely sampled under radiographic guidance from prophylactic mastectomy fresh specimens of high-risk women, then inserted into separate vascularized murine biochambers. The murine hosts were concurrently implanted with Tamoxifen, oestrogen or placebo pellets, and the high and low MD biochamber tissues maintained in the murine host environment for 3 months, before the high and low MD biochamber tissues were harvested for histologic and radiographic analyses. The radiographic density of high MD tissue maintained in murine biochambers was decreased in Tamoxifen-treated mice compared to oestrogen-treated mice (p = 0.02). Tamoxifen treatment of high MD tissue in SCID mice led to a decrease in stromal (p = 0.009), and an increase in adipose (p = 0.023) percent areas, compared to placebo-treated mice. No histologic or radiographic differences were observed in low MD biochamber tissue with any treatment. High MD biochamber tissues maintained in mice implanted with Tamoxifen, oestrogen or placebo pellets had dynamic and measurable histologic compositional and radiographic changes. This further validates the dynamic nature of the MD xenograft model, and suggests the biochamber model may be useful for assessing the underlying molecular pathways of Tamoxifen-reduced MD, and in testing of other pharmacologic interventions in a preclinical model of high MD.

Cardiometabolic effects of genetic upregulation of the interleukin 1 receptor antagonist: A Mendelian randomisation analysis

Background To investigate potential cardiovascular and other effects of long-term pharmacological interleukin 1 (IL-1) inhibition, we studied genetic variants that produce inhibition of IL-1, a master regulator of inflammation. Methods We created a genetic score combining the effects of alleles of two common variants (rs6743376 and rs1542176) that are located upstream of IL1RN, the gene encoding the IL-1 receptor antagonist (IL-1Ra; an endogenous inhibitor of both IL-1α and IL-1β); both alleles increase soluble IL-1Ra protein concentration. We compared effects on inflammation biomarkers of this genetic score with those of anakinra, the recombinant form of IL-1Ra, which has previously been studied in randomised trials of rheumatoid arthritis and other inflammatory disorders. In primary analyses, we investigated the score in relation to rheumatoid arthritis and four cardiometabolic diseases (type 2 diabetes, coronary heart disease, ischaemic stroke, and abdominal aortic aneurysm; 453 411 total participants). In exploratory analyses, we studied the relation of the score to many disease traits and to 24 other disorders of proposed relevance to IL-1 signalling (746 171 total participants). Findings For each IL1RN minor allele inherited, serum concentrations of IL-1Ra increased by 0·22 SD (95% CI 0·18–0·25; 12·5%; p=9·3 × 10−33), concentrations of interleukin 6 decreased by 0·02 SD (−0·04 to −0·01; −1·7%; p=3·5 × 10−3), and concentrations of C-reactive protein decreased by 0·03 SD (−0·04 to −0·02; −3·4%; p=7·7 × 10−14). We noted the effects of the genetic score on these inflammation biomarkers to be directionally concordant with those of anakinra. The allele count of the genetic score had roughly log-linear, dose-dependent associations with both IL-1Ra concentration and risk of coronary heart disease. For people who carried four IL-1Ra-raising alleles, the odds ratio for coronary heart disease was 1·15 (1·08–1·22; p=1·8 × 10−6) compared with people who carried no IL-1Ra-raising alleles; the per-allele odds ratio for coronary heart disease was 1·03 (1·02–1·04; p=3·9 × 10−10). Per-allele odds ratios were 0·97 (0·95–0·99; p=9·9 × 10−4) for rheumatoid arthritis, 0·99 (0·97–1·01; p=0·47) for type 2 diabetes, 1·00 (0·98–1·02; p=0·92) for ischaemic stroke, and 1·08 (1·04–1·12; p=1·8 × 10−5) for abdominal aortic aneurysm. In exploratory analyses, we observed per-allele increases in concentrations of proatherogenic lipids, including LDL-cholesterol, but no clear evidence of association for blood pressure, glycaemic traits, or any of the 24 other disorders studied. Modelling suggested that the observed increase in LDL-cholesterol could account for about a third of the association observed between the genetic score and increased coronary risk. Interpretation Human genetic data suggest that long-term dual IL-1α/β inhibition could increase cardiovascular risk and, conversely, reduce the risk of development of rheumatoid arthritis. The cardiovascular risk might, in part, be mediated through an increase in proatherogenic lipid concentrations. Funding UK Medical Research Council, British Heart Foundation, UK National Institute for Health Research, National Institute for Health Research Cambridge Biomedical Research Centre, European Research Council, and European Commission Framework Programme 7.

Genetic determinants of heel bone properties: Genome-wide association meta-analysis and replication in the GEFOS/GENOMOS consortium

Quantitative ultrasound of the heel captures heel bone properties that independently predict fracture risk and, with bone mineral density (BMD) assessed by X-ray (DXA), may be convenient alternatives for evaluating osteoporosis and fracture risk. We performed a meta-analysis of genome-wide association (GWA) studies to assess the genetic determinants of heel broadband ultrasound attenuation (BUA; n 5 14 260), velocity of sound (VOS; n 5 15 514) and BMD (n 5 4566) in 13 discovery cohorts. Independent replication involved seven cohorts with GWA data (in silico n 5 11 452) and new genotyping in 15 cohorts (de novo n 5 24 902). In combined random effects, meta-analysis of the discovery and replication cohorts, nine single nucleotide polymorphisms (SNPs) had genome-wide significant (P &lt; 5 3 108) associations with heel bone properties. Alongside SNPs within or near previously identified osteoporosis susceptibility genes including ESR1 (6q25.1: rs4869739, rs3020331, rs2982552), SPTBN1 (2p16.2: rs11898505), RSPO3 (6q22.33: rs7741021), WNT16 (7q31.31: rs2908007), DKK1 (10q21.1: rs7902708) and GPATCH1 (19q13.11: rs10416265), we identified a new locus on chromosome 11q14.2 (rs597319 close to TMEM135, a gene recently linked to osteoblastogenesis and longevity) significantly associated with both BUA and VOS (P &lt; 8.23 3 1014). In meta-analyses involving 25 cohorts with up to 14 985 fracture cases, six of 10 SNPs associated with heel bone properties at P &lt; 5 3 106 also had the expected direction of association with any fracture (P &lt; 0.05), including threeSNPswithP &lt; 0.005: 6q22.33 (rs7741021), 7q31.31 (rs2908007) and 10q21.1 (rs7902708). In conclusion, thisGWAstudy reveals the effect of several genescommon to central DXA-derivedBMDand heel ultrasound/DXAmeasures and points to anewgenetic locus with potential implications for better understanding of osteoporosis pathophysiology.

Genome-wide association study of intraocular pressure identifies the GLCCI1/ICA1 region as a glaucoma susceptibility locus

To discover quantitative trait loci for intraocular pressure, amajor risk factor for glaucoma and the only modifiable one,weperformed agenome-wide association studyonadiscoverycohort of2175individualsfromSydney, Australia. We found a novel association between intraocular pressure and a common variant at 7p21 near to GLCCI1 and ICA1. The findings in this region were confirmed through two UK replication cohorts totalling 4866 individuals (rs59072263, Pcombined = 1.10 × 10-8). A copy of the G allele at this SNP is associated with an increase in mean IOP of 0.45 mmHg (95%CI = 0.30-0.61 mmHg). These results lend support to the implication of vesicle trafficking and glucocorticoid inducibility pathways in the determination of intraocular pressure and in the pathogenesis of primary open-angle glaucoma.

Identification of multiple risk variants for ankylosing spondylitis through high-density genotyping of immune-related loci

Ankylosing spondylitis is a common, highly heritable inflammatory arthritis affecting primarily the spine and pelvis. In addition to HLA-B*27 alleles, 12 loci have previously been identified that are associated with ankylosing spondylitis in populations of European ancestry, and 2 associated loci have been identified in Asians. In this study, we used the Illumina Immunochip microarray to perform a case-control association study involving 10,619 individuals with ankylosing spondylitis (cases) and 15,145 controls. We identified 13 new risk loci and 12 additional ankylosing spondylitis-associated haplotypes at 11 loci. Two ankylosing spondylitis-associated regions have now been identified encoding four aminopeptidases that are involved in peptide processing before major histocompatibility complex (MHC) class I presentation. Protective variants at two of these loci are associated both with reduced aminopeptidase function and with MHC class I cell surface expression.

Common variants at the MHC locus and at chromosome 16q24.1 predispose to Barrett's Esophagus

Barrett's esophagus is an increasingly common disease that is strongly associated with reflux of stomach acid and usually a hiatus hernia, and it strongly predisposes to esophageal adenocarcinoma (EAC), a tumor with a very poor prognosis. We report the first genome-wide association study on Barrett's esophagus, comprising 1,852 UK cases and 5,172 UK controls in the discovery stage and 5,986 cases and 12,825 controls in the replication stage. Variants at two loci were associated with disease risk: chromosome 6p21, rs9257809 (P combined = 4.09 × 10-9; odds ratio (OR) = 1.21, 95% confidence interval (CI) =1.13-1.28), within the major histocompatibility complex locus, and chromosome 16q24, rs9936833 (P combined = 2.74 × 10-10; OR = 1.14, 95% CI = 1.10-1.19), for which the closest protein-coding gene is FOXF1, which is implicated in esophageal development and structure. We found evidence that many common variants of small effect contribute to genetic susceptibility to Barrett's esophagus and that SNP alleles predisposing to obesity also increase risk for Barrett's esophagus. © 2012 Nature America, Inc. All rights reserved.

Genome-wide association study of ankylosing spondylitis identifies non-MHC susceptibility loci

To identify susceptibility loci for ankylosing spondylitis, we undertook a genome-wide association study in 2,053 unrelated ankylosing spondylitis cases among people of European descent and 5,140 ethnically matched controls, with replication in an independent cohort of 898 ankylosing spondylitis cases and 1,518 controls. Cases were genotyped with Illumina HumHap370 genotyping chips. In addition to strong association with the major histocompatibility complex (MHC; P 10 800), we found association with SNPs in two gene deserts at 2p15 (rs10865331; combined P = 1.9 × 10 19) and 21q22 (rs2242944; P = 8.3 × 10 20), as well as in the genes ANTXR2 (rs4333130; P = 9.3 × 10 8) and IL1R2 (rs2310173; P = 4.8 × 10 7). We also replicated previously reported associations at IL23R (rs11209026; P = 9.1 × 10 14) and ERAP1 (rs27434; P = 5.3 × 10 12). This study reports four genetic loci associated with ankylosing spondylitis risk and identifies a major role for the interleukin (IL)-23 and IL-1 cytokine pathways in disease susceptibility. © 2010 Nature America, Inc. All rights reserved.

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with diseaseIRGM for Crohns disease, HLA for Crohns disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetesalthough in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases. © 2010 Macmillan Publishers Limited. All rights reserved.

Genome-wide association study identifies eight loci associated with blood pressure

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5 million genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N 71,225 European ancestry, N 12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N = 29,136). We identified association between systolic or diastolic blood pressure and common variants in eight regions near the CYP17A1 (P = 7 × 10 24), CYP1A2 (P = 1 × 10 23), FGF5 (P = 1 × 10 21), SH2B3 (P = 3 × 10 18), MTHFR (P = 2 × 10 13), c10orf107 (P = 1 × 10 9), ZNF652 (P = 5 × 10 9) and PLCD3 (P = 1 × 10 8) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis

Objective. The heritability of RA has been estimated to be ∼55%, of which the MHC contributes about one-third. HLA-DRB1 alleles are strongly associated with RA, but it is likely that significant non-DRB1 MHC genetic susceptibility factors are involved. Previously, we identified two three-marker haplotypes in a 106-kb region in the MHC class III region immediately centromeric to TNF, which are strongly associated with RA on HLA-DRB1*0404 haplotypes. In the present study, we aimed to refine these associations further using a combination of genotyping and gene expression studies. Methods. Thirty-nine nucleotide polymorphisms (SNPs) were genotyped in 95 DRB1*0404 carrying unrelated RA cases, 125 DRB1*0404 - carrying healthy controls and 87 parent-case trio RA families in which the affected child carried HLA-DRB1*04. Quantitative RT-PCR was used to assess the expression of the positional candidate MHC class III genes APOM, BAT2, BAT3, BAT4, BAT5, AIF1, C6orf47, CSNK2β and LY6G5C, and the housekeeper genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β2-microglobulin (B2M) in 31 RA cases and 21 ethnically, age- and sex-matched healthy controls. Synovial membrane specimens from RA, PsA and OA cases were stained by an indirect immunoperoxidase technique using a mouse-anti-human AIF1 monoclonal antibody. Results. Association was observed between RA and single markers or two marker haplotypes involving AIF1, BAT3 and CSNK. AIF1 was also significantly overexpressed in RA mononuclear cells (1.5- to 1.9-fold difference, P = 0.02 vs HPRT, P = 0.002 vs B2M). AIF1 protein was clearly expressed by synovial macrophages in all the inflammatory synovial samples in contrast to the non-inflammatory OA samples. Conclusions. The results of the genotyping and expression studies presented here suggest a role for AIF1 in both the aetiology and pathogenesis of RA.

Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.