Sheila Fisher

There is increasing evidence that genome-wide association (GWA) studies represent a powerful approach to the identification of genes involved in common human diseases. We describe a joint GWA study (using the Affymetrix GeneChip 500K Mapping Array Set) undertaken in the British population, which has examined ∼2,000 individuals for each of 7 major diseases and a shared set of ∼3,000 controls. Case-control comparisons identified 24 independent association signals at P < 5 × 10-7: 1 in bipolar disorder, 1 in coronary artery disease, 9 in Crohn’s disease, 3 in rheumatoid arthritis, 7 in type 1 diabetes and 3 in type 2 diabetes. On the basis of prior findings and replication studies thus-far completed, almost all of these signals reflect genuine susceptibility effects. We observed association at many previously identified loci, and found compelling evidence that some loci confer risk for more than one of the diseases studied. Across all diseases, we identified a large number of further signals (including 58 loci with single-point P values between 10-5 and 5 × 10-7) likely to yield additional susceptibility loci. The importance of appropriately large samples was confirmed by the modest effect sizes observed at most loci identified. This study thus represents a thorough validation of the GWA approach. It has also demonstrated that careful use of a shared control group represents a safe and effective approach to GWA analyses of multiple disease phenotypes; has generated a genome-wide genotype database for future studies of common diseases in the British population; and shown that, provided individuals with non-European ancestry are excluded, the extent of population stratification in the British population is generally modest. Our findings offer new avenues for exploring the pathophysiology of these important disorders. We anticipate that our data, results and software, which will be widely available to other investigators, will provide a powerful resource for human genetics research. With the advent of many more markers in the human genome, it has become possible to search for genes associated with human disease without having to narrow down candidate regions of the genome first. In a ground-breaking publication, the Wellcome Trust Case Control Consortium reports an exciting genome-wide association study of some 17,000 individuals for seven common familial diseases. The analysis confirms previously identified loci and provides strong evidence for many novel disease susceptibility genes. An exciting genome-wide association study in the British population for seven common diseases. This analysis confirms previously identified loci and provides strong evidence for many novel disease susceptibility loci.

We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Mark Daly and colleagues present results of a combined analysis of data from three recent genome-wide association studies for Crohn's disease, followed by replication in a large independent sample collection. Their results confirm 11 previously reported risk loci and provide genome-wide significant evidence for 21 new loci associated with the disease. Several risk factors for Crohn's disease have been identified in recent genome-wide association studies. To advance gene discovery further, we combined data from three studies on Crohn's disease (a total of 3,230 cases and 4,829 controls) and carried out replication in 3,664 independent cases with a mixture of population-based and family-based controls. The results strongly confirm 11 previously reported loci and provide genome-wide significant evidence for 21 additional loci, including the regions containing STAT3, JAK2, ICOSLG, CDKAL1 and ITLN1. The expanded molecular understanding of the basis of this disease offers promise for informed therapeutic development.

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests approximately 900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.

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.

Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

Prostate cancer is the second most common cause of male cancer deaths in the United States. However, the full range of prostate cancer genomic alterations is incompletely characterized. Here we present the complete sequence of seven primary human prostate cancers and their paired normal counterparts. Several tumours contained complex chains of balanced (that is, 'copy-neutral') rearrangements that occurred within or adjacent to known cancer genes. Rearrangement breakpoints were enriched near open chromatin, androgen receptor and ERG DNA binding sites in the setting of the ETS gene fusion TMPRSS2-ERG, but inversely correlated with these regions in tumours lacking ETS fusions. This observation suggests a link between chromatin or transcriptional regulation and the genesis of genomic aberrations. Three tumours contained rearrangements that disrupted CADM2, and four harboured events disrupting either PTEN (unbalanced events), a prostate tumour suppressor, or MAGI2 (balanced events), a PTEN interacting protein not previously implicated in prostate tumorigenesis. Thus, genomic rearrangements may arise from transcriptional or chromatin aberrancies and engage prostate tumorigenic mechanisms.

A genome-wide association scan in individuals with Crohn's disease by the Wellcome Trust Case Control Consortium detected strong association at four novel loci. We tested 37 SNPs from these and other loci for association in an independent case-control sample. We obtained replication for the autophagy-inducing IRGM gene on chromosome 5q33.1 (replication P = 6.6 × 10−4, combined P = 2.1 × 10−10) and for nine other loci, including NKX2-3, PTPN2 and gene deserts on chromosomes 1q and 5p13.

Background Genetic predisposition to inflammatory bowel disease (IBD) has been shown by epidemiological and linkage studies. Genetic linkage of IBD to chromosome 16 has been previously observed and replicated in independent populations. The recently identified NOD2 gene is a good positional and functional candidate gene since it is located in the region of linkage on chromosome 16q12, and activates nuclear factor (NF) kappaB in response to bacterial lipopolysaccharides. Methods We sequenced the coding region of the NOD2 gene and genotyped an insertion polymorphism affecting the leucine-rich region of the protein product in 512 individuals with IBD from 309 German or British families, 369 German trios (ie, German patients with sporadic IBD and their unaffected parents), and 272 normal controls. We then tested for association with Crohn's disease and ulcerative colitis. Findings Family-based association analyses were consistently positive in 95 British and 99 German affected sibling pairs with Crohn's disease (combined p<0.0001); the association was confirmed in the 304 German trios with Crohn's disease. No association was seen in the 115 sibling pairs and 65 trios with ulcerative colitis. The genotype-specific disease risks conferred by heterozygous and homozygous mutant genotypes were 2.6 (95% CI 1.5-4.5) and 42.1 (4.3-infinity), respectively. Interpretation The insertion mutation in the NOD2 gene confers a substantially increased susceptibility to Crohn's disease but not to ulcerative colitis.

Age-related macular degeneration (AMD) is a multifactorial disease and a prevalent cause of visual impairment in developed countries.Risk factors include environmental components and genetic determinants.The complement factor H (CFH) has been the first major susceptibility gene for AMD identified within 1q32.Here, we focused on a second region of interest in 10q26 where a recent meta-analysis revealed strongest evidence for linkage to AMD at a genome-wide significance level.Within an interval of 22 Mb, we have analyzed 93 single nucleotide polymorphisms for allelic association with AMD in two independent case-control cohorts of German origin (AMD combined n 5 1166; controls combined n 5 945).Significant association was found across a 60 kb region of high linkage disequilibrium harboring two genes PLEKHA1 and hypothetical LOC387715.The strongest association (P 5 10 234 ) centered over a frequent coding polymorphism, Ala69Ser, at LOC387715, strongly implicating this gene in the pathogenesis of AMD.Besides abundant expression in placenta, we demonstrate weak expression of LOC387715 in the human retina.At present, however, there is no functional information on this gene, which appears to have evolved recently within the primate lineage.The joint contribution of the common risk allele at LOC387715, Ala69Ser, and at CFH, Tyr402His, was assessed in our case-control population, which suggests an additive model indicating an independent contribution of the two gene loci to disease risk.Our data show a disease odds ratio of 57.6 (95% CI: 37.2, 89.0) conferred by homozygosity for risk alleles at both CFH and LOC387715 when compared with the baseline non-risk genotype.

Mutations in the NOD2 gene are strongly associated with susceptibility to Crohn's disease (CD). We analyzed a large cohort of European patients with inflammatory bowel disease to determine which mutations confer susceptibility, the degree of risk conferred, their prevalence in familial and sporadic forms of the disease, and whether they are associated with site of disease.Individuals were genotyped for 4 NOD2 mutations: P268S, R702W, G908R, and 3020insC. Allelic transmission distortion to 531 CD- and 337 ulcerative colitis-affected offspring was assessed by the transmission disequilibrium test. Association was also tested in an independent cohort of 995 patients with inflammatory bowel disease and 290 controls. Cases were stratified by disease site and compared across NOD2 genotypes.R702W, G908R, and 3020insC were strongly associated with CD but not with ulcerative colitis. Linkage disequilibrium was observed between P268S and the other mutations, forming 3 independent disease haplotypes. Genotype relative risks were 3.0 for mutation heterozygotes and 23.4 for homozygotes or compound heterozygotes. The frequency of NOD2 mutations was higher in cases from families affected only with CD and was significantly increased in ileal-specific disease cases compared with colon-specific disease (26.9% vs. 12.7%, P = 0.0004).The R702W, G908R, and 3020insC mutations are strong independent risk factors for CD and are associated particularly with ileal disease.

We sequenced all protein-coding regions of the genome (the "exome") in two family members with combined hypolipidemia, marked by extremely low plasma levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. These two participants were compound heterozygotes for two distinct nonsense mutations in ANGPTL3 (encoding the angiopoietin-like 3 protein). ANGPTL3 has been reported to inhibit lipoprotein lipase and endothelial lipase, thereby increasing plasma triglyceride and HDL cholesterol levels in rodents. Our finding of ANGPTL3 mutations highlights a role for the gene in LDL cholesterol metabolism in humans and shows the usefulness of exome sequencing for identification of novel genetic causes of inherited disorders. (Funded by the National Human Genome Research Institute and others.).

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.

Whole-exome sequencing (WES) has emerged as a transformative technology for biological discovery, but technical difficulties have so far prevented its widespread clinical use. Here, Eliezer Van Allen and colleagues are able to perform production-scale WES on small amounts of clinically acquired formalin-fixed, paraffin-embedded tumor tissues. Using a newly created WES clinical interpretation algorithm, they apply the complete clinical WES framework prospectively to patients and demonstrate how it can be used to directly affect patient care. Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.

Abstract Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP–ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2Q60P). MEK2Q60P conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal–regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma. Significance: This study represents an initial clinical genomic study of acquired resistance to combined RAF/MEK inhibition in BRAF-mutant melanoma, using WES and RNA-seq. The presence of diverse resistance mechanisms suggests that serial biopsies and genomic/molecular profiling at the time of resistance may ultimately improve the care of patients with resistant BRAF-mutant melanoma by specifying tailored targeted combinations to overcome specific resistance mechanisms. Cancer Discov; 4(1); 61–8. ©2013 AACR. See related commentary by Solit and Rosen, p. 27 This article is highlighted in the In This Issue feature, p. 1

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.

We report results of a nonsynonymous SNP scan for ulcerative colitis and identify a previously unknown susceptibility locus at ECM1. We also show that several risk loci are common to ulcerative colitis and Crohn's disease (IL23R, IL12B, HLA, NKX2-3 and MST1), whereas autophagy genes ATG16L1 and IRGM, along with NOD2 (also known as CARD15), are specific for Crohn's disease. These data provide the first detailed illustration of the genetic relationship between these common inflammatory bowel diseases.

Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.

A genome-wide association scan of nonsynonymous DNA polymorphisms identified association of a threonine-to-alanine substitution (T300A) in the autophagy-related 16-like gene ATG16L1 with Crohn's disease. We investigated this association in independent U.K. cohorts of Crohn's disease and ulcerative colitis.The T300A variant (rs2241880) was genotyped in an independent sample of 727 Crohn's disease and 877 ulcerative colitis cases, and in 579 controls. We then performed an extension analysis combining these data with the U.K. data from the initial study to give a total of 1236 U.K. Crohn's disease cases and 1235 controls to estimate disease risk and test for interaction with the CARD15 and IBD5 risk loci and for association with disease subtypes.The association of T300A was replicated in the independent sample of 727 Crohn's disease cases (P = .001), and was strongly associated in the extended analysis of 1236 Crohn's cases (P = 2.4 x 10(-6)). The 300A/A genotype conferred a 1.65-fold risk of Crohn's disease, with a 2.2-fold risk of ileal disease. Analysis of the interaction of ATG16L1 with CARD15 and IBD5 indicated that all 3 loci contribute independently to disease risk. Homozygosity for the risk allele at all 3 loci conferred a combined risk of 20.4 (95% confidence interval: 8.71, 47.7) for Crohn's disease. The ATG16L1 risk genotype showed a modest but significant association with ulcerative colitis (P = .026).The association of ATG16L1 with Crohn's disease and possibly with ulcerative colitis supports a role for autophagy in the pathogenesis of inflammatory bowel disease.

Cell transplantation offers a potential therapeutic approach to the repair and regeneration of damaged vascular and cardiac tissue after acute myocardial infarction (AMI). This has resulted in multiple randomised controlled trials (RCTs) across the world.To determine the safety and efficacy of autologous adult bone marrow stem cells as a treatment for acute myocardial infarction (AMI), focusing on clinical outcomes.This Cochrane review is an update of a previous version (published in 2012). We searched the Cochrane Central Register of Controlled Trials (CENTRAL 2015, Issue 2), MEDLINE (1950 to March 2015), EMBASE (1974 to March 2015), CINAHL (1982 to March 2015) and the Transfusion Evidence Library (1980 to March 2015). In addition, we searched several international and ongoing trial databases in March 2015 and handsearched relevant conference proceedings to January 2011.RCTs comparing autologous bone marrow-derived cells with no cells in patients diagnosed with AMI were eligible.Two review authors independently screened all references, assessed the risk of bias of the included trials and extracted data. We conducted meta-analyses using random-effects models throughout. We analysed outcomes at short-term (less than 12 months) and long-term (12 months or more) follow-up. Dichotomous outcomes are reported as risk ratio (RR) and continuous outcomes are reported as mean difference (MD) or standardised MD (SMD). We performed sensitivity analyses to evaluate the results in the context of the risk of selection, performance and attrition bias. Exploratory subgroup analysis investigated the effects of baseline cardiac function (left ventricular ejection fraction, LVEF) and cell dose, type and timing of administration, as well as the use of heparin in the final cell solution.Forty-one RCTs with a total of 2732 participants (1564 cell therapy, 1168 controls) were eligible for inclusion. Cell treatment was not associated with any changes in the risk of all-cause mortality (34/538 versus 32/458; RR 0.93, 95% CI 0.58 to 1.50; 996 participants; 14 studies; moderate quality evidence), cardiovascular mortality (23/277 versus 18/250; RR 1.04, 95% CI 0.54 to 1.99; 527 participants; nine studies; moderate quality evidence) or a composite measure of mortality, reinfarction and re-hospitalisation for heart failure (24/262 versus 33/235; RR 0.63, 95% CI 0.36 to 1.10; 497 participants; six studies; moderate quality evidence) at long-term follow-up. Statistical heterogeneity was low (I(2) = 0% to 12%). Serious periprocedural adverse events were rare and were generally unlikely to be related to cell therapy. Additionally, cell therapy had no effect on morbidity, quality of life/performance or LVEF measured by magnetic resonance imaging. Meta-analyses of LVEF measured by echocardiography, single photon emission computed tomography and left ventricular angiography showed evidence of differences in mean LVEF between treatment groups although the mean differences ranged between 2% and 5%, which are accepted not to be clinically relevant. Results were robust to the risk of selection, performance and attrition bias from individual studies.The results of this review suggest that there is insufficient evidence for a beneficial effect of cell therapy for AMI patients. However, most of the evidence comes from small trials that showed no difference in clinically relevant outcomes. Further adequately powered trials are needed and until then the efficacy of this intervention remains unproven.

Rationale: Cell-based therapies are a promising intervention for the treatment of heart failure (HF) secondary to ischemic and nonischemic cardiomyopathy. However, the clinical efficacy of such new treatment requires further evaluation. Objective: To assess available clinical evidence on the safety and efficacy of cell-based therapies for HF. Methods and Results: Electronic databases (CENTRAL, DARE, NHSEED &amp; HTA, PubMed, MEDLINE, EMBASE, CINAHL, LILACS, KoreaMed, PakMediNet, IndMed, and the Transfusion Evidence Library) were searched for relevant randomized controlled trials to June 2014. Trials of participants with HF and where the administration of any dose of autologous cells by any delivery route was compared with no intervention or placebo were eligible for inclusion. Primary outcomes were defined as mortality and rehospitalization as a result of HF. Secondary outcomes included performance status, quality of life, incidence of arrhythmias, brain natriuretic peptide levels, left ventricular ejection fraction, myocardial perfusion, and adverse events. Thirty-one independent trials (1521 participants) were included. The treatment significantly reduced the risk of mortality and rehospitalization caused by HF. There was a significant improvement in favor of stem cell treatment in performance status and exercise capacity, left ventricular ejection fraction, and quality of life. The treatment was also associated with a reduction of brain natriuretic peptide levels and no increase in the incidence of arrhythmias. However, there was considerable risk of performance, selection, and reporting bias among the included trials. Conclusions: This study shows evidence that autologous cell therapy may be beneficial for patients having HF, but further evidence is required.

A promising approach to the treatment of chronic ischaemic heart disease and congestive heart failure is the use of stem cells. The last decade has seen a plethora of randomised controlled trials developed worldwide, which have generated conflicting results.The critical evaluation of clinical evidence on the safety and efficacy of autologous adult bone marrow-derived stem/progenitor cells as a treatment for chronic ischaemic heart disease and congestive heart failure.We searched CENTRAL in the Cochrane Library, MEDLINE, Embase, CINAHL, LILACS, and four ongoing trial databases for relevant trials up to 14 December 2015.Eligible studies were randomised controlled trials comparing autologous adult stem/progenitor cells with no cells in people with chronic ischaemic heart disease and congestive heart failure. We included co-interventions, such as primary angioplasty, surgery, or administration of stem cell mobilising agents, when administered to treatment and control arms equally.Two review authors independently screened all references for eligibility, assessed trial quality, and extracted data. We undertook a quantitative evaluation of data using random-effects meta-analyses. We evaluated heterogeneity using the I2 statistic and explored substantial heterogeneity (I2 greater than 50%) through subgroup analyses. We assessed the quality of the evidence using the GRADE approach. We created a 'Summary of findings' table using GRADEprofiler (GRADEpro), excluding studies with a high or unclear risk of selection bias. We focused our summary of findings on long-term follow-up of mortality, morbidity outcomes, and left ventricular ejection fraction measured by magnetic resonance imaging.We included 38 randomised controlled trials involving 1907 participants (1114 cell therapy, 793 controls) in this review update. Twenty-three trials were at high or unclear risk of selection bias. Other sources of potential bias included lack of blinding of participants (12 trials) and full or partial commercial sponsorship (13 trials).Cell therapy reduced the incidence of long-term mortality (≥ 12 months) (risk ratio (RR) 0.42, 95% confidence interval (CI) 0.21 to 0.87; participants = 491; studies = 9; I2 = 0%; low-quality evidence). Periprocedural adverse events associated with the mapping or cell/placebo injection procedure were infrequent. Cell therapy was also associated with a long-term reduction in the incidence of non-fatal myocardial infarction (RR 0.38, 95% CI 0.15 to 0.97; participants = 345; studies = 5; I2 = 0%; low-quality evidence) and incidence of arrhythmias (RR 0.42, 95% CI 0.18 to 0.99; participants = 82; studies = 1; low-quality evidence). However, we found no evidence that cell therapy affects the risk of rehospitalisation for heart failure (RR 0.63, 95% CI 0.36 to 1.09; participants = 375; studies = 6; I2 = 0%; low-quality evidence) or composite incidence of mortality, non-fatal myocardial infarction, and/or rehospitalisation for heart failure (RR 0.64, 95% CI 0.38 to 1.08; participants = 141; studies = 3; I2 = 0%; low-quality evidence), or long-term left ventricular ejection fraction when measured by magnetic resonance imaging (mean difference -1.60, 95% CI -8.70 to 5.50; participants = 25; studies = 1; low-quality evidence).This systematic review and meta-analysis found low-quality evidence that treatment with bone marrow-derived stem/progenitor cells reduces mortality and improves left ventricular ejection fraction over short- and long-term follow-up and may reduce the incidence of non-fatal myocardial infarction and improve New York Heart Association (NYHA) Functional Classification in people with chronic ischaemic heart disease and congestive heart failure. These findings should be interpreted with caution, as event rates were generally low, leading to a lack of precision.

Although often considered "minimal" organisms, mycoplasmas show a wide range of diversity with respect to host environment, phenotypic traits, and pathogenicity. Here we report the complete genomic sequence and proteogenomic map for the piscine mycoplasma Mycoplasma mobile, noted for its robust gliding motility. For the first time, proteomic data are used in the primary annotation of a new genome, providing validation of expression for many of the predicted proteins. Several novel features were discovered including a long repeating unit of DNA of approximately 2435 bp present in five complete copies that are shown to code for nearly identical yet uniquely expressed proteins. M. mobile has among the lowest DNA GC contents (24.9%) and most reduced set of tRNAs of any organism yet reported (28). Numerous instances of tandem duplication as well as lateral gene transfer are evident in the genome. The multiple available complete genome sequences for other motile and immotile mycoplasmas enabled us to use comparative genomic and phylogenetic methods to suggest several candidate genes that might be involved in motility. The results of these analyses leave open the possibility that gliding motility might have arisen independently more than once in the mycoplasma lineage.

A genetic contribution to the development of age-related macular degeneration (AMD) is well established. Several genome-wide linkage studies have identified a number of putative susceptibility loci for AMD but only a few of these regions have been replicated in independent studies. Here, we perform a meta-analysis of six AMD genome screens using the genome-scan meta-analysis method, which allows linkage results from several studies to be combined, providing greater power to identify regions that show only weak evidence for linkage in individual studies. Results from non-parametric analysis for a broad AMD clinical phenotype (including two studies with quantitative traits) were extracted. For each study, 120 genomic bins of approximately 30 cM were defined and ranked according to maximum evidence for linkage within each bin. Bin ranks were weighted according to study size and summed across all studies; the summed rank (SR) for each bin was assessed empirically for significance using permutation methods. A high SR indicates a region with consistent evidence for linkage across studies. The strongest evidence for an AMD susceptibility locus was found on chromosome 10q26 where genome-wide significant linkage was observed (P=0.00025). Several other regions met the empirical significance criteria for bins likely to contain linked loci including adjacent pairs of bins on chromosomes 1q, 2p, 3p and 16. Several of the regions identified here showed only weak evidence for linkage in the individual studies. These results will help prioritize regions for future positional and functional candidate gene studies in AMD.

Crohn's disease and ulcerative colitis (the inflammatory bowel diseases) have a strong genetic component. Although over 20 putative susceptibility loci have been identified by individual genome scans, the majority of these loci have not been replicated. Many individual studies are at the lower limit of acceptable power for complex disease linkage analysis. Genome scan meta-analysis (GSMA), by use of sample sizes an order of magnitude greater than individual linkage studies, has increased power to detect novel loci, may confirm or refute regions detected in smaller individual studies, and enables regions to be prioritized for further gene identification efforts. Genome scan data (markers, significance scores) were obtained from 10 separate studies and meta-analysis was performed using the GSMA method. These studies comprised 1952 inflammatory bowel disease, 1068 Crohn's disease and 457 ulcerative colitis affected relative pairs. Study results were divided into 34 cM chromosomal bins, ranked, weighted by study size, summed across studies and bin-by-bin significance obtained by simulation. A region on chromosome 6p (containing the HLA) met genome wide significance for inflammatory bowel disease. Loci meeting suggestive significance for inflammatory bowel disease were 2q, 3q, 5q, 7q and 16 (NOD2/CARD15 region); Crohn's disease, 2q, 3q, 6p, 16 (NOD2/CARD15 region), 17q, 19p; and ulcerative colitis, 2q. Clustering of adjacent bins was observed for chromosomes 6p, 16, 19p. The meta-analysis has identified novel loci and prioritized genomic regions for further gene identification studies.

Background & Aims: Identification of inflammatory bowel disease (IBD) susceptibility genes is key to understanding pathogenic mechanisms. Recently, the North American IBD Genetics Consortium provided compelling evidence for an association between ileal Crohn’s disease (CD) and the IL23R gene using genome-wide association scanning. External replication is a priority, both to confirm this finding in other populations and to validate this new technique. We tested for association between IL23R and IBD in a large independent UK panel to determine the size of the effect and explore subphenotype correlation and interaction with CARD15. Methods: Eight single nucleotide polymorphism markers in IL23R tested in the North American study were genotyped in 1902 cases of Crohn’s disease (CD), 975 cases of ulcerative colitis (UC), and 1345 controls using MassARRAY. Data were analyzed using χ2 statistics, and subgroup association was sought. Results: A highly significant association with CD was observed, with the strongest signal at coding variant Arg381Gln (allele frequency, 2.5% in CD vs 6.2% in controls [P = 1.1 × 10−12]; odds ratio, 0.38; 95% confidence interval, 0.29–0.50). A weaker effect was seen in UC (allele frequency, 4.6%; odds ratio, 0.73; 95% confidence interval, 0.55–0.96). Analysis accounting for Arg381Gln suggested that other loci within IL23R also influence IBD susceptibility. Within CD, there were no subphenotype associations or evidence of interaction with CARD15. Conclusions: This study shows an association between IL23R and all subphenotypes of CD with a smaller effect on UC. This extends the findings of the North American study, providing clear evidence that genome-wide association scanning can successfully identify true complex disease genes. Background & Aims: Identification of inflammatory bowel disease (IBD) susceptibility genes is key to understanding pathogenic mechanisms. Recently, the North American IBD Genetics Consortium provided compelling evidence for an association between ileal Crohn’s disease (CD) and the IL23R gene using genome-wide association scanning. External replication is a priority, both to confirm this finding in other populations and to validate this new technique. We tested for association between IL23R and IBD in a large independent UK panel to determine the size of the effect and explore subphenotype correlation and interaction with CARD15. Methods: Eight single nucleotide polymorphism markers in IL23R tested in the North American study were genotyped in 1902 cases of Crohn’s disease (CD), 975 cases of ulcerative colitis (UC), and 1345 controls using MassARRAY. Data were analyzed using χ2 statistics, and subgroup association was sought. Results: A highly significant association with CD was observed, with the strongest signal at coding variant Arg381Gln (allele frequency, 2.5% in CD vs 6.2% in controls [P = 1.1 × 10−12]; odds ratio, 0.38; 95% confidence interval, 0.29–0.50). A weaker effect was seen in UC (allele frequency, 4.6%; odds ratio, 0.73; 95% confidence interval, 0.55–0.96). Analysis accounting for Arg381Gln suggested that other loci within IL23R also influence IBD susceptibility. Within CD, there were no subphenotype associations or evidence of interaction with CARD15. Conclusions: This study shows an association between IL23R and all subphenotypes of CD with a smaller effect on UC. This extends the findings of the North American study, providing clear evidence that genome-wide association scanning can successfully identify true complex disease genes. See editorial on page 2045; CME quiz on page 1999. It is widely recognized that knowledge regarding the genetic basis of inflammatory bowel disease (IBD) and other complex diseases will provide key insights into pathogenic mechanisms. It is this fact that has spurred efforts to identify disease susceptibility genes. Of the many complex diseases investigated using molecular genetic techniques, Crohn’s disease (CD) is exceptional in that specific genetic variants unequivocally associated with disease susceptibility have been successfully identified.1Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nunez G. Cho J.H. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature. 2001; 411: 603-606Google Scholar, 2Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O’Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.Nature. 2001; 411: 599-603Google Scholar Nonetheless, characterization of the unknown number of remaining CD genes is required to complete the picture and remains a priority. CD is one of the 2 common and related forms of IBD, the other being ulcerative colitis (UC). Within the United Kingdom, they have a combined prevalence of approximately 4/1000.3Stone M.A. Mayberry J.F. Baker R. Prevalence and management of inflammatory bowel disease: a cross-sectional study from central England.Eur J Gastroenterol Hepatol. 2003; 15: 1275-1280Google Scholar Both are known to have a significant genetic contribution to their etiology, but this is stronger for CD than UC.4Tysk C. Lindberg E. Jarnerot G. Floderus-Myrhed B. Ulcerative colitis and Crohn’s disease in an unselected population of monozygotic and dizygotic twins A study of heritability and the influence of smoking.Gut. 1988; 29: 990-996Google Scholar The epidemiologic evidence also suggests that CD and UC share some susceptibility genes. In 2001, fine mapping of a widely replicated linkage region on chromosome 16 led to the identification of CARD15 as a major CD susceptibility gene, with mutations leading to dysregulation of innate immune pathways.1Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nunez G. Cho J.H. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature. 2001; 411: 603-606Google Scholar, 2Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O’Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.Nature. 2001; 411: 599-603Google ScholarCARD15 genes have subsequently been shown in meta-analysis to predominantly determine susceptibility to ileal CD. Variants within a number of other genes have been associated with CD, UC, or both,5Reinhard C. Rioux J.D. Role of the IBD5 susceptibility locus in the inflammatory bowel diseases.Inflamm Bowel Dis. 2006; 12: 227-238Google Scholar, 6Yamazaki K. McGovern D. Ragoussis J. Paolucci M. Butler H. Jewell D. Cardon L. Takazoe M. Tanaka T. Ichimori T. Saito S. Sekine A. Iida A. Takahashi A. Tsunoda T. Lathrop M. Nakamura Y. Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn’s disease.Hum Mol Genet. 2005; 14: 3499-3506Google Scholar, 7Ho G.-T. Nimmo E.R. Tenesa A. Fennell J. Drummond H. Mowat C. Arnott I.D. Satsangi J. Allelic variations of the multidrug resistance gene determine susceptibility and disease behavior in ulcerative colitis.Gastroenterology. 2005; 128: 288-296Abstract Full Text Full Text PDF Scopus (168) Google Scholar, 8Franchimont D. Vermeire S. El Housni H. Pierik M. Van Steen K. Gustot T. Quertinmont E. Abramowicz M. Van Gossum A. Deviere J. Rutgeerts P. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn’s disease and ulcerative colitis.Gut. 2004; 53: 987-992Google Scholar, 9Stoll M. Corneliussen B. Costello C.M. Waetzig G.H. Mellgard B. Koch W.A. Rosenstiel P. Albrecht M. Croucher P.J. Seegert D. Nikolaus S. Hampe J. Lengauer T. Pierrou S. Foelsch U.R. Mathew C.G. Lagerstrom-Fermer M. Schreiber S. Genetic variation in DLG5 is associated with inflammatory bowel disease.Nat Genet. 2004; 36: 476-480Google Scholar although their exact roles in IBD susceptibility require clarification and, in some cases, replication. To date, pinpointing of disease genes has depended on detailed evaluation of candidates implicated by their function or patterns of expression or by fine mapping within large regions identified in the course of genome-wide linkage scans. Across the range of common diseases, productivity of such approaches has been limited. Most complex disease genetic studies, including many in IBD, have been beset by poor reproducibility of results and slow progress in identifying disease genes. This has been attributed to a range of factors, some of the most important being the low resolution of sib-pair linkage analysis, use of inappropriate statistical thresholds for significance, and poor matching of controls due to population admixture.10Cardon L.R. Bell J.I. Association study designs for complex diseases.Nat Rev Genet. 2001; 2: 91-99Google Scholar One powerful new method for the identification of complex disease genes is genome-wide association scanning, genotyping large panels of affected individuals and appropriately matched population controls for hundreds of thousands of polymorphic markers across the genome and using appropriately stringent statistical thresholds for significance.11Risch N. Merikangas K. The future of genetic studies of complex human diseases.Science. 1996; 273: 1516-1517Google Scholar Within the past year, such studies have become technically and financially possible using sets of markers that capture most of the common variation across the genome using knowledge regarding human haplotype structure available from the International HapMap Project (http://www.hapmap.org).12Barrett J.C. Cardon L.R. Evaluating coverage of genome-wide association studies.Nat Genet. 2006; 38: 659-662Google Scholar Systematic whole-genome association studies, in comparison with the previous gold standard of linkage analysis, should provide substantially increased power and resolution for detection of complex disease susceptibility genes.13Hirschhorn J.N. Daly M.J. Genome-wide association studies for common diseases and complex traits.Nat Rev Genet. 2005; 6: 95-108Google Scholar Recently, the results of a 308,332-marker genome scan in a North American panel of 547 non-Jewish case patients with CD and 548 controls were reported. Case patients were selected as having ileal CD to reduce heterogeneity.14Duerr R.H. Taylor K.D. Brant S.R. Rioux J.D. Silverberg M.S. Daly M.J. Steinhart A.H. Abraham C. Regueiro M. Griffiths A. Dassopoulos T. Bitton A. Yang H. Targan S. Datta L.W. Kistner E.O. Schumm L.P. Lee A. Gregersen P.K. Barmada M.M. Rotter J.I. Nicolae D.L. Cho J.H. A Genome-wide association study identifies IL23R as an inflammatory bowel disease gene.Science. 2006; 314: 1461-1463Google Scholar Three markers showed a highly significant association with CD, 2 of which were in CARD15. The third marker was a rare coding variant rs11209026c (1142G→A; Arg381Gln) found in the interleukin 23 receptor (IL23R) gene on chromosome 1 (P = 5.05 × 10−9). Nine other markers showed association with P < .0001 either within IL23R or in the intergenic area with the adjacent IL12RB2 gene. Internal replication was achieved in the index study using both a Jewish CD case-control cohort (peak P value, 3.36 × 10−13) and family-based methodologies, the latter in addition suggesting association with UC in a small non-Jewish cohort. This finding indicates that IL23R may have a general role in the etiology of IBD.14Duerr R.H. Taylor K.D. Brant S.R. Rioux J.D. Silverberg M.S. Daly M.J. Steinhart A.H. Abraham C. Regueiro M. Griffiths A. Dassopoulos T. Bitton A. Yang H. Targan S. Datta L.W. Kistner E.O. Schumm L.P. Lee A. Gregersen P.K. Barmada M.M. Rotter J.I. Nicolae D.L. Cho J.H. A Genome-wide association study identifies IL23R as an inflammatory bowel disease gene.Science. 2006; 314: 1461-1463Google Scholar The aims of the current study were to seek replication of the association between IL23R and IBD in a large independent North European cohort representing the full range of CD and UC phenotypes, examine in detail genotype-phenotype relationships, explore evidence for epistasis with the known CD susceptibility gene CARD15, and provide accurate estimates of disease risk for associated variants. Replication of the association in an independent cohort would serve 2 important purposes. First, it is key to confirming the veracity of the original finding and the applicability of these findings in populations outside North America. Further, strong independent replication of the key finding of one of the first published genome-wide association scans would provide proof of principle that this novel methodology can be used to identify risk variants for complex diseases. A total of 2877 individuals with IBD (1902 with CD and 975 with UC) were recruited in 5 centers across England and Scotland. The study was approved by the research ethics committees at each center. Standard clinical, radiologic, and histologic diagnostic criteria were applied.15Lennard-Jones J.E. Classification of inflammatory bowel disease.Scand J Gastroenterol Suppl. 1989; 170: 2-6Google Scholar Phenotypic details were obtained by retrospective case notes review. CD phenotype was classified by age at diagnosis, location, and behavior of disease. Only one member of multiply affected families was included. A total of 1.75% were of Jewish origin, and 2.25% were nonwhite. Demographic and subphenotype data are presented in Table 1.Table 1Demographic Details of 2877 Individuals With IBD Used in Case-Control PanelCD (n = 1902)UC (n = 975)Median age at diagnosis (y)2638.9Gender (F/M)1153/745480/495Smoking at diagnosis (%) Never58.455.0 Ex9.430.3 Current32.214.7Jewish ancestry (%)1.751.9Nonwhite (%)2.253.25Surgery (%)61.8Location/extent (%)32.7 ileal16.5 rectum only31.8 colonic35.0 distal to35.5 ileocolonic splenic flexure27.1 perianal48.5 proximal to splenic flexureBehavior (%)36.5 stenosing17.15 penetrating Open table in a new tab Control allele frequencies were obtained from 1345 individuals recruited across Britain as part of the 1958 British birth cohort.16Power C. Elliott J. Cohort profile: 1958 British birth cohort (National Child Development Study).Int J Epidemiol. 2006; 35: 34-41Google Scholar Cases and controls were categorized into 12 broad geographical regions within Great Britain to minimize confounding due to variation in allele frequencies across the country.17Clayton D.G. Walker N.M. Smyth D.J. Pask R. Cooper J.D. Maier L.M. Smink L.J. Lam A.C. Ovington N.R. Stevens H.E. Nutland S. Howson J.M.M. Faham M. Moorhead M. Jones H.B. Falkowski M. Hardenbol P. Willis T.D. Todd J.A. Population structure, differential bias and genomic control in a large-scale, case-control association study.Nat Genet. 2005; 37: 1243-1246Google Scholar Genotyping of cases was undertaken with iPLEX chemistry on a matrix-assisted laser desorption/ionization time-of-flight MassARRAY platform (Sequenom, San Diego, CA). Cases were genotyped for 8 IL23R markers reported in the index study, including the nonsynonymous single nucleotide polymorphism (SNP) rs11209026 encoding amino acid change Arg381Gln (primer sequences in Supplementary Table 1; see supplemental material online at www.gastrojournal.org). Two of the North American markers (rs7517847, rs2201841) were omitted due to their location within a sequence of interspersed low-complexity repeats. Genotyping of controls was undertaken at the Wellcome Trust Sanger Institute using the Illumina 550K chip (Illumina, San Diego, CA). Concordance of genotype calls between the different platforms was confirmed by genotyping 87 control DNAs for all 8 markers using the MassARRAY platform with strong concordance of calls between technologies—98.99% for the 8 markers overall. There was 100% concordance for 3 markers, including the coding variant Arg381Gln (Supplementary Table 2; see supplemental material online at www.gastrojournal.org). The data for 1594 cases of CD genotyped for CARD15 mutations in earlier studies were used to undertake analysis for evidence of interaction between CARD15 and IL23R.18Waller S. Tremelling M. Bredin F. Godfrey L. Howson J. Parkes M. Evidence for association of OCTN genes and IBD5 with ulcerative colitis.Gut. 2006; 55: 809-814Google Scholar, 19Pearce A.V. Fisher S.A. Prescott N.J. Onnie C.M. Pattni R. Green P. Forbes A. Mansfield J. Sanderson J. Schreiber S. Lewis C.M. Mathew C.G. Investigation of association of the DLG5 gene with phenotypes of inflammatory bowel disease in the British population.Int J Colorectal Dis. 2007; 22: 419-424Google Scholar, 20Arnott I.D. Nimmo E.R. Drummond H.E. Fennell J. Smith B.R. MacKinlay E. Morecroft J. Anderson N. Kelleher D. O’Sullivan M. McManus R. Satsangi J. NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn’s disease patients: evidence for genetic heterogeneity within Europe?.Genes Immun. 2004; 5: 417-425Google Scholar, 21Ahmad T. Tamboli C.P. Jewell D. Colombel J.F. Clinical relevance of advances in genetics and pharmacogenetics of IBD.Gastroenterology. 2004; 126: 1533-1549Google Scholar Allele frequencies were compared between cases and controls and between phenotypic subgroups using χ2 tests of 2 × 2 tables. Odds ratios were calculated for the minor allele at each SNP; confidence intervals (CIs) were calculated using Woolf’s method.22Woolf B. On estimating the relation between blood group and disease.Ann Hum Genet. 1955; 19: 251-253Google Scholar Pairwise SNP linkage disequilibrium coefficients were estimated using Haploview.23Barrett J.C. Fry B. Maller J. Daly M.J. Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Google Scholar Conditional association analysis was implemented using COCAPHASE, a module of the UNPHASED program.24Dudbridge F. Pedigree disequilibrium tests for multilocus haplotypes.Genet Epidemiol. 2003; 25: 115-121Google Scholar This method tests for equality of odds ratios for haplotypes identical at conditioning loci. The Mantel–Haenszel test for association conditioning on geographical region was implemented using PLINK (http://pngu.mgh.harvard.edu/∼purcell/plink/). Median age at disease diagnosis between groups was compared using the Wilcoxon rank sum test. Age at diagnosis was dichotomized according to the Montreal classification.25Silverberg M.S. Satsangi J. Ahmad T. Arnott I.D. Bernstein C.N. Brant S.R. Caprilli R. Colombel J.F. Gasche C. Geboes K. Jewell D.P. Karban A. Loftus Jr, E.V. Pena A.S. Riddell R.H. Sachar D.B. Schreiber S. Steinhart A.H. Targan S.R. Vermeire S. Warren B.F. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology.Can J Gastroenterol. 2005; 19: 5-36Crossref Scopus (2406) Google Scholar Unless specified otherwise, all analyses were performed using R version 2.2 for Windows (http://www.R-project.org). All genotypes were in Hardy–Weinberg equilibrium in both cases and controls (P > .05). A highly significant association with CD was observed across the region (Table 2). The strongest association was observed at the nonsynonymous SNP Arg381Gln, where the frequency of the A allele was 2.5% in CD compared with 6.2% in controls (P = 1.1 ×10−12). The odds ratio for this protective allele was 0.38 (95% CI, 0.29–0.50). Alternatively, the common wild-type homozygous GG genotype can be considered as the risk genotype with an odds ratio of 2.70. To minimize potential confounding from regional differences in allele frequencies, a Mantel–Haenszel test was performed across 12 regional strata. Mantel–Haenszel odds ratios was very similar to those obtained from pooled data for all SNPs. For example, the Mantel–Haenszel odds ratio was 0.36 (95% CI, 0.25–0.51) for Arg381Gln.Table 2Case-Control Allele Frequencies and Disease Odds Ratios (95% Confidence Intervals) for CD and UCSNPAlleleControlsCDPOdds ratio (95% CI)UCPOdds ratio (95% CI)rs1004819T0.3070.3831.1 × 10−81.41 (1.23–1.56)0.348.007131.20 (1.05–1.37)rs10489629G0.4480.3721.8 × 10−80.73 (0.66–0.82)0.43.260.93 (0.82–1.05)rs11465804G0.0580.0257.2 × 10−110.41 (0.31–0.53)0.046.0810.77 (0.58–1.02)rs11209026A0.0620.0251.1 × 10−120.38 (0.29–0.50)0.046.02910.73 (0.55–0.96)rs1343151T0.3320.2661.1 × 10−70.73 (0.65–0.82)0.315.260.92 (0.81–1.06)rs10889677A0.3150.3983.4 × 10−101.45 (1.28–1.61)0.358.00421.22 (1.07–1.39)rs11209032A0.3200.3901.3 × 10−71.35 (1.22–1.52)0.3524.0321.16 (1.01–1.32)rs1495965G0.4470.5173.4 × 10−71.32 (1.19–1.47)0.457.571.04 (0.92–1.18) Open table in a new tab Several SNPs also showed significant association with UC (Table 2). The strongest signal was observed with common SNPs rs1004819 (P = .0071) and rs10889677 (P = .0042). The frequency of Arg381Gln was only marginally different between cases and controls (UC, 0.046; controls, 0.062; P = .029), with an odds ratio of 0.73 (95% CI, 0.55–0.96). The nonsynonymous SNP Arg381Gln was in tight linkage disequilibrium with one other SNP (rs11465804, r2 = 0.85) but weak linkage disequilibrium with all 6 other SNPs (r2 = 0.03–0.1). A separate test for CD association was performed for each SNP conditioning on Arg381Gln by conditional regression modeling. This showed a significant association at all SNPs (P < .001) except rs11465804, with the strongest residual association detected at rs10889677 (P = 4.6 × 10−8). Hence, the nonsynonymous SNP does not account for all the association signal at this locus. Data were then analyzed for evidence of significant genotype-phenotype correlations based on age at onset of CD, disease location, and disease behavior (Table 3). No significant subgroup association was observed. In particular, the subgroup of subjects with CD affecting the colon only without small bowel disease (n = 539) appeared to be as strongly associated as those with exclusively ileal/small bowel involvement (n = 668) (minor allele frequencies, 2.3% and 2.0%, respectively). The age at disease onset ranged from 12 to 67 years in patients with CD who carried the A allele of Arg381Gln and from 0 to 80 years in wild-type GG cases. There was no difference in the median age of onset between these 2 groups (AA/AG: median, 28 years [n = 85]; GG: median, 26 years [n = 1650]; P = .26). Stratification of cases by age at diagnosis according to the Montreal classification25Silverberg M.S. Satsangi J. Ahmad T. Arnott I.D. Bernstein C.N. Brant S.R. Caprilli R. Colombel J.F. Gasche C. Geboes K. Jewell D.P. Karban A. Loftus Jr, E.V. Pena A.S. Riddell R.H. Sachar D.B. Schreiber S. Steinhart A.H. Targan S.R. Vermeire S. Warren B.F. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology.Can J Gastroenterol. 2005; 19: 5-36Crossref Scopus (2406) Google Scholar revealed similar genotype frequencies in all groups (Table 3). For UC, subgroup analysis by disease extent, smoking history, and sex also revealed no significant subgroup association. Age at onset of UC ranged from 14 to 79 years in cases who carried the A allele of Arg381Gln and from 2 to 81 years in wild-type GG cases, with no difference in the median age of onset between the 2 groups (AA/AG: median, 34 years [n = 72]; GG: median, 33 years [n = 708]; P = .14) (Table 4). A total of 1540 subjects with CD were fully genotyped for the 3 CARD15 mutations (G908R, L1007fs, R702W) (Table 3). The frequency of Arg381Gln in 460 cases carrying at least one CARD15 mutation (2.2%) was not significantly different from that in 1081 cases who carried none (2.7%; P = .47). None of the 3 cases who were homozygous for the rare A allele also carried a CARD15 mutation.Table 3Arg381Gln Genotype and Allele Frequencies in CD Cases Stratified by Known Phenotypic Subgroups and CARD15 StatusAAAGGGTotalFreq(A)Sex Male1366907270.026 Female249106811190.024Smoking history No1287297580.020 Yes1214144360.026 Ex011271280.004Disease location Pure colorectal disease1235155390.023 Pure ileal disease0315335640.027 Ileocolonic disease1256426680.020 Any colorectal disease246110111490.022 Any ileal disease154111511700.024Perianal disease Yes2204544760.025 No159120512650.024Disease behavior Stenosing1326046370.027 Penetrating2152482650.036 Inflammatory only0307147440.020Surgery Yes251103510880.025 No1316456770.024Age at diagnosis (y) 16 or younger181972060.024 17–40260112911910.027 Older than 400143243380.021CARD15 statusaSamples are subdivided by CARD15 status into those homozygous wild-type (−/ −), those heterozygous for CD-associated variants (−/+), and those homozygous or compound heterozygous for CD-associated variants (+/+). −/ −352102510800.027 −/+0153423570.021 +/+05981030.024a Samples are subdivided by CARD15 status into those homozygous wild-type (−/ −), those heterozygous for CD-associated variants (−/+), and those homozygous or compound heterozygous for CD-associated variants (+/+). Open table in a new tab Table 4Arg381Gln Genotype and Allele Frequencies in UC Cases Stratified by Known Phenotypic SubgroupsAAAGGGTotalFreq(A)Sex Male1414474890.044 Female2434364810.049Smoking history No0283013290.043 Yes0781880.040 Ex1201601810.061Disease extent Rectum only1121341470.048 Distal to splenic flexure1242863110.042 Proximal to splenic flexure1433874310.052Age at diagnosis (y) 16 or younger0239410.025 17–401414434850.044 Older than 400282282560.055 Open table in a new tab This study provides unequivocal confirmation of association between variants in the IL23R gene and IBD, suggesting a major effect on overall susceptibility to CD and a more modest effect on UC. Importantly, this study also shows the association at IL23R for the first time in a non-American population. The strength of this association at IL23R and the fact that it reaches such a magnitude in 2 independent data sets leaves no doubt that it is a true finding. In addition, this is one of the first instances of highly significant, independent replication of data derived from a genome-wide association scan and provides important validation of this technique as a hypothesis-free method for the identification of complex disease genes. As with the North American genome-wide scan, the strongest evidence for association was seen at the nonsynonymous SNP Arg381Gln, where the frequency of the A allele was 2.5% in CD compared with 6.2% in controls (P = 1.1 × 10−12). These allele frequencies are similar to those seen in the North American panel.14Duerr R.H. Taylor K.D. Brant S.R. Rioux J.D. Silverberg M.S. Daly M.J. Steinhart A.H. Abraham C. Regueiro M. Griffiths A. Dassopoulos T. Bitton A. Yang H. Targan S. Datta L.W. Kistner E.O. Schumm L.P. Lee A. Gregersen P.K. Barmada M.M. Rotter J.I. Nicolae D.L. Cho J.H. A Genome-wide association study identifies IL23R as an inflammatory bowel disease gene.Science. 2006; 314: 1461-1463Google Scholar There was no evidence that IL23R variants associate with any particular subphenotype of CD based on disease behavior or location. Hence, there was no difference in minor allele frequency even between the extremes of pure ileal/small bowel CD and pure colonic CD (2.7% and 2.3%, respectively). Likewise, analysis based on disease behavior did not show any specific subgroup associations (Table 3). This negative result is interesting because it contrasts with the other confirmed CD susceptibility locus CARD15, which seems to have definite associations with ileal disease.26Economou M. Trikalinos T.A. Loizou K.T. Tsianos E.V. Ioannidis J.P.A. Differential effects of NOD2 variants on Crohn’s disease risk and phenotype in diverse populations: a metaanalysis.Am J Gastroenterol. 2004; 99: 2393-2404Google Scholar These findings are extended by the observation of association with UC overall but not with any known UC subphenotype group, suggesting that IL23R variants may exert a rather generic effect on chronic intestinal inflammation, although the effect size in UC does appear to be smaller than in CD. It is noteworthy that the odds ratio confidence interval at Arg381Gln for UC (0.73 [95% CI, 0.55–0.96]) does not overlap with that for CD (0.38 [95% CI, 0.29–0.50]), suggesting a significantly less marked protective effect of the rare allele for UC compared with CD. Based on data from our large, independent panel of CD cases, it is possible to provide an accurate estimate of the size of the effect conferred by IL23R variants with regard to the risk of CD. We estimated an odds ratio of 0.38 (95% CI, 0.29–0.49) for Arg381Gln. This is likely to be a more accurate estimate than that provided in the index report from the North American study (odds ratio, 0.26; 95% CI, 0.15–0.43) due to the well-recognized bias of the so-called “winner’s curse,” which leads to overestimation of effect size in discovery panels.27Lohmueller K.E. Pearce C.L. Pike M. Lander E.S. Hirschhorn J.N. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease.Nat Genet. 2003; 33: 177-182Google Scholar Characterizing the exact effect size is important to permit sample size calculation for any further attempts at replication. Where the effect size is overestimated, there is a risk that apparently appropriately powered studies will fail to observe the effect and erroneously conclude that it is a fa

Identifying shared and disease-specific susceptibility loci for Crohn's disease (CD) and ulcerative colitis (UC) would help define the biologic relationship between the inflammatory bowel diseases. More than 30 CD susceptibility loci have been identified. These represent important candidate susceptibility loci for UC. Loci discovered by the index genome scans in CD have previously been tested for association with UC, but those identified in the recent meta-analysis await such investigation. Furthermore, the recently identified UC locus at ECM1 requires formal testing for association with CD.We analyzed 45 single nucleotide polymorphisms, tagging 29 of the loci recently associated with CD in 2527 UC cases and 4070 population controls. We also genotyped the UC-associated ECM1 variant rs11205387 in 1560 CD patients and 3028 controls.Nine regions showed association with UC at a threshold corrected for the 29 loci tested (P < .0017). The strongest association (P = 4.13 x 10(-8); odds ratio = 1.27) was identified with a 170-kilobase region on chromosome 1q32 that contains 3 genes. We also found association with JAK2 and replicated a recently reported association with STAT3, further implicating the role of this signaling pathway in inflammatory bowel disease. Additional novel UC susceptibility genes were LYRM4 and CDKAL1. Twenty of the loci were not associated with UC, and several appear to be specific to CD. ECM1 variation was not associated with CD.Collectively, these data help define the genetic relationship between CD and UC and characterize common, as well as disease-specific mechanisms of pathogenesis.

Background Stem cell therapy offers a promising approach to the regeneration of damaged vascular and cardiac tissue after acute myocardial infarction (AMI). This has resulted in multiple randomised controlled trials (RCTs) worldwide. Objectives To critically evaluate evidence from RCTs on the effectiveness of adult bone marrow‐derived stem cells (BMSC) to treat acute myocardial infarction (AMI). Search methods This Cochrane review is an update of a previous one (published in 2008). MEDLINE (1950 to January 2011), EMBASE (1974 to January 2011), the Cochrane Central Register of Controlled Trials (CENTRAL) (Issue 1, 2011), CINAHL (1982 to January 2011) and the Transfusion Evidence Library (1980 to January 2011) were searched. In addition, several international and ongoing trial databases were searched and handsearching of relevant conference proceedings undertaken to January 2011. Selection criteria RCTs comparing autologous stem/progenitor cells with no autologous stem/progenitor cells in patients diagnosed with AMI were eligible. Data collection and analysis Two authors independently screened all references, assessed trial quality and extracted data. Meta‐analyses using a random‐effects model were conducted and heterogeneity was explored for the primary outcome using sub‐group analyses. Main results Thirty‐three RCTs (1765 participants) were eligible for inclusion. Stem/progenitor cell treatment was not associated with statistically significant changes in the incidence of mortality (RR 0.70, 95% CI 0.40 to 1.21) or morbidity (the latter measured by re‐infarction, hospital re‐admission, restenosis and target vessel revascularisation). A considerably high degree of heterogeneity has been observed among the included trials. In short‐term follow up, stem cell treatment was observed to improve left ventricular ejection fraction (LVEF) significantly (WMD 2.87, 95% CI 2.00 to 3.73). This improvement in LVEF was maintained over long‐term follow up of 12 to 61 months (WMD 3.75, 95% CI 2.57 to 4.93). With certain measurements and at certain times, stem cell treatment was observed to reduce left ventricular end systolic and end diastolic volumes (LVESV & LVEDV) and infarct size significantly in long‐term follow up. There was a positive correlation between mononuclear cell dose infused and the effect on LVEF measured by magnetic resonance imaging. A correlation between timing of stem cell treatment and effect on LVEF measured by left ventricular angiography was also observed. Authors' conclusions Despite the high degree of heterogeneity observed, the results of this systematic review suggest that moderate improvement in global heart function is significant and sustained long‐term. However, because mortality rates after successful revascularization of the culprit arteries are very low, larger number of participants would be required to assess the full clinical effect of this treatment. Standardisation of methodology, cell dosing and cell product formulation, timing of cell transplantation and patient selection may also be required in order to reduce the substantial heterogeneity observed among the included studies.

Background The evidence for vitamin D and other agents that experimentally modulate T regulatory cells (Tregs) for the treatment of patients with autoimmune or allergic diseases has not been established. Objective We have undertaken a systematic review of randomised controlled trials to assess the efficacy of vitamin D, vitamin A, niacin and short-chain fatty acids in enhancing absolute Treg numbers and phenotypes in patients with inflammatory or autoimmune disease. Methods This systematic review was conducted using a predefined protocol (PROSPERO International prospective register of systematic reviews, ID = CRD42016048648/ CRD42016048646). Randomised controlled trials of patients with inflammatory or autoimmune disease or healthy participants which compared either oral vitamin D or vitamin A or short-chain fatty acids with control or placebo and measured the absolute concentration of proportion of Tregs were eligible for inclusion. Searches of electronic databases (CENTRAL, MEDLINE, EMBASE, CINAHL, PUBMED and Web of Science) identified eight eligible independent trials (seven autoimmune disease trials, one trial of healthy subjects). Data were extracted by two reviewers and the risk of study bias was assessed using Cochrane Collaboration methodology. Results Planned meta-analysis was not possible due to the heterogeneous nature of the studies. Nevertheless, in five trials of autoimmune disorders which measured the proportion of Tregs, a higher proportion was observed in the vitamin D group compared to controls at 12 months in all but one trial. In the trial of healthy subjects, a significant difference was reported, with a higher percentage of Tregs observed in the vitamin D group (at 12 weeks, mean 6.4% (SD 0.8%) (vitamin D) vs 5.5% (1.0%) (placebo). There were no trials to assess the efficacy of vitamin A, niacin and short-chain fatty acids in enhancing absolute Treg numbers. Conclusions Vitamin D supplementation may increase Treg/CD3 ratios in both healthy individuals and patients with autoimmune disorders and may increase Treg function. There remains a need for further suitably powered clinical studies aimed at enhancing Treg numbers and/or function.

Background A promising approach to the treatment of chronic ischaemic heart disease (IHD) and heart failure is the use of stem cells. The last decade has seen a plethora of randomised controlled trials (RCTs) developed worldwide which have generated conflicting results. Objectives The critical evaluation of clinical evidence on the safety and efficacy of autologous adult bone marrow‐derived stem cells (BMSC) as a treatment for chronic ischaemic heart disease (IHD) and heart failure. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library, 2013, Issue 3), MEDLINE (from 1950), EMBASE (from 1974), CINAHL (from 1982) and the Transfusion Evidence Library (from 1980), together with ongoing trial databases, for relevant trials up to 31st March 2013. Selection criteria Eligible studies included RCTs comparing autologous adult stem/progenitor cells with no autologous stem/progenitor cells in participants with chronic IHD and heart failure. Co‐interventions such as primary angioplasty, surgery or administration of stem cell mobilising agents, were included where administered to treatment and control arms equally. Data collection and analysis Two review authors independently screened all references for eligibility, assessed trial quality and extracted data. We undertook a quantitative evaluation of data using fixed‐effect meta‐analyses. We evaluated heterogeneity using the I² statistic; we explored considerable heterogeneity (I² > 75%) using a random‐effects model and subgroup analyses. Main results We include 23 RCTs involving 1255 participants in this review. Risk of bias was generally low, with the majority of studies reporting appropriate methods of randomisation and blinding, Autologous bone marrow stem cell treatment reduced the incidence of mortality (risk ratio (RR) 0.28, 95% confidence interval (CI) 0.14 to 0.53, P = 0.0001, 8 studies, 494 participants, low quality evidence) and rehospitalisation due to heart failure (RR 0.26, 95% CI 0.07 to 0.94, P = 0.04, 2 studies, 198 participants, low quality evidence) in the long term (≥12 months). The treatment had no clear effect on mortality (RR 0.68, 95% CI 0.32 to 1.41, P = 0.30, 21 studies, 1138 participants, low quality evidence) or rehospitalisation due to heart failure (RR 0.36, 95% CI 0.12 to 1.06, P = 0.06, 4 studies, 236 participants, low quality evidence) in the short term (< 12 months), which is compatible with benefit, no difference or harm. The treatment was also associated with a reduction in left ventricular end systolic volume (LVESV) (mean difference (MD) ‐14.64 ml, 95% CI ‐20.88 ml to ‐8.39 ml, P < 0.00001, 3 studies, 153 participants, moderate quality evidence) and stroke volume index (MD 6.52, 95% CI 1.51 to 11.54, P = 0.01, 2 studies, 62 participants, moderate quality evidence), and an improvement in left ventricular ejection fraction (LVEF) (MD 2.62%, 95% CI 0.50% to 4.73%, P = 0.02, 6 studies, 254 participants, moderate quality evidence), all at long‐term follow‐up. Overall, we observed a reduction in functional class (New York Heart Association (NYHA) class) in favour of BMSC treatment during short‐term follow‐up (MD ‐0.63, 95% CI ‐1.08 to ‐0.19, P = 0.005, 11 studies, 486 participants, moderate quality evidence) and long‐term follow‐up (MD ‐0.91, 95% CI ‐1.38 to ‐0.44, P = 0.0002, 4 studies, 196 participants, moderate quality evidence), as well as a difference in Canadian Cardiovascular Society score in favour of BMSC (MD ‐0.81, 95% CI ‐1.55 to ‐0.07, P = 0.03, 8 studies, 379 participants, moderate quality evidence). Of 19 trials in which adverse events were reported, adverse events relating to the BMSC treatment or procedure occurred in only four individuals. No long‐term adverse events were reported. Subgroup analyses conducted for outcomes such as LVEF and NYHA class revealed that (i) route of administration, (ii) baseline LVEF, (iii) cell type, and (iv) clinical condition are important factors that may influence treatment effect. Authors' conclusions This systematic review and meta‐analysis found moderate quality evidence that BMSC treatment improves LVEF. Unlike in trials where BMSC were administered following acute myocardial infarction (AMI), we found some evidence for a potential beneficial clinical effect in terms of mortality and performance status in the long term (after at least one year) in people who suffer from chronic IHD and heart failure, although the quality of evidence was low.

Recipients of allogeneic haematopoietic stem cell transplants (HSCT) can develop acute or chronic, or both forms of graft-versus-host disease (a/cGvHD), whereby immune cells of the donor attack host tissues. Steroids are the primary treatment, but patients with severe, refractory disease have limited options and a poor prognosis. Mesenchymal stromal cells (MSCs) exhibit immunosuppressive properties and are being tested in clinical trials for their safety and efficacy in treating many immune-mediated disorders. GvHD is one of the first areas in which MSCs were clinically applied, and it is important that the accumulating evidence is systematically reviewed to assess whether their use is favoured.To determine the evidence for the safety and efficacy of MSCs for treating immune-mediated inflammation post-transplantation of haematopoietic stem cells.We searched for randomised controlled trials (RCTs) in the Cochrane Central Register of Controlled Trials (CENTRAL, the Cochrane Library 2018, Issue 12), MEDLINE (from 1946), Embase (from 1974), CINAHL (from 1937), Web of Science: Conference Proceedings Citation Index-Science (CPCI-S) (from 1990) and ongoing trial databases to 6 December 2018. No constraints were placed on language or publication status.We included RCTs of participants with a haematological condition who have undergone an HSCT as treatment for their condition and were randomised to MSCs (intervention arm) or no MSCs (comparator arm), to prevent or treat GvHD. We also included RCTs which compared different doses of MSCs or MSCs of different sources (e.g. bone marrow versus cord). We included MSCs co-transplanted with haematopoietic stem cells as well as MSCs administered post-transplantation of haematopoietic stem cells.We used standard methodological procedures expected by Cochrane.We employed a random-effects model for all analyses due to expected clinical heterogeneity arising from differences in participant characteristics and interventions.We identified 12 completed RCTs (879 participants), and 13 ongoing trials (1532 enrolled participants planned). Of 12 completed trials, 10 compared MSCs versus no MSCs and two compared different doses of MSCs. One trial was in people with thalassaemia major, the remaining trials were for haematological malignancies. Seven trials administered MSCs to prevent GvHD, whereas five trials gave MSCs to treat GvHD.In the comparison of MSCs with no MSCs, cells were administered at a dose of between 105 and 107 cells/kg in either a single dose (six trials) or in multiple doses (four trials) over a period of three days to four months. The dose-comparison trials compared 2 x 106 cells/kg with 8 x 106 cells/kg in two infusions, or 1 x 106 cells/kg with 3 x 106 cells/kg in a single infusion.The median duration of follow-up in seven trials which administered MSCs prophylactically ranged from 10 to 60 months. In three trials of MSCs as treatment for aGvHD, participants were followed up for 90 or 100 days. In two trials of MSCs as treatment for cGvHD, the mean duration of follow-up was 13.4 months (MSC group) and 23.6 months (control group) in one trial, and 56 weeks in the second trial. Five trials included adults only, six trials included adults and children, and one trial included children only. In eight trials which reported the gender distribution, the percentage of females ranged from 20% to 59% (median 35.8%).The overall quality of the included studies was low: randomisation methods were poorly reported and several of the included studies were subject to a high risk of performance bias and reporting bias. One trial which started in 2008 has not been published and the progress of this trial in unknown, leading to potential publication bias. The quality of evidence was therefore low or very low for all outcomes due to a high risk of bias as well as imprecision due to the low number of overall participants, and in some cases evidence based on a single study. We found that MSCs may make little or no difference in the risk of all-cause mortality in either prophylactic trials (HR 0.85, 95% CI 0.50 to 1.42; participants = 301; studies = 5; I2 = 34% ; low-quality evidence) or therapeutic trials (HR 1.12, 95% CI 0.80 to 1.56; participants = 244; studies = 1; very low-quality evidence), and no difference in the risk of relapse of malignant disease (prophylactic trials: RR 1.08, 95% CI 0.73 to 1.59; participants = 323; studies = 6; I2 = 0%; low-quality evidence) compared with no MSCs. MSCs were well-tolerated, no infusion-related toxicity or ectopic tissue formation was reported. No study reported health-related quality of life. In prophylactic trials, MSCs may reduce the risk of chronic GvHD (RR 0.66, 95% CI 0.49 to 0.89; participants = 283; studies = 6; I2 = 0%; low-quality evidence). This means that only 310 (95% CI 230 to 418) in every 1000 patients in the MSC arm are expected to develop chronic GvHD compared to 469 in the control arm. However, MSCs may make little or no difference to the risk of aGvHD (RR 0.86, 95% CI 0.63 to 1.17; participants = 247; studies = 6; I2 = 0%; low-quality evidence). In GvHD therapeutic trials, we are very uncertain whether MSCs improve complete response of either aGvHD (RR 1.16, 95% CI 0.79 to 1.70, participants = 260, studies = 1; very low-quality evidence) or cGvHD (RR 5.00, 95%CI 0.75 to 33.21, participants = 40, studies = 1; very low-quality evidence).In two trials which compared different doses of MSCs, we found no evidence of any differences in outcomes.MSCs are an area of intense research activity, and an increasing number of trials have been undertaken or are planned. Despite a number of reports of positive outcomes from the use of MSCs for treating acute GvHD, the evidence to date from RCTs has not supported the conclusion that they are an effective therapy. There is low-quality evidence that MSCs may reduce the risk of cGvHD. New trial evidence will be incorporated into future updates of this review, which may better establish a role for MSCs in the prevention or treatment of GvHD.

Understanding of the genetic basis of autoimmune diseases is currently incomplete. Cytokine gene polymorphisms warrant consideration as factors explaining variation in the human immune and inflammatory responses and as candidate susceptibility genes for related pathological states. Interleukin 12 (IL-12) is a key regulator of the polarisation of immune responses to T helper 1 or 2 categories and plays a role in autoimmune and infectious diseases. Using a bioinformatic strategy, we aligned cDNA and expressed sequence tag sequences to identify putative polymorphic regions of the IL-12 p40 gene. Position 1188 in the 3' untranslated region (UTR) was polymorphic with the frequency of the common allele around 80% in healthy UK Caucasoids. PCR genotyping of multiple Caucasoid groups and an African group showed significant population variation. In a case-control design, the polymorphism was not associated with rheumatoid arthritis, Felty's syndrome or large granular lymphocyte syndrome with arthritis or multiple sclerosis. A nonsignificant increase in the B allele frequency was observed in the rare large granular lymphocyte syndrome without arthritis (odds ratio 2.02 95% CI 0.95-4.3). This new genetic marker could be useful in anthropological studies and should be investigated in other autoimmune, allergic, inflammatory and infectious diseases.

Abstract Objective Levels of interleukin‐6 (IL‐6) have been shown to correlate with the fever and disease activity of systemic juvenile idiopathic arthritis (JIA). In a previous case–control study, a significant association between the IL‐6 −174 nucleotide variant and systemic JIA was noted, and HeLa cell transfection assays show functional differences in levels of transcription of the IL‐6 −174 alleles. The present study was undertaken to confirm the previous findings and to assess possible association with variations of the A n T n tract in the promoter. Methods We studied a cohort of JIA families from 3 countries, using transmission disequilibrium testing. Genotyping of the −174 nucleotide variant was done by restriction fragment length polymorphism, heteroduplex analysis, or allelic discrimination. The A n T n tract at −392 to −373 was typed using DNA sequencing. Statistical analysis was performed using the programs Transmit and EHplus. Results There was a significant excess transmission of the −174G allele in the systemic JIA families ( P = 0.041). The excess transmission was only to systemic JIA patients with age at onset &gt;5 years ( P = 0.007). No significant association with the other subtypes was found. No A n T n alleles or −174/A n T n haplotypes were significantly associated with systemic JIA. Conclusion This study confirms that the IL‐6 –174 nucleotide variant is significantly associated with systemic JIA. The significant excess transmission to patients with age at onset &gt;5 years but not to those with age at onset ≤5 years suggests that there may be genetic heterogeneity between the 2 groups.

Common germline genetic variation in the 3' region of myosin IXB (MYO9B) has been associated recently with susceptibility to celiac disease, with a hypothesis that MYO9B variants might influence intestinal permeability. These findings suggested the current study investigating a possible further role for MYO9B variation in inflammatory bowel disease.Eight single-nucleotide polymorphisms (SNPs) were selected to tag common haplotypes from the 35-kb 3' region of MYO9B. These included the strongest celiac disease-associated variants reported in a Dutch cohort. These SNPs were studied in 3 independently collected and genotyped case-control cohorts of European descent (UK, Dutch, and Canadian/Italian), comprising in total 2717 inflammatory bowel disease patients (1197 with Crohn's disease, 1520 with ulcerative colitis) and 4440 controls.Common variation in MYO9B was associated with susceptibility to inflammatory bowel disease in all 3 cohorts examined (most associated SNP, rs1545620; meta-analysis P = 1.9 x 10(-6); odds ratio, 1.2), with the same alleles showing association as reported for celiac disease.MYO9B genetic variants predispose to inflammatory bowel disease. Interestingly, rs1545620 is a nonsynonymous variant leading to an amino acid change (Ala1011Ser) in the third calmodulin binding IQ domain of MYO9B. Unlike previous variants (in other genes) reported to predispose to inflammatory bowel disease, the association at MYO9B was considerably stronger with ulcerative colitis, although weaker association with Crohn's disease also was observed. These data imply shared causal mechanisms underlying intestinal inflammatory diseases.

DNA polymorphisms in a region on chromosome 5q33.1 which contains two genes, immunity related GTPase related family, M (IRGM) and zinc finger protein 300 (ZNF300), are associated with Crohn's disease (CD). The deleted allele of a 20 kb copy number variation (CNV) upstream of IRGM was recently shown to be in strong linkage disequilibrium (LD) with the CD-associated single nucleotide polymorphisms and is itself associated with CD (P < 0.01). The deletion was correlated with increased or reduced expression of IRGM in transformed cells in a cell line-dependent manner, and has been proposed as a likely causal variant. We report here that small insertion/deletion polymorphisms in the promoter and 5' untranslated region of IRGM are, together with the CNV, strongly associated with CD (P = 1.37 x 10(-5) to 1.40 x 10(-9)), and that the CNV and the 5'-untranslated region variant -308(GTTT)(5) contribute independently to CD susceptibility (P = 2.6 x 10(-7) and P = 2 x 10(-5), respectively). We also show that the CD risk haplotype is associated with a significant decrease in IRGM expression (P < 10(-12)) in untransformed lymphocytes from CD patients. Further analysis of these variants in a Japanese CD case-control sample and of IRGM expression in HapMap populations revealed that neither the IRGM insertion/deletion polymorphisms nor the CNV was associated with CD or with altered IRGM expression in the Asian population. This suggests that the involvement of the IRGM risk haplotype in the pathogenesis of CD requires gene-gene or gene-environment interactions which are absent in Asian populations, or that none of the variants analysed are causal, and that the true causal variants arose after the European-Asian split.

To evaluate bone marrow stem cell treatment (BMSC) in patients with ischemic heart disease (IHD) and no option of revascularization.Autologous BMSC therapy has emerged as a novel approach to treat patients with acute myocardial infarction or chronic ischemia and heart failure following percutaneous or surgical revascularization, respectively. However, the effect of the treatment has not been systematic evaluated in patients who are not eligible for revascularization.MEDLINE (1950-2012), EMBASE (1980-2012), CENTRAL (The Cochrane Library 2012, Issue 8) and ongoing trial databases were searched for relevant randomized controlled trials. Trials where participants were diagnosed with IHD, with no option for revascularization and who received any dose of stem cells by any delivery route were selected for inclusion. Study and participant characteristics, details of the intervention and comparator, and outcomes measured were recorded by two reviewers independently. Primary outcome measures were defined as mortality and measures of angina; secondary outcomes were heart failure, quality of life measures, exercise/performance and left ventricular ejection fraction (LVEF).Nine trials were eligible for inclusion. BMSC treatment significantly reduced the risk of mortality (Relative Risk 0.33; 95% Confidence Interval 0.17 to 0.65; P = 0.001). Patients who received BMSC showed a significantly greater improvement in CCS angina class (Mean Difference -0.55; 95% Confidence Interval -1.00 to -0.10; P = 0.02) and significantly fewer angina episodes per week at the end of the trial (Mean Difference -5.21; 95% Confidence Interval -7.35 to -3.07; P<0.00001) than those who received no BMSC. In addition, the treatment significantly improved quality of life, exercise/performance and LVEF in these patients.BMSC treatment has significant clinical benefit as stand-alone treatment in patients with IHD and no other treatment option. These results require confirmation in large well-powered trials with long-term follow-up to fully evaluate the clinical efficacy of this treatment.

Aims To investigate whether there are important sources of heterogeneity between the findings of different clinical trials which administer autologous stem cell treatment for acute myocardial infarction (AMI) and to evaluate what factors may influence the long-term effects of this treatment. Methods and Results MEDLINE (1950-January 2011), EMBASE (1974-January 2011), CENTRAL (The Cochrane Library 2011, Issue 1), CINAHL (1982-January 2011), and ongoing trials registers were searched for randomised trials of bone marrow stem cells as treatment for AMI. Hand-searching was used to screen recent, relevant conference proceedings (2005–2010/11). Meta-analyses were conducted using random-effects models and heterogeneity between subgroups was assessed using chi-squared tests. Planned analyses included length of follow-up, timing of cell infusion and dose, patient selection, small trial size effect, methodological quality, loss of follow-up and date of publication. Thirty-three trials with a total of 1,765 participants were included. There was no evidence of bias due to publication or time-lag, methodological quality of included studies, participant drop-out, duration of follow-up or date of the first disclosure of results. However, in long-term follow-ups the treatment seemed more effective when administered at doses greater than 108 cells and to patients with more severe heart dysfunction. Conclusions Evaluation of heterogeneity between trials has not identified significant sources of bias in this study. However, clinical differences between trials are likely to exist which should be considered when undertaking future trials.

ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a ∼16.8 cM interval in chromosomal region 3q22-24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain-which can activate p53-of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans.

The introduction of oral direct anti-Xa anticoagulants apixaban and rivaroxaban has significantly impacted the treatment and prevention of thromboembolic disease. Clinical scenarios exist in which a quantitative assessment for degree of anticoagulation due to these agents would aid management. The purpose of this work was to evaluate the chromogenic antifactor Xa assay calibrated with heparin standards at our institution for assessment of intensity of anticoagulation with rivaroxaban or apixaban in addition to its current use for unfractionated heparin or low-molecular-weight heparin. We also aimed to propose expected steady state peak and trough antifactor Xa activities for these agents based upon dosing regimens approved for nonvalvular atrial fibrillation. Antifactor Xa activity correlated very strongly with apixaban and rivaroxaban concentration in both spiked samples and treated patient plasma samples ( r 2 = .99, P &lt; .001). This correlation was observed over a broad range (20-500 ng/mL) of drug concentrations, as sample dilution with pooled normal plasma significantly extended the range of quantitative assessment. Based on drug concentrations previously published in pharmacokinetic studies, the expected steady state peak and trough antifactor Xa activity ranges for apixaban are 1.80 to 2.20 IU/mL and 0.70 to 1.10 IU/mL, respectively. For rivaroxaban, these ranges are 3.80 to 6.20 IU/mL and 0.60 to 1.00 IU/mL, respectively. In conclusion, our findings demonstrate that heparin-calibrated antifactor Xa activity correlates strongly with apixaban and rivaroxaban concentration. The dilution of samples allowed for this correlation to be extended over the majority of on-therapy drug concentrations.

A common haplotype spanning 250 kb in the cytokine gene cluster on chromosome 5q31 has recently been reported to be strongly associated with Crohn disease (CD) in Canadian families. We have replicated this finding by both the transmission-disequilibrium test (TDT) (P=.016) and in a case-control association study (P=.008) in a large European cohort of patients with CD, although the increase in disease risk was small (odds ratio 1.49 for homozygotes, 95% CI 1.11–2.0). No association was detected in families or individuals with ulcerative colitis (UC). Stratification of offspring with CD in the TDT sample by mutation status in the CD susceptibility gene CARD15 showed that the association with the 5q31 risk haplotype was present only in offspring with at least one of the known CARD15 disease susceptibility alleles (P=.044). The 5q31 risk haplotype frequency was 53.1% in unrelated individuals with CD who had one or two CARD15 mutations versus 43.7% in control subjects (P=.0001) but was not significantly elevated in individuals with CD who had no CARD15 mutations (45.4%, P=.41). Kaplan-Meier survival analysis of age at disease onset showed a significantly earlier onset in homozygotes for the 5q31 risk haplotype (P=.0019). These findings suggest that genetic variants at the 5q31 (IBD5) locus may hasten the onset of Crohn disease and cooperate with CARD15 in disease causation. A common haplotype spanning 250 kb in the cytokine gene cluster on chromosome 5q31 has recently been reported to be strongly associated with Crohn disease (CD) in Canadian families. We have replicated this finding by both the transmission-disequilibrium test (TDT) (P=.016) and in a case-control association study (P=.008) in a large European cohort of patients with CD, although the increase in disease risk was small (odds ratio 1.49 for homozygotes, 95% CI 1.11–2.0). No association was detected in families or individuals with ulcerative colitis (UC). Stratification of offspring with CD in the TDT sample by mutation status in the CD susceptibility gene CARD15 showed that the association with the 5q31 risk haplotype was present only in offspring with at least one of the known CARD15 disease susceptibility alleles (P=.044). The 5q31 risk haplotype frequency was 53.1% in unrelated individuals with CD who had one or two CARD15 mutations versus 43.7% in control subjects (P=.0001) but was not significantly elevated in individuals with CD who had no CARD15 mutations (45.4%, P=.41). Kaplan-Meier survival analysis of age at disease onset showed a significantly earlier onset in homozygotes for the 5q31 risk haplotype (P=.0019). These findings suggest that genetic variants at the 5q31 (IBD5) locus may hasten the onset of Crohn disease and cooperate with CARD15 in disease causation. Crohn disease (CD [MIM 266600]) and ulcerative colitis (UC [MIM 191390]) are two forms of chronic inflammatory bowel disease (IBD) with distinct clinical features. CD affects any part of the gastrointestinal tract, whereas in UC the inflammation is confined to the colon and rectum. Genetic linkage studies identified a susceptibility locus for CD on chromosome 16 (Hugot et al. Hugot et al., 1996Hugot JP Laurent-Puig P Gower-Rousseau C Olson JM Lee JC Beaugerie L Naom I Dupas JL Van Gossum A Orholm M Bonaiti-Pellie C Weissenbach J Mathew CG Lennard-Jones JE Cortot A Colombel JF Thomas G Mapping of a susceptibility locus for Crohn's disease on chromosome 16.Nature. 1996; 379: 821-823Crossref PubMed Scopus (833) Google Scholar), and subsequent positional cloning and candidate gene studies revealed mutations in the NOD2 gene (now known as CARD15) at this locus that were strongly associated with CD (Hampe et al. Hampe et al., 2001Hampe J Cuthbert A Croucher PJ Mirza MM Mascheretti S Fisher S Frenzel H King K Hasselmeyer A MacPherson AJ Bridger S van Deventer S Forbes A Nikolaus S Lennard-Jones JE Foelsch UR Krawczak M Lewis C Schreiber S Mathew CG Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations.Lancet. 2001; 357: 1925-1928Abstract Full Text Full Text PDF PubMed Scopus (985) Google Scholar; Hugot et al. Hugot et al., 2001Hugot JP Chamaillard M Zouali H Lesage S Cezard JP Belaiche J Almer S Tysk C O’Morain CA Gassull M Binder V Finkel Y Cortot A Modigliani R Laurent-Puig P Gower-Rousseau C Macry J Colombel JF Sahbatou M Thomas G Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.Nature. 2001; 411: 599-603Crossref PubMed Scopus (4485) Google Scholar; Ogura et al. Ogura et al., 2001Ogura Y Bonen DK Inohara N Nicolae DL Chen FF Ramos R Britton H Moran T Karaliuskas R Duerr RH Achkar JP Brant SR Bayless TM Kirschner BS Hanauer SB Nunez G Cho JH A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature. 2001; 411: 603-606Crossref PubMed Scopus (3992) Google Scholar). A genomewide search in Canadian families found evidence of linkage to CD on chromosome 5q31 (Rioux et al. Rioux et al., 2000Rioux JD Silverberg MS Daly MJ Steinhart AH McLeod RS Griffiths AM Green T Brettin TS Stone V Bull SB Bitton A Williams CN Greenberg GR Cohen Z Lander ES Hudson TJ Siminovitch KA Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci.Am J Hum Genet. 2000; 66: 1863-1870Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar), and linkage disequilibrium (LD) mapping of this locus detected association of a common haplotype in the 5q31 cytokine gene cluster with CD (Rioux et al. Rioux et al., 2001Rioux JD Daly MJ Silverberg MS Lindblad K Steinhart H Cohen Z Delmonte T et al.Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.Nat Genet. 2001; 29: 223-228Crossref PubMed Scopus (678) Google Scholar). This haplotype contained 11 SNPs that were in almost complete LD with each other and were strongly associated with disease. The recent report (Dahlman et al. Dahlman et al., 2002Dahlman I Eaves IA Kosoy R Morrison VA Heward J Gough SC Allahabadia A Franklyn JA Tuomilehto J Tuomilehto-Wolf E Cucca F Guja C Ionescu-Tirgoviste C Stevens H Carr P Nutland S McKinney P Shield JP Wang W Cordell HJ Walker N Todd JA Concannon P Parameters for reliable results in genetic association studies in common disease.Nat Genet. 2002; 30: 149-150Crossref PubMed Scopus (198) Google Scholar) of a lack of confirmation of a published association between a genetic variant in the IL12B gene and type 1 diabetes (Morahan et al. Morahan et al., 2001Morahan G Huang D Ymer SI Cancilla MR Stephen K Dabadghao P Werther G Tait BD Harrison LC Colman PG Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele.Nat Genet. 2001; 27: 218-221Crossref PubMed Scopus (273) Google Scholar) exemplifies the importance of independent replication of such findings in complex disorders. We therefore sought to replicate the association of the 5q31 locus with CD in a large sample of European patients. We also investigated whether this association extends to UC, since common genes may contribute to susceptibility to both of these IBD subphenotypes (Watts and Satsangi Watts and Satsangi, 2002Watts DA Satsangi J The genetic jigsaw of inflammatory bowel disease.Gut. 2002; : iii31-iii36PubMed Google Scholar). Two SNPs (C2063G and C2198G) that lie ∼67 kb apart on the common disease-associated haplotype were genotyped in 267 unrelated individuals and confirmed to be in strong LD (Δ=0.87; D′=0.91). C2063G, which is a nonsynonymous change in a GENSCAN predicted gene (Rioux et al. Rioux et al., 2001Rioux JD Daly MJ Silverberg MS Lindblad K Steinhart H Cohen Z Delmonte T et al.Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.Nat Genet. 2001; 29: 223-228Crossref PubMed Scopus (678) Google Scholar), was then genotyped in a set of 427 British and 138 German families (Hampe et al. Hampe et al., 1999Hampe J Schreiber S Shaw SH Lau KF Bridger S Macpherson AJS Cardon LR Sakul H Harris TJ Buckler A Hall J Stokkers P van Deventer SJJ Nürnberg P Mirza MM Lee JCW Lennard-Jones JE Mathew CG Curran ME A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort.Am J Hum Genet. 1999; 64: 808-816Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) containing a total of 511 offspring with CD and 320 with UC. All genotyping was performed using the TaqMan biallelic discrimination system (Livak et al. Livak et al., 1995Livak KJ Marmaro J Todd JA Towards fully automated genome-wide polymorphism screening.Nat Genet. 1995; 9: 341-342Crossref PubMed Scopus (286) Google Scholar) on an ABI 7700 DNA analyzer. Allelic transmission distortion to these two disease phenotypes was assessed by the transmission-disequilibrium test (TDT) (Spielman et al. Spielman et al., 1993Spielman RS McGinnis RE Ewens WJ Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM).Am J Hum Genet. 1993; 52: 506-516PubMed Google Scholar), implemented using TRANSMIT (Clayton Clayton, 1999Clayton D A generalization of the transmission/disequilibrium test for uncertain-haplotype transmission.Am J Hum Genet. 1999; 65: 1170-1177Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). An association test using transmission to all affected offspring was performed by a bootstrap simulation across families (TRANSMIT). A χ2 test confirmed that genotypes were in Hardy-Weinberg equilibrium. Excess transmission of the G allele of the C2063G SNP to affected offspring was observed in CD (P=.016 (table 1) but not in UC. The SNP was then genotyped in an independent set of unrelated British individuals with CD (n=684) and UC (n=388) (Cuthbert et al. Cuthbert et al., 2002Cuthbert AP Fisher SA Mirza MM King K Hampe J Croucher PJ Mascheretti S Sanderson J Forbes A Mansfield J Schreiber S Lewis CM Mathew CG The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease.Gastroenterology. 2002; 122: 867-874Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar) and in 701 British control subjects. The frequency of the G allele in patients with CD was significantly higher than in controls (P=.008) but was not increased in patients with UC (table 1). The CD odds ratios (ORs) for the CG and GG genotypes were 1.19 (95% CI 0.93–1.52) and 1.49 (95% CI 1.11–2.01), respectively.Table 1TDT and Case-Control Analyses for the C2063G SNP at 5q31Value for AnalysisTDTCases and ControlsPhenotypeNTDTaNTDT = number of affected offspring.ObsbObs = observed number of transmissions of G allele to affected offspring.ExpcExp = expected number of transmissions of G allele to affected offspring.PTDTdPTDT = P value from TDT association of allele G.NCCeNCC = number of case and control subjects.CCCGGGFreq(G)PCCfPCC = P value from test for difference in frequency of allele G in cases and controls, assessed with a χ2 test.CD511484458.9.01668419032716748.3%.008UC320249263.3.0633881461727040.2%NSgNS = not significant (P>.05).Control…………70123133413643.2%…a NTDT = number of affected offspring.b Obs = observed number of transmissions of G allele to affected offspring.c Exp = expected number of transmissions of G allele to affected offspring.d PTDT = P value from TDT association of allele G.e NCC = number of case and control subjects.f PCC = P value from test for difference in frequency of allele G in cases and controls, assessed with a χ2 test.g NS = not significant (P>.05). Open table in a new tab In view of the strong contribution of mutations in the CARD15 gene to the risk of CD, we investigated the possibility of interaction between this gene and the 5q31 locus in CD. Both the TDT and the case-control sample were genotyped for the C2063G SNP and for the three SNPs in the CARD15 gene (R702W, G908R, and 1007fs) that are independently associated with disease risk (Hugot et al. Hugot et al., 2001Hugot JP Chamaillard M Zouali H Lesage S Cezard JP Belaiche J Almer S Tysk C O’Morain CA Gassull M Binder V Finkel Y Cortot A Modigliani R Laurent-Puig P Gower-Rousseau C Macry J Colombel JF Sahbatou M Thomas G Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.Nature. 2001; 411: 599-603Crossref PubMed Scopus (4485) Google Scholar; Cuthbert et al. Cuthbert et al., 2002Cuthbert AP Fisher SA Mirza MM King K Hampe J Croucher PJ Mascheretti S Sanderson J Forbes A Mansfield J Schreiber S Lewis CM Mathew CG The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease.Gastroenterology. 2002; 122: 867-874Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). In the TDT sample, stratification of offspring with CD by their CARD15 mutation status showed some evidence of association in offspring carrying at least one CARD15 disease-susceptibility allele (DSA) (n=203; P=.044) but no association in offspring without the three mutations (n=259; P=.20). No significant differences in the distribution of CARD15 and 5q31 genotypes were observed between familial and sporadically ascertained patients with CD (P=.7). A single individual with CD was therefore randomly selected from each family, and these were combined with the sporadically ascertained patients with CD, providing a total of 943 unrelated patients with CD and 676 control subjects. These patients with CD and control subjects were stratified according to whether they had no, one, or two CARD15 DSAs, and the frequency of the C2063G SNP was determined in these three groups (table 2). By use of a χ2 test of independence of ordinal data, the distribution of C2063G genotypes was shown to be associated with the number of CARD15 mutations in patients with CD (P=.001) but not in control subjects (P>.5). Individuals with CD who had one or two CARD15 DSAs had a significantly higher frequency of the 2063G allele than those with none (53.1% vs. 45.4%; P=.0018). The frequency of 2063G in all unrelated individuals with CD (48.0%) was significantly higher than in control subjects (43.7%; P=.016). The association of this haplotype with CD was much stronger in affected individuals with one or two CARD15 DSAs (53.1% vs. 43.7% in control subjects; P=.0001) but was not significant in case subjects with CD who had no CARD15 DSAs (45.4% vs. 43.7%; P=.41). There was no significant difference in G allele frequency in control subjects with different CARD15 genotypes. Logistic regression analysis by Splus v5.1 was then used to estimate ORs for each genotype and for combinations of genotypes, modeling case/control status on 5q31 and CARD15 genotypes (table 2). Risk owing to 5q31 was modeled by the number of alleles (zero, one, or two), and independent risks were calculated for CARD15 genotypes, giving an OR of 1.12 for each copy of the 5q31 G allele and ORs of 2.47 and 22.05 for CARD15 −/+ and +/+, respectively. ORs for specific 5q31/CARD15 genotypes were obtained by multiplying the baseline contributions for each locus, and 95% CIs were obtained directly from logistic regression parameter estimates. CARD15 contributed most of the disease risk (P<10-10); the increased risk owing to 5q31 genotypes in individuals with CD carrying no CARD15 DSAs was not significant. The combined population attributable risk for these two loci was 31%.Table 2Case-Control Genotypes for the 5q31 and CARD15 Loci, with Disease Risk Estimated by Logistic RegressionControls(N=676)aN = number of case and control subjects.CD Cases(N=943)aN = number of case and control subjects.5q31/CARD15 OR (95% CI)CARD15GenotypeCCCGGGFreq(G)CCCGGGFreq(G)CCCGGG−/−18628211143.5%19029613345.4%11.12 (.97–1.29)1.25 (.94–1.67)−/+33392244.1%591236952.0%2.47 (1.90–3.22)2.77 (2.06–3.71)3.10 (2.12–4.53)+/+021…11412156.8%22.05 (7.30–66.67)24.70 (8.13–75.06)27.67 (8.89–86.04)Note.—CARD15 genotypes are defined by the presence or absence of three CD DSAs: −/− (wild type), −/+ (heterozygous for mutation), and +/+ (homozygous or compound heterozygous for mutation).a N = number of case and control subjects. Open table in a new tab Note.— CARD15 genotypes are defined by the presence or absence of three CD DSAs: −/− (wild type), −/+ (heterozygous for mutation), and +/+ (homozygous or compound heterozygous for mutation). The strongest evidence for linkage to 5q31 has been found in families with early-onset CD (Rioux et al. Rioux et al., 2000Rioux JD Silverberg MS Daly MJ Steinhart AH McLeod RS Griffiths AM Green T Brettin TS Stone V Bull SB Bitton A Williams CN Greenberg GR Cohen Z Lander ES Hudson TJ Siminovitch KA Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci.Am J Hum Genet. 2000; 66: 1863-1870Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). We therefore considered the effects of CARD15 and 5q31 loci on age at disease onset in 500 independent individuals with CD. Survival curves (years until age at CD onset) were calculated using Kaplan-Meier survival analysis (Splus v5.1) and were compared using a Mantel-Haenszel test (fig. 1). A significantly earlier age at onset occurred in the 2063 GG patients with CD than in CC/CG patients with CD (P=.0019); an earlier age at onset in patients with CD carrying at least one CARD15 DSA was marginally significant (P=.048). The age at disease onset in patients with CD with the genotype A−/− (2063 CC/CG, no CARD15 DSAs) was therefore compared with each of the two-locus early-onset high-risk genotypes, A−/+ (CC/CG, at least one CARD15 DSA), A+/− (GG, no CARD15 DSAs), and A+/+ (GG, at least one CARD15 DSA) (table 3). A significantly earlier age at onset occurred in both 2063 GG patients with CD who also carried at least one CARD15 DSA (A+/+; median age 21 years; P=.003) and those who did not carry a CARD15 DSA (A+/−; median age 23 years; P=.002), compared with the baseline genotype (A−/−; median age 27 years). The age at onset in 2063 GG patients with CD who also carried at least one CARD15 DSA (A−/+) was not significantly different from those without a CARD15 DSA. In 2063 CC/CG patients with CD, the effect of CARD15 on age at onset was only marginally significant (P=.058). Disease-onset risk ratios, as defined by the ratio of the relative disease-onset rates (observed/expected number of cases) for the risk and baseline genotypes (table 3), were calculated. The relative disease onset risk ratios between two-locus risk genotypes and the baseline genotype are 1.23 (A−/+), 1.49 (A+/−), and 1.59 (A+/+). By age 21 years, 58.1% of 2063 GG patients with CD who also carried at least one CARD15 DSA had developed the disease (95% CI 40.5–70.6) compared with 27.5% of patients with CD who did not carry a 5q31 or CARD15 risk genotype (95% CI 21.6–32.9). By age 36 years, 93% of individuals with the high-risk genotype at both loci had developed the disease. We have shown elsewhere that the presence of CARD15 mutations was associated with the ileal form of CD (Cuthbert et al. Cuthbert et al., 2002Cuthbert AP Fisher SA Mirza MM King K Hampe J Croucher PJ Mascheretti S Sanderson J Forbes A Mansfield J Schreiber S Lewis CM Mathew CG The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease.Gastroenterology. 2002; 122: 867-874Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). We therefore investigated the possibility of an association of the 5q31 haplotype with the site of disease in 551 independent individuals with CD. No association was found; the frequency of the 2063G allele was 48.7% in affected individuals with ileal disease and 48.4% in affected individuals with colon-specific disease (P=.99).Table 3Disease Onset Age Distribution by CARD15/5q31 Genotypes and Comparison of Baseline and Risk GenotypesAge at Onset(years)No. of CasesDisease OnsetRisk GenotypeMeanMedian (95% CI)Observed (N)ExpectedaExpected number of cases, under the assumption of no difference in age at disease onset between risk genotype (2063 GG or one or two CARD15 DSAs) and baseline genotype (no or one copy of 2063 G allele and no CARD15 DSAs). (PbP value obtained from test of difference between two survival curves (risk genotype vs. baseline genotype).)A−/−30.127 (25–29)244…A−/+27.123 (20–26)121105.0 (.058)A+/−25.223 (22–25)7352.9 (.002)A+/+24.121 (19–24)4328.6 (.003)a Expected number of cases, under the assumption of no difference in age at disease onset between risk genotype (2063 GG or one or two CARD15 DSAs) and baseline genotype (no or one copy of 2063 G allele and no CARD15 DSAs).b P value obtained from test of difference between two survival curves (risk genotype vs. baseline genotype). Open table in a new tab Our family- and population-based tests for association of genetic variation at the cytokine gene cluster on chromosome 5q31 and disease support the primary evidence for the existence of a CD susceptibility gene at this locus (IBD5) (Rioux et al. Rioux et al., 2001Rioux JD Daly MJ Silverberg MS Lindblad K Steinhart H Cohen Z Delmonte T et al.Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.Nat Genet. 2001; 29: 223-228Crossref PubMed Scopus (678) Google Scholar). We found no evidence of association with UC, which was not tested in the original study. The disease risk conferred by this locus in our large sample of individuals with CD was only 1.49 in 2063G homozygotes, which is substantially less than the sixfold risk predicted by Rioux et al (Rioux et al., 2001Rioux JD Daly MJ Silverberg MS Lindblad K Steinhart H Cohen Z Delmonte T et al.Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.Nat Genet. 2001; 29: 223-228Crossref PubMed Scopus (678) Google Scholar). The low risk is consistent with the absence of significant linkage to this locus in the genome scan from the British and German populations (Hampe et al. Hampe et al., 1999Hampe J Schreiber S Shaw SH Lau KF Bridger S Macpherson AJS Cardon LR Sakul H Harris TJ Buckler A Hall J Stokkers P van Deventer SJJ Nürnberg P Mirza MM Lee JCW Lennard-Jones JE Mathew CG Curran ME A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort.Am J Hum Genet. 1999; 64: 808-816Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) and underlines the difficulty in detection of linkage at loci of modest effect (Risch and Merikangas Risch and Merikangas, 1996Risch N Merikangas K The future of genetic studies of complex human diseases.Science. 1996; 273: 1516-1517Crossref PubMed Scopus (4160) Google Scholar). The most striking properties of the 5q31 locus, however, were its significantly higher frequency in our sample of 943 patients with CD who had one or two CARD15 mutations and its association with a younger age at disease onset. The early onset is consistent with the original linkage study (Rioux et al. Rioux et al., 2000Rioux JD Silverberg MS Daly MJ Steinhart AH McLeod RS Griffiths AM Green T Brettin TS Stone V Bull SB Bitton A Williams CN Greenberg GR Cohen Z Lander ES Hudson TJ Siminovitch KA Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci.Am J Hum Genet. 2000; 66: 1863-1870Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar), which showed a higher LOD score at this locus in families with at least one affected sibling with a diagnosis at age 16 years or younger. These authors did not find evidence of interaction of the 5q31 disease risk haplotype with CARD15 in 84 CD trios but pointed out that larger samples might be required to detect it (Vermeire et al. Vermeire et al., 2002Vermeire S Wild G Kocher K Cousineau J Dufresne L Bitton A Langelier D Pare P Lapointe G Cohen A Daly MJ Rioux JD CARD15 genetic variation in a Quebec population: prevalence, genotype-phenotype relationship, and haplotype structure.Am J Hum Genet. 2002; 71: 74-83Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The identity of the actual disease susceptibility gene at the 5q31 locus is unknown, but our findings suggest that it may interact with CARD15 in a common pathophysiological pathway. The 5q31 locus contains the genes for the cytokines IL4, IL5, and IL13, and CARD15 appears to be involved in bacterial lipopolysaccharide-induced activation of the transcription factor NF-κB in mononuclear phagocytes (Ogura et al. Ogura et al., 2001Ogura Y Bonen DK Inohara N Nicolae DL Chen FF Ramos R Britton H Moran T Karaliuskas R Duerr RH Achkar JP Brant SR Bayless TM Kirschner BS Hanauer SB Nunez G Cho JH A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature. 2001; 411: 603-606Crossref PubMed Scopus (3992) Google Scholar). Since abnormal regulation of monocyte activation by IL4 and IL13 has been described in CD (Schreiber et al. Schreiber et al., 1995Schreiber S Heinig T Panzer U Reinking R Bouchard A Stahl PD Raedler A Impaired response of activated mononuclear phagocytes to interleukin 4 in inflammatory bowel disease.Gastroenterology. 1995; 108: 21-33Abstract Full Text PDF PubMed Scopus (119) Google Scholar), it is possible that a hypomorphic allele in one of these regulatory cytokines in combination with a mutation in CARD15 may lead to an early and persistent proinflammatory response in the gastrointestinal tract. Thus, the 5q31 locus may contain a modifier gene which hastens disease onset in some genetically susceptible individuals. Such models must remain speculative until the function of CARD15 is more precisely defined and the identity of the CD susceptibility gene at 5q31 is known. These loci have roles in other inflammatory disorders; genetic and functional studies have implicated the 5q31 cytokine gene cluster in asthma (Cookson Cookson, 2002Cookson WO Asthma genetics.Chest. 2002; 121: 7S-13SCrossref PubMed Scopus (83) Google Scholar), and mutations in CARD15 have been detected in Blau syndrome, which is a Mendelian disorder characterized by granulomatous arthritis (Miceli-Richard et al. Miceli-Richard et al., 2001Miceli-Richard C Lesage S Rybojad M Prieur AM Manouvrier-Hanu S Hafner R Chamaillard M Zouali H Thomas G Hugot JP CARD15 mutations in Blau syndrome.Nat Genet. 2001; 29: 19-20Crossref PubMed Scopus (751) Google Scholar). The connection between these two loci in CD may, therefore, be of broader significance in the etiology of chronic inflammatory disease. We thank Prof. J. Lennard-Jones and Drs. S. Bridger and J. Lee, for patient ascertainment, and A. Craggs and J. Hirst, for help in obtaining blood samples. This work was supported by the Wellcome Trust, the Generation Trust, the European Union Fifth Framework Programme, and the Deutsche Forschungsgemeinschaft.

Upregulation of p53 protein induces either growth arrest or apoptosis in response to cellular injury This is signaled from a highly conserved p53 domain between codons 64 and 92, where a functional polymorphism results in either a proline (p53-72P) or an arginine (p53-72R) at codon 72. Preliminary studies suggest that p53-72R may be a risk factor for cervical cancer and, consistent with this, preferential mutation and retention of the p53-72R allele has also been demonstrated in other cancers of squamous cell origin. Here we examine the relationship between allelic forms of p53 and nonmelanoma skin cancer, by determining the correlation with susceptibility to sunburn, which is a known risk factor, and then by p53 sequence analysis of a large series of tumors. We found a significant positive association between p53-72R and susceptibility to sunburn, as assessed by skin phototype and minimal erythemal dose following solar-simulated radiation (p = 0.0001 for trend). We also found a significant association between p53-72R homozygosity and nonmelanoma skin cancer in renal transplant recipients (basal cell carcinoma, p < 0.01; squamous cell carcinoma, p < 0.05) but not in immunocompetent patients compared with skin type matched controls. p53 sequence data revealed mutations in 30 of 70 (42.9%) nonmelanoma skin cancers, 28 (93%) of which were in the p53-72R allele. Loss of heterozygosity occurred more frequently in p53-72RP than in p53-72RR tumors (p = 0.0001) with preferential loss of p53-72P in heterozygotes (p = 0.016), irrespective of the mutant status of the concomitant allele. Together these data infer functional differences between polymorphic forms of p53 that are likely to be relevant to skin carcinogenesis.

Allelic variants of the ATP-binding cassette, subfamily B member 1 (ABCB1), also known as the multidrug resistance gene (MDR1) that encodes the membrane-bound efflux transporter P-glycoprotein 170 (PGP-170), have been associated with inflammatory bowel disease but with conflicting results.The present study examined the association of ABCB1 C3435T and G2677T/A in a large British case-control cohort of 828 Crohn's disease, 580 ulcerative colitis (UC) cases, and 285 healthy controls. The effect of these variants was further examined with respect to phenotypic and epidemiological characteristics. A meta-analysis was carried out of our results and those from 8 previously published association studies of the C3435T variant in inflammatory bowel disease.The 2677T allele was significantly increased in British UC cases compared with controls (45.2% vs. 39.6%; P = 0.034). In particular, the TT genotype was significantly associated with severe UC (odds ratio [OR] 1.90; 95% CI 1.01-3.55) and the use of steroids in UC (OR 1.77; 95% CI 1.08-2.88). No significant association was seen with C3435T and UC, Crohn's disease, or any clinical subgroup. A meta-analysis of 9 association studies of C3435T showed a significant association of the 3435T allele with UC (OR 1.12; 95% CI 1.02-1.23; P = 0.013) but not with CD.These results indicate that ABCB1 sequence variants are associated with a small increase in the risk of developing UC and may influence disease behavior.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal system known as the inflammatory bowel diseases (IBD). Recently, Stoll and colleagues reported a novel finding of genetic variation in the DLG5 gene that is associated with IBD (CD and UC combined). We present here a study of the genetic variation described in that report in two well-powered, independent case–control cohorts and one family-based collection, and confirm the proposed association between IBD and the R30Q variant of DLG5 in two of the three studies. We are, however, unable to replicate the other proposed association to the common haplotype described in Stoll et al and suggest that this other finding could conceivably have been partially a statistical fluctuation and partially a result of LD with the replicated R30Q association. This study provides support for the hypothesis that DLG5 constitutes a true IBD risk factor of modest effect.

Thalassaemia major is a genetic disease characterised by a reduced ability to produce haemoglobin. Management of the resulting anaemia is through red blood cell transfusions.Repeated transfusions result in an excessive accumulation of iron in the body (iron overload), removal of which is achieved through iron chelation therapy. Desferrioxamine mesylate (desferrioxamine) is one of the most widely used iron chelators. Substantial data have shown the beneficial effects of desferrioxamine, although adherence to desferrioxamine therapy is a challenge. Alternative oral iron chelators, deferiprone and deferasirox, are now commonly used. Important questions exist about whether desferrioxamine, as monotherapy or in combination with an oral iron chelator, is the best treatment for iron chelation therapy.To determine the effectiveness (dose and method of administration) of desferrioxamine in people with transfusion-dependent thalassaemia.To summarise data from trials on the clinical efficacy and safety of desferrioxamine for thalassaemia and to compare these with deferiprone and deferasirox.We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register. We also searched MEDLINE, EMBASE, CENTRAL (The Cochrane Library), LILACS and other international medical databases, plus ongoing trials registers and the Transfusion Evidence Library (www.transfusionevidencelibrary.com). All searches were updated to 5 March 2013.Randomised controlled trials comparing desferrioxamine with placebo, with another iron chelator, or comparing two schedules or doses of desferrioxamine, in people with transfusion-dependent thalassaemia.Six authors working independently were involved in trial quality assessment and data extraction. For one trial, investigators supplied additional data upon request.A total of 22 trials involving 2187 participants (range 11 to 586 people) were included. These trials included eight comparisons between desferrioxamine alone and deferiprone alone; five comparisons between desferrioxamine combined with deferiprone and deferiprone alone; eight comparisons between desferrioxamine alone and desferrioxamine combined with deferiprone; two comparisons of desferrioxamine with deferasirox; and two comparisons of different routes of desferrioxamine administration (bolus versus continuous infusion). Overall, few trials measured the same or long-term outcomes. Seven trials reported cardiac function or liver fibrosis as measures of end organ damage; none of these included a comparison with deferasirox.Five trials reported a total of seven deaths; three in patients who received desferrioxamine alone, two in patients who received desferrioxamine and deferiprone. A further death occurred in a patient who received deferiprone in another who received deferasirox alone. One trial reported five further deaths in patients who withdrew from randomised treatment (deferiprone with or without desferrioxamine) and switched to desferrioxamine alone.One trial planned five years of follow up but was stopped early due to the beneficial effects of a reduction in serum ferritin levels in those receiving combined desferrioxamine and deferiprone treatment compared with deferiprone alone. The results of this and three other trials suggest an advantage of combined therapy with desferrioxamine and deferiprone over monotherapy to reduce iron stores as measured by serum ferritin. There is, however, no evidence for the improved efficacy of combined desferrioxamine and deferiprone therapy against monotherapy from direct or indirect measures of liver iron.Earlier trials measuring the cardiac iron load indirectly by measurement of the magnetic resonance imaging T2* signal had suggested deferiprone may reduce cardiac iron more quickly than desferrioxamine. However, meta-analysis of two trials showed a significantly lower left ventricular ejection fraction in patients who received desferrioxamine alone compared with those who received combination therapy using desferrioxamine with deferiprone.Adverse events were recorded by 18 trials. These occurred with all treatments, but were significantly less likely with desferrioxamine than deferiprone in one trial, relative risk 0.45 (95% confidence interval 0.24 to 0.84) and significantly less likely with desferrioxamine alone than desferrioxamine combined with deferiprone in two other trials, relative risk 0.33 (95% confidence interval 0.13 to 0.84). In particular, four studies reported permanent treatment withdrawal due to adverse events from deferiprone; only one of these reported permanent withdrawals associated with desferrioxamine. Adverse events also occurred at a higher frequency in patients who received deferasirox than desferrioxamine in one trial. Eight trials reported local adverse reactions at the site of desferrioxamine infusion including pain and swelling. Adverse events associated with deferiprone included joint pain, gastrointestinal disturbance, increases in liver enzymes and neutropenia; adverse events associated with deferasirox comprised increases in liver enzymes and renal impairment. Regular monitoring of white cell counts has been recommended for deferiprone and monitoring of liver and renal function for deferasirox.In summary, desferrioxamine and the oral iron chelators deferiprone and deferasirox produce significant reductions in iron stores in transfusion-dependent, iron-overloaded people. There is no evidence from randomised clinical trials to suggest that any one of these has a greater reduction of clinically significant end organ damage, although in two trials, combination therapy with desferrioxamine and deferiprone showed a greater improvement in left ventricular ejection fraction than desferrioxamine used alone.Desferrioxamine is the recommended first-line therapy for iron overload in people with thalassaemia major and deferiprone or deferasirox are indicated for treating iron overload when desferrioxamine is contraindicated or inadequate. Oral deferasirox has been licensed for use in children aged over six years who receive frequent blood transfusions and in children aged two to five years who receive infrequent blood transfusions. In the absence of randomised controlled trials with long-term follow up, there is no compelling evidence to change this conclusion.Worsening iron deposition in the myocardium in patients receiving desferrioxamine alone would suggest a change of therapy by intensification of desferrioxamine treatment or the use of desferrioxamine and deferiprone combination therapy.Adverse events are increased in patients treated with deferiprone compared with desferrioxamine and in patients treated with combined deferiprone and desferrioxamine compared with desferrioxamine alone. People treated with all chelators must be kept under close medical supervision and treatment with deferiprone or deferasirox requires regular monitoring of neutrophil counts or renal function respectively. There is an urgent need for adequately-powered, high-quality trials comparing the overall clinical efficacy and long-term outcomes of deferiprone, deferasirox and desferrioxamine.

Thalassaemia major is a genetic disease characterised by a reduced ability to produce haemoglobin. Management of the resulting anaemia is through red blood cell transfusions.Repeated transfusions result in an excessive accumulation of iron in the body (iron overload), removal of which is achieved through iron chelation therapy. A commonly used iron chelator, deferiprone, has been found to be pharmacologically efficacious. However, important questions exist about the efficacy and safety of deferiprone compared to another iron chelator, desferrioxamine.To summarise data from trials on the clinical efficacy and safety of deferiprone and to compare the clinical efficacy and safety of deferiprone with desferrioxamine for thalassaemia.We searched the Cochrane Cystic fibrosis and Genetic Disorders Group's Haemoglobinopathies trials Register and MEDLINE, EMBASE, CENTRAL (The Cochrane Library), LILACS and other international medical databases, plus registers of ongoing trials and the Transfusion Evidence Library (www.transfusionevidencelibrary.com). We also contacted the manufacturers of deferiprone and desferrioxamine.All searches were updated to 05 March 2013.Randomised controlled trials comparing deferiprone with another iron chelator; or comparing two schedules or doses of deferiprone, in people with transfusion-dependent thalassaemia.Two authors independently assessed trials for risk of bias and extracted data. Missing data were requested from the original investigators.A total of 17 trials involving 1061 participants (range 13 to 213 participants per trial) were included. Of these, 16 trials compared either deferiprone alone with desferrioxamine alone, or a combined therapy of deferiprone and desferrioxamine with either deferiprone alone or desferrioxamine alone; one compared different schedules of deferiprone. There was little consistency between outcomes and limited information to fully assess the risk of bias of most of the included trials.Four trials reported mortality; each reported the death of one individual receiving deferiprone with or without desferrioxamine. One trial reported five further deaths in patients who withdrew from randomised treatment (deferiprone with or without desferrioxamine) and switched to desferrioxamine alone. Seven trials reported cardiac function or liver fibrosis as measures of end organ damage.Earlier trials measuring the cardiac iron load indirectly by magnetic resonance imaging (MRI) T2* signal had suggested deferiprone may reduce cardiac iron more quickly than desferrioxamine. However, a meta-analysis of two trials suggested that left ventricular ejection fraction was significantly reduced in patients who received desferrioxamine alone compared with combination therapy. One trial, which planned five years of follow up, was stopped early due to the beneficial effects of combined treatment compared with deferiprone alone in terms of serum ferritin levels reduction.The results of this and three other trials suggest an advantage of combined therapy over monotherapy to reduce iron stores as measured by serum ferritin. There is, however, no conclusive or consistent evidence for the improved efficacy of combined deferiprone and desferrioxamine therapy over monotherapy from direct or indirect measures of liver iron. Both deferiprone and desferrioxamine produce a significant reduction in iron stores in transfusion-dependent, iron-overloaded people. There is no evidence from randomised controlled trials to suggest that either has a greater reduction of clinically significant end organ damage.Evidence of adverse events were observed in all treatment groups. Occurrence of any adverse event was significantly more likely with deferiprone than desferrioxamine in one trial, RR 2.24 (95% CI 1.19 to 4.23). Meta-analysis of a further two trials showed a significant increased risk of adverse events associated with combined deferiprone and desferrioxamine compared with desferrioxamine alone, RR 3.04 (95% CI 1.18 to 7.83). The most commonly reported adverse event was joint pain, which occurred significantly more frequently in patients receiving deferiprone than desferrioxamine, RR 2.64 (95% CI 1.21 to 5.77). Other common adverse events included gastrointestinal disturbances as well as neutropenia or leucopenia, or both.In the absence of data from randomised controlled trials, there is no evidence to suggest the need for a change in current treatment recommendations; namely that deferiprone is indicated for treating iron overload in people with thalassaemia major when desferrioxamine is contraindicated or inadequate. Intensified desferrioxamine treatment (by either subcutaneous or intravenous route) or use of other oral iron chelators, or both, remains the established treatment to reverse cardiac dysfunction due to iron overload. Indeed, the US Food and Drug Administration (FDA) recently only gave support for deferiprone to be used as a last resort for treating iron overload in thalassaemia, myelodysplasia and sickle cell disease. However, there is evidence that adverse events are increased in patients treated with deferiprone compared with desferrioxamine and in patients treated with combined deferiprone and desferrioxamine compared with desferrioxamine alone. There is an urgent need for adequately-powered, high-quality trials comparing the overall clinical efficacy and long-term outcome of deferiprone with desferrioxamine.

Background Iron deficiency is a significant cause of deferral in people wishing to donate blood. If iron removed from the body through blood donation is not replaced, then donors may become iron deficient. All donors are screened at each visit for low haemoglobin (Hb) levels. However, some deferred blood donors do not return to donate. Deferred first‐time donors are even less likely to return. Interventions that reduce the risk of provoking iron deficiency and anaemia in blood donors will therefore increase the number of blood donations. Currently, iron supplementation for blood donors is not a standard of care in many blood services. A systematic review is required to answer specific questions regarding the efficacy and safety of iron supplementation in blood donors. Objectives To assess the efficacy and safety of iron supplementation to reduce deferral, iron deficiency and/or anaemia in blood donors. Search methods We ran the search on 18 November 2013. We searched Cochrane Injuries Group Specialised Register, CENTRAL, PubMed, MEDLINE (OvidSP), EMBASE (OvidSP), CINAHL (EBSCO Host) and six other databases. We also searched clinical trials registers and screened guidelines reference lists. Selection criteria Randomised controlled trials (RCTs) comparing iron supplementation versus placebo or control, oral versus parenteral iron supplementation, iron supplementation versus iron‐rich food supplements, and different doses, treatment durations and preparations of iron supplementation in healthy blood donors. Autologous blood donors were excluded. Data collection and analysis We combined data using random‐effects meta‐analyses. We evaluated heterogeneity using the I2 statistic; we explored considerable heterogeneity (I2 > 75%) in subgroup analyses. We carried out sensitivity analyses to assess the impact of trial quality on the results. Main results Thirty RCTs (4704 participants) met the eligibility criteria, including 19 comparisons of iron supplementation and placebo or control; one comparison of oral and parenteral iron supplementation; four comparisons of different doses of iron supplementation; one comparison of different treatment durations of iron supplementation; and 12 comparisons of different iron supplementation preparations. Many studies were of low or uncertain methodological quality and therefore at high or uncertain risk of bias. We therefore rated the quality of the evidence for our outcomes as moderate. There was a statistically significant reduction in deferral due to low haemoglobin in donors who received iron supplementation compared with donors who received no iron supplementation, both at the first donation visit after commencement of iron supplementation (risk ratio (RR) 0.34; 95% confidence interval (CI) 0.21 to 0.55; four studies; 1194 participants; P value < 0.0001) and at subsequent donations (RR 0.25; 95% CI 0.15 to 0.41; three studies; 793 participants; P value < 0.00001). Supplementation also resulted in significantly higher haemoglobin levels (mean difference (MD) 2.36 g/L; 95% CI 0.06 to 4.66; eight studies; 847 participants, P value =0.04), and iron stores, including serum ferritin (MD 13.98 ng/mL; 95% CI 8.92 to 19.03; five studies; 640 participants; P value < 0.00001) and transferrin saturation (MD 3.91%; 95% CI 2.02 to 5.80; four studies; 344 participants; P value < 0.0001) prior to further donation. The differences were maintained after subsequent donation(s). Adverse effects were widely reported and were more frequent in donors who received iron supplementation (RR 1.60; 95% CI 1.23 to 2.07; four studies; 1748 participants; P value = 0.0005). Adverse effects included constipation, diarrhoea, nausea, vomiting and taste disturbances, and some participants stopped treatment due to side effects. Authors' conclusions There is moderate quality evidence that rates of donor deferral due to low haemoglobin are considerably less in those taking iron supplements compared with those without iron supplementation, both at the first donation visit and at subsequent donation. Iron‐supplemented donors also show elevated haemoglobin and iron stores. These beneficial effects are balanced by more frequent adverse events in donors who receive iron supplementation than in those who do not; this is likely to limit acceptability and compliance. The long‐term effects of iron supplementation without measurement of iron stores are unknown. These considerations are likely to preclude widespread use of iron supplementation by tablets. Blood services may consider targeted use of supplementation in those at greatest risk of iron deficiency, personalised donation intervals and providing dietary advice.

ObjectivesThe activated partial thromboplastin time (aPTT) test has been used for years to monitor parenteral direct thrombin inhibitors (DTIs) and unfractionated heparin. Because the aPTT correlates poorly with unfractionated heparin levels, we hypothesized that the aPTT may not be the best test for monitoring parenteral DTIs.

Anaemia affects 60–80 % of patients admitted to intensive care units (ICUs). Allogeneic red blood cell (RBC) transfusions remain the mainstay of treatment for anaemia but are associated with risks and are costly. Our objective was to assess the efficacy and safety of iron supplementation by any route, in anaemic patients in adult ICUs. Electronic databases (CENTRAL, MEDLINE, EMBASE) were searched through March 2016 for randomized controlled trials (RCT)s comparing iron by any route with placebo/no iron. Primary outcomes were red blood cell transfusions and mean haemoglobin concentration. Secondary outcomes included mortality, infection, ICU and hospital length of stay, mean difference (MD) in iron biomarkers, health-related quality of life and adverse events. Five RCTs recruiting 665 patients met the inclusion criteria; intravenous iron was tested in four of the RCTs. There was no difference in allogeneic RBC transfusion requirements (relative risk 0.87, 95 % confidence interval (CI) 0.70 to 1.07, p = 0.18, five trials) or mean number of RBC units transfused (MD -0.45, 95 % CI -1.34 to 0.43, p = 0.32, two trials) in patients receiving or not receiving iron. Similarly, there was no difference between groups in haemoglobin at short-term (up to 10 days) (MD -0.25, 95 % CI -0.79 to 0.28, p = 0.35, three trials) or mid-term follow up (last measured time point in hospital or end of trial) (MD 0.21, 95 % CI -0.13 to 0.55, p = 0.23, three trials). There was no difference in secondary outcomes of mortality, in-hospital infection, or length of stay. Risk of bias was generally low although three trials had high risk of attrition bias; only one trial had low risk of bias across all domains. Iron supplementation does not reduce RBC transfusion requirements in critically ill adults, but there is considerable heterogeneity between trials in study design, nature of interventions, and outcomes. Well-designed trials are needed to investigate the optimal iron dosing regimens and strategies to identify which patients are most likely to benefit from iron, together with patient-focused outcomes. PROSPERO International prospective register of systematic reviews CRD42015016627 . Registered 2 March 2015.

DLG5 p.R30Q has been reported to be associated with Crohn disease (CD), but this association has not been replicated in most studies. A recent analysis of gender-stratified data from two case-control studies and two population cohorts found an association of DLG5 30Q with increased risk of CD in men but not in women and found differences between 30Q population frequencies for males and females. Male-female differences in population allele frequencies and male-specific risk could explain the difficulty in replicating the association with CD.DLG5 R30Q genotype data were collected for patients with CD and controls from 11 studies that did not include gender-stratified allele counts in their published reports and tested for male-female frequency differences in controls and for case-control frequency differences in men and in women.The data showed no male-female allele frequency differences in controls. An exact conditional test gave marginal evidence that 30Q is associated with decreased risk of CD in women (p = 0.049, OR = 0.87, 95% CI 0.77 to 1.00). There was also a trend towards reduced 30Q frequencies in male patients with CD compared with male controls, but this was not significant at the 0.05 level (p = 0.058, OR = 0.87, 95% CI 0.74 to 1.01). When data from this study were combined with previously published, gender-stratified data, the 30Q allele was found to be associated with decreased risk of CD in women (p = 0.010, OR = 0.86, 95% CI 0.76 to 0.97), but not in men.DLG5 30Q is associated with a small reduction in risk of CD in women.

Allogeneic haematopoietic stem cell transplant (HSCT) recipients are at increased risk of morbidity and mortality, often due to the development of acute or chronic graft-versus-host disease (GVHD). Low numbers or proportions of regulatory T cells (Tregs) have been reported in patients who develop GVHD. We undertook a systematic review of studies that reported the Treg composition of HSCT grafts in patients with haematological malignancies. Fourteen eligible studies were identified, eight of which stratified patients by Tregs (absolute dose or ratio to CD3+ or CD4+ cells). Meta-analyses showed that high levels of Tregs in the grafts were associated with improved overall survival [hazard ratio (HR) 0·42, 95% confidence interval (CI) 0·23-0·74, P = 0·003, 2 studies], with a significant reduction in non-relapse mortality (HR 0·30, 95% CI 0·14-0·64, P = 0·002, 2 studies) and a reduced risk of acute GVHD (relative risk (RR) 0·59, 95% CI 0·40-0·89, P = 0·01, 6 studies). The consistency of these findings strongly suggests that the Treg composition of HSCT grafts has a powerful effect on the success of allogeneic HSCT. The major challenge is to translate these findings into better selection of allografts and future donors to provide a substantial improvement in allogeneic HSCT outcomes and practice.

Vasovagal reactions (VVRs) in blood donors have significant implications for the welfare of donors, donor retention and the management of donor sessions. We present a systematic review of interventions designed to prevent or reduce VVRs in blood donors. Electronic databases were searched for eligible randomised trials to March 2015. Data on study design and outcomes were extracted and pooled using random effects meta-analyses. Sixteen trials met the inclusion criteria: five trials (12 042 participants) of pre-donation water, eight trials (3500 participants) of applied muscle tension (AMT) and one trial each of AMT combined with water, caffeine, audio-visual distraction and/or social support. In donors receiving pre-donation water, the relative risk (RR) compared with controls for VVRs was 0·79 [95% confidence interval (CI) 0·70-0·89, P < 0·0001] and the mean difference (MD) in severity of VVRs measured with the Blood Donation Reactions Inventory (BDRI) score was -0·32 (95% CI -0·51 to -0·12, P < 0·0001). Excluding trials with a high risk of selection bias, the RR for VVRs was 0·70 (95% CI 0·45-1·11, P = 0·13). In donors who received AMT, there was no difference in the risk of chair recline in response to donor distress from controls (RR 0·76, 95% CI 0·53-1·10, P = 0·15), although the MD in BDRI score was -0·07 (95% CI -0·11 to -0·03, P = 0·0005). There was insufficient data to perform meta-analysis for other interventions. Current evidence on interventions to prevent or reduce VVRs in blood donors is indeed limited and does not provide strong support for the administration of pre-donation water or AMT during donation. Further large trials are required to reliably evaluate the effect of these and other interventions in the prevention of VVRs.

A genetic contribution to the development of systemic lupus erythematosus (SLE) is well established. Several genome-wide linkage scans have identified a number of putative susceptibility loci for SLE, some of which have been replicated in independent samples. This study aimed to identify the regions showing the most consistent evidence for linkage by applying the genome scan meta-analysis (GSMA) method. The study identified two genome-wide suggestive regions on 6p21.1–q15 and 20p11–q13.13 (P-value=0.0056 and P-value=0.0044, respectively) and a region with P-value<0.01 on 16p13–q12.2. The region on chromosome 6 contains the human leukocyte antigen cluster, and the chromosome 16 and 20 regions have been replicated in several cohorts. The potential importance of the identified genomic regions are also highlighted. These results, in conjunction with data emerging from dense single nucleotide polymorphism typing of specific regions or future genome-wide association studies will help guide efforts to identify the actual predisposing genetic variation contributing to this complex genetic disease.

Autoimmune diseases are chronic disorders initiated by a loss of immunologic tolerance to self-antigens. They cluster within families, and patients may be diagnosed with more than one disease, suggesting pleiotropic genes are involved in the aetiology of different diseases. To identify potential loci, which confer susceptibility to autoimmunity independent of disease phenotype, we pooled results from genome-wide linkage studies, using the genome scan meta-analysis method (GSMA). The meta-analysis included 42 independent studies for 11 autoimmune diseases, using 7350 families with 18 291 affected individuals. In addition to the HLA region, which showed highly significant genome-wide evidence for linkage, we obtained suggestive evidence for linkage on chromosome 16, with peak evidence at 10.0–19.8 Mb. This region may harbour a pleiotropic gene (or genes) conferring risk for several diseases, although no such gene has been identified through association studies. We did not identify evidence for linkage at several genes known to confer increased risk to different autoimmune diseases (PTPN22, CTLA4), even in subgroups of diseases consistently found to be associated with these genes. The relative risks conferred by variants in these genes are modest (<1.5 in most cases), and even a large study like this meta-analysis lacks power to detect linkage. This study illustrates the concept that linkage and association studies have power to identify very different types of disease-predisposing variants.

This unit describes a protocol for the targeted enrichment of exons from randomly sheared genomic DNA libraries using an in-solution hybrid selection approach for sequencing on an Illumina Genome Analyzer II. The steps for designing and ordering a hybrid selection oligo pool are reviewed, as are critical steps for performing the preparation and hybrid selection of an Illumina paired-end library. Critical parameters, performance metrics, and analysis workflow are discussed.

Regularly transfused people with sickle cell disease (SCD) and people with thalassaemia (who are transfusion-dependent or non-transfusion-dependent) are at risk of iron overload. Iron overload can lead to iron toxicity in vulnerable organs such as the heart, liver and endocrine glands; which can be prevented and treated with iron chelating agents. The intensive demands and uncomfortable side effects of therapy can have a negative impact on daily activities and well-being, which may affect adherence.To identify and assess the effectiveness of interventions (psychological and psychosocial, educational, medication interventions, or multi-component interventions) to improve adherence to iron chelation therapy in people with SCD or thalassaemia.We searched CENTRAL (the Cochrane Library), MEDLINE, Embase, CINAHL, PsycINFO, Psychology and Behavioral Sciences Collection, Web of Science Science & Social Sciences Conference Proceedings Indexes and ongoing trial databases (01 February 2017). We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register (12 December 2017).For trials comparing medications or medication changes, only randomised controlled trials (RCTs) were eligible for inclusion.For studies including psychological and psychosocial interventions, educational Interventions, or multi-component interventions, non-RCTs, controlled before-after studies, and interrupted time series studies with adherence as a primary outcome were also eligible for inclusion.Three authors independently assessed trial eligibility, risk of bias and extracted data. The quality of the evidence was assessed using GRADE.We included 16 RCTs (1525 participants) published between 1997 and 2017. Most participants had β-thalassaemia major; 195 had SCD and 88 had β-thalassaemia intermedia. Mean age ranged from 11 to 41 years. One trial was of medication management and 15 RCTs were of medication interventions. Medications assessed were subcutaneous deferoxamine, and two oral-chelating agents, deferiprone and deferasirox.We rated the quality of evidence as low to very low across all outcomes identified in this review.Three trials measured quality of life (QoL) with validated instruments, but provided no analysable data and reported no difference in QoL.Deferiprone versus deferoxamineWe are uncertain whether deferiprone increases adherence to iron chelation therapy (four trials, very low-quality evidence). Results could not be combined due to considerable heterogeneity (participants' age and different medication regimens). Medication adherence was high (deferiprone (85% to 94.9%); deferoxamine (71.6% to 93%)).We are uncertain whether deferiprone increases the risk of agranulocytosis, risk ratio (RR) 7.88 (99% confidence interval (CI) 0.18 to 352.39); or has any effect on all-cause mortality, RR 0.44 (95% CI 0.12 to 1.63) (one trial; 88 participants; very low-quality evidence).Deferasirox versus deferoxamineWe are uncertain whether deferasirox increases adherence to iron chelation therapy, mean difference (MD) -1.40 (95% CI -3.66 to 0.86) (one trial; 197 participants; very-low quality evidence). Medication adherence was high (deferasirox (99%); deferoxamine (100%)). We are uncertain whether deferasirox decreases the risk of thalassaemia-related serious adverse events (SAEs), RR 0.95 (95% CI 0.41 to 2.17); or all-cause mortality, RR 0.96 (95% CI 0.06 to 15.06) (two trials; 240 participants; very low-quality evidence).We are uncertain whether deferasirox decreases the risk of SCD-related pain crises, RR 1.05 (95% CI 0.68 to 1.62); or other SCD-related SAEs, RR 1.08 (95% CI 0.77 to 1.51) (one trial; 195 participants; very low-quality evidence).Deferasirox film-coated tablet (FCT) versus deferasirox dispersible tablet (DT)Deferasirox FCT may make little or no difference to adherence, RR 1.10 (95% CI 0.99 to 1.22) (one trial; 173 participants; low-quality evidence). Medication adherence was high (FCT (92.9%); DT (85.3%)).We are uncertain if deferasirox FCT increases the incidence of SAEs, RR 1.22 (95% CI 0.62 to 2.37); or all-cause mortality, RR 2.97 (95% CI 0.12 to 71.81) (one trial; 173 participants; very low-quality evidence).Deferiprone and deferoxamine combined versus deferiprone alone We are uncertain if deferiprone and deferoxamine combined increases adherence to iron chelation therapy (very low-quality evidence). Medication adherence was high (deferiprone 92.7% (range 37% to 100%) to 93.6% (range 56% to 100%); deferoxamine 70.6% (range 25% to 100%).Combination therapy may make little or no difference to the risk of SAEs, RR 0.15 (95% CI 0.01 to 2.81) (one trial; 213 participants; low-quality evidence).We are uncertain if combination therapy decreases all-cause mortality, RR 0.77 (95% CI 0.18 to 3.35) (two trials; 237 participants; very low-quality evidence).Deferiprone and deferoxamine combined versus deferoxamine aloneDeferiprone and deferoxamine combined may have little or no effect on adherence to iron chelation therapy (four trials; 216 participants; low-quality evidence). Medication adherence was high (deferoxamine 91.4% to 96.1%; deferiprone: 82.4%)Deferiprone and deferoxamine combined, may have little or no difference in SAEs or mortality (low-quality evidence). No SAEs occurred in three trials and were not reported in one trial. No deaths occurred in two trials and were not reported in two trials.Deferiprone and deferoxamine combined versus deferiprone and deferasirox combinedDeferiprone and deferasirox combined may improve adherence to iron chelation therapy, RR 0.84 (95% CI 0.72 to 0.99) (one trial; 96 participants; low-quality evidence). Medication adherence was high (deferiprone and deferoxamine: 80%; deferiprone and deferasirox: 95%).We are uncertain if deferiprone and deferasirox decreases the incidence of SAEs, RR 1.00 (95% CI 0.06 to 15.53) (one trial; 96 participants; very low-quality evidence).There were no deaths in the trial (low-quality evidence).Medication management versus standard careWe are uncertain if medication management improves health-related QoL (one trial; 48 participants; very low-quality evidence). Adherence was only measured in one arm of the trial.The medication comparisons included in this review had higher than average adherence rates not accounted for by differences in medication administration or side effects.Participants may have been selected based on higher adherence to trial medications at baseline. Also, within the clinical trial context, there is increased attention and involvement of clinicians, thus high adherence rates may be an artefact of trial participation.Real-world, pragmatic trials in community and clinic settings are needed that examine both confirmed or unconfirmed adherence strategies that may increase adherence to iron chelation therapy.Due to lack of evidence this review cannot comment on intervention strategies for different age groups.

To evaluate the safety (risk of infection) and efficacy (transfusion requirements, changes in haemoglobin (Hb)) of iron therapy in adult intensive care unit (ICU) patients. We systematically searched seven databases for all relevant studies until January 2018 and included randomized (RCT) studies comparing iron, by any route, with placebo/no iron. 805 participants from 6 RCTs were included. Iron therapy, by any route, did not decrease the risk of requirement for a red blood cell (RBC) transfusion (Risk ratio (RR) 0.91, 95% CI 0.80 to 1.04, p = 0.15) or mean number of RBCs transfused per participant (mean difference (MD) -0.30, 95% CI -0.68 to 0.07, p = 0.15). Iron therapy did increase mean Hb concentration (MD 0.31 g/dL, 95% CI 0.04 to 0.59, p = 0.03). There was no difference in infection (RR 0.95, 95% CI 0.79 to 1.19, p = 0.44). Trial Sequential Analysis suggests that the required participant numbers to detect or reject a clinically important effect of iron therapy on transfusion requirements or infection in ICU patients has not yet been reached. Iron therapy results in a modest increase in Hb. The current evidence is inadequate to exclude an important effect on transfusion requirements or infection.

The choice of an analytical procedure and the determination of an appropriate sampling strategy are here treated as a decision theory problem in which sampling and analytical costs are balanced against possible end-user losses due to measurement error. Measurement error is taken here to include both sampling and analytical variances, but systematic errors are not considered. The theory is developed in detail for the case exemplified by a simple accept or reject decision following an analytical measurement on a batch of material, and useful approximate formulae are given for this case. Two worked examples are given, one involving a batch production process and the other a land reclamation site.

Inflammatory bowel disease (IBD) is a multifactorial disorder, with both genetic and environmental factors contributing to the two clinical phenotypes of Crohn's disease (CD) and ulcerative colitis (UC). The underlying genetic model is thought to involve multiple genes with complex interactions between disease loci, and the NOD2 gene on chromosome 16 has recently been identified as a CD susceptibility locus. Several genome-wide linkage studies have identified candidate regions, but there has been little replication across studies. Here we investigate the role of sex-specific loci in susceptibility to IBD. Linkage data from our previously reported genome search and follow-up study were stratified by the sex of the affected sib pair. Non-parametric linkage analysis was performed using Genehunter Plus. Simulation studies were used to assess the significance of differences in LOD scores between male and female families for each chromosome. Several regions of sex-specific linkage were identified, including existing and novel candidate loci. The major histocompatibility region on chromosome 6p, referred to as IBD3, showed evidence of male-specific linkage with a maximum LOD score of 5.9 in both CD and UC male-affected families. Regions on chromosomes 11, 14 and 18 showed strong evidence of linkage in male-affected families but not in female-affected families. No evidence of sex-specific linkage was found in the IBD1 or IBD2 candidate regions of chromosomes 16 and 12. The existence of sex-specific linkage is further evidence of the complex mechanisms involved in IBD and will facilitate future studies to identify susceptibility genes.

To determine whether there are any unusual patterns of transmission of susceptibility to inflammatory bowel disease (IBD) within multiplex families.Individuals with IBD were recruited for genome-wide screening of susceptibility genes. The extent of familial aggregation and blood relationships in multiplex families were determined by questionnaires given to participants followed up by confirmation of disease diagnosis by participants' physicians.Of 135 families identified in which both a parent and a child had IBD, 93 involved transmission of susceptibility to disease from mother to child versus 42 examples of transmission from father to child (p = 0.00001, exact two-tailed binomial test). This distortion in transmission on the basis of the sex of the parent was observed only among non-Jewish pairs with Crohn's disease (CD), in which, of 33 parent-child pairs with CD, disease susceptibility was transmitted from the mother 28 times (p = 0.00007).Susceptibility to CD in a subset of patients may involve a gene that is imprinted.

A common haplotype spanning 250 kb on chromosome 5q31 is strongly associated with Crohn disease (CD). Recently, two functional variants within the SLC22A4 and SLC22A5 genes at this locus (IBD5), L503F (c.1507C > T) and G-207C (c.-207G > C), have been proposed to contribute directly to susceptibility to CD. However, extensive linkage disequilibrium at the IBD5 locus has complicated efforts to distinguish causal variants from association of the general risk haplotype. We genotyped the SLC22A4 and SLC22A5 variants and other polymorphisms across the risk haplotype in four populations of European origin, and applied regression-based haplotype analysis to over 1,200 fully genotyped case-control pairs, modeling case/control status on the presence of one or more SNPs to test for conditional association and to identify risk haplotypes. We found highly significant association of SNPs at the IBD5 locus with Crohn disease in all populations tested. However, the frequencies of L503F and G-207C in individuals who did not carry the general IBD5 risk haplotype were not significantly different in cases and controls, with associated disease odds ratios (ORs) of 0.90 (95% CI, 0.57-1.40) and 0.90 (95% CI, 0.65-1.23), respectively. Haplotype analysis showed that addition of the SLC22A4 and SLC22A5 variants to a null model that included the background risk haplotype did not significantly improve the model fit. In addition to the common risk haplotype, several rare haplotypes had an increased frequency in cases compared to controls. This study suggests that the molecular basis for Crohn disease susceptibility at the IBD5 locus remains to be defined, and highlights the challenge of the identification of causal variants in a complex disease in regions of extensive linkage disequilibrium.

A strong association has been confirmed between age-related macular degeneration (AMD) and variants at two independent loci including Tyr402His in the complement factor H (CFH) on 1q32 and Ser69Ala at LOC387715, a hypothetical gene on chromosome 10q26. The contribution of both loci to AMD was investigated in an isolated north-west Russian population.Together with a PLEKHA1 variant at 10q26, the CFH Tyr402His and LOC387715 Ser69Ala polymorphisms were genotyped in 155 patients with AMD and 151 age-matched controls. chi(2) and Mantel-Haenszel (M-H) score tests were used to test for association. Sex-adjusted ORs were calculated.The frequency of the Tyr402His C allele was significantly higher in patients with AMD compared with controls (p(M-H)=0.0035). The increased risk observed in patients homozygous for the C allele (OR(HOM)=2.71, 95% CI 1.25 to 5.90) in this indigenous Russian population was considerably lower than that observed in previous western Caucasian populations. A significant increase in the frequency of the LOC387715 variant was observed in patients with late-stage AMD compared with controls (p(M-H)=0.007), with a homozygous OR of 3.47 (95% CI 1.01 to 11.9), although this association was not seen with early-stage AMD.The CFH gene contributes to AMD in this Russian population, although the risk conferred is considerably lower in this population than that found in other Western populations. A contribution of LOC387715 to disease in this population is also likely to be of weak effect.

SUMMARY Background/Objectives Blood donors attending a donation session may be deemed ineligible to donate blood due to a failure to meet low haemoglobin (Hb) thresholds. Several studies have identified factors associated with a donor falling below these Hb thresholds. A review of these factors will inform future prospective studies and form the basis for predictive models of deferral due to low Hb. Materials/Methods Studies were identified by searching MEDLINE , EMBASE , The Cochrane Library and the WHO International Clinical Trials Registry from 1980 to September 2012. Demographic data, donor history, haematological/biological factors and the primary outcome of deferral due to low Hb were extracted. Analyses were descriptive and quantitative; pooled odds ratios (ORs) were obtained by meta‐analysis. Results Fifty‐five studies met the inclusion criteria. A consistently higher rate of low Hb deferral was reported in females compared with males; meta‐analysis showed a significantly greater risk of deferral due to low Hb in females compared with males in studies with universal Hb thresholds for males and females ( OR 14·91, 95% confidence interval ( CI ) 12·82–17·34) and in studies with sex‐specific Hb thresholds ( OR 8·19, 95% CI 4·88–13·74). Greater rates of deferral due to low Hb were also associated with increasing age, higher ambient temperature, low body weight, shorter inter‐donation interval and in donors of Hispanic or African descent. Conclusion This work will help to define the criteria that should be considered in any large scale study of blood donor deferral, especially those that measure or aim to change failure to meet low Hb thresholds.

Cell therapy provides a promising approach in the treatment of chronic ischaemic heart disease (IHD) and heart failure (HF) secondary to IHD. Preclinical and clinical studies have suggested that cell therapies may potentially reverse left ventricular dysfunction in chronic IHD and congestive heart failure (CHF). The cell therapy procedure involves stem or progenitor cells being collected from the recipient, either harvested from bone marrow (BM) or through mobilisation of BM cells into circulation by a growth factor stimulant, most commonly granulocyte colony-stimulating factor. In both procedures, the cells are infused directly into the recipient’s coronary arteries or heart. Delivery of cells to the coronary arteries is made via a special balloon catheter during angioplasty, whereas administration of cells into the heart muscle is made during an angioplasty-like procedure using electromechanical mapping and direct intramyocardial injection or during cardiac surgery (eg, coronary artery bypass grafting). This treatment is currently only available in research settings, but if long-term effectiveness is confirmed, it may become available to some or all people with chronic heart disease since BM and peripheral blood harvest is a standard procedure used in BM transplantation. Clinical trials and systematic reviews of BM-derived cells administered to patients with IHD or CHF have yielded divergent results, and therefore the mechanism of action of such therapies remains unclear. In this Cochrane Corner we report the main findings of our recent Cochrane systematic review of BM-derived cell therapy in patients with chronic IHD and CHF1 which incorporates evidence from a total of 38 eligible randomised controlled trials (RCTs). Review methods followed the Cochrane Handbook for Systematic Reviews of Interventions .2 We searched CENTRAL in The Cochrane Library , MEDLINE, Embase, CINAHL and …

Blood donors attending a donation session may be deferred from donating blood due to a failure to meet low hemoglobin (Hb) thresholds. This costs the blood donor service and donors valuable time and resources. In addition, donors who are deferred may have more symptoms, and as a direct and/or indirect effect of their experience, return rates of donors deferred for low Hb are reduced, even in repeat donors. It is therefore vital that low Hb deferral (LHD) is minimized. The aim of this updated systematic review is to expand the evidence base for factors which affect a donor's risk of deferral due to low Hb. Studies were identified by searching MEDLINE, Embase, The Cochrane Library, and the WHO International Clinical Trials Registry to March 2019. Demographic data, donor history, hematological/biological factors, and the primary outcome of deferral due to low Hb were extracted. Our primary outcome was deferral due to low Hb. Analyses were descriptive and quantitative; pooled odds ratios (ORs) and 95% confidence intervals (CIs) were obtained by meta-analysis using random-effects models. A total of 116 studies met the inclusion criteria. Meta-analysis showed a significantly greater risk of LHD in females compared with males in studies applying universal Hb thresholds for males and females (OR 14.62 95% CI 12.43-17.19) and in those which used sex-specific thresholds (OR 5.73, 95% CI 4.36-7.53). Higher rates of LHD were also associated with increasing age in men, low body weight, shorter interdonation interval, donors of Hispanic or African descent, higher ambient temperature, donors with low ferritin levels, and donation in a fixed donor center. There was conflicting evidence on the effect of new and repeat donor status, and blood group. This work has strengthened the evidence of the previous review in identifying factors that should be considered in studies of donor deferral and highlighting areas in need of further study, including ABO and Rh blood groups, previous platelet donation, diet, smoking, time of day, and genetic data. These factors may lead to individually tailored donation criteria for safe and efficient donation in the future.

Rheumatoid arthritis (RA) is the most common disabling autoimmune disease, affecting approximately 1% of the population. The disease etiology is unknown, but it involves inflammation and immune dysregulation and is influenced by genetic variation at both HLA and other, as-yet-unidentified genetic loci. Corticotropin-releasing hormone (CRH; or corticotropin-releasing factor), a primary regulator of the hypothalamic-pituitary-adrenal axis and a key element in the response to stress and inflammation, is a strong candidate gene for RA. We examined the role of DNA variation across the region containing this gene in multicase families with RA.We genotyped fluorescently labeled simple tandem repeat genetic markers from chromosome 8q13 in 295 families with multiple cases of RA. Singlepoint and multipoint nonparametric linkage analysis and association analysis using transmission disequilibrium testing (TDT) were also used.Single-point linkage analysis using a microsatellite within 30 kb of the CRH locus (CRH.PCR at position 8q13) showed a significant excess of allele sharing in 295 United Kingdom RA families with at least 2 affected members (MapMaker/Sibs logarithm of odds [LOD] 1.4; P = 5.5x10(-3); mean identity by descent [ibd] sharing 55.9%). To provide a more detailed linkage map, a multipoint analysis was conducted with an additional 7 dinucleotide microsatellite markers (average heterozygosity 0.75) flanking the CRH locus. Significant linkage was detected over a 22-cM region between D8S285 and D8S530, with the maximum singlepoint LOD score of 1.77 at D8S1723 (MapMaker/Sibs P = 2.2x10(-3); mean ibd sharing 59.3%). Multipoint analysis showed strongest evidence for linkage at the same marker (multipoint LOD 1.78, P = 2.1x10(-3), mean ibd sharing 55.8%). TDT analysis showed significant association at the CRH locus (P = 2.6x10(-3)). CRH has a sibling relative risk of 1.14, and contributes <10% to the sibling relative risk of RA.With the exception of HLA, this is the strongest evidence yet of a genetic locus that is both linked to and associated with RA, and provides an avenue for further genetic characterization and potentially novel therapeutic intervention.

Ancestral mutations in BRCA1 and BRCA2 are common in people of Ashkenazi Jewish descent and are associated with a substantially increased risk of breast and ovarian cancer. Women considering mutation testing usually have several personal and family cancer characteristics, so predicting mutation status from one factor alone could be misleading. The aim of this study was to develop a simple algorithm to estimate the probability that an Ashkenazi Jewish woman carries an ancestral mutation, based on multiple predictive factors. We studied Ashkenazi Jewish women with a personal or family history of breast or ovarian cancer and living in Melbourne or Sydney, Australia, or with a previous diagnosis of breast or ovarian cancer and living in the UK. DNA samples were tested for the germline mutations 185delAG and 5382insC in BRCA1, and 6174delT in BRCA2. Logistic regression was used to identify, and to estimate the predictive strength of, major determinants. A mutation was detected in 64 of 424 women. An algorithm was developed by combining our findings with those from similar analyses of a large study of unaffected Jewish women in Washington. Starting with a baseline score, a multiple of 0.5 (based on the logistic regression estimates) is added for each predictive feature. The sum is the estimated log odds ratio that a woman is a carrier, and is converted to a probability by using a table. There was good internal consistency. This simple algorithm might be useful in the clinical and genetic counselling setting. Comparison and validation in other settings should be sought.

Three common mutations in the CARD15 (NOD2) gene are known to be associated with susceptibility to Crohn disease (CD), and genetic data suggest a gene dosage model with an increased risk of 2–4-fold in heterozygotes and 20–40-fold in homozygotes. However, the discovery of numerous rare variants of CARD15 indicates that some heterozygotes for the common mutations have a rare mutation on the other CARD15 allele, which would support a recessive model for CD. We addressed this issue by screening CARD15 for mutations in 100 CD patients who were heterozygous for one of the three common mutations. We also developed a strategy for evaluating potential disease susceptibility alleles (DSAs) that involves assessing the degree of evolutionary conservation of involved residues, predicted effects on protein structure and function, and genotyping in a large sample of cases and controls. The evolutionary analysis was aided by sequencing the entire coding region of CARD15 in three primates (chimp, gibbon, and tamarin) and aligning the human sequence with these and orthologs from other species. We found that 11 of the 100 CD patients screened had a second potential pathogenic mutation within the exonic and periexonic sequences examined. Assuming that there are no additional pathogenic mutations in noncoding regions, our study suggests that most carriers of the common DSAs are true heterozygotes, and supports previous evidence for a gene dosage model. Four novel nonsynonymous mutations were detected, one of which would produce premature termination of translation c.2686C>T (p.Arg896X). Two potential DSAs—c.2107C>T (p.Arg703Cys) and g.2238T>A (c.74–7T>A)—were significantly associated with CD in the case control sample. Analysis of the evolution of CARD15 revealed strong conservation of the encoded protein, with identity to the human sequence ranging from 99.1% in the chimp to 44.5% in fugu. Higher primates possess an open reading frame (ORF) upstream of the putative initiation site in other species that encodes a further 27 N-terminal amino acids, while four regions of high conservation are observed outside of the known domains of CARD15, indicative of additional residues of functional importance. The strategy developed here may have general application to the assessment of mutation pathogenicity and genetic models in other complex disorders. Hum Mutat 27(1), 44–54, 2006. © 2005 Wiley-Liss, Inc.

This article reports a well-powered age-related macular degeneration (AMD) case–control association study in the HMCN1 gene, showing that common variants do not account for a substantial proportion of AMD cases. Thus, the consistent linkage peak observed by several genome-wide linkage scans within the 1q32 region is unlikely to be attributed to polymorphisms at the HMCN1 locus. In addition, the analysis provides comprehensive data suggesting that low-frequency variants encoding possible functional amino acid polymorphisms in the HMCN1 gene may not contribute substantially to disease, although HMCN1 mutations may still confer disease susceptibility in a small subset of patients. Interestingly, the HMCN1 p.Gln5346Arg mutation, which is thought to be a causal mutation in a large AMD pedigree segregating the disease as a single-gene trait, appears to occur in our control cohort as a low-frequency polymorphism with an allele frequency of approximately 0.0026. Hum Mutat 28(4), 406–413, 2007. © 2007 Wiley-Liss, Inc.

Objectives Genetic variants at the CARD15 and IBD5 loci are strongly associated with Crohn's disease (CD), but evidence of the effect of these variants on the clinical expression of CD is conflicting and has often been hampered by small sample sizes. We studied 630 well-characterized patients to clarify the genotype/phenotype relationship in CD. Methods Patients and healthy controls were genotyped for three common mutations in CARD15 and a marker of the IBD5 risk haplotype. Allele frequencies were compared between phenotypic subgroups using χ2 or Fisher's exact tests. Genotype/phenotype analysis was carried out using multinomial logistic regression modelling allowing for adjustment for correlated or confounding factors. Results The mean age at diagnosis was significantly lower in carriers of the CARD15 or IBD5 risk alleles. After correction for age and smoking, CARD15 mutations were strongly associated with both ileal disease (P=8.8×10−6) and stenotic disease (P=0.003), but the association with stenotic disease appeared to be due to a confounding effect with ileal disease. CARD15 mutations were also associated with the presence of granulomas (P=5.7×10−5), which remained significant after adjustment for age at diagnosis and disease location (P=0.0047). The IBD5 risk haplotype frequency was significantly elevated in cases with perianal disease (P=0.028) and axial spondyloarthropathy (P=0.012). Conclusion Genetic variants at the CARD15 and IBD5 loci have diverse effects on clinical expression in CD. CARD15 mutations are significantly correlated with the presence of granulomas.

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To identify and assess the effectiveness of interventions to improve adherence to iron chelation therapy compared to standard care in people with SCD or thalassaemia including: identifying and assessing the effectiveness of different types of interventions (psychological and psychosocial, educational, medication interventions, or multi‐component interventions); identifying and assessing the effectiveness of interventions specific to different age groups (children, adolescents, adults).

The importance of CARD15 mutations in susceptibility to Crohn’s disease (CD)1Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O’Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Interleukin-23 drives innate and T cell-mediated intestinal inflammation.J Exp Med. 2001; 203: 2473-2483Google Scholar, 2Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nunez G. Cho J.H. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature. 2001; 411: 603-606Crossref PubMed Scopus (4171) Google Scholar, 3Hampe J. Cuthbert A. Croucher P.J.P. Mirza M.M. Mascheretti S. Fisher S. Frenzel H. King K. Hasselmeyer A. MacPherson A.J.S. Bridger S. van Deventer S. Forbes A. Nikolaus S. Lennard-Jones J.E. Foelsch U.R. Krawczak M. Lewis C. Schreiber S. Mathew C.G. Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations.Lancet. 2001; 357: 1925-1928Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar and its role in the activation of nuclear factor-κB provides a compelling case for the involvement of other genes in this signaling pathway. We therefore read with interest the recent report by McGovern et al4McGovern D.P. Butler H. Ahmad T. Paolucci M. van Heel D.A. Negoro K. Hysi P. Ragoussis J. Travis S.P. Cardon L.R. Jewell D.P. TUCAN (CARD8) genetic variants and inflammatory bowel disease.Gastroenterology. 2006; 131: 1190-1196Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar of a novel association between a variant in the CARD8 gene and inflammatory bowel disease (IBD). The CARD8 protein, also known as TUCAN (tumour–up-regulated CARD-containing antagonist of caspase nine) is a 48-kDa peptide (TUCAN-48; T48) expressed mainly in monocytes, placenta, lymph nodes, and spleen; it interacts directly with caspase-1 and can induce apoptosis. Recently, a second, larger, alternatively spliced isoform of TUCAN, TUCAN-54 (T54), that is over-expressed in some cancer tissues and suppresses caspase-mediated apoptosis, has been described.5Yamamoto M. Torigoe T. Kamiguchi K. Hirohashi Y. Nakanishi K. Nabeta C. Asanuma H. Tsuruma T. Sato T. Hata F. Ohmura T. Yamaguchi K. Kurotaki T. Hirata K. Sato N. A novel isoform of TUCAN is overexpressed in human cancer tissues and suppresses both caspase-8- and caspase-9-mediated apoptosis.Cancer Res. 2005; 65: 8706-8714Crossref PubMed Scopus (25) Google Scholar We undertook screening of the CARD8 gene for sequence variants by sequencing all exons, splice sites, 5′ and 3′ untranslated regions (UTRs) in 24 unrelated IBD patients. Six known single nucleotide polymorphisms (SNPs) were detected: rs10500299 (T54:c.1102G/A or T48:c.938 G/A), rs13745718 (T54:c.1278A/G or T48:c.1114A/G), rs8112588 (T54:c.1395C/T or T48:c.1231C/T), rs2288876 (T54:c.96T/C or T48:c.-29T/C), rs2043211 which causes a premature STOP codon at Cys10 of T48 or a Phe>Ile at residue 52 of T54; and rs12984612 (T54: c.-295A/C only). In addition, 2 novel variants were detected: an insertion AA frameshift mutation at residue 43 of T48 (or residue 98 of T54), resulting in a premature stop codon 25 amino acids downstream, and a -318G/T substitution in exon 1 of T54 5′UTR. The stop codon and frame shift mutations apart, none of the other variants would be predicted to alter the amino acid sequence of the protein. Evaluation of linkage disequilibrium (LD) in an extended sample of 47 patients revealed strong LD (r2 = 0.93) between T54 c.-318G/T and rs12984612, and therefore T54 c.-318G/T was identified as a tagging SNP for rs12984612. All other pairwise values of r2 were <0.6 and no further tagging SNPs could be identified. One SNP, rs8112588, was observed in only 1 individual out of 47 and was therefore not considered further as our study would provide insufficient power to detect association. The remaining 6 SNPs were genotyped in a total of 1399 IBD patients (851 CD, 548 ulcerative colitis [UC]) recruited from Guy’s, King’s and St Thomas’ Hospitals and St Mark’s Hospital (London, England) and 652 normal healthy controls, as previously described.6Cuthbert A.P. Fisher S.A. Mirza M.M. King K. Hampe J. Croucher P.J.P. Mascheretti S. Sanderson J. Forbes A. Mansfield J. Schreiber S. Lewis C.M. Mathew C.G. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease.Gastroenterology. 2002; 131: 1190-1196Google Scholar, 7Onnie C.M. Fisher S.A. Pattni R. Sanderson J. Forbes A. Lewis C.M. Mathew C.F. Associations of allelic variants of the Multidrug Resistance Gene (ABCB1 or MDR1) and inflammatory bowel disease and their effects on disease behaviour: a case-control study and meta-analysis.Inflamm Bowel Dis. 2006; 12: 263-271Crossref PubMed Scopus (74) Google Scholar No evidence for association was found with SNPs T54 c.-318G/T, rs2288876, T48 p.43 Valfs, rs10500299 or rs3745718. In particular, the frequency of the T48 p.43 Valfs frameshift mutation was 5.1% in IBD cases compared with 6.2% in controls (P = .24). However, a significant increase in the frequency of the stop codon mutation C10X (rs2043211) was observed in both CD (33.3%) and UC cases (32.7%) compared with controls (29.1%) with a combined P value of 0.012 for IBD (Table 1). Our results are in contrast to those of McGovern et al; in their study, the common allele, which encodes a cysteine, rather than the rare stop codon allele, was associated with an increased risk of CD, with the frequency of the stop codon being 35.6% in the Oxford controls compared with 29.1% in the controls from our study. We therefore genotyped C10X_rs2043211 in a second cohort of 791 IBD cases (494 CD, 298 UC) from Newcastle, England,6Cuthbert A.P. Fisher S.A. Mirza M.M. King K. Hampe J. Croucher P.J.P. Mascheretti S. Sanderson J. Forbes A. Mansfield J. Schreiber S. Lewis C.M. Mathew C.G. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease.Gastroenterology. 2002; 131: 1190-1196Google Scholar and 764 British controls, including 249 from Newcastle8Hartland S. Newton J.L. Griffin S.M. Donaldson P.T. A functional polymorphism in the Interleukin-1 receptor gene is associated with increased risk of Helicobacter pylori infection but not with gastric cancer.Dig Dis Sci. 2004; 49: 1545-1550Crossref PubMed Scopus (32) Google Scholar and 515 from the 1958 British Birth Cohort (www.cls.ioe.ac.uk). In this second, independent, case-control cohort, the frequency of the stop codon allele was significantly higher in CD (35%) and UC (35.1%) compared with controls (30.4%) (P = .0037 for IBD; Table 1). Combining our original and replication cohorts, the overall evidence for an association with IBD was highly significant (P = .0003). The pooled odds ratio for the stop codon allele was 1.21 (95% confidence interval [CI], 1.09–1.34) for IBD; similar risks were observed for CD and UC.Table 1CARD8 rs2043211 (C10X) Stop Codon Allele Frequencies in Original, Replication, and Pooled Case Control Cohorts (P value for difference in allele frequency between cases and controls)StudyControlsCDUCIBDN%N%PN%PN%POriginal65229.180933.3.01650232.7.069131133.1.012Replication72930.449535.1.01929736.2.01379235.5.0037Pooled138129.8130434.0.001179934.0.0046210334.0.0003 Open table in a new tab In light of the conflicting results between our cohorts and that of McGovern et al,4McGovern D.P. Butler H. Ahmad T. Paolucci M. van Heel D.A. Negoro K. Hysi P. Ragoussis J. Travis S.P. Cardon L.R. Jewell D.P. TUCAN (CARD8) genetic variants and inflammatory bowel disease.Gastroenterology. 2006; 131: 1190-1196Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar we undertook a meta-analysis across all 3 studies. Using a random effects model,9DerSimonian R. Laird N. Meta-analysis in clinical trials.Control Clin Trials. 1986; 7: 177-188Abstract Full Text PDF PubMed Scopus (29196) Google Scholar the combined odds ratio for IBD associated with the rare stop codon allele was 1.06 (95% CI, 0.81–1.39; P = .65). The corresponding odds ratios for CD and UC were 1.05 (95% CI, 0.78–1.40; P = .77) and 1.09 (95% CI, 0.84–1.40; P = .52), respectively. In summary, our results do not support the conclusions made by McGovern et al4McGovern D.P. Butler H. Ahmad T. Paolucci M. van Heel D.A. Negoro K. Hysi P. Ragoussis J. Travis S.P. Cardon L.R. Jewell D.P. TUCAN (CARD8) genetic variants and inflammatory bowel disease.Gastroenterology. 2006; 131: 1190-1196Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar of an association of the common allele encoding cysteine with CD. Furthermore, we have shown a significant association of the less common allele of this SNP encoding the stop codon with IBD in 2 independent studies. None of the other variants detected from screening of the CARD8 gene have shown any evidence for association. On the basis of the combined evidence across studies, we conclude that CARD8/TUCAN is not likely to be a major susceptibility locus for IBD. TUCAN (CARD8) Genetic Variants and Inflammatory Bowel DiseaseGastroenterologyVol. 131Issue 4PreviewBackground & Aims: The identification of the association between Crohn’s disease (CD) and NOD2 (CARD15) confirmed both the heritability of CD and highlighted the role of the nuclear factor κB (NFκB) pathway in disease pathogenesis. Other susceptibility loci exist. TUCAN (CARD8) is located beneath a CD peak of linkage on chromosome 19q. TUCAN is expressed in the gut and is a negative regulator of NFκB, making it an excellent candidate gene for gastrointestinal inflammation. Methods: Ten single nucleotide polymorphisms (SNP) across TUCAN were genotyped in 365 controls, 372 patients with CD, and 373 patients with ulcerative colitis. Full-Text PDF

Guidelines to treat anaemia with intravenous (IV) iron have focused on elective surgical patients with little attention paid to those undergoing non-elective/emergency surgery. Whilst these patients may experience poor outcomes because of their presenting illness, observational data suggests that untreated anaemia may also be a contributing factor to poor outcomes. We conducted a systematic review to investigate the safety and efficacy of IV iron in patients undergoing non-elective surgery.We followed a pre-defined review protocol and included randomised controlled trials (RCTs) in patients undergoing non-elective surgery who received IV iron. Primary outcomes were all-cause infection and mean difference in haemoglobin (Hb) at follow-up. Secondary outcomes included transfusion requirements, hospital length of stay (LOS), health-related quality of life (HRQoL), mortality and adverse events.Three RCTs (605 participants) were included in this systematic review of which two, in both hip fracture (HF) patients, provided data for meta-analysis. Both of these RCTs were at low risk of bias. We found no evidence of a difference in the risk of infection (RR 0.99, 95% CI 0.55 to 1.80, I2 = 9%) or in the Hb concentration at 'short-term' (≤ 7 days) follow-up (mean difference - 0.32 g/L, 95% CI - 3.28 to 2.64, I2 = 37%). IV iron did not reduce the risk of requiring a blood transfusion (RR 0.90, 95% CI 0.73 to 1.11, p = 0.46, I2 = 0%), and we observed no difference in mortality, LOS or adverse events. One RCT reported on HRQoL and found no difference between treatment arms.We found no conclusive evidence of an effect of IV iron on clinically important outcomes in patients undergoing non-elective surgery. Further adequately powered trials to evaluate its benefit in emergency surgical specialties with a high burden of anaemia are warranted.This systematic review was registered on PROSPERO (CRD42018096288).

A single-nucleotide polymorphism (C1858T) causing an amino acid substitution (R620W) in the lymphoid protein tyrosine phosphatase gene PTPN22 has been implicated in type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, juvenile idiopathic arthritis and Hashimoto's thyroiditis, thus revealing a general role for this gene in autoimmune disease. We investigated the association of the C1858T variant in an additional autoimmune disease population by performing a case-control study of 514 British individuals with inflammatory bowel disease (IBD) [294 with Crohn's disease (CD) and 220 with ulcerative colitis (UC)] and 374 normal controls. No significant differences in genotype or allele frequencies were observed between IBD, CD or UC and controls, indicating that PTPN22 does not influence risk of IBD.

It has been proposed that genetic susceptibility loci for rheumatoid arthritis (RA) may be shared with other autoimmune/inflammatory diseases. Recently, common variation in the CARD15 (NOD2) gene on chromosome 16q12 has been associated with Crohn's disease (CD) in several independent populations. CARD15 is an excellent functional and positional candidate gene for RA.Genomic DNA was obtained from 392 RA cases and 471 ethnically matched healthy controls. All samples were genotyped for two polymorphisms in CARD15, 1007fs and R702W, using 5' nuclease reporter assays. Allele frequencies were compared between cases and controls using the chi(2) test. Estimated haplotype frequencies across the two mutations were determined using the EH program.The allele frequency of the 1007fs variant in RA cases was 1.8% compared with 1.6% in normal controls (not significant). The frequency of the R702W variant was 4.0% in both cases and controls. Haplotypes carrying either of the two mutations accounted for 5.6% of possible haplotypes. A haplotype carrying both mutations was rare, with estimated frequency <0.01%. This study provided high power to detect an association of similar magnitude to that in Crohn's disease. These data therefore exclude the possibility that the contribution of these mutations to RA is comparable to that seen in CD.Within defined statistical parameters, we excluded a role for the CARD15 1007fs and R702W variants in RA susceptibility. These data do not preclude a role for other polymorphisms in the CARD15 gene in RA susceptibility. Results from other autoimmune and inflammatory diseases will reveal whether the CARD15 gene is in fact a common autoimmune susceptibility locus.

PSORS1 on chromosome 6p21.3, which contains the MHC, is a major susceptibility locus for psoriasis vulgaris. This region is characterized by strong linkage disequilibrium and contains the corneodesmosin (CSDN) gene, an attractive candidate for psoriasis susceptibility based on its putative biological function in keratinocyte adhesion, and HLA-Cw6, an established marker for psoriasis susceptibility. We compared two genetically independent populations in order to define the major psoriasis susceptibility gene, a British Caucasian population comprising parent-offspring trios analysed by the transmission disequilibrium test (TDT) and a Japanese case-control population. All individuals were investigated for CDSN polymorphism (+619, +1236, +1240 and +1243) and HLA-C association. Our data confirms strong association with HLA-Cw6 and CDSN allele 5 (+619T, +1240G, +1243C) in the Caucasian cohort (TDT, P = 5.4 x 10(-6)) and in addition defines this region further by identifying a high-risk CDSN haplotype (allele 5 and +1236T, P = 8.5 x 10(-8)). In contrast no association was observed in the Japanese cohort for any HLA-C or CDSN alleles. This data supports a role for the CDSN gene in Caucasian populations with psoriasis. However the lack of association with HLA-Cw6 and CDSN alleles in Japanese psoriasis patients may be because Japanese patients exhibit a form of psoriasis similar to late onset or Type II psoriasis vulgaris in contrast to early onset or Type I disease characterizing our Caucasian population.

Many genome-wide linkage studies in multiple sclerosis (MS) have been performed, but results are disappointing, with linkage confirmed only in the HLA region. We combined results from all available, non-overlapping genome-wide linkage studies in MS using the Genome Search Meta-Analysis method (GSMA). The GSMA is a rank-based analysis, which assesses the strongest evidence for linkage within bins of traditionally 30 cM width on the autosomes and X chromosome. Genome-wide evidence for linkage was confirmed on chromosome 6p (HLA region; P=0.00004). Suggestive evidence for linkage was found on chromosomes 10q (P=0.0077), 18p (P=0.0054) and 20p (P=0.0079). To explore how these results could be affected by bin definition, we analysed the data using different bin widths (20 and 40 cM) and using a shifted 30 cM bin by moving bin boundaries by 15 cM. Consistently significant results were obtained for the 6p region. The regions on 10q and 18p provided suggestive evidence for linkage in some analyses, and, interestingly, a region on 6q, that showed only nominal significance in the original analysis, yielded increased, suggestive significance in two of the additional analyses. These regions may provide targets to focus candidate gene studies or to prioritise results from genome-wide association studies.

Rheumatoid arthritis (RA) is a common disabling autoimmune disease with a complex genetic component. We have previously described linkage of a region of chromosome 8q12.3 with RA and association of the microsatellite marker CRHRA1 with RA in 295 affected sibling-pair families. In the current study we aimed to physically link the RA-associated marker with the corticotropin-releasing hormone (CRH) candidate gene, and to examine the genomic region for additional short tandem repeat (STR) genetic markers in order to clarify the association with RA.We examined the association of 2 STR markers with disease in the original 295 multicase families and in a cohort of 131 simplex families to refine our understanding of this genetic region in disease susceptibility in sporadic and familial RA. Genomic library screening and sequencing were used to generate physical sequences in the CRH genomic region. Bioinformatic analysis of the sequence flanking the CRH structural gene was used to screen for additional STRs and other genetic features. Genotyping was carried out using a standard fluorescence approach. Estimations of haplotype frequencies were performed to assess linkage disequilibrium. The transmission disequilibrium test was performed using TRANSMIT.Physical cloning and sequencing analyses identified the genomic region linking the CRHRA1 marker and the CRH structural locus. Moreover, we identified a further STR, CRHRA2, which was in strong linkage disequilibrium with CRHRA1 (P = 4.0 x 10(-14)). A haplotype, CRHRA1*10;CRHRA2*14, was preferentially carried by unaffected parents at a frequency of 8.6% compared with the expected frequency of 3.1%. This haplotype was overtransmitted in the multiply affected families (P = 0.0077) and, similarly, in the simplex families (P = 0.024). Combined analysis of both family cohorts confirmed significant evidence for linkage (P = 4.9 x 10(-4)) and association (P = 5.5 x 10(-3)) for this haplotype with RA.In demonstrating significant linkage disequilibrium between these 2 markers, we have refined the disease-associated region to a single haplotype and confirmed the significance of this region in our understanding of the genetics of RA.

The Genome Search Meta-Analysis (GSMA) method enables researchers to pool results across genome-wide linkage studies, to increase the power to detect linkage. RESULTS from individual studies must be extracted, with the maximum evidence for linkage placed into bins, usually of 30 cM width, and ranked within the study. Ranks are then summed across studies, with high summed ranks potentially showing evidence for linkage in the meta-analysis.In this paper we study the properties of the GSMA method considering two different issues: (1) data binning from genome-wide results when indexed markers or graphs are available, based on either predefined boundary markers, or equal-length bins; (2) the use of selected instead of genome-wide results, using simulation to estimate power and type I error rates of GSMA. This is relevant when published papers show only summary results (e.g. with NPL score >1).Using digitizing software to extract linkage statistics from graphs and assigning equal bin length is accurate, with the resulting ranking of bins similar to those defined through boundary markers. Simulation results show that power can fall substantially when genome-wide results are not available, particularly when only results from a single marker are available in a linked region. However there is no increase in false positive findings.The GSMA method is robust across different bin definitions and methods of data presentation and extraction. Using studies based on only the top ranked bins does not produce false positive results, but lacks power to detect genes conferring a modest increase in risk. Therefore, we advise that effort should be made to obtain genome-wide results from investigators or from published papers to avoid limiting the utility of the GSMA.

Although Illumina shot-gun reads cover most genomes almost completely, sequences with extreme base compositions are often underrepresented or missing. Bias can potentially be introduced at any step during the library construction in the lab, on the Illumina instrument, in data processing or at the sequence analysis stage. Here we set out to evaluate sources of bias and ameliorate the effects. To dissect the library construction process, we developed a panel of qPCR assays for loci ranging from 6% to 90% GC that work well in a pool of three microbial DNA samples of different base composition: Plasmodium falciparum (19% GC), Escherichia coli (51% GC) and Rhodobacter sphaeroides (69% GC). We also developed qPCR assays for loci in the human genome that represent four categories of underrepresented sequence motifs as well as GC-rich promoters known to be underrepresented or missing in 'whole' genome sequencing data sets. We tracked the relative abundance of these loci throughout the standard Illumina library protocol and saw no significant introduction of bias in the initial steps including shearing, end repair, adaptor ligation and size selection. However, GC-rich and extremely GC-poor sequences were depleted during the subsequent PCR-enrichment step. Using qPCR as a readout, we tested different PCR enzymes, the addition of betaine and/or DMSO, and thermocycling profile variations. The choice of PCR instrument itself and the ramp rate had a significant effect on the GC profile of the PCR product, especially when using the recommended amplification conditions (Phusion HF and 10s denaturation per cycle). Our optimized conditions produce PCR-amplified libraries that display little systematic bias between 15% and 80% GC that resulted during sample preparation. We saw significantly improved representation of challenging human sequence motifs both in the PCR-amplified library (qPCR assay) and in the final Illumina reads. Our conditions are also more reliable and robust because they minimize the effect of PCR instrument and ramp rate. These conditions are currently being implemented in the Sequencing Platform at the Broad Institute. Finally, we still observe some bias in the sequencing readout, which is introduced by steps subsequent to sample preparation, including cluster generation and sequencing. These sources of bias are the object of ongoing investigations.

The calculation of the power and sample size required for association studies is essential, particularly for follow-up of genome-wide association studies, where much genotyping is required to replicate the original finding and identify the true disease susceptibility mutation.In this paper, we derive equations for estimation of sample sizes for the transmission disequilibrium test (TDT) and for case-control studies, in the presence of allelic heterogeneity and indirect association - where the genotyped tagging SNP is in linkage disequilibrium (LD) with the true mutation. Using data from NOD2 and PTPN22, we show that the true sample sizes required to detect association may be incorrect when calculated under the assumption of a single mutation and complete LD with the genotyped marker.The true sample sizes may be lower when allelic heterogeneity acts in a recessive model across mutations, or increased when mutations lie on different alleles of a common tagging SNP.Calculating power and sample size under a range of realistic models of LD and allelic heterogeneity is essential to ensure that association studies have sufficient power to detect mutations.

There has been considerable recent success in the detection of gene-disease associations. We consider here the development of tools that facilitate the more detailed characterization of the effect of a genetic variant on disease. We replace the simplistic classification of individuals according to a single binary disease indicator with classification according to a number of subphenotypes. This more accurately reflects the underlying biological complexity of the disease process, but it poses additional analytical difficulties. Notably, the subphenotypes that make up a particular disease are typically highly associated, and it becomes difficult to distinguish which genes might be causing which subphenotypes. Such problems arise in many complex diseases. Here, we concentrate on an application to Crohn disease (CD). We consider this problem as one of model selection based upon log-linear models, fitted in a Bayesian framework via reversible-jump Metropolis-Hastings approach. We evaluate the performance of our suggested approach with a simple simulation study and then apply the method to a real data example in CD, revealing a sparse disease structure. Most notably, the associated NOD2.908G→R mutation appears to be directly related to more severe disease behaviors, whereas the other two associated NOD2 variants, 1007L→FS and 702R→W, are more generally related to disease in the small bowel (ileum and jejenum). The ATG16L1.300T→A variant appears to be directly associated with only disease of the small bowel. There has been considerable recent success in the detection of gene-disease associations. We consider here the development of tools that facilitate the more detailed characterization of the effect of a genetic variant on disease. We replace the simplistic classification of individuals according to a single binary disease indicator with classification according to a number of subphenotypes. This more accurately reflects the underlying biological complexity of the disease process, but it poses additional analytical difficulties. Notably, the subphenotypes that make up a particular disease are typically highly associated, and it becomes difficult to distinguish which genes might be causing which subphenotypes. Such problems arise in many complex diseases. Here, we concentrate on an application to Crohn disease (CD). We consider this problem as one of model selection based upon log-linear models, fitted in a Bayesian framework via reversible-jump Metropolis-Hastings approach. We evaluate the performance of our suggested approach with a simple simulation study and then apply the method to a real data example in CD, revealing a sparse disease structure. Most notably, the associated NOD2.908G→R mutation appears to be directly related to more severe disease behaviors, whereas the other two associated NOD2 variants, 1007L→FS and 702R→W, are more generally related to disease in the small bowel (ileum and jejenum). The ATG16L1.300T→A variant appears to be directly associated with only disease of the small bowel.

Heart failure (HF) is the major cause of mortality worldwide. For more than a decade, cell-based therapies have been developed as treatment for heart disease as an alternative to current therapies. Trials and systematic reviews have assessed the safety and efficacy of cell therapies in a diverse number of participants and clinical settings. The present study collated and synthesized evidence from all systematic reviews related to cell-based therapies and HF. A total of 11 systematic reviews were identified through searches of electronic databases up to June 2014. We set out to answer 2 key questions on the efficacy of cell therapies in HF: (1) What is the overall effect of cell therapies on primary outcomes such as left ventricular ejection fraction (LVEF) and mortality? (2) How important is it to define the clinical setting and length of follow-up when assessing cell therapies and HF? There seems to be enough evidence to suggest that cell therapies have a moderate, long-lasting effect on LVEF, but the reduction on the risk of mortality observed by some systematic reviews needs to be confirmed in larger, statistically powered clinical trials. Additionally, and in order to strengthen conclusions, it is important to assess clinical evidence for defined clinical settings and to standardize the length of follow-up when comparing outcome data across several trials and systematic reviews.

Mutations in the NOD2 (CARD15) gene predispose to Crohn's disease (CD), a human chronic inflammatory bowel disorder, and can cause Blau syndrome. During an investigation of an apparent correlation between a frameshifting mutation in the canonical first exon of NOD2 of marmoset and tamarin species and their susceptibility to chronic colitis, we found that, contrary to previous reports, the basal levels of NOD2 transcripts in tissues relevant to CD arise from a distinct novel promoter and first exon. The canonical first exon, by contrast, seems to be of negligible transcriptional importance under physiological conditions, and its reading frame has been disrupted twice during primate evolution. Thus the main NOD2/CARD15 protein isoform produced in humans and other primates is 27 amino acids shorter than previously reported, starting at a conserved methionine in exon 2. We show that there is no significant association between variants in the novel NOD2 promoter region and CD.

The chemokine gene cluster [CCL22, CX3CL1, CCL17] (previously known as [SCYA22, SCYD1, SCYA17]) is a candidate locus for one of the susceptibility genes for inflammatory bowel disease that are located in the peri-centromeric region of chromosome 16. Screening for sequence variation at this locus led to the detection of 14 single nucleotide polymorphisms (SNPs). An efficient experimental and computational approach was developed to estimate allele frequencies and pairwise linkage disequilibrium relationships between SNPs at this locus, and to test them for association with inflammatory bowel disease. The 12 common SNPs were assigned to 5 distinct linkage disequilibrium groups. Genotyping of one SNP from each linkage disequilibrium group in a large cohort of families with inflammatory bowel disease did not provide convincing evidence of association with either Crohn's disease or ulcerative colitis. We describe an efficient experimental design from SNP screening to association testing. This strategy can be used to test candidate genes for involvement in susceptibility to complex disease.

&lt;p&gt;PDF file 171K, Contains Supplementary Data and Methods&lt;/p&gt;

&lt;p&gt;PDF file 550K, Contains supplementary figures 1-3&lt;/p&gt;

&lt;p&gt;XLSX file 3709K, Alterations with Significantly Enriched CCF from Pretreatment to Resistant&lt;/p&gt;

&lt;p&gt;XLSX file 12K, Candidate Resistance Genes from RNAi and ORF Functional Screens&lt;/p&gt;

&lt;p&gt;XLSX file 90K, Alterations in Each Patient in Genes that Scored in Functional Screens&lt;/p&gt;

&lt;p&gt;XLSX file 31K, Coverage and Metrics&lt;/p&gt;

&lt;p&gt;XLS file 6188K, Annotated SNVs and Indels in All Samples&lt;/p&gt;

&lt;p&gt;XLSX file 3569K, Change in Cancer Cell Fraction for all SNVs and Indels in All Samples&lt;/p&gt;

&lt;p&gt;XLS file 6188K, Annotated SNVs and Indels in All Samples&lt;/p&gt;

&lt;p&gt;XLSX file 3709K, Alterations with Significantly Enriched CCF from Pretreatment to Resistant&lt;/p&gt;