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2.  Plasma concentrations of soluble IL-2 receptor α (CD25) are increased in type 1 diabetes and associated with reduced C-peptide levels in young patients 
Diabetologia  2013;57:366-372.
Type 1 diabetes is a common autoimmune disease that has genetic and environmental determinants. Variations within the IL2 and IL2RA (also known as CD25) gene regions are associated with disease risk, and variation in expression or function of these proteins is likely to be causal. We aimed to investigate if circulating concentrations of the soluble form of CD25, sCD25, an established marker of immune activation and inflammation, were increased in individuals with type 1 diabetes and if this was associated with the concentration of C-peptide, a measure of insulin production that reflects the degree of autoimmune destruction of the insulin-producing beta cells.
We used immunoassays to measure sCD25 and C-peptide in peripheral blood plasma from patient and control samples.
We identified that sCD25 was increased in patients with type 1 diabetes compared with controls and replicated this result in an independent set of 86 adult patient and 80 age-matched control samples (p = 1.17 × 10−3). In 230 patients under 20 years of age, with median duration-of-disease of 6.1 years, concentrations of sCD25 were negatively associated with C-peptide concentrations (p = 4.8 × 10−3).
The 25% increase in sCD25 in patients, alongside the inverse association between sCD25 and C-peptide, probably reflect the adverse effects of an on-going, actively autoimmune and inflammatory immune system on beta cell function in patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-013-3113-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC3890035  PMID: 24264051
Autoimmune; Blood; Case–control; CD25; C-peptide; IL-2; IL-2RA Immunoassay; Peripheral; sCD25; Soluble cytokine receptor; Type 1 diabetes
3.  Evidence of Gene-Gene Interaction and Age-at-Diagnosis Effects in Type 1 Diabetes 
Diabetes  2012;61(11):3012-3017.
The common genetic loci that independently influence the risk of type 1 diabetes have largely been determined. Their interactions with age-at-diagnosis of type 1 diabetes, sex, or the major susceptibility locus, HLA class II, remain mostly unexplored. A large collection of more than 14,866 type 1 diabetes samples (6,750 British diabetic individuals and 8,116 affected family samples of European descent) were genotyped at 38 confirmed type 1 diabetes-associated non-HLA regions and used to test for interaction of association with age-at-diagnosis, sex, and HLA class II genotypes using regression models. The alleles that confer susceptibility to type 1 diabetes at interleukin-2 (IL-2), IL2/4q27 (rs2069763) and renalase, FAD-dependent amine oxidase (RNLS)/10q23.31 (rs10509540), were associated with a lower age-at-diagnosis (P = 4.6 × 10−6 and 2.5 × 10−5, respectively). For both loci, individuals carrying the susceptible homozygous genotype were, on average, 7.2 months younger at diagnosis than those carrying the protective homozygous genotypes. In addition to protein tyrosine phosphatase nonreceptor type 22 (PTPN22), evidence of statistical interaction between HLA class II genotypes and rs3087243 at cytotoxic T-lymphocyte antigen 4 (CTLA4)/2q33.2 was obtained (P = 7.90 × 10−5). No evidence of differential risk by sex was obtained at any loci (P ≥ 0.01). Statistical interaction effects can be detected in type 1 diabetes although they provide a relatively small contribution to our understanding of the familial clustering of the disease.
PMCID: PMC3478521  PMID: 22891215
4.  Bayesian refinement of association signals for 14 loci in 3 common diseases 
Nature genetics  2012;44(12):1294-1301.
To further investigate susceptibility loci identified by genome-wide association studies, we genotyped 5,500 SNPs across 14 associated regions in 8,000 samples from a control group and 3 diseases: type 2 diabetes (T2D), coronary artery disease (CAD) and Graves’ disease. We defined, using Bayes theorem, credible sets of SNPs that were 95% likely, based on posterior probability, to contain the causal disease-associated SNPs. In 3 of the 14 regions, TCF7L2 (T2D), CTLA4 (Graves’ disease) and CDKN2A-CDKN2B (T2D), much of the posterior probability rested on a single SNP, and, in 4 other regions (CDKN2A-CDKN2B (CAD) and CDKAL1, FTO and HHEX (T2D)), the 95% sets were small, thereby excluding most SNPs as potentially causal. Very few SNPs in our credible sets had annotated functions, illustrating the limitations in understanding the mechanisms underlying susceptibility to common diseases. Our results also show the value of more detailed mapping to target sequences for functional studies.
PMCID: PMC3791416  PMID: 23104008
5.  Postthymic Expansion in Human CD4 Naive T Cells Defined by Expression of Functional High-Affinity IL-2 Receptors* 
As the thymus involutes with age, the maintenance of peripheral naive T cells in humans becomes strongly dependent on peripheral cell division. However, mechanisms that orchestrate homeostatic division remain unclear. In this study we present evidence that the frequency of naive CD4 T cells that express CD25 (IL-2 receptor α-chain) increases with age on subsets of both CD31+ and CD31− naive CD4 T cells. Analyses of TCR excision circles from sorted subsets indicate that CD25+ naive CD4 T cells have undergone more rounds of homeostatic proliferation than their CD25− counterparts in both the CD31+ and CD31− subsets, indicating that CD25 is a marker of naive CD4 T cells that have preferentially responded to survival signals from self-Ags or cytokines. CD25 expression on CD25− naive CD4 T cells can be induced by IL-7 in vitro in the absence of TCR activation. Although CD25+ naive T cells respond to lower concentrations of IL-2 as compared with their CD25− counterparts, IL-2 responsiveness is further increased in CD31− naive T cells by their expression of the signaling IL-2 receptor β-chain CD122, forming with common γ-chain functional high-affinity IL-2 receptors. CD25 plays a role during activation: CD25+ naive T cells stimulated in an APC-dependent manner were shown to produce increased levels of IL-2 as compared with their CD25− counterparts. This study establishes CD25+ naive CD4 T cells, which are further delineated by CD31 expression, as a major functionally distinct immune cell subset in humans that warrants further characterization in health and disease.
PMCID: PMC3614027  PMID: 23418630
6.  Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys 
Science translational medicine  2011;3(81):81ra36.
The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was an about one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanism of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.
PMCID: PMC3718279  PMID: 21543722
7.  A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk 
Nature  2010;467(7314):460-464.
Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases. Here, we used integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)1-driven inflammatory network (iDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and was regulated in multiple tissues by a locus on rat chromosome 15q25. At this locus, Epstein-Barr virus induced gene 2 (Ebi2 or Gpr183), which we localised to macrophages and is known to control B lymphocyte migration2,3, regulated the iDIN. The human chromosome 13q32 locus, orthologous to rat 15q25, controlled the human equivalent of iDIN, which was conserved in monocytes. For the macrophage-associated autoimmune disease type 1 diabetes (T1D) iDIN genes were more likely to associate with T1D susceptibility than randomly selected immune response genes (P = 8.85 × 10−6). The human locus controlling the iDIN, was associated with the risk of T1D at SNP rs9585056 (P = 7.0 × 10−10, odds ratio = 1.15), which was one of five SNPs in this region associated with EBI2 expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.
PMCID: PMC3657719  PMID: 20827270
8.  Functional IL6R 358Ala Allele Impairs Classical IL-6 Receptor Signaling and Influences Risk of Diverse Inflammatory Diseases 
PLoS Genetics  2013;9(4):e1003444.
Inflammation, which is directly regulated by interleukin-6 (IL-6) signaling, is implicated in the etiology of several chronic diseases. Although a common, non-synonymous variant in the IL-6 receptor gene (IL6R Asp358Ala; rs2228145 A>C) is associated with the risk of several common diseases, with the 358Ala allele conferring protection from coronary heart disease (CHD), rheumatoid arthritis (RA), atrial fibrillation (AF), abdominal aortic aneurysm (AAA), and increased susceptibility to asthma, the variant's effect on IL-6 signaling is not known. Here we provide evidence for the association of this non-synonymous variant with the risk of type 1 diabetes (T1D) in two independent populations and confirm that rs2228145 is the major determinant of the concentration of circulating soluble IL-6R (sIL-6R) levels (34.6% increase in sIL-6R per copy of the minor allele 358Ala; rs2228145 [C]). To further investigate the molecular mechanism of this variant, we analyzed expression of IL-6R in peripheral blood mononuclear cells (PBMCs) in 128 volunteers from the Cambridge BioResource. We demonstrate that, although 358Ala increases transcription of the soluble IL6R isoform (P = 8.3×10−22) and not the membrane-bound isoform, 358Ala reduces surface expression of IL-6R on CD4+ T cells and monocytes (up to 28% reduction per allele; P≤5.6×10−22). Importantly, reduced expression of membrane-bound IL-6R resulted in impaired IL-6 responsiveness, as measured by decreased phosphorylation of the transcription factors STAT3 and STAT1 following stimulation with IL-6 (P≤5.2×10−7). Our findings elucidate the regulation of IL-6 signaling by IL-6R, which is causally relevant to several complex diseases, identify mechanisms for new approaches to target the IL-6/IL-6R axis, and anticipate differences in treatment response to IL-6 therapies based on this common IL6R variant.
Author Summary
Interleukin-6 (IL-6) is a complex cytokine, which plays a critical role in the regulation of inflammatory responses. Genetic variation in the IL-6 receptor gene is associated with the risk of several human diseases with an inflammatory component, including coronary heart disease, rheumatoid arthritis, and asthma. A common non-synonymous single nucleotide polymorphism in this gene (Asp358Ala) has been suggested to be the causal variant in this region by affecting the circulatory concentrations of soluble IL-6R (sIL-6R). In this study we extend the genetic association of this variant to type 1 diabetes and provide evidence that this variant exerts its functional mechanism by regulating the balance between sIL-6R (generated through cleavage of the surface receptor and by alternative splicing of a soluble IL6R isoform) and membrane-bound IL-6R. These data show for the first time that the minor allele of this non-synonymous variant (Ala358) directly controls the surface levels of IL-6R on individual immune cells and that these differences in protein levels translate into a functional impairment in IL-6R signaling. These findings may have implications for clinical trials targeting inflammatory mechanisms involving IL-6R signaling and may provide tools for identifying patients with specific benefit from therapeutic intervention in the IL-6R signaling pathway.
PMCID: PMC3617094  PMID: 23593036
9.  Gene-Based Vaccination with a Mismatched Envelope Protects against Simian Immunodeficiency Virus Infection in Nonhuman Primates 
Journal of Virology  2012;86(15):7760-7770.
The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.
PMCID: PMC3421661  PMID: 22593152
10.  FUT2 Nonsecretor Status Links Type 1 Diabetes Susceptibility and Resistance to Infection 
Diabetes  2011;60(11):3081-3084.
FUT2 encodes the α(1,2) fucosyltransferase that determines blood group secretor status. Homozygotes (A/A) for the common nonsense mutation rs601338A>G (W143X) are nonsecretors and are unable to express histo-blood group antigens in secretions and on mucosal surfaces. This mutation has been reported to provide resistance to Norovirus and susceptibility to Crohn’s disease, and hence we aimed to determine if it also affects risk of type 1 diabetes.
rs601338A>G was genotyped in 8,344 patients with type 1 diabetes, 10,008 control subjects, and 3,360 type 1 diabetic families. Logistic regression models were used to analyze the case-control collection, and conditional logistic regression was used to analyze the family collection.
The nonsecretor A/A genotype of rs601338A>G was found to confer susceptibility to type 1 diabetes in both the case-control and family collections (odds ratio for AA 1.29 [95% CI 1.20–1.37] and relative risk for AA 1.22 [95% CI = 1.12–1.32]; combined P = 4.3 × 10−18), based on a recessive effects model.
Our findings linking FUT2 and type 1 diabetes highlight the intriguing relationship between host resistance to infections and susceptibility to autoimmune disease.
PMCID: PMC3198057  PMID: 22025780
11.  Type 1 Diabetes-associated IL2RAvariationlowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T-cell function 
Numerous reports have demonstrated that CD4+CD25+regulatory T cells (Tregs) from individuals with a range of human autoimmune diseases, including Type 1 diabetes (T1D),are deficient in theirability to control autologous pro-inflammatory responses when compared to non-diseased, control individuals. Treg dysfunction could be a primary, causal event or may result from perturbations in the immune system during disease development.Polymorphisms in genes associated with Treg function, such as IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in autoimmunity, a link between IL2RA gene polymorphisms and Treg function has not been examined. We addressed this by examining the impact of an IL2RA haplotype associated with T1D on Treg fitness and suppressive function. Studies were conducted using healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease-associated IL2RA haplotype correlates with diminished interleukin (IL)-2-responsiveness in antigen-experienced CD4+ T cells, as measured by phosphorylation of STAT5a, and is associated with lower levels of FoxP3 expression by Tregs, and a reduction in their ability to suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular mechanisms through which polymorphisms in the IL-2RA gene impact upon immune regulation, and consequently upon susceptibility to autoimmune and inflammatory diseases.
PMCID: PMC3378653  PMID: 22461703
12.  Evidence That HLA Class I and II Associations With Type 1 Diabetes, Autoantibodies to GAD and Autoantibodies to IA-2, Are Distinct 
Diabetes  2011;60(10):2635-2644.
A major feature of type 1 diabetes is the appearance of islet autoantibodies before diagnosis. However, although the genetics of type 1 diabetes is advanced, the genetics of islet autoantibodies needs further investigation. The primary susceptibility loci in type 1 diabetes, the HLA class I and II genes, are believed to determine the specificity and magnitude of the autoimmune response to islet antigens. We investigated the association of glutamic acid decarboxylase autoantibodies (GADA) and insulinoma-associated antigen-2 autoantibodies (IA-2A) with the HLA region.
Associations of GADA and IA-2A with HLA-DRB1, HLA-DQB1, HLA-B, HLA-C, HLA-A, MICA, and 3,779 single nucleotide polymorphisms (SNPs) were analyzed in 2,531 childhood-onset case subjects (median time since diagnosis 5 years). All analyses were adjusted for age-at-diagnosis and duration of diabetes.
GADA and IA-2A were associated with an older age-at-diagnosis (P < 10−19). For GADA, the primary association was with HLA-DQB1 (P = 9.00 × 10−18), with evidence of a second independent effect in the HLA class I region with SNP, rs9266722 (P = 2.84 × 10−6). HLA-DRB1 had the strongest association with IA-2A (P = 1.94 × 10−41), with HLA-A*24 adding to the association, albeit negatively (P = 1.21 × 10−10). There was no evidence of association of either IA-2A or GADA with the highly type 1 diabetes predisposing genotype, HLA-DRB1*03/04.
Despite genetic association of type 1 diabetes and the islet autoantibodies localizing to the same HLA class II genes, HLA-DRB1 and HLA-DQB1, the effects of the class II alleles and genotypes involved are quite different. Therefore, the presence of autoantibodies is unlikely to be causal, and their role in pathogenesis remains to be established.
PMCID: PMC3178284  PMID: 21831970
13.  Genetic Analysis of Adult-Onset Autoimmune Diabetes 
Diabetes  2011;60(10):2645-2653.
In contrast with childhood-onset type 1 diabetes, the genetics of autoimmune diabetes in adults are not well understood. We have therefore investigated the genetics of diabetes diagnosed in adults positive for autoantibodies.
GAD autoantibodies (GADAs), insulinoma-associated antigen-2 antibodies (IA-2As), and islet cell autoantibodies were measured at time of diagnosis. Autoantibody-positive diabetic subjects (n = 1,384) and population-based control subjects (n = 2,235) were genotyped at 20 childhood-onset type 1 diabetes loci and FCRL3, GAD2, TCF7L2, and FTO.
PTPN22 (1p13.2), STAT4 (2q32.2), CTLA4 (2q33.2), HLA (6p21), IL2RA (10p15.1), INS (11p15.5), ERBB3 (12q13.2), SH2B3 (12q24.12), and CLEC16A (16p13.13) were convincingly associated with autoimmune diabetes in adults (P ≤ 0.002), with consistent directions of effect as reported for pediatric type 1 diabetes. No evidence of an HLA-DRB1*03/HLA-DRB1*04 (DR3/4) genotype effect was obtained (P = 0.55), but it remained highly predisposing (odds ratio 26.22). DR3/4 was associated with a lower age at diagnosis of disease, as was DR4 (P = 4.67 × 10−6) but not DR3. DR3 was associated with GADA positivity (P = 6.03 × 10−6) but absence of IA-2A (P = 3.22 × 10−7). DR4 was associated with IA-2A positivity (P = 5.45 × 10−6).
Our results are consistent with the hypothesis that the genetics of autoimmune diabetes in adults and children are differentiated by only relatively few age-dependent genetic effects. The slower progression toward autoimmune insulin deficiency in adults is probably due to a lower genetic load overall combined with subtle variation in the HLA class II gene associations and autoreactivity.
PMCID: PMC3178303  PMID: 21873553
14.  Proteome-Wide Analysis of Disease-Associated SNPs That Show Allele-Specific Transcription Factor Binding 
PLoS Genetics  2012;8(9):e1002982.
A causative role for single nucleotide polymorphisms (SNPs) in many genetic disorders has become evident through numerous genome-wide association studies. However, identification of these common causal variants and the molecular mechanisms underlying these associations remains a major challenge. Differential transcription factor binding at a SNP resulting in altered gene expression is one possible mechanism. Here we apply PWAS (“proteome-wide analysis of SNPs”), a methodology based on quantitative mass spectrometry that enables rapid screening of SNPs for differential transcription factor binding, to 12 SNPs that are highly associated with type 1 diabetes at the IL2RA locus, encoding the interleukin-2 receptor CD25. We report differential, allele-specific binding of the transcription factors RUNX1, LEF1, CREB, and TFAP4 to IL2RA SNPs rs12722508*A, rs12722522*C, rs41295061*A, and rs2104286*A and demonstrate the functional influence of RUNX1 at rs12722508 by reporter gene assay. Thus, PWAS may be able to contribute to our understanding of the molecular consequences of human genetic variability underpinning susceptibility to multi-factorial disease.
Author Summary
Genome-wide association studies (GWAS) are a powerful approach to identifying genes contributing to risk of disease. However, individual mapped single nucleotide polymorphisms (SNPs) may not map close to a gene, and it can be difficult to distinguish marker SNPs from causal SNPs. Furthermore, the molecular mechanism responsible for disease association is usually not clear. Here we develop a method termed “proteome-wide analysis of SNPs” (PWAS) that identifies differentially binding transcription factors (TFs) and thereby helps to unravel the molecular mechanisms by which the SNPs may exert their effect on gene regulation. We use quantitative interaction proteomics to identify proteins with allele-specific binding. Applied to fine-mapped SNPs conferring risk in type 1 diabetes, PWAS revealed preferential binding of common transcription factors to certain disease-associated SNPs, suggesting they could be causal. In general, a proportion of causal SNPs are likely to function by mimicking binding motifs for transcription factors, increasing their occupancy and modulating gene expression. In addition, PWAS is streamlined and can be used as an informative follow-up approach to GWAS results.
PMCID: PMC3459973  PMID: 23028375
15.  Extra-binomial variation approach for analysis of pooled DNA sequencing data 
Bioinformatics  2012;28(22):2898-2904.
Motivation: The invention of next-generation sequencing technology has made it possible to study the rare variants that are more likely to pinpoint causal disease genes. To make such experiments financially viable, DNA samples from several subjects are often pooled before sequencing. This induces large between-pool variation which, together with other sources of experimental error, creates over-dispersed data. Statistical analysis of pooled sequencing data needs to appropriately model this additional variance to avoid inflating the false-positive rate.
Results: We propose a new statistical method based on an extra-binomial model to address the over-dispersion and apply it to pooled case-control data. We demonstrate that our model provides a better fit to the data than either a standard binomial model or a traditional extra-binomial model proposed by Williams and can analyse both rare and common variants with lower or more variable pool depths compared to the other methods.
Availability: Package ‘extraBinomial’ is on
Supplementary information: Supplementary data are available at Bioinformatics Online.
PMCID: PMC3496343  PMID: 22976083
16.  Seven newly identified loci for autoimmune thyroid disease 
Human Molecular Genetics  2012;21(23):5202-5208.
Autoimmune thyroid disease (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT), is one of the most common of the immune-mediated diseases. To further investigate the genetic determinants of AITD, we conducted an association study using a custom-made single-nucleotide polymorphism (SNP) array, the ImmunoChip. The SNP array contains all known and genotype-able SNPs across 186 distinct susceptibility loci associated with one or more immune-mediated diseases. After stringent quality control, we analysed 103 875 common SNPs (minor allele frequency >0.05) in 2285 GD and 462 HT patients and 9364 controls. We found evidence for seven new AITD risk loci (P < 1.12 × 10−6; a permutation test derived significance threshold), five at locations previously associated and two at locations awaiting confirmation, with other immune-mediated diseases.
PMCID: PMC3490518  PMID: 22922229
17.  High-Sensitivity Cardiac Troponin-I Is Elevated in Patients with Rheumatoid Arthritis, Independent of Cardiovascular Risk Factors and Inflammation 
PLoS ONE  2012;7(6):e38930.
We examined the hypothesis that cardiac-specific troponin-I (cTn-I), a biomarker of myocardial injury, is elevated in patients with rheumatoid arthritis (RA).
RA patients have an increased incidence of heart failure (HF). Chronic myocardial injury in RA may be a mechanism for the development of HF.
We compared cTn-I concentrations measured by high-sensitivity immunoassay in 164 patients with RA and 90 controls, excluding prior or active heart failure. We examined the relationship between cTn-I concentrations and cardiovascular risk factors, inflammation, and coronary artery calcium score (CACS), a measure of coronary atherosclerosis.
cTn-I concentrations were 49% higher in patients with RA (median 1.15 pg/mL [IQR 0.73–1.92] than controls (0.77 pg/mL [0.49–1.28](P<0.001). The difference remained statistically significant after adjustment for demographic characteristics (P = 0.002), further adjustment for cardiovascular (CV) risk factors (P = 0.004), inflammatory markers (P = 0.008), and in a comprehensive model of CV risk factors and inflammatory markers (P = 0.03). In patients with RA, cTn-I concentrations were positively correlated with age (rho = 0.359), Framingham risk score (FRS) (rho = 0.366), and systolic blood pressure (rho = 0.248 (all P values ≤0.001)), but not with measures of inflammation or RA drug therapies. cTn-I was significantly correlated with CACS in RA in univariate analysis, but not after adjustment for age, race, sex and FRS (P = 0.79). Further model adjustments for renal function and coronary artery disease confirmed the significance of the findings.
High-sensitivity cTn-I concentrations are elevated in patients with RA without heart failure, independent of cardiovascular risk profile and inflammatory markers. Elevated troponin concentrations in RA may indicate subclinical, indolent myocardial injury.
PMCID: PMC3386265  PMID: 22761714
18.  DNA Vaccination Elicits Protective Immune Responses against Pandemic and Classic Swine Influenza Viruses in Pigs ▿ † 
Clinical and Vaccine Immunology : CVI  2011;18(11):1987-1995.
Swine influenza is a highly contagious viral infection in pigs that significantly impacts the pork industry due to weight loss and secondary infections. There is also the potential of a significant threat to public health, as was seen in 2009 when the pandemic H1N1 influenza virus strain emerged from reassortment events among avian, swine, and human influenza viruses within pigs. As classic and pandemic H1N1 strains now circulate in swine, an effective vaccine may be the best strategy to protect the pork industry and public health. Current inactivated-virus vaccines available for swine influenza protect only against viral strains closely related to the vaccine strain, and egg-based production of these vaccines is insufficient to respond to large outbreaks. DNA vaccines are a promising alternative since they can potentially induce broad-based protection with more efficient production methods. In this study we evaluated the potentials of monovalent and trivalent DNA vaccine constructs to (i) elicit both humoral and gamma interferon (IFN-γ) responses and (ii) protect pigs against viral shedding and lung disease after challenge with pandemic H1N1 or classic swine H1N1 influenza virus. We also compared the efficiency of a needle-free vaccine delivery method to that of a conventional needle/syringe injection. We report that DNA vaccination elicits robust serum antibody and cellular responses after three immunizations and confers significant protection against influenza virus challenge. Needle-free delivery elicited improved antibody responses with the same efficiency as conventional injection and should be considered for development as a practical alternative for vaccine administration.
PMCID: PMC3209026  PMID: 21918118
19.  Inherited Variation in Vitamin D Genes Is Associated With Predisposition to Autoimmune Disease Type 1 Diabetes 
Diabetes  2011;60(5):1624-1631.
Vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] <50 nmol/L) is commonly reported in both children and adults worldwide, and growing evidence indicates that vitamin D deficiency is associated with many extraskeletal chronic disorders, including the autoimmune diseases type 1 diabetes and multiple sclerosis.
We measured 25(OH)D concentrations in 720 case and 2,610 control plasma samples and genotyped single nucleotide polymorphisms from seven vitamin D metabolism genes in 8,517 case, 10,438 control, and 1,933 family samples. We tested genetic variants influencing 25(OH)D metabolism for an association with both circulating 25(OH)D concentrations and disease status.
Type 1 diabetic patients have lower circulating levels of 25(OH)D than similarly aged subjects from the British population. Only 4.3 and 18.6% of type 1 diabetic patients reached optimal levels (≥75 nmol/L) of 25(OH)D for bone health in the winter and summer, respectively. We replicated the associations of four vitamin D metabolism genes (GC, DHCR7, CYP2R1, and CYP24A1) with 25(OH)D in control subjects. In addition to the previously reported association between type 1 diabetes and CYP27B1 (P = 1.4 × 10−4), we obtained consistent evidence of type 1 diabetes being associated with DHCR7 (P = 1.2 × 10−3) and CYP2R1 (P = 3.0 × 10−3).
Circulating levels of 25(OH)D in children and adolescents with type 1 diabetes vary seasonally and are under the same genetic control as in the general population but are much lower. Three key 25(OH)D metabolism genes show consistent evidence of association with type 1 diabetes risk, indicating a genetic etiological role for vitamin D deficiency in type 1 diabetes.
PMCID: PMC3292339  PMID: 21441443
20.  The TRIM5 Gene Modulates Penile Mucosal Acquisition of Simian Immunodeficiency Virus in Rhesus Monkeys ▿ †  
Journal of Virology  2011;85(19):10389-10398.
There is considerable variability in host susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, but the host genetic determinants of that variability are not well understood. In addition to serving as a block for cross-species retroviral infection, TRIM5 was recently shown to play a central role in limiting primate immunodeficiency virus replication. We hypothesized that TRIM5 may also contribute to susceptibility to mucosal acquisition of simian immunodeficiency virus (SIV) in rhesus monkeys. We explored this hypothesis by establishing 3 cohorts of Indian-origin rhesus monkeys with different TRIM5 genotypes: homozygous restrictive, heterozygous permissive, and homozygous permissive. We then evaluated the effect of TRIM5 genotype on the penile transmission of SIVsmE660. We observed a significant effect of TRIM5 genotype on mucosal SIVsmE660 acquisition in that no SIV transmission occurred in monkeys with only restrictive TRIM5 alleles. In contrast, systemic SIV infections were initiated after preputial pocket exposures in monkeys that had at least one permissive TRIM5 allele. These data demonstrate that host genetic factors can play a critical role in restricting mucosal transmission of a primate immunodeficiency virus. In addition, we used our understanding of TRIM5 to establish a novel nonhuman primate penile transmission model for AIDS mucosal pathogenesis and vaccine research.
PMCID: PMC3196432  PMID: 21775457
21.  Statistical colocalization of monocyte gene expression and genetic risk variants for type 1 diabetes 
Human Molecular Genetics  2012;21(12):2815-2824.
One mechanism by which disease-associated DNA variation can alter disease risk is altering gene expression. However, linkage disequilibrium (LD) between variants, mostly single-nucleotide polymorphisms (SNPs), means it is not sufficient to show that a particular variant associates with both disease and expression, as there could be two distinct causal variants in LD. Here, we describe a formal statistical test of colocalization and apply it to type 1 diabetes (T1D)-associated regions identified mostly through genome-wide association studies and expression quantitative trait loci (eQTLs) discovered in a recently determined large monocyte expression data set from the Gutenberg Health Study (1370 individuals), with confirmation sought in an additional data set from the Cardiogenics Transcriptome Study (558 individuals). We excluded 39 out of 60 overlapping eQTLs in 49 T1D regions from possible colocalization and identified 21 coincident eQTLs, representing 21 genes in 14 distinct T1D regions. Our results reflect the importance of monocyte (and their derivatives, macrophage and dendritic cell) gene expression in human T1D and support the candidacy of several genes as causal factors in autoimmune pancreatic beta-cell destruction, including AFF3, CD226, CLECL1, DEXI, FKRP, PRKD2, RNLS, SMARCE1 and SUOX, in addition to the recently described GPR183 (EBI2) gene.
PMCID: PMC3363338  PMID: 22403184
22.  Tests for Genetic Interactions in Type 1 Diabetes 
Diabetes  2011;60(3):1030-1040.
Interactions between genetic and environmental factors lead to immune dysregulation causing type 1 diabetes and other autoimmune disorders. Recently, many common genetic variants have been associated with type 1 diabetes risk, but each has modest individual effects. Familial clustering of type 1 diabetes has not been explained fully and could arise from many factors, including undetected genetic variation and gene interactions.
To address this issue, the Type 1 Diabetes Genetics Consortium recruited 3,892 families, including 4,422 affected sib-pairs. After genotyping 6,090 markers, linkage analyses of these families were performed, using a novel method and taking into account factors such as genotype at known susceptibility loci.
Evidence for linkage was robust at the HLA and INS loci, with logarithm of odds (LOD) scores of 398.6 and 5.5, respectively. There was suggestive support for five other loci. Stratification by other risk factors (including HLA and age at diagnosis) identified one convincing region on chromosome 6q14 showing linkage in male subjects (corrected LOD = 4.49; replication P = 0.0002), a locus on chromosome 19q in HLA identical siblings (replication P = 0.006), and four other suggestive loci.
This is the largest linkage study reported for any disease. Our data indicate there are no major type 1 diabetes subtypes definable by linkage analyses; susceptibility is caused by actions of HLA and an apparently random selection from a large number of modest-effect loci; and apart from HLA and INS, there is no important susceptibility factor discoverable by linkage methods.
PMCID: PMC3046821  PMID: 21266329
23.  An Allele of IKZF1 (Ikaros) Conferring Susceptibility to Childhood Acute Lymphoblastic Leukemia Protects Against Type 1 Diabetes 
Diabetes  2011;60(3):1041-1044.
IKZF1 encoding Ikaros, an essential regulator of lymphopoiesis and immune homeostasis, has been implicated in the development of childhood acute lymphoblastic leukemia (C-ALL). Because recent genome-wide association (GWA) studies have linked a region of the 3′-UTR of IKZF1 with C-ALL susceptibility, we tested whether IKZF1 is associated with the autoimmune disease type 1 diabetes.
rs10272724 (T>C) near IKZF1 at 7p12 was genotyped in 8,333 individuals with type 1 diabetes, 9,947 control subjects, and 3,997 families of European ancestry. Association was tested using logistic regression in the case-control data and by the transmission disequilibrium test in the families. Expression data for IKZF1 by rs10272724 genotype were obtained using quantitative PCR of mRNA/cDNA generated from peripheral blood mononuclear cells from 88 individuals, whereas expression data for five other neighboring genes were obtained from the online Genevar dataset.
The minor allele of rs10272724 (C) was found to be protective from type 1 diabetes (odds ratio 0.87 [95% CI 0.83–0.91]; P = 1.1 × 10−11). rs10272724 was not correlated with levels of two transcripts of IKZF1 in peripheral blood mononuclear cells.
The major susceptibility genotype for C-ALL confers protection from type 1 diabetes. Our finding strengthens the link between autoimmunity and lymphoid cancers. Further investigation is warranted for the genetic effect marked by rs10272724, its impact on IKZF1, and the role of Ikaros and other family members, Ailios (IKZF3) and Eos (IKZF4), in autoimmunity.
PMCID: PMC3046822  PMID: 21270240
24.  Evidence that Cd101 is an autoimmune diabetes gene in NOD mice 
We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 (B6) mice accounts for the protection from type 1 diabetes (T1D) provided by the Idd10 region, a <1 Mb region on mouse chromosome 3. Here, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including FoxP3+ regulatory CD4+ T cells, CD11c+ dendritic cells and Gr1+ myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Since CD101 has been associated with T regulatory and antigen presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.
PMCID: PMC3128927  PMID: 21613616
Rodent; Diabetes; Autoimmunity
25.  Identification of Cd101 as a susceptibility gene for Novosphingobium aromaticivorans - induced liver autoimmunity 
Environmental and genetic factors define the susceptibility of an individual to autoimmune disease. Although common genetic pathways affect general immunological tolerance mechanisms in autoimmunity, the effects of such genes could vary under distinct immune challenges within different tissues. Here we demonstrate this by observing that autoimmune type 1 diabetes (T1D) protective haplotypes at the susceptibility region 10 (Idd10) introgressed from chromosome 3 of B6 and A/J mice onto the NOD background increase the severity of autoimmune primary biliary cirrhosis (PBC) induced by infection with Novosphingobium aromaticivorans (N. aro), an ubiquitous alphaproteobacterium, when compared to mice having the NOD and NOD.CAST Idd10 T1D susceptible haplotypes. Substantially increased liver pathology in mice having the B6 and A/J Idd10 haplotypes correlates with reduced expression of CD101 on dendritic cells (DCs), macrophages and granulocytes following infection, delayed clearance of N. aro and the promotion of overzealous, IFN-γ- and IL-17-dominated T cell responses essential for the adoptive transfer of liver lesions. CD101-knockout mice generated on the B6 background also exhibit substantially more severe N.aro-induced liver disease correlating with increased IFN-γ and IL-17 responses compared to wild type mice. These data strongly support the hypothesis that allelic variation of the Cd101 gene, located in the Idd10 region, alters the severity of liver autoimmunity induced by N. aro.
PMCID: PMC3134939  PMID: 21613619
rodent; cytokines; dendritic cells; T cells; autoimmunity; costimulation; inflammation

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