Publications by authors named "Graeme Stewart"

77 Publications

Regulation of the methylome in differentiation from adult stem cells may underpin vitamin D risk in MS.

Genes Immun 2020 11 9;21(5):335-347. Epub 2020 Oct 9.

Centre for Immunology and Allergy Research, Westmead Institute for Medical Research, The University of Sydney, 176 Hawkesbury Rd, Westmead, NSW, 2145, Australia.

Multiple lines of evidence indicate Multiple Sclerosis (MS) is affected by vitamin D. This effect may be mediated by methylation in immune cell progenitors. We aimed to determine (1) if haematopoietic stem cell methylation constrains methylation in daughter cells and is variable between individuals, and (2) the interaction of methylation with the vitamin D receptor binding sites. We interrogated genomic methylation levels from matching purified CD34+ haematopoietic stem cells and progeny CD14+ monocytes and CD56+ NK cells from 11 individuals using modified reduced representation bisulfite sequencing. Differential methylation of Vitamin D Receptor binding sites and MS risk genes was assessed from this and using pyrosequencing for the vitamin D regulated MS risk gene ZMIZ1. Although DNA methylation states at CpG islands and other sites are almost entirely recapitulated between progenitor and progeny immune cells, significant variation was detected at some regions between cell subsets and individuals; including around the MS risk genes HLA DRB1 and the vitamin D repressor NCOR2. Methylation of the vitamin D responsive MS risk gene ZMIZ1 was associated with risk SNP and disease. We conclude that DNA methylation settings in adult haematopoietic stem cells may contribute to individual variation in vitamin D responses in immune cells.
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http://dx.doi.org/10.1038/s41435-020-00114-4DOI Listing
November 2020

Amyloid Cardiomyopathy.

Heart Lung Circ 2020 Apr 17;29(4):575-583. Epub 2019 Dec 17.

Cardiology Department, St. Vincent's Hospital, Sydney NSW, Australia; Molecular Cardiology Division, Victor Chang Cardiac Research Institute, Sydney NSW, Australia; St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney NSW, Australia. Electronic address:

Amyloid cardiomyopathy is emerging as an important and under-recognised cause of heart failure and cardiac arrhythmias, especially in older adults. This disorder is characterised by extracellular deposition of amyloid fibrils that form due to misfolding of secreted light chains (AL) or transthyretin protein (ATTR). In ATTR, amyloid aggregates typically result from excessive accumulation of wild-type transthyretin (ATTRwt) or from protein structural defects caused by TTR gene variants (ATTRv). Amyloid fibril deposition may predominantly affect the heart or show multi-system involvement. Previously considered to be rare and inexorably progressive with no specific therapy, there has been enormous recent interest in ATTR cardiomyopathy due to upwardly-revised estimates of disease prevalence together with development of disease-modifying interventions. Because of this, there is a clinical imperative to have a high index of suspicion to identify potential cases and to be aware of contemporary diagnostic methods and treatment options. Genetic testing should be offered to all patients with proven ATTR to access the benefits of new therapies specific to ATTRv and allow predictive testing of family members. With heightened awareness of amyloid cardiomyopathy and expanded use of genetic testing, a substantial rise in the numbers of asymptomatic individuals who are carriers of pathogenic variants is expected, and optimal strategies for monitoring and treatment of these individuals at risk need to be determined. Pre-emptive administration of fibril-modifying therapies provides an unprecedented opportunity for disease prevention and promises to change amyloid cardiomyopathy from being a fatal to a treatable disorder.
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http://dx.doi.org/10.1016/j.hlc.2019.11.019DOI Listing
April 2020

The interaction of Multiple Sclerosis risk loci with Epstein-Barr virus phenotypes implicates the virus in pathogenesis.

Sci Rep 2020 01 13;10(1):193. Epub 2020 Jan 13.

Centre for Immunology and Allergy Research, Westmead Institute for Medical Research, University of Sydney, Sydney, Australia.

Translating the findings of genome wide association studies (GWAS) to new therapies requires identification of the relevant immunological contexts to interrogate for genetic effects. In one of the largest GWAS, more than 200 risk loci have been identified for Multiple Sclerosis (MS) susceptibility. Infection with Epstein-Barr virus (EBV) appears to be necessary for the development of Multiple Sclerosis (MS). Many MS risk loci are associated with altered gene expression in EBV infected B cells (LCLs). We have interrogated this immunological context to identify interaction between MS risk loci and EBV DNA copy number, intrinsic growth rate and EBV encoded miRNA expression. The EBV DNA copy number was associated with significantly more risk alleles for MS than for other diseases or traits. EBV miRNAs BART4-3p and BART3-5p were highly associated with EBV DNA copy number and MS risk loci. The poliovirus receptor (PVR) risk SNP was associated with EBV DNA copy number, PVR and miRNA expression. Targeting EBV miRNAs BART4-3p and BART3-5p, and the gene PVR, may provide therapeutic benefit in MS. This study also indicates how immunological context and risk loci interactions can be exploited to validate and develop novel therapeutic approaches.
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http://dx.doi.org/10.1038/s41598-019-55850-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957475PMC
January 2020

Transcribed B lymphocyte genes and multiple sclerosis risk genes are underrepresented in Epstein-Barr Virus hypomethylated regions.

Genes Immun 2020 02 16;21(2):91-99. Epub 2019 Oct 16.

Centre for Immunology and Allergy Research, Westmead Institute for Medical Research, The University of Sydney, 176 Hawkesbury Rd, Westmead, NSW, 2145, Australia.

Epstein-Barr Virus (EBV) infection appears to be necessary for the development of Multiple Sclerosis (MS), although the specific mechanisms are unknown. More than 200 single-nucleotide polymorphisms (SNPs) are known to be associated with the risk of developing MS. About a quarter of these are also highly associated with proximal gene expression in B cells infected with EBV (lymphoblastoid cell lines-LCLs). The DNA of LCLs is hypomethylated compared with both uninfected and activated B cells. Since methylation can affect gene expression, and so cell differentiation and immune evasion, we hypothesised that EBV-driven hypomethylation may affect the interaction between EBV infection and MS. We interrogated an existing dataset comprising three individuals with whole-genome bisulfite sequencing data from EBV transformed B cells and CD40L-activated B cells. DNA methylation surrounding MS risk SNPs associated with gene expression in LCLs (LCLeQTL) was less likely to be hypomethylated than randomly selected chromosomal regions. Differential methylation was independent of genomic features such as promoter regions, but genes preferentially expressed in EBV-infected B cells, including the LCLeQTL genes, were underrepresented in the hypomethylated regions. Our data does not indicate MS genetic risk is affected by EBV hypomethylation.
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http://dx.doi.org/10.1038/s41435-019-0089-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182534PMC
February 2020

Evidence from genome wide association studies implicates reduced control of Epstein-Barr virus infection in multiple sclerosis susceptibility.

Genome Med 2019 04 30;11(1):26. Epub 2019 Apr 30.

Centre for Immunology and Allergy Research, Westmead Institute for Medical Research, University of Sydney, Sydney, Australia.

Background: Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40.

Methods: We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed.

Results: These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype.

Conclusions: These data indicate targeting EBV may be of therapeutic benefit in MS.
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http://dx.doi.org/10.1186/s13073-019-0640-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492329PMC
April 2019

Transthyretin Val122Ile amyloidosis associated with isolated gastrointestinal disease and bowel rupture in a Caucasian woman.

Pathology 2019 Jun 16;51(4):436-439. Epub 2019 Apr 16.

Haematology Department, Eastern Health, Victorian and Tasmanian Amyloidosis Service, Monash University Eastern Health Clinical School, Melbourne, Vic, Australia.

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http://dx.doi.org/10.1016/j.pathol.2018.12.423DOI Listing
June 2019

The latitude-dependent autoimmune disease risk genes ZMIZ1 and IRF8 regulate mononuclear phagocytic cell differentiation in response to vitamin D.

Hum Mol Genet 2019 01;28(2):269-278

Centre for Immunology and Allergy Research, Westmead Institute for Medical Research, University of Sydney, Westmead NSW, Australia.

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.
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http://dx.doi.org/10.1093/hmg/ddy324DOI Listing
January 2019

Association of Regulatory T-Cell Expansion With Progression of Amyotrophic Lateral Sclerosis: A Study of Humans and a Transgenic Mouse Model.

JAMA Neurol 2018 06;75(6):681-689

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia.

Importance: Neuroinflammation appears to be a key modulator of disease progression in amyotrophic lateral sclerosis (ALS) and thereby a promising therapeutic target. The CD4+Foxp3+ regulatory T-cells (Tregs) infiltrating into the central nervous system suppress neuroinflammation and promote the activation of neuroprotective microglia in mouse models of ALS. To our knowledge, the therapeutic association of host Treg expansion with ALS progression has not been studied in vivo.

Objective: To assess the role of Tregs in regulating the pathophysiology of ALS in humans and the therapeutic outcome of increasing Treg activity in a mouse model of the disease.

Design, Setting, And Participants: This prospective multicenter human and animal study was performed in hospitals, outpatient clinics, and research institutes. Clinical and function assessment, as well as immunological studies, were undertaken in 33 patients with sporadic ALS, and results were compared with 38 healthy control participants who were consecutively recruited from the multidisciplinary ALS clinic at Westmead Hospital between February 1, 2013, and December 31, 2014. All data analysis on patients with ALS was undertaken between January 2015 and December 2016. Subsequently, we implemented a novel approach to amplify the endogenous Treg population using peripheral injections of interleukin 2/interleukin 2 monoclonal antibody complexes (IL-2c) in transgenic mice that expressed mutant superoxide dismutase 1 (SOD1), a gene associated with motor neuron degeneration.

Main Outcomes And Measures: In patients with ALS, Treg levels were determined and then correlated with disease progression. Circulating T-cell populations, motor neuron size, glial cell activation, and T-cell and microglial gene expression in spinal cords were determined in SOD1G93A mice, as well as the association of Treg amplification with disease onset and survival time in mice.

Results: The cohort of patients with ALS included 24 male patients and 9 female patients (mean [SD] age at assessment, 58.9 [10.9] years). There was an inverse correlation between total Treg levels (including the effector CD45RO+ subset) and rate of disease progression (R = -0.40, P = .002). Expansion of the effector Treg population in the SOD1G93A mice was associated with a significant slowing of disease progression, which was accompanied by an increase in survival time (IL-2c-treated mice: mean [SD], 160.6 [10.8] days; control mice: mean [SD], 144.9 [10.6] days; P = .003). Importantly, Treg expansion was associated with preserved motor neuron soma size and marked suppression of astrocytic and microglial immunoreactivity in the spinal cords of SOD1G93A mice, as well as elevated neurotrophic factor gene expression in spinal cord and peripheral nerves.

Conclusions And Relevance: These findings establish a neuroprotective effect of Tregs, possibly mediated by suppression of toxic neuroinflammation in the central nervous system. Strategies aimed at enhancing the Treg population and neuroprotective activity from the periphery may prove therapeutically useful for patients with ALS.
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http://dx.doi.org/10.1001/jamaneurol.2018.0035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885208PMC
June 2018

GPR65 inhibits experimental autoimmune encephalomyelitis through CD4 T cell independent mechanisms that include effects on iNKT cells.

Immunol Cell Biol 2018 02 19;96(2):128-136. Epub 2017 Dec 19.

Immunology Division, Garvan Institute of Medical Research, Sydney, NSW, Australia.

The G protein-coupled receptor 65 (GPR65) gene has been genetically associated with several autoimmune diseases, including multiple sclerosis (MS). GPR65 is predominantly expressed in lymphoid organs and is activated by extracellular protons. In this study, we tested whether GPR65 plays a functional role in demyelinating autoimmune disease. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we found that Gpr65-deficient mice develop exacerbated disease. CD4 helper T cells are key drivers of EAE pathogenesis, however, Gpr65 deficiency in these cells did not contribute to the observed exacerbated disease. Instead, Gpr65 expression levels were found to be highest on invariant natural killer T (iNKT) cells. EAE severity in Gpr65-deficient mice was normalized in the absence of iNKT cells (CD1d-deficient mice), suggesting that GPR65 signals in iNKT cells are important for suppressing autoimmune disease. These findings provide functional support for the genetic association of GPR65 with MS and demonstrate GPR65 signals suppress autoimmune activity in EAE.
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http://dx.doi.org/10.1111/imcb.1031DOI Listing
February 2018

Differences in common heritable blood immune cell populations may underlie MS susceptibility and progression.

Mult Scler J Exp Transl Clin 2016 Jan-Dec;2:2055217316637087. Epub 2016 Mar 11.

Centre for Immunology and Allergy Research, Westmead Institute for Medical Research, University of Sydney, Australia.

A promising new avenue of MS research that may lead to a better understanding of pathogenesis, progression and therapeutic response, and to development of new therapies, comes from the recent identification of defined immune cell populations that are highly heritable. Such stable populations have been identified in three recent papers using extensive flow cytometric panels to investigate twin and family cohorts. They showed that while most of the variation in immune cell populations between individuals was not heritable, some was. This heritability was sometimes very high, and the authors concluded that it likely contributes to variability in response among individuals for disease and drug response traits.
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http://dx.doi.org/10.1177/2055217316637087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433329PMC
March 2016

The low EOMES/TBX21 molecular phenotype in multiple sclerosis reflects CD56+ cell dysregulation and is affected by immunomodulatory therapies.

Clin Immunol 2016 Feb 4;163:96-107. Epub 2016 Jan 4.

Centre for Immunology and Allergy Research, Westmead Institute for Medical Research, University of Sydney, Sydney, New South Wales 2145, Australia. Electronic address:

Multiple Sclerosis (MS) is an autoimmune disease treated by therapies targeting peripheral blood cells. We previously identified that expression of two MS-risk genes, the transcription factors EOMES and TBX21 (ET), was low in blood from MS and stable over time. Here we replicated the low ET expression in a new MS cohort (p<0.0007 for EOMES, p<0.028 for TBX21) and demonstrate longitudinal stability (p<10(-4)) and high heritability (h(2)=0.48 for EOMES) for this molecular phenotype. Genes whose expression correlated with ET, especially those controlling cell migration, further defined the phenotype. CD56+ cells and other subsets expressed lower levels of Eomes or T-bet protein and/or were under-represented in MS. EOMES and TBX21 risk SNP genotypes, and serum EBNA-1 titres were not correlated with ET expression, but HLA-DRB1*1501 genotype was. ET expression was normalised to healthy control levels with natalizumab, and was highly variable for glatiramer acetate, fingolimod, interferon-beta, dimethyl fumarate.
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http://dx.doi.org/10.1016/j.clim.2015.12.015DOI Listing
February 2016

Class II HLA interactions modulate genetic risk for multiple sclerosis.

Nat Genet 2015 Oct 7;47(10):1107-1113. Epub 2015 Sep 7.

Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.

Association studies have greatly refined the understanding of how variation within the human leukocyte antigen (HLA) genes influences risk of multiple sclerosis. However, the extent to which major effects are modulated by interactions is poorly characterized. We analyzed high-density SNP data on 17,465 cases and 30,385 controls from 11 cohorts of European ancestry, in combination with imputation of classical HLA alleles, to build a high-resolution map of HLA genetic risk and assess the evidence for interactions involving classical HLA alleles. Among new and previously identified class II risk alleles (HLA-DRB1*15:01, HLA-DRB1*13:03, HLA-DRB1*03:01, HLA-DRB1*08:01 and HLA-DQB1*03:02) and class I protective alleles (HLA-A*02:01, HLA-B*44:02, HLA-B*38:01 and HLA-B*55:01), we find evidence for two interactions involving pairs of class II alleles: HLA-DQA1*01:01-HLA-DRB1*15:01 and HLA-DQB1*03:01-HLA-DQB1*03:02. We find no evidence for interactions between classical HLA alleles and non-HLA risk-associated variants and estimate a minimal effect of polygenic epistasis in modulating major risk alleles.
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http://dx.doi.org/10.1038/ng.3395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874245PMC
October 2015

The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function.

PLoS One 2015 11;10(6):e0127080. Epub 2015 Jun 11.

Centre for Immunology and Allergy Research, Westmead Millennium Institute, University of Sydney, Sydney, New South Wales, Australia.

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127080PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465929PMC
April 2016

Dual-specificity phosphatase 5 regulates nuclear ERK activity and suppresses skin cancer by inhibiting mutant Harvey-Ras (HRasQ61L)-driven SerpinB2 expression.

Proc Natl Acad Sci U S A 2014 Dec 8;111(51):18267-72. Epub 2014 Dec 8.

Cancer Research-UK Stress Response Laboratory, Division of Cancer Research, Medical Research Institute, Ninewells Hospital & Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom;

Ectopic expression of dual-specificity phosphatase 5 (DUSP5), an inducible mitogen-activated protein (MAP) kinase phosphatase, specifically inactivates and anchors extracellular signal-regulated kinase (ERK)1/2 in the nucleus. However, the role of endogenous DUSP5 in regulating the outcome of Ras/ERK kinase signaling under normal and pathological conditions is unknown. Here we report that mice lacking DUSP5 show a greatly increased sensitivity to mutant Harvey-Ras (HRas(Q61L))-driven papilloma formation in the 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) model of skin carcinogenesis. Furthermore, mouse embryo fibroblasts (MEFs) from DUSP5(-/-) mice show increased levels of nuclear phospho-ERK immediately after TPA stimulation and fail to accumulate total ERK in the nucleus compared with DUSP5(+/+) cells. Surprisingly, a microarray analysis reveals that only a small number of Ras/ERK-dependent TPA-responsive transcripts are up-regulated on deletion of DUSP5 in MEFs and mouse skin. The most up-regulated gene on DUSP5 loss encodes SerpinB2, an inhibitor of extracellular urokinase plasminogen activator and deletion of DUSP5 acts synergistically with mutant HRas(Q61L) and TPA to activate ERK-dependent SerpinB2 expression at the transcriptional level. SerpinB2 has previously been implicated as a mediator of DMBA/TPA-induced skin carcinogenesis. By analyzing DUSP5(-/-), SerpinB2(-/-) double knockout mice, we demonstrate that deletion of SerpinB2 abrogates the increased sensitivity to papilloma formation seen on DUSP5 deletion. We conclude that DUSP5 performs a key nonredundant role in regulating nuclear ERK activation, localization, and gene expression. Furthermore, our results suggest an in vivo role for DUSP5 as a tumor suppressor by modulating the oncogenic potential of activated Ras in the epidermis.
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http://dx.doi.org/10.1073/pnas.1420159112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280588PMC
December 2014

Antibodies to myelin oligodendrocyte glycoprotein in bilateral and recurrent optic neuritis.

Neurol Neuroimmunol Neuroinflamm 2014 Dec 29;1(4):e40. Epub 2014 Oct 29.

Authors' affiliations are listed at the end of the article.

Objective: We examined a cohort of adults with aquaporin-4 (AQP4) antibody-negative neuromyelitis optica/neuromyelitis optica spectrum disorder (NMO/NMOSD) for antibodies to myelin oligodendrocyte glycoprotein (MOG).

Methods: We performed a flow cytometry cell-based assay using live human lentivirus-transduced cells expressing full-length surface MOG. Serum was tested in 23 AQP4 antibody-negative NMO/NMOSD patients with bilateral and/or recurrent optic neuritis (BON, n = 11), longitudinally extensive transverse myelitis (LETM, n = 10), and sequential BON and LETM (n = 2), as well as in patients with multiple sclerosis (MS, n = 76) and controls (n = 52).

Results: MOG antibodies were detected in 9/23 AQP4 antibody-negative patients with NMO/NMOSD, compared to 1/76 patients with MS and 0/52 controls (p < 0.001). MOG antibodies were detected in 8/11 patients with BON, 0/10 patients with LETM, and 1/2 patients with sequential BON and LETM. Six of 9 MOG antibody-positive patients had a relapsing course. MOG antibody-positive patients had prominent optic disc swelling and were more likely to have a rapid response to steroid therapy and relapse on steroid cessation than MOG antibody-negative patients (p = 0.034 and p = 0.029, respectively). While 8/9 MOG antibody-positive patients had good follow-up visual acuity, one experienced sustained visual impairment, 3 had retinal nerve fiber layer thinning, and one had residual spinal disability.

Conclusions: MOG antibodies have a strong association with BON and may be a useful clinical biomarker. MOG antibody-associated BON is a relapsing disorder that is frequently steroid responsive and often steroid dependent. Failure to recognize the disorder early and institute immunotherapy promptly may be associated with sustained impairment.

Classification Of Evidence: This study provides Class II evidence that MOG antibodies are associated with AQP4 antibody-negative BON (sensitivity 69%, 95% confidence interval [CI] 42%-87%; specificity 99%, 95% CI 93.7%-99.8%).
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http://dx.doi.org/10.1212/NXI.0000000000000040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215392PMC
December 2014

The autoimmune disease-associated transcription factors EOMES and TBX21 are dysregulated in multiple sclerosis and define a molecular subtype of disease.

Clin Immunol 2014 Mar 15;151(1):16-24. Epub 2014 Jan 15.

Institute for Immunology and Allergy Research, Westmead Millennium Institute University of Sydney, Sydney, New South Wales 2145, Australia. Electronic address:

We have identified a marked over-representation of transcription factors controlling differentiation of T, B, myeloid and NK cells among the 110 MS genes now known to be associated with multiple sclerosis (MS). To test if the expression of these genes might define molecular subtypes of MS, we interrogated their expression in blood in three independent cohorts of untreated MS (from Sydney and Adelaide) or clinically isolated syndrome (CIS, from San Francisco) patients. Expression of the transcription factors (TF) controlling T and NK cell differentiation, EOMES, TBX21 and other TFs was significantly lower in MS/CIS compared to healthy controls in all three cohorts. Expression was tightly correlated between these TFs, with other T/NK cell TFs, and to another downregulated gene, CCL5. Expression was stable over time, but did not predict disease phenotype. Optimal response to therapy might be indicated by normalization of expression of these genes in blood.
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http://dx.doi.org/10.1016/j.clim.2014.01.003DOI Listing
March 2014

Association of IFNL3 rs12979860 and rs8099917 with biochemical predictors of interferon responsiveness in chronic hepatitis C virus infection.

PLoS One 2013 29;8(10):e77530. Epub 2013 Oct 29.

Universitätsklinikum Leipzig, Sektion Hepatologie, Klinik und Poliklinik für Gastroenterologie und Rheumatologie, Leipzig, Germany.

Background & Aims: Genetic variations near the interferon lambda 3 gene (IFNL3, IL28B) are the most powerful predictors for sustained virologic response (SVR) in patients with chronic hepatitis C virus (HCV) infection, compared to other biochemical or histological baseline parameters. We evaluated whether the interplay of both IFNL3 polymorphisms rs12979860 and rs8099917 together with non-genetic clinical factors contributes to the predictive role of these genetic variants.

Methods: The cohort comprised 1,402 patients of European descent with chronic HCV type 1 infection. 1,298 patients received interferon-based antiviral therapy, and 719 (55%) achieved SVR. The IFNL3 polymorphisms were genotyped by polymerase chain reaction and melting curve analysis.

Results: A significant correlation was found between the IFNL3 polymorphisms and biochemical as well as virologic predictors of treatment outcome such as ALT, GGT, cholesterol, and HCV RNA levels. In multivariate regression analysis, IFLN3 SNPs, HCV RNA levels, and the GGT/ALT ratio were independent predictors of SVR. Dependent on the GGT/ALT ratio and on the HCV RNA concentration, significant variations in the likelihood for achieving SVR were observed in both, carriers of the responder as well as non-responder alleles.

Conclusions: Our data support a clear association between IFNL3 genotypes and baseline parameters known to impact interferon responsiveness. Improved treatment outcome prediction was achieved when these predictors were considered in combination with the IFNL3 genotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077530PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812277PMC
August 2014

The CYP27B1 variant associated with an increased risk of autoimmune disease is underexpressed in tolerizing dendritic cells.

Hum Mol Genet 2014 Mar 24;23(6):1425-34. Epub 2013 Oct 24.

Westmead Millennium Institute.

Genome-wide association studies have identified a linkage disequilibrium (LD) block on chromosome 12 associated with multiple sclerosis (MS), type 1 diabetes and other autoimmune diseases. This block contains CYP27B1, which catalyzes the conversion of 25 vitamin D3 (VitD3) to 1,25VitD3. Fine-mapping analysis has failed to identify which of the 17 genes in this block is most associated with MS. We have previously used a functional approach to identify the causal gene. We showed that the expression of several genes in this block in whole blood is highly associated with the MS risk allele, but not CYP27B1. Here, we show that CYP27B1 is predominantly expressed in dendritic cells (DCs). Its expression in these cells is necessary for their response to VitD, which is known to upregulate pathways involved in generating a tolerogenic DC phenotype. Here, we utilize a differentiation protocol to generate inflammatory (DC1) and tolerogenic (DC2) DCs, and show that for the MS risk allele CYP27B1 is underexpressed in DCs, especially DC2s. Of the other Chr12 LD block genes expressed in these cells, only METT21B expression was as affected by the genotype. Another gene associated with autoimmune diseases, CYP24A1, catabolizes 1,25 VitD3, and is predominantly expressed in DCs, but equally between DC1s and DC2s. Overall, these data are consistent with the hypothesis that reduced VitD pathway gene upregulation in DC2s of carriers of the risk haplotype of CYP27B1 contributes to autoimmune diseases. These data support therapeutic approaches aimed at targeting VitD effects on DCs.
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http://dx.doi.org/10.1093/hmg/ddt529DOI Listing
March 2014

IL7Rα expression and upregulation by IFNβ in dendritic cell subsets is haplotype-dependent.

PLoS One 2013 16;8(10):e77508. Epub 2013 Oct 16.

Institute for Immunology and Allergy Research, Westmead Millennium Institute, Sydney, New South Whales, Australia ; Department of Medicine, University of Sydney, Sydney, New South Whales, Australia.

The IL7Rα gene is unequivocally associated with susceptibility to multiple sclerosis (MS). Haplotype 2 (Hap 2) confers protection from MS, and T cells and dendritic cells (DCs) of Hap 2 exhibit reduced splicing of exon 6, resulting in production of relatively less soluble receptor, and potentially more response to ligand. We have previously shown in CD4 T cells that IL7Rα haplotypes 1 and 2, but not 4, respond to interferon beta (IFNβ), the most commonly used immunomodulatory drug in MS, and that haplotype 4 (Hap 4) homozygotes have the highest risk of developing MS. We now show that IL7R expression increases in myeloid cells in response to IFNβ, but that the response is haplotype-dependent, with cells from homozygotes for Hap 4 again showing no response. This was shown using freshly derived monocytes, in vitro cultured immature and mature monocyte-derived dendritic cells, and by comparing homozygotes for the common haplotypes, and relative expression of alleles in heterozygotes (Hap 4 vs not Hap 4). As for T cells, in all myeloid cell subsets examined, Hap 2 homozygotes showed a trend for reduced splicing of exon 6 compared to the other haplotypes, significantly so in most conditions. These data are consistent with increased signaling being protective from MS, constitutively and in response to IFNβ. We also demonstrate significant regulation of immune response, chemokine activity and cytokine biosynthesis pathways by IL7Rα signaling in IFNβ -treated myeloid subsets. IFNβ-responsive genes are over-represented amongst genes associated with MS susceptibility. IL7Rα haplotype may contribute to MS susceptibility through reduced capacity for IL7Rα signalling in myeloid cells, especially in the presence of IFNβ, and is currently under investigation as a predictor of therapeutic response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077508PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3797747PMC
August 2014

Ribosomal protein S6 mRNA is a biomarker upregulated in multiple sclerosis, downregulated by interferon treatment, and affected by season.

Mult Scler 2014 May 14;20(6):675-85. Epub 2013 Oct 14.

Westmead Millennium Institute, University of Sydney, Sydney, New South Wales, Australia.

Background: Multiple Sclerosis (MS) is an immune-mediated disease of the central nervous system which responds to therapies targeting circulating immune cells.

Objective: Our aim was to test if the T-cell activation gene expression pattern (TCAGE) we had previously described from whole blood was replicated in an independent cohort.

Methods: We used RNA-seq to interrogate the whole blood transcriptomes of 72 individuals (40 healthy controls, 32 untreated MS). A cohort of 862 control individuals from the Brisbane Systems Genetics Study (BSGS) was used to assess heritability and seasonal expression. The effect of interferon beta (IFNB) therapy on expression was evaluated.

Results: The MS/TCAGE association was replicated and rationalized to a single marker, ribosomal protein S6 (RPS6). Expression of RPS6 was higher in MS than controls (p<0.0004), and lower in winter than summer (p<4.6E-06). The seasonal pattern correlated with monthly UV light index (R=0.82, p<0.002), and was also identified in the BSGS cohort (p<0.0016). Variation in expression of RPS6 was not strongly heritable. RPS6 expression was reduced by IFNB therapy.

Conclusions: These data support investigation of RPS6 as a potential therapeutic target and candidate biomarker for measuring clinical response to IFNB and other MS therapies, and of MS disease heterogeneity.
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http://dx.doi.org/10.1177/1352458513507819DOI Listing
May 2014

Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis.

Nat Genet 2013 Nov 29;45(11):1353-60. Epub 2013 Sep 29.

1] John P. Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Miami, Florida, USA. [2].

Using the ImmunoChip custom genotyping array, we analyzed 14,498 subjects with multiple sclerosis and 24,091 healthy controls for 161,311 autosomal variants and identified 135 potentially associated regions (P < 1.0 × 10(-4)). In a replication phase, we combined these data with previous genome-wide association study (GWAS) data from an independent 14,802 subjects with multiple sclerosis and 26,703 healthy controls. In these 80,094 individuals of European ancestry, we identified 48 new susceptibility variants (P < 5.0 × 10(-8)), 3 of which we found after conditioning on previously identified variants. Thus, there are now 110 established multiple sclerosis risk variants at 103 discrete loci outside of the major histocompatibility complex. With high-resolution Bayesian fine mapping, we identified five regions where one variant accounted for more than 50% of the posterior probability of association. This study enhances the catalog of multiple sclerosis risk variants and illustrates the value of fine mapping in the resolution of GWAS signals.
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http://dx.doi.org/10.1038/ng.2770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832895PMC
November 2013

IFNL3 mediates interaction between innate immune cells: Implications for hepatitis C virus pathogenesis.

Innate Immun 2014 Aug 17;20(6):598-605. Epub 2013 Sep 17.

Institute for Immunology and Allergy Research, Westmead Millennium Institute, University of Sydney, Sydney, Australia.

Common IFN lambda 3 (IFNL3) variants have been demonstrated to affect spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infection. The functional basis of these genetic variants has yet to be determined. Data examining the effect of IFNL3, specifically, in innate immune cells is lacking. Here, we determined the expression of IFNL3 and its receptor IFNLR1 in blood immune cell subsets and in HCV-infected livers. Next we assessed their sensitivity to IFNL3. All participants were genotyped for the IFNL3 SNPs rs8099917 and rs12979860. Importantly, unstimulated blood immune cells express significantly higher levels of IFNL3 than HCV liver biopsies. Plasmacytoid dendritic cells (pDCs) are the predominant producers of IFNLR1, especially in response to IFN-α. PBMCs, monocytes and pDCs all respond to IFNL3 based on MxA up-regulation. No differences in IFNL3 expression levels between rs8099917 or rs12979860 genotypes were detected. This is the first study to show peripheral blood pDCs to be the main producers of IFNL3, especially compared with HCV-infected livers. This makes innate immune cells the key players in determining the functional significance of INFL3 polymorphisms in patients with HCV.
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http://dx.doi.org/10.1177/1753425913503385DOI Listing
August 2014

MANBA, CXCR5, SOX8, RPS6KB1 and ZBTB46 are genetic risk loci for multiple sclerosis.

Brain 2013 Jun;136(Pt 6):1778-82

Neuropsychiatric Genetics Group, Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany.

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.
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http://dx.doi.org/10.1093/brain/awt101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3673463PMC
June 2013

Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection.

Gastroenterology 2012 Nov 27;143(5):1244-1252.e12. Epub 2012 Jul 27.

Service d'Hépato-gastroentérologie, Hôpital Saint-Joseph, Marseilles, France.

Background & Aims: Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection.

Methods: We studied well-characterized HCV-infected patients of European descent who underwent liver biopsies before treatment. We defined various liver fibrosis phenotypes on the basis of METAVIR scores, with and without taking the duration of HCV infection into account. Our GWA analyses were conducted on a filtered primary cohort of 1161 patients using 780,650 single nucleotide polymorphisms (SNPs). We genotyped 96 SNPs with P values <5 × 10(-5) from an independent replication cohort of 962 patients. We then assessed the most interesting replicated SNPs using DNA samples collected from 219 patients who participated in separate GWA studies of HCV clearance.

Results: In the combined cohort of 2342 HCV-infected patients, the SNPs rs16851720 (in the total sample) and rs4374383 (in patients who received blood transfusions) were associated with fibrosis progression (P(combined) = 8.9 × 10(-9) and 1.1 × 10(-9), respectively). The SNP rs16851720 is located within RNF7, which encodes an antioxidant that protects against apoptosis. The SNP rs4374383, together with another replicated SNP, rs9380516 (P(combined) = 5.4 × 10(-7)), were linked to the functionally related genes MERTK and TULP1, which encode factors involved in phagocytosis of apoptotic cells by macrophages.

Conclusions: Our GWA study identified several susceptibility loci for HCV-induced liver fibrosis; these were linked to genes that regulate apoptosis. Apoptotic control might therefore be involved in liver fibrosis.
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http://dx.doi.org/10.1053/j.gastro.2012.07.097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756935PMC
November 2012

A genome-wide association study in progressive multiple sclerosis.

Mult Scler 2012 Oct 28;18(10):1384-94. Epub 2012 Mar 28.

Institute of Experimental Neurology and Department of Neurology, Division of Neuroscience, San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy.

Background: The role played by genetic factors in influencing the clinical course of multiple sclerosis (MS) is not yet well established.

Objective: We aimed to identify genetic variants associated with progressive MS (PrMS).

Methods: We conducted a genome-wide association study (GWAS) in 197 patients with PrMS and 234 controls of Italian origin. We tested the top 20 single nucleotide polymorphisms (SNPs) with suggestive evidence of association (p-value<10(-4)) in two independent sets of primary progressive MS cases and controls.

Results: We identified a risk-associated SNP in the HLA region in linkage disequilibrium (LD) with DRB1*1501 and DQB*0602 loci, with genome-wide significance (rs3129934(T), p (combined)=6.7×10(-16), OR=2.34, 95% CI=1.90-2.87), and a novel locus on chromosome 7q35 with suggestive evidence of association (rs996343(G), p (combined)=2.4×10(-5), OR=0.70, 95% CI=0.59-0.83) which maps within a human endogenous retroviral (HERV) element. The new locus did not have a 'cis' effect on RNA expression in lymphoblastic cell lines, but pathway analyses of 'trans' effects point to an expression regulation of genes involved in neurodegeneration, including glutamate metabolism (p<0.01) and axonal guidance signalling (p<0.02).

Conclusions: We have confirmed the established association with the HLA region and, despite the low statistical power of the study, we found suggestive evidence for association with a novel locus on chromosome 7, with a putative regulatory role.
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http://dx.doi.org/10.1177/1352458512439118DOI Listing
October 2012

Combined effects of different interleukin-28B gene variants on the outcome of dual combination therapy in chronic hepatitis C virus type 1 infection.

Hepatology 2012 Jun 18;55(6):1700-10. Epub 2012 Apr 18.

Sektion Hepatologie, Klinik und Poliklinik für Gastroenterologie und Rheumatologie, Universitätsklinikum Leipzig, Leipzig, Germany.

Unlabelled: In patients with chronic hepatitis C virus (HCV) infection, several variants of the interleukin-28B (IL28B) gene have been shown to correlate significantly with a sustained virologic response (SVR). Recent evidence shows that determination of one single IL28B polymorphism, rs12979860, is sufficient for predicting treatment outcome. We examined whether the combined determination of the IL28B single-nucleotide polymorphisms (SNPs), rs12979860, rs8099917, rs12980275, and rs8103142, might improve the prediction of SVR in patients with HCV. In the study cohort, 54% of 942 patients with chronic HCV type 1 infection had SVR. The IL28B SNPs, rs12979860CC and rs8099917TT, correlated significantly with SVR (68% and 62%). The SNPs, rs12980275 and rs8103142, were in strong linkage disequilibrium with rs12979860 and were not included in further analysis. In homozygous carriers of the rs12979860 responder allele C, additional genotyping of the rs8099917 SNP had no effect on response prediction, whereas in carriers of the rs12979860 nonresponder allele, the rs8099917 SNP improved the response prediction. In heterozygous carriers of the rs12979860 nonresponder T allele, SVR rates were 55% in the presence of the rs8099917TT genotype and 40% in patients carrying the rs8099917 TG or GG genotype. Analysis of an independent confirmation cohort of 377 HCV type 1-infected patients verified the significant difference in SVR rates between the combined genotypes, rs12979860CT/rs8099917TT and rs12979860CT/rs8099917TG (38% versus 21%; P = 0.018).

Conclusion: Treatment outcome prediction could not be improved in homozygous carriers of the IL28B rs12979860 C responder allele by the additional determination of the rs8099917 SNP. There is evidence that a significant proportion of heterozygous carriers of the rs12979860 T nonresponder allele can profit with respect to SVR prediction by further determination of the rs8099917 SNP. (HEPATOLOGY 2012;55:1700-1710).
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http://dx.doi.org/10.1002/hep.25582DOI Listing
June 2012

Genome-wide meta-analysis identifies novel multiple sclerosis susceptibility loci.

Ann Neurol 2011 Dec;70(6):897-912

Program in Translational NeuroPsychiatric Genomics, Institute for the Neurosciences, Department of Neurology, Brigham and Women's Hospital, Boston, MA 02115, USA.

Objective: To perform a 1-stage meta-analysis of genome-wide association studies (GWAS) of multiple sclerosis (MS) susceptibility and to explore functional consequences of new susceptibility loci.

Methods: We synthesized 7 MS GWAS. Each data set was imputed using HapMap phase II, and a per single nucleotide polymorphism (SNP) meta-analysis was performed across the 7 data sets. We explored RNA expression data using a quantitative trait analysis in peripheral blood mononuclear cells (PBMCs) of 228 subjects with demyelinating disease.

Results: We meta-analyzed 2,529,394 unique SNPs in 5,545 cases and 12,153 controls. We identified 3 novel susceptibility alleles: rs170934(T) at 3p24.1 (odds ratio [OR], 1.17; p = 1.6 × 10(-8)) near EOMES, rs2150702(G) in the second intron of MLANA on chromosome 9p24.1 (OR, 1.16; p = 3.3 × 10(-8)), and rs6718520(A) in an intergenic region on chromosome 2p21, with THADA as the nearest flanking gene (OR, 1.17; p = 3.4 × 10(-8)). The 3 new loci do not have a strong cis effect on RNA expression in PBMCs. Ten other susceptibility loci had a suggestive p < 1 × 10(-6) , some of these loci have evidence of association in other inflammatory diseases (ie, IL12B, TAGAP, PLEK, and ZMIZ1).

Interpretation: We have performed a meta-analysis of GWAS in MS that more than doubles the size of previous gene discovery efforts and highlights 3 novel MS susceptibility loci. These and additional loci with suggestive evidence of association are excellent candidates for further investigations to refine and validate their role in the genetic architecture of MS.
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http://dx.doi.org/10.1002/ana.22609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247076PMC
December 2011

IL28B, HLA-C, and KIR variants additively predict response to therapy in chronic hepatitis C virus infection in a European Cohort: a cross-sectional study.

PLoS Med 2011 Sep 13;8(9):e1001092. Epub 2011 Sep 13.

Storr Liver Unit, Westmead Millennium Institute, University of Sydney, Sydney, Australia.

Background: To date, drug response genes have not proved as useful in clinical practice as was anticipated at the start of the genomic era. An exception is in the treatment of chronic hepatitis C virus (HCV) genotype 1 infection with pegylated interferon-alpha and ribavirin (PegIFN/R). Viral clearance is achieved in 40%-50% of patients. Interleukin 28B (IL28B) genotype predicts treatment-induced and spontaneous clearance. To improve the predictive value of this genotype, we studied the combined effect of variants of IL28B with human leukocyte antigen C (HLA-C), and its ligands the killer immunoglobulin-like receptors (KIR), which have previously been implicated in HCV viral control.

Methods And Findings: We genotyped chronic hepatitis C (CHC) genotype 1 patients with PegIFN/R treatment-induced clearance (n = 417) and treatment failure (n = 493), and 234 individuals with spontaneous clearance, for HLA-C C1 versus C2, presence of inhibitory and activating KIR genes, and two IL28B SNPs, rs8099917 and rs12979860. All individuals were Europeans or of European descent. IL28B SNP rs8099917 "G" was associated with absence of treatment-induced clearance (odds ratio [OR] 2.19, p = 1.27×10(-8), 1.67-2.88) and absence of spontaneous clearance (OR 3.83, p = 1.71×10(-14), 2.67-5.48) of HCV, as was rs12979860, with slightly lower ORs. The HLA-C C2C2 genotype was also over-represented in patients who failed treatment (OR 1.52, p = 0.024, 1.05-2.20), but was not associated with spontaneous clearance. Prediction of treatment failure improved from 66% with IL28B to 80% using both genes in this cohort (OR 3.78, p = 8.83×10(-6), 2.03-7.04). There was evidence that KIR2DL3 and KIR2DS2 carriage also altered HCV treatment response in combination with HLA-C and IL28B.

Conclusions: Genotyping for IL28B, HLA-C, and KIR genes improves prediction of HCV treatment response. These findings support a role for natural killer (NK) cell activation in PegIFN/R treatment-induced clearance, partially mediated by IL28B.
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http://dx.doi.org/10.1371/journal.pmed.1001092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172251PMC
September 2011

Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways.

J Biol Chem 2011 Nov 9;286(44):38018-38026. Epub 2011 Sep 9.

CR-UK Stress Response Laboratory, Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom. Electronic address:

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.
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http://dx.doi.org/10.1074/jbc.M111.255844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207433PMC
November 2011