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Mirna Šimurina

University of Zagreb

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Gordan Lauc

Genos (Croatia)

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Jerko Štambuk

Science Research Laboratory

Irena Trbojević‐Akmačić

Science Research Laboratory

Tim D. Spector

King's College London

Marija Vilaj

Science Research Laboratory

Maja Pučić‐Baković

Genos (Croatia)

Tamara Pavić

University of Zagreb

Toma Keser

University of Zagreb

Jasminka Krištić

Science Research Laboratory

Ivan Gudelj

Science Research Laboratory

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University of Edinburgh

Western General Hospital

Genos (Croatia)

University of Zagreb

King's College London

Science Research Laboratory

MRC Institute of Genetics and Molecular Medicine

Leiden University Medical Center

Istituti di Ricovero e Cura a Carattere Scientifico

Edinburgh Cancer Research

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SP086GLYCOSILATION PROFILE OF IMMUNOGLOBULIN G IN EARLY CHRONIC KIDNEY DISEASE

Nephrology Dialysis Transplantation (2015)

Clara Barrios Jonas Zierer Ivan Gudelj Jerko Štambuk Ivo Ugrina

10.1093/ndt/gfv188.49

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Decreased Immunoglobulin G Core Fucosylation, A Player in Antibody-dependent Cell-mediated Cytotoxicity, is Associated with Autoimmune Thyroid Diseases

Molecular & Cellular Proteomics (2020)

Tiphaine Martin Mirna Šimurina Marta Ząbczyńska Marina Martinić Kavur Magdalena Rydlewska

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels. Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels. Autoimmune thyroid diseases (AITD) 1The abbreviations used are:AITDAutoimmune thyroid diseaseADCCantibody-dependent cell-mediated cytotoxicityFcãRsFcã-receptorsFUT8Fucosyltransferase 8 (Alpha (1,6) Fucosyltransferase)GDGraves' diseaseGlcNAcN-acetylglucosamineGWASgenome-wide association studiesHTHashimoto's thyroiditisIgGImmunoglobulin GIKZF1IKAROS Family Zinc Finger 1LDlinkage disequilibriumvNKNatural killer cellNSFN-Ethylmaleimide Sensitive FactorPBMCperipheral blood mononuclear cellsTgthyroglobulinTgAbthyroglobulin antibodiesTPOthyroid peroxidaseTPOAbThyroid peroxidase antibodiesTSAbantibodies against TSH receptorsTSHthyroid-stimulating hormoneTSHRTSH receptorTWAStranscriptome-wide association studies. 1The abbreviations used are:AITDAutoimmune thyroid diseaseADCCantibody-dependent cell-mediated cytotoxicityFcãRsFcã-receptorsFUT8Fucosyltransferase 8 (Alpha (1,6) Fucosyltransferase)GDGraves' diseaseGlcNAcN-acetylglucosamineGWASgenome-wide association studiesHTHashimoto's thyroiditisIgGImmunoglobulin GIKZF1IKAROS Family Zinc Finger 1LDlinkage disequilibriumvNKNatural killer cellNSFN-Ethylmaleimide Sensitive FactorPBMCperipheral blood mononuclear cellsTgthyroglobulinTgAbthyroglobulin antibodiesTPOthyroid peroxidaseTPOAbThyroid peroxidase antibodiesTSAbantibodies against TSH receptorsTSHthyroid-stimulating hormoneTSHRTSH receptorTWAStranscriptome-wide association studies. are a class of chronic, organ-specific autoimmune disorders that disturb the function of the thyroid gland. They affect close to 5% of the European population (with a gender disparity) and so, represent the most common group of autoimmune diseases (1Wang B. Shao X. Song R. Xu D. Zhang J.-A. The emerging role of epigenetics in autoimmune thyroid diseases.Front. Immunol. 2017; 8: 113-174PubMed Google Scholar). AITD encompass a spectrum of conditions including Hashimoto's thyroiditis (HT) and Graves' disease (GD). One of the features of AITD is the production of autoantibodies against components of thyroid cells that are also detected in the bloodstream. Autoimmune thyroid disease antibody-dependent cell-mediated cytotoxicity Fcã-receptors Fucosyltransferase 8 (Alpha (1,6) Fucosyltransferase) Graves' disease N-acetylglucosamine genome-wide association studies Hashimoto's thyroiditis Immunoglobulin G IKAROS Family Zinc Finger 1 linkage disequilibriumv Natural killer cell N-Ethylmaleimide Sensitive Factor peripheral blood mononuclear cells thyroglobulin thyroglobulin antibodies thyroid peroxidase Thyroid peroxidase antibodies antibodies against TSH receptors thyroid-stimulating hormone TSH receptor transcriptome-wide association studies. Autoimmune thyroid disease antibody-dependent cell-mediated cytotoxicity Fcã-receptors Fucosyltransferase 8 (Alpha (1,6) Fucosyltransferase) Graves' disease N-acetylglucosamine genome-wide association studies Hashimoto's thyroiditis Immunoglobulin G IKAROS Family Zinc Finger 1 linkage disequilibriumv Natural killer cell N-Ethylmaleimide Sensitive Factor peripheral blood mononuclear cells thyroglobulin thyroglobulin antibodies thyroid peroxidase Thyroid peroxidase antibodies antibodies against TSH receptors thyroid-stimulating hormone TSH receptor transcriptome-wide association studies. The autoantibodies are produced against the three core thyroid proteins: thyroid peroxidase (TPO), thyroglobulin (Tg), and the thyroid-stimulating hormone receptor (TSHR). Except for antibodies against TSH receptors (TSAb), which are known to stimulate the production of thyroid hormones by binding TSH receptors in GD (2De Groot L. Graves' disease and the manifestations of thyrotoxicosis.in: Feingold K.R. Anawalt B. Boyce A. Chrousos G. Dungan K. Grossman A. Hershman J.M. Kaltsas G. Koch C. Kopp P. Korbonits M. McLachlan R. Morley J.E. New M. Perreault L. Purnell J. Rebar R. Singer F. Trence D.L. Vinik A. Wilson D.P. Thyroid Disease Manager. MDText.com, Inc., South Dartmouth, MA2015Google Scholar), little is known about the role of two other thyroid autoantibodies, thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb). Circulating TPOAb is the most common and diagnostically useful marker of AITD, detectable in the serum of most HT (95%) and GD (85%) patients (3Mariotti S. Caturegli P. Piccolo P. Barbesino G. Pinchera A. Antithyroid peroxidase autoantibodies in thyroid diseases.J. Clin. Endocrinol. Metabolism. 1990; 71: 661-669Crossref PubMed Scopus (247) Google Scholar). In recent years, using TPOAb as a marker has been challenged because it appears in ∼10% of apparently healthy individuals (4Hollowell J.G. Staehling N.W. Flanders W.D. Hannon W.H. Gunter E.W. Spencer C.A. Braverman L.E. Serum TSH, T sub4/sub, and Thyroid Antibodies in the United States Population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III).J. Clin. Endocrinol. Metabolism. 2002; 87: 489-499Crossref PubMed Scopus (2939) Google Scholar). Even though autoantibodies are often a hallmark of autoimmune disorders, they can appear years before the first symptoms (5Seeling M. Brückner C. Nimmerjahn F. Differential antibody glycosylation in autoimmunity: sweet biomarker or modulator of disease activity?.Nat. Rev. Rheumatol. 2017; 13: 621-630Crossref PubMed Scopus (108) Google Scholar), which poses the question about their causative role. Some evidence exists that autoantibodies can trigger autoimmunity, and IgG isotype seems to be connected with the development of autoimmune diseases potentially through regulation of IgG effector functions by alternative glycosylation (5Seeling M. Brückner C. Nimmerjahn F. Differential antibody glycosylation in autoimmunity: sweet biomarker or modulator of disease activity?.Nat. Rev. Rheumatol. 2017; 13: 621-630Crossref PubMed Scopus (108) Google Scholar). However, it is yet to be determined if anti-thyroid antibodies cause AITD, or whether additional control mechanisms, such as post-translational modifications, are required to trigger the disease onset. The most abundant and diverse form of post-translational modification is glycosylation, the attachment of sugar moieties to proteins, and various glycans are involved in virtually all physiological processes (6Kobata A. The N-linked sugar chains of human immunoglobulin G: their unique pattern, and their functional roles.Biochim. Biophys. Acta. 2008; 1780: 472-478Crossref PubMed Scopus (88) Google Scholar). Glycans attached to Immunoglobulin G (IgG) are indispensable for its effector function and control of inflammation (7Shade K.-T. Anthony R. Antibody glycosylation and inflammation.Antibodies. 2013; 2: 392-414Crossref Scopus (79) Google Scholar, 8Novokmet M. Lukić E. Vučković F. Đurić Ž Keser T. Rajšl K. Remondini D. Castellani G. Gašparović H. Gornik O. Lauc G. Changes in IgG and total plasma protein glycomes in acute systemic inflammation.Sci. Reports. 2014; 4: 4347Crossref PubMed Scopus (97) Google Scholar, 9Marth J.D. Grewal P.K. Mammalian glycosylation in immunity.Nat. Rev. Immunol. 2008; 8: 874-887Crossref PubMed Scopus (526) Google Scholar, 10Maverakis E. Kim K. Shimoda M. Gershwin M.E. Patel F. Wilken R. Raychaudhuri S. Ruhaak L.R. Lebrilla C.B. Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: a critical review.J. Autoimmun. 2015; 57: 1-13Crossref PubMed Scopus (286) Google Scholar). There are two glycosylation sites within the fragment crystallizable (Fc) of IgG that affect the molecule's 3D-conformation and affinity for binding to Fcγ-receptors (FcγRs) on a range of immune cells (6Kobata A. The N-linked sugar chains of human immunoglobulin G: their unique pattern, and their functional roles.Biochim. Biophys. Acta. 2008; 1780: 472-478Crossref PubMed Scopus (88) Google Scholar, 11Subedi G.P. Barb A.W. The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc γ receptor.MAbs. 2016; 8: 1512-1524Crossref PubMed Scopus (112) Google Scholar, 12Arnold J.N. Wormald M.R. Sim R.B. Rudd P.M. Dwek R.A. The impact of glycosylation on the biological function and structure of human immunoglobulins.Annu. Rev. Immunol. 2007; 25: 21-50Crossref PubMed Scopus (993) Google Scholar). Additional N-glycosylation sites are present in ∼20% of IgG fragment antigen binding (Fab) and play a role in immunity, such as the affinity of epitope-binding site (13Bondt A. Rombouts Y. Selman M.H.J. Hensbergen P.J. Reiding K.R. Hazes J.M.W. Dolhain R.J.E.M. Wuhrer M. Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.Mol. Cell. Proteomics. 2014; 13: 3029-3039Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 14Spiegelberg H.L. Abel C.A. Grey H.M. Fishkin B.G. Localization of the carbohydrate within the variable region of light and heavy chains of human γG myeloma proteins.Biochemistry. 1970; 9: 4217-4223Crossref PubMed Scopus (87) Google Scholar, 15Abel C.A. Spiegelberg H.L. Grey H.M. Carbohydrate content of fragments and polypeptide chains of human .gamma.G-myeloma proteins of different heavy-chain subclasses.Biochemistry. 1968; 7: 1271-1278Crossref PubMed Scopus (90) Google Scholar, 16van de Bovenkamp F.S. Hafkenscheid L. Rispens T. Rombouts Y. The emerging importance of IgG Fab glycosylation in immunity.J. Immunol. 2016; 196: 1435-1441Crossref PubMed Scopus (166) Google Scholar). Previous analysis of IgG glycosylation with other autoimmune diseases showed a reduction of IgG galactosylation and sialylation, which trigger inflammatory response (17Jennewein M.F. Alter G. The immunoregulatory roles of antibody glycosylation.Trends Immunol. 2017; 38: 358-372Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 18Vučkovic F. Krištic J. Gudelj I. Teruel M. Keser T. Pezer M. Pučic-Bakovic M. Štambuk Trbojevic-Akmačic J.I. Barrios C. Pavić T. Menni C. Wang Y. Zhou Y. Cui L. Song H. Zeng Q. Guo X. Pons-Estel B.A. McKeigue P. Leslie Patrick A. Gornik O. Spector T.D. Harjaček M. Alarcon-Riquelme M. Molokhia M. Wang W. Lauc G. Association of systemic lupus erythematosus with decreased immunosuppressive potential of the IgG glycome.Arthritis Rheumatol. 2015; 67: 2978-2989Crossref PubMed Scopus (165) Google Scholar, 19Trbojević-Akmačić I. Ventham N.T. Theodoratou E. Vučković F. Kennedy N.A. Krištić J. Nimmo E.R. Kalla R. Drummond H. Štambuk Dunlop J.M.G. Novokmet M. Aulchenko Y. Gornik O. Campbell H. Pučić Baković M. Satsangi J. Lauc G. Consortium IBD-BIOM Inflammatory bowel disease associates with proinflammatory potential of the immunoglobulin G glycome.Inflammatory Bowel Dis. 2015; 21: 1Crossref PubMed Scopus (130) Google Scholar, 20Harre U. Lang S.C. Pfeifle R. Rombouts Y. Fruhbeisser S. Amara K. Bang H. Lux A. Koeleman C.A. Baum W. Dietel K. Grohn F. Malmstrom V. Klareskog L. Kronke G. Kocijan R. Nimmerjahn F. Toes R.E.M. Herrmann M. Scherer H.U. Schett G. Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss.Nat. Commun. 2015; 6: 6651Crossref PubMed Scopus (189) Google Scholar). In relation to AITD, two small studies (62 and 146 patients respectively) looked at the TgAb glycosylation and reported differences between AITD, papillary thyroid cancers, and controls (21Yuan S. Li Q. Zhang Y. Huang C. Wu H. Li Y. Liu Y. Yu N. Zhang H. Lu G. Gao Y. Gao Y. Guo X. Changes in anti-thyroglobulin IgG glycosylation patterns in Hashimoto's thyroiditis patients.J. Clin. Endocrinol. Metabolism. 2015; 100: 717-724Crossref PubMed Scopus (29) Google Scholar, 22Zhao L. Liu M. Gao Y. Huang Y. Lu G. Gao Y. Guo X. She B. Glycosylation of sera thyroglobulin antibody in patients with thyroid diseases.Eur. J. Endocrinol. 2013; 168: 585-592Crossref PubMed Scopus (15) Google Scholar). First, TgAb IgG from HT patients showed higher levels of core fucose than the control group, as well as of terminal sialic acid and mannose (21Yuan S. Li Q. Zhang Y. Huang C. Wu H. Li Y. Liu Y. Yu N. Zhang H. Lu G. Gao Y. Gao Y. Guo X. Changes in anti-thyroglobulin IgG glycosylation patterns in Hashimoto's thyroiditis patients.J. Clin. Endocrinol. Metabolism. 2015; 100: 717-724Crossref PubMed Scopus (29) Google Scholar). On the other hand, it was observed that among HT, GD, and papillary thyroid cancer groups, HT patients had significantly lower core fucose content on TgAb than the other two groups (22Zhao L. Liu M. Gao Y. Huang Y. Lu G. Gao Y. Guo X. She B. Glycosylation of sera thyroglobulin antibody in patients with thyroid diseases.Eur. J. Endocrinol. 2013; 168: 585-592Crossref PubMed Scopus (15) Google Scholar). Furthermore, because recent genome-wide association studies (GWAS) identified novel loci associated with IgG glycosylation, which were known to be strongly associated with autoimmune conditions (23Lauc G. Huffman J.E. Pučić M. Zgaga L. Adamczyk B. Mužinić A. Novokmet M. Polašek O. Gornik O. Krištić J. Keser T. Vitart V. Scheijen B. Uh H.W. Molokhia M. Patrick A.L. McKeigue P. Kolčić I. Lukić I.K. Swann O. van Leeuwen F.N. Ruhaak L.R. Houwing-Duistermaat J.J. Slagboom P.E. Beekman M. de Craen A.J. Deelder A.M. Zeng Q. Wang W. Hastie N.D. Gyllensten U. Wilson J.F. Wuhrer M. Wright A.F. Rudd P.M. Hayward C. Aulchenko Y. Campbell H. Rudan I. Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.PLoS Genet. 2013; 9: e1003225Crossref PubMed Scopus (188) Google Scholar, 24Shen X. Klarić L. Sharapov S. Mangino M. Ning Z. Di Wu Trbojević-Akmačić I. Pučić-Baković M. Rudan I. Polašek O. Hayward C. Spector T.D. Wilson J.F. Lauc G. Aulchenko Y.S. Multivariate discovery and replication of five novel loci associated with Immunoglobulin G N-glycosylation.Nat. Communications. 2017; 8: 447Crossref PubMed Scopus (47) Google Scholar) and the heritability of AITD is estimated to 55–75% (25Brix T.H. Kyvik K.O. Hegedüs L. A population-based of chronic autoimmune hypothyroidism in Danish twins.JCEM. 2000; 85: 536-539Crossref PubMed Scopus (208) Google Scholar, 26Brix T.H. Kyvik K.O. Christensen K. Hegedüs L. Evidence for a major role of heredity in Graves' disease: a population-based study of two Danish twin cohorts.J. Clin. Endocrinol. Metabolism. 2001; 86: 930-934Crossref PubMed Scopus (409) Google Scholar, 27Hansen P.S. Brix T.H. Bennedbæk F.N. Bonnema S.J. Iachine I. Kyvik K.O. Hegedüs L. The relative importance of genetic and environmental factors in the aetiology of thyroid nodularity: A study of healthy Danish twins.Clin. Endocrinol. 2006; 62: 380-386Crossref Scopus (21) Google Scholar), the next logical step was to examine if there was any common genetic background between those features. No data is currently available on common genetic variants associated with IgG glycosylation traits and AITD, and no large study on associations of the glycosylation of total IgG with the level of thyroid autoantibodies or with AITD has been performed. Our goal was to determine if there are any IgG or peripheral blood mononuclear cells glycan structures associated with the AITD or TPOAb positivity and examine if there are any common heritable factors between AITD and glycan structures. We investigated the association of total serum or plasma IgG glycome composition with TPOAb level in two cohorts: TwinsUK discovery cohort of 2297 individuals (988 controls, 1309 TPOAb positive) and Croatian replication cohort (73 controls, 90 TPOAb positive). We then focused our analysis of the IgG glycosylation on individuals with AITD in discovery cohort (988 controls, 203 AITD), and compared them to Polish replication cohort (114 control, 105 HT). In a fraction of the samples from Polish cohort we analyzed peripheral blood mononuclear cells glycosylation. Additionally, we looked for possible common genetic background between AITD and N-glycans, and at the gene expression of relevant genes. In total, we found an association between the decreased level of IgG core fucosylation and PBMCs antennary α1,2 fucosylation with TPOAb level as well as with AITD. We observed the association of significantly affected IgG N-glycan traits with FUT8 and IKZF1 genes (responsible for IgG fucosylation), but we could not identify SNPs or a general dysregulation of gene expression in whole blood; suggesting a restricted dysregulation of glycosylation in a subpopulation of B cells. The discovery sample consisted of twins from the UK Adult Twin Registry (TwinsUK cohort). The TwinsUK cohort is comprised of virtually 12,000 monozygotic and dizygotic twins unselected for any disease or trait. The cohort is from Northern European/UK ancestry and has been shown to be representative of singleton populations and the UK population in general (29Spector T.D. Williams F.M. The UK Adult Twin Registry (TwinsUK).Twin Res Hum Genet. 2006; 9: 899-906Crossref PubMed Google Scholar) (30Moayyeri A. Hammond C.J. Valdes A.M. Spector T.D. Cohort Profile: TwinsUK and Healthy Ageing Twin Study.Int. J. Epidemiol. 2013; 42: 76-85Crossref PubMed Scopus (174) Google Scholar). The project was approved by the local Ethics Committee, and informed consent was obtained from all participants. A more detailed description of the subjects and analyses performed on the TwinsUK cohort can be found in the supplemental Table S1. TwinsUK glycomic, transcriptomic, genetic data and GWAS results are publicly available upon request on the department website (http://www.twinsuk.ac.uk/data-access/accessmanagement/). The blood collected for the detection of different biological markers (TPOAb levels, plasma IgG N-glycan traits, transcriptomic data, and genetic variants) may have come from different time points for the same individuals between 1997 and 2013. As aging is an important modulator of IgG glycosylation (31Krištić J. Vučković F. Menni C. Klarić L. Keser T. Beceheli I. Pučić-Baković M. Novokmet M. Mangino M. Thaqi K. Rudan P. Novokmet N. Sarac J. Missoni S. Kolčić I. Polašek O. Rudan I. Campbell H. Hayward C. Aulchenko Y. Valdes A. Wilson J.F. Gornik O. Primorac D. Zoldoš V. Spector T. Lauc G. Glycans are a novel biomarker of chronological and biological ages.J. Gerontol. 2013; 69: 779-789Crossref Scopus (206) Google Scholar) and evolution of AITD, we restricted our sample collection/selection protocol in such a way that the largest absolute time difference between different samples collected from the same individuals is five years. This was performed in order to have enough subjects, but with a weak, potential aging effect between two samples. The criteria are not used on genomic data as it is almost stable relative to aging. For the current study, only twins who have phenotype data collected within the same year as the collection of plasma used for the IgG glycosylation profiling were selected in order to account for the potential effects of aging. Consequently, only 2,297 twins have glycan data and TPOAb level measured by Roche immunoassay and 392 twins from Abbott immunoassay (Table I.1 in supplemental Table S1). The blood to assess gene expression has been collected between 2009 and 2011. For the current analysis, only twins who have phenotype data collected within a 5-year range around the collection of blood used for the gene expression were selected in order to account for the potential effects of aging. Consequently, gene expression data was collected in only 180 twins that have gene expression data and AITD status, 199 twins with TPOAb level and 326 twins with IgG N-glycan traits (Table I.2 in supplemental Table S1). Two other cohorts were used to test the findings obtained in the TwinsUK cohort. Both cohorts are described in the Table I.1 in supplemental Table S1. The recruitment in the two replicated cohorts was performed to have age and gender-matched population between cases and controls. Croatian replication cohort includes 73 control individuals and 90 case individuals from Croatia. Individuals were considered as a control if they have TPOAb and TgAb levels within reference ranges (0–4.11 IU/ml for TPOAb and 0–5.61 IU/ml for TgAb). On the other hand, subjects were considered as case individuals if they have a high level of TPOAb or TgAb (with the antibody level at least double the upper limit of its reference range) and with normal complete blood count. The blood was collected via venous puncture for the detection of different biological markers from March to June 2015. The study was approved by the Ethics Committee of Genos Ltd. Informed consent for participation in the study was obtained from all participants. The second replication cohort consisted of patients from the Endocrinology Department of the University Hospital in Krakow and Jagiellonian University staff, Poland, and included 219 individuals (105 HT patients and 114 healthy donors)—a cohort of mainly females (103 HT and 106 in control group) with an average age of 36 (Table I.1 supplemental Table S1). All HT patients have been on levothyroxine sodium substitutive therapy and were diagnosed based on a high TSH level and the TPOAb and/or TgAb positivity. Moreover, patients do not have any other concomitant autoimmune diseases, other thyroid diseases and history of cancers. The control group consisted of 114 individuals with a negative history of HT, other autoimmune diseases, other thyroid diseases and cancers with negative/low antibody titers of TPOAb, TgAb and anti-TSHR and TSH level within the reference range. Having a small number of men in this cohort, we ran an analysis of IgG N-glycan traits with only data from women. After quality control of data obtained after IgG glycome analysis, 106 control subjects and 103 HT patients were analyzed concerning IgG glycosylation profiling and to keep the age and gender match. 24 blood samples from 219 collected from the Polish replication cohort were also used to analyze PBMC glycosylation: 11 patients with HT and 13 healthy donors. The groups were matched for sex and age. Informed consent for participation in the study was obtained from all participants. The blood was collected via venous puncture for the detection of different biological markers from May 2014 to July 2015. The study was approved by the Ethics Committee of the Jagiellonian University Medical College in Krakow. The study was performed using TPOAb level as a continuous variable and a clinical AITD definition as a binary trait. Not having a clinical diagnostic for all twins, TPOAb level, and TSH level were used to define individuals with AITD and healthy groups. Individuals were considered to have AITD if they had a positive TPOAb titer (3 times more than the threshold set by the manufacturer (18 IU/ml for Abbott assay and 100 IU/ml for Roche assay)) or had a TSH level more than 10 mIU/L. We considered subjects to have HT when they have a TSH level ≥10 mIU/L or a positive TPOAb titer as decribed previously with TSH level more than 4 mIU/L. We considered individuals as heathy controls if they had a TSH level in the normal range and a negative TPOAb titer, with no previous clinical diagnosis of thyroid disease and not treated with thyroid medications or steroids. Individuals with a history of thyroid cancer or thyroid surgery (because of benign conditions) were excluded. The sera to assess TPOAb and TSH levels have been collected by a trained nurse or phlebotomist using venipuncture and a SafetyLokTM Blood Collection Kit (21G3/4 Needles) and plain 10 ml serum-separating tube vacutainer (no additives) during twin visits. After collection from the study subject, whole blood was held at 22 °C for 50 min at room temperature for a clot to form and serum separated within 60 min of collection. Processing of blood was performed using a refrigerated (4 °C) clinical centrifuge at 3000 × g for 10 min with the serum supernatant subsequently collected, transferred to a 2 ml screw capped Nunc Cryotubes and immediately frozen at −80 °C and kept frozen in 2 ml screw capped Nunc Cryotube at −80 °C until use. The quantitative determination of TSH and TPOAb (only IgG class) levels was done on the sera by a chemiluminescent microparticle immunoassay (ARCHITECT® Anti-TPO or TSH (ABBOTT Diagnostics Division, Wiesbaden, Germany, 2005)) (a TPOAb titer >6 IU/ml considered positive; normal range of TSH level is between 0.4 mIU/L and 4 mIU/L). For samples analyzed by an electrochemiluminescence immunoassay "ECLIA" (Elecsys and cobas e analyzers, (Roche Diagnostics, Indianapolis, lN, 2010)) (a TPOAb titer >34 IU/ml considered positive; normal range of TSH level is between 0.4 mIU/L and 4 mIU/L). Sera samples for Croatian cohort were collected via venous puncture from March to June 2015 into 10 ml serum-separating vacutainer tube without additives, centrifuged at 3500 rpm for 10 min, after which the serum was transferred to 2 ml Eppendorf tubes and kept frozen at −20 °C until use. Quantitative determination of TSH, free T3, free T4, TPOAb and TgAb was performed by a chemiluminescent micro-particle immunoassay (ARCHITECT® TSH, free T3, free T4, Anti-TPO or Anti-Tg (ABBOTT Diagnostics Division, Wiesbaden, Germany, 2015)). Sera and PBMCs for Polish cohort were collected via venous puncture for the detection of different biological markers from May 2014 to July 2015 using S-Monovette Serum Tubes 2.7 ml that contains Clotting Activator/Serum (Sarstedt, 05.1557.001) for collection. Blood samples were left at room temperature for five hours for blood coagulation and centrifuged at 1200 × g for 10 min at 4 °C. Serum samples were stored in deep freezing in Safe Lock Tubes 2 ml (Eppendorf, 0030123.344). The TSH level in sera samples were determined by immunoradiometric assay (IRMA) (DIAsource TSH-IRMA Kit (DIAsource ImmunoAssays S.A., Louvain-la-Neuve, Belgium)). Anti-TPO and anti-Tg levels were measured by radioimmunoassays (RIAs) (BRAHMS anti-TPOn RIA, BRAHMS anti-Tgn RIA (BRAHMS GmbH, Hennigsdorf, Germany)). The sera, as well as PBMC samples, were kept frozen at −70 °C until the analysis of glycosylation. The IgG glycosylation profiling was applied on total IgG glycome from blood (combined Fc and Fab glycans and all IgG subclasses). It is noted that glycosylation patterns of total IgG reflect mostly IgG Fc glycans because of the small proportion of IgG Fab glycans in the total IgG N-glycan structures (16van de Bovenkamp F.S. Hafkenscheid L. Rispens T. Rombouts Y. The emerging importance of IgG Fab glycosylation in immunity.J. Immunol. 2016; 196: 1435-1441Crossref PubMed Scopus (166) Google Scholar). It was performed in Genos Glycoscience Research Laboratory in Croatia using UPLC analysis of 2AB-labeled glycans. The IgG glycosylation of the discovery cohort was performed in four batches whereas the replications cohorts were performed in one batch per cohort. However, the protocol for replication cohorts followed the same protocol than the protocol used for the two last batches of the discovery cohort and described below. Effects of technical confounders (96-well plate and run-day batch effects) are minimized by careful experimental design (blocking on case-control status, sex and age, whereas the rest was randomized) (32Ugrina I. Campbell H. Vučkovic F. Laboratory experimental design for a glycomic study.Methods Mol. Biol. 2017; 1503: 13-19Crossref PubMed Scopus (5) Google Scholar). Additionally, batch-effects are controlled by introducing in-house plasma standards to each 96-well plate (32Ugrina I. Campbell H. Vučkovic F. Laboratory experimental design for a glycomic study.Methods Mol. Biol. 2017; 1503: 13-19Crossref PubMed Scopus (5) Google Scholar). The IgG was isolated using 96-well protein G monolithic plates (BIA Separations, Ljubljana, Slovenia) as described previously (33Pučić M. Knežević A. Vidič J. Adamczyk B. Novokmet M. Polašek O. Gornik O. Šupraha Goreta -S. Wormald M.R. Redžić I. Campbell H. Wright A. Hast

Fucosylation

10.1074/mcp.ra119.001860

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IgG glycosylation and DNA methylation are interconnected with smoking

Biochimica et Biophysica Acta (BBA) - General Subjects (2017)

Annika Wahl Silva Kasela Elena Carnero‐Montoro Maarten van Iterson Jerko Štambuk

Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic.With the use of two different analytical methods for assessing IgG glycosylation, we aim to elucidate the link between DNA methylation and glycosylation of IgG by means of epigenome-wide association studies. In total, 3000 individuals from 4 cohorts were analyzed.The overlap of the results from the two glycan measurement panels yielded DNA methylation of 7 CpG-sites on 5 genomic locations to be associated with IgG glycosylation: cg25189904 (chr.1, GNG12); cg05951221, cg21566642 and cg01940273 (chr.2, ALPPL2); cg05575921 (chr.5, AHRR); cg06126421 (6p21.33); and cg03636183 (chr.19, F2RL3). Mediation analyses with respect to smoking revealed that the effect of smoking on IgG glycosylation may be at least partially mediated via DNA methylation levels at these 7 CpG-sites.Our results suggest the presence of an indirect link between DNA methylation and IgG glycosylation that may in part capture environmental exposures.An epigenome-wide analysis conducted in four population-based cohorts revealed an association between DNA methylation and IgG glycosylation patterns. Presumably, DNA methylation mediates the effect of smoking on IgG glycosylation.

CpG site

Epigenome

N-linked glycosylation

10.1016/j.bbagen.2017.10.012

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N-glikozilacija imunoglobulina G u upalnim bolestima crijeva

Mirna Šimurina

The cause of inflammatory bowel disease (IBD) is still poorly understood and the most prominent forms Crohn’s disease (CD) and ulcerative colitis (UC) are sometimes hard to distinguish. Glycosylation of immunoglobulin G (IgG) has been associated with CD and UC. IgG Fc-glycosylation affects IgG effector functions. The changes in IgG Fc-glycosylation associated with UC and CD, as well as with disease characteristics in different patient groups, were evaluated, and this is the first study of inflammatory bowel disease where subclass-specific IgG gycosylation was analyzed. A total of 2 357 plasma samples were collected in retrospective cohort that contained samples from UC (1056) and CD patients (874) as well as healthy controls (HC, 427). So far, this is the largest study of IgG glycosylation in IBD. Subclass-specific IgG Fc-glycosylation (tryptic glycopeptides) was analyzed by liquid chromatography coupled to mass spectrometry (nanoLC-MS). Associations between disease status (UC vs HC, CD vs HC, and UC vs CD) and glycopetide traits and associations between clinical characteristics and glycopeptide traits, was performed. Patients with CD or UC had lower levels of IgG galactosylation than controls. Fucosylation of IgG was increased in patients with CD vs controls but decreased in patients with UC vs controls. Decreased galactosylation associated with more severe CD or UC, including the need for surgery. In a retrospective analysis of plasma samples from patients with CD or UC, we associated levels of IgG Fc-glycosylation with disease (compared to controls) and its clinical features. Through better patient classification, these findings could increase our understanding of mechanisms of CD and UC pathogenesis and be used to develop diagnostics or guide treatment.

Fucosylation

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The Association Between Low Back Pain and Composition of IgG Glycome

Scientific Reports (2016)

Maxim B. Freidin Toma Keser Ivan Gudelj Jerko Štambuk Dunja Vučenović

Abstract Low back pain (LBP) is a common debilitating condition which aetiology and pathogenesis are poorly understood. We carried out a first so far analysis of associations between LBP and plasma IgG N-glycome in a sample of 4511 twins from TwinsUK database assessed for LBP, lumbar disc degeneration (LDD) as its possible cause, and IgG-glycan levels. Using weighted correlation network analysis, we established a correlation between LBP and glycan modules featured by glycans that either promote or block antibody-dependent cell-mediated cytotoxicity (ADCC). The levels of four glycan traits representing two of those modules were statistically significantly different in monozygotic twins discordant for LBP. Also, the trend to higher prevalence of systemic inflammatory disorders was shown for twins with low level of fucosylated glycans and high level of non-fucosylated glycans. Core fucosylation of IgG is a “safety switch” reducing ADCC, thus our results suggest the involvement of ADCC and associated inflammation in pathogenesis of LBP. No correlation between LDD scores and glycans was found assuming that the inflammation may not be a part of LDD. These data provide a new insight into understanding the complex pathophysiology of LBP and suggest glycan levels as a possible biomarker for inflammation-related subtypes of LBP.

Glycome

Fucosylation

Pathogenesis

Monozygotic twin

10.1038/srep26815

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Global variability of the human IgG glycome

bioRxiv (Cold Spring Harbor Laboratory) (2019)

Jerko Štambuk Natali Nakić Frano Vučković Maja Pučić‐Baković Genadij Razdorov

Abstract Immunoglobulin G (IgG) is the most abundant serum antibody and is a key determinant of the humoral immune response. Its structural characteristics and effector functions are modulated through the attachment of various sugar moieties called glycans. IgG N-glycome patterns change with the age of individual and in different diseases. Variability of IgG glycosylation within a population is well studied and is affected by a combination of genetic and environmental factors. However, global inter-population differences in IgG glycosylation have never been properly addressed. Here we present population-specific N-glycosylation patterns of whole IgG, analysed in 5 different populations totalling 10,482 IgG glycomes, and of IgG’s fragment crystallisable region (Fc), analysed in 2,530 samples from 27 populations sampled across the world. We observed that country of residence associates with many N-glycan features and is a strong predictor of monogalactosylation variability. IgG galactosylation also strongly correlated with the development level of a country, defined by United Nations health and socioeconomic development indicators. We found that subjects from developing countries had low IgG galactosylation levels, characteristic for inflammation and ageing. Our results suggest that citizens of developing countries may be exposed to country-specific environmental factors that can cause low-grade chronic inflammation and the apparent increase in biological age.

Glycome

10.1101/535237

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Glycosylation Profile of IgG in Moderate Kidney Dysfunction

Journal of the American Society of Nephrology (2015)

C. Pérez Barrios Jonas Zierer Ivan Gudelj Jerko Štambuk Ivo Ugrina

Glycans constitute the most abundant and diverse form of the post-translational modifications, and animal studies have suggested the involvement of IgG glycosylation in mechanisms of renal damage. Here, we explored the associations between IgG glycans and renal function in 3274 individuals from the TwinsUK registry. We analyzed the correlation between renal function measured as eGFR and 76 N-glycan traits using linear regressions adjusted for covariates and multiple testing in the larger population. We replicated our results in 31 monozygotic twin pairs discordant for renal function. Results from both analyses were then meta-analyzed. Fourteen glycan traits were associated with renal function in the discovery sample (P<6.5×10−4) and remained significant after validation. Those glycan traits belong to three main glycosylation features: galactosylation, sialylation, and level of bisecting N-acetylglucosamine of the IgG glycans. These results show the role of IgG glycosylation in kidney function and provide novel insight into the pathophysiology of CKD and potential diagnostic and therapeutic targets.

10.1681/asn.2015010109

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Defining the genetic control of human blood plasma N-glycome using genome-wide association study

Human Molecular Genetics (2019)

Sodbo Sharapov Yakov A. Tsepilov Lucija Klarić Massimo Mangino Gaurav Thareja

Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area.

Glycome

Genome-wide Association Study

International HapMap Project

Human genetics

10.1093/hmg/ddz054

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Heritability of Human Plasma N-Glycome

Journal of Proteome Research (2019)

Olga O. Zaytseva Maxim B. Freidin Toma Keser Jerko Štambuk Ivo Ugrina

The N-glycosylation profile of total human plasma proteins could be a useful biomarker for various pathological states. Reliable high-throughput methods for such profiling have been developed. However, studies of relative importance of genetic and environmental factors in regulating plasma N-glycome are scarce. The aim of our study was to determine the role of genetic factors in phenotypic variation of plasma N-glycan profile through the estimates of its heritability. Thirty-nine total plasma N-glycome traits were analyzed in 2816 individuals from the TwinsUK data set. For the majority of the traits, high heritability estimates (>50%) were obtained pointing at a significant contribution of genetic factors in plasma N-glycome variation, especially for glycans mostly attached to immunoglobulins. We have also found several structures with higher environmental contribution to their variation.

Glycome

10.1021/acs.jproteome.9b00348

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The N-glycosylation of immunoglobulin G as a novel biomarker of Parkinson's disease

Glycobiology (2017)

Alyce Russell Mirna Šimurina Monique Garcia Mislav Novokmet Youxin Wang

The use of the emerging “omics” technologies for large scale population screening is promising in terms of predictive, preventive and personalized medicine. For Parkinson's disease, it is essential that an accurate diagnosis is obtained and disease progression can be monitored. Immunoglobulin G (IgG) has the ability to exert both anti-inflammatory and pro-inflammatory effects, and the N-glycosylation of the fragment crystallizable portion of IgG is involved in this process. This study aimed to determine whether the IgG glycome could be a candidate biomarker for Parkinson's disease. Ninety-four community-based individuals with Parkinson's disease and a sex-, age- and ethnically-matched cohort of 102 individuals with mixed phenotypes, representative of a “normally” aged Caucasian controls, were investigated. Plasma IgG glycans were analyzed by ultra-performance liquid chromatography. Overall, seven glycan peaks and 11 derived traits had statistically significant differences (P < 8.06 × 10−4) between Parkinson's disease cases and healthy controls. Out of the seven significantly different glycan peaks, four were selected by Akaike's Information Criterion to be included in the logistic regression model, with a sensitivity of 87.2% and a specificity of 92.2%. The study suggested that there may be a reduced capacity for the IgG to inhibit Fcγ-RIIIa binding, which would allow an increased ability for the IgG to cause antibody-dependent cell cytotoxicity and a possible state of low-grade inflammation in individuals with Parkinson's disease.

Glycome

10.1093/glycob/cwx022

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Citations (129)