Genome-Wide Association Study Finds New Loci Affecting N-Glycosylation of Human Blood Plasma Proteins

Genome-wide association study finds new loci affecting N-glycosylation of human blood plasma proteins

Sodbo Zh. Sharapov1,2, Yakov A. Tsepilov1,2, Lucija Klaric3,4, Massimo Mangino5,6, Gaurav Thareja7, Alexandra S. Shadrina2, Mirna Simurina8, Concetta Dagostino9, Julia Dmitrieva10, Marija Vilaj4, FranoVuckovic4, Tamara Pavic8, Jerko Stambuk4, Irena Trbojevic-Akmacic4, Jasminka Kristic4, Jelena Simunovic4, Ana Momcilovic4, Harry Campbell11,12, Margaret Doherty13,14, Malcolm G Dunlop12, Susan M Farrington12, Maja Pucic-Bakovic4, Christian Gieger15, Massimo Allegri16, Edouard Louis17, Michel Georges10, Karsten Suhre7, Tim Spector5, Frances MK Williams5 1Institute of Cytology and Genetics SB RAS, Prospekt Lavrentyeva 10, Novosibirsk, 630090, Russia 2Novosibirsk State University, 1, Pirogova str., Novosibirsk, 630090, Russia 3MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XU, UK 4Genos Glycoscience Research Laboratory, Borongajska cesta 83h, 10000 Zagreb, Croatia 5Department of Twin Research and Genetic Epidemiology, School of Life Course Sciences, King’s College London, St Thomas’ Campus, Lambeth Palace Road, London, SE1 7EH, UK 6NIHR Biomedical Research Centre at Guy’s and St Thomas’ Foundation Trust, London SE1 9RT, UK. 7Department of Physiology and Biophysics, Weill Cornell Medicine-Qatar, Education City, PO 24144 Doha, Qatar 8Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovacica 1, 10 000 Zagreb, Croatia 9Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126 Parma, Italy 10Unit of Animal Genomics, WELBIO, GIGA-R & Faculty of Veterinary Medicine, University of Liège (B34), 1 Avenue de l’Hôpital, Liège 4000, Belgium 11Centre for Global Health Research, Usher Institute of Population Health Sciences and Informatics, The University of Edinburgh, Edinburgh EH8 9AG, UK 12Colon Cancer Genetics Group, MRC Human Genetics Unit, MRC Institute of Genetics & Molecular Medicine, Western General Hospital, The University of Edinburgh, Edinburgh EH4 2XU, UK 13Institute of Technology Sligo, Department of Life Sciences, Sligo, Ireland 14National Institute for Bioprocessing Research & Training, Dublin, Ireland 15Institute of Epidemiology II, Research Unit of Molecular Epidemiology, Helmholtz Centre Munich, German Research Center for Environmental Health, Ingolstädter Landstr. 1, D-85764, Neuherberg, Germany 16Pain Therapy Department Policlinico Monza Hospital, 20090 Monza, Italy 17CHU-Liège and Unit of Gastroenterology, GIGA-R & Faculty of Medicine, University of Liège, 1 Avenue de l’Hôpital, Liège 4000, Belgium 18PolyOmica, Het Vlaggeschip 61, 5237 PA 's-Hertogenbosch, The Netherlands

Gordan Lauc4,8,*, Yurii Aulchenko1,2,18,* * These authors jointly supervised this work and contributed equally. Correspondence and requests for materials should be addressed to YA (email: [email protected], Tel: +7 (383) 363-49-63*1228, Fax: +7(383) 333-12-78)

Glycosylation – covalent attachment of carbohydrates (glycans) to a substrate - is a common and structurally diverse cotranslational and posttranslational modification of proteins. Glycans influence the physical properties of proteins (solubility, conformation, folding, stability, trafficking, etc.) 1–3 as well as their biological functions including protein-protein, cell-cell, cell-matrix, and host-pathogen interactions 1,2,4,5. Alteration in glycosylation is observed in a number of human diseases such as type 1 and 2 diabetes 6, rheumatoid arthritis 7, Parkinson's disease 8 and cancer 9–11. Glycans are considered potential therapeutic targets 12 and biomarkers for early diagnosis and disease prognosis 13–16, making glycobiology a promising field for future clinical applications. Defining the genetic factors underlying glycosylation will potientially provide novel approaches to diagnostic and pharmaceutical applications. Genome-wide association studies (GWAS) provide important tools because of their hypothesis-free search of genes involved in regulation of glycosylation.

Here we present a genetic study of the human blood plasma proteins N-glycosylation measured by Ultra Performance Liquid Chromatography (UPLC) technology in up to 3,811 people. We studied association between 8.5 million genetic polymorphisms on human autosomes and 113 relative abundances of N-glycan structures. We discovered and replicated twelve associated loci, seven of which were new. To prioritize potentially causal genes in the found loci we performed an extensive in silico functional study including analysis of eQTL effects, tissue/cell type expression enrichment analyses and gene-set enrichment analysis. The majority of 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 reflect other, more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3 (plays role in the degradation for misfolded luminal glycoproteins), CHCHD10 (encodes a mitochondrial protein that is enriched at cristae junctions in the intermembrane space), TMEM121, IGH (encodes immunoglobulin heavy chains), and IKZF1 (considered an important regulator of lymphocyte differentiation). These genes can be considered candidates for future experimental research. Additionally, we demonstrated a clear overlap in genetic control of glycosylation of plasma proteins and immunoglobulin G 18,19, leading is to future studies that will distinguish global, cell-, tissue-, and protein-specific pathways of protein glycosylation.

Acknowledgements

This work was supported by the European Community’s Seventh Framework Programme funded project PainOmics (Grant agreement # 602736), by the European Structural and Investment Funds IRI grant (#KK.01.2.1.01.0003) and Croatian National Centre of Research Excellence in Personalized Healthcare grant (#KK.01.1.1.01.0010). The work of ASh was supported by the Russian Ministry of Science and Education under the 5-100 Excellence Programme. The work of SSh, YT and YA were supported by the Federal Agency of Scientific Organizations via the Institute of Cytology and Genetics (project #0324-2019-0040). The work of LK was supported by the RCUK Innovation Fellowship from the National Productivity Investment Fund (MR/R026408/1). Karsten Suhre and Gaurav Thareja are supported by ‘Biomedical Research Program’ funds at Weill Cornell Medicine - Qatar, a program funded by the Qatar Foundation. We thank all staff at Weill Cornell Medicine - Qatar and Hamad Medical Corporation, and especially all study participants who made the QMDiab study possible.The SOCCS study was supported by grants from Cancer Research UK (C348/A3758, C348/A8896, C348/ A18927); Scottish Government Chief Scientist Office (K/OPR/2/2/D333, CZB/4/94); Medical Research Council (G0000657-53203, MR/K018647/1); Centre Grant from CORE as part of the Digestive Cancer Campaign (http://www.corecharity.org.uk). TwinsUK is funded by the Wellcome Trust, Medical Research Council, European Union, the National Institute for Health Research (NIHR)-funded BioResource, Clinical Research Facility and Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London.

References: 1. Ohtsubo, K. & Marth, J. D. Glycosylation in Cellular Mechanisms of Health and Disease. Cell 126, 855–867 (2006).

2. Skropeta, D. The effect of individual N-glycans on enzyme activity. Bioorg. Med. Chem. 17, 2645–2653 (2009). 3. Takeuchi, H. et al. O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking. J. Biol. Chem. 292, 15964–15973 (2017).

4. Lauc, G., Pezer, M., Rudan, I. & Campbell, H. Mechanisms of disease: The human N-glycome. Biochim. Biophys. Acta 1860, 1574–1582 (2015).

5. Poole, J., Day, C. J., von Itzstein, M., Paton, J. C. & Jennings, M. P. Glycointeractions in bacterial pathogenesis. Nat. Rev. Microbiol. 16, 440–452 (2018).

6. Lemmers, R. F. H. et al. IgG glycan patterns are associated with type 2 diabetes in independent European populations. Biochim. Biophys. Acta - Gen. Subj. 1861, 2240–2249 (2017).

7. Gudelj, I. et al. Low galactosylation of IgG associates with higher risk for future diagnosis of rheumatoid arthritis during 10 years of follow-up. Biochim. Biophys. Acta - Mol. Basis Dis. 1864, 2034–2039 (2018).

8. Russell, A. C. et al. The N-glycosylation of immunoglobulin G as a novel biomarker of Parkinson’s disease. Glycobiology 27, 501–510 (2017).

9. Vajaria, B. N. & Patel, P. S. Glycosylation: a hallmark of cancer? Glycoconj. J. 34, 147–156 (2017).

10. Munkley, J. & Elliott, D. J. Hallmarks of glycosylation in cancer. Oncotarget 7, 35478–35489 (2016).

11. Taniguchi, N. & Kizuka, Y. Glycans and cancer: role of N-glycans in cancer biomarker, progression and metastasis, and therapeutics. Adv. Cancer Res. 126, 11–51 (2015).

12. RodrÍguez, E., Schetters, S. T. T. & van Kooyk, Y. The tumour glyco-code as a novel immune checkpoint for immunotherapy. Nat. Rev. Immunol. 18, 204–211 (2018).

13. Thanabalasingham, G. et al. Mutations in HNF1A result in marked alterations of plasma glycan profile. Diabetes 62, 1329–1337 (2013).

14. Adamczyk, B., Tharmalingam, T. & Rudd, P. M. Glycans as cancer biomarkers. Biochim. Biophys. Acta - Gen. Subj. 1820, 1347–1353 (2012).

15. Shinohara, Y., Furukawa, J. & Miura, Y. in General Methods in Biomarker Research and their Applications (eds. Preedy, V. R. & Patel, V. B.) 111–140 (Springer Netherlands, 2015). doi:10.1007/978-94-007-7696-8_23

16. Peng, W. et al. Clinical application of quantitative glycomics. Expert Rev. Proteomics 15, 1007–1031 (2018).

17. Huffman, J. E. et al. Polymorphisms in B3GAT1, SLC9A9 and MGAT5 are associated with variation within the human plasma N-glycome of 3533 European adults. Hum. Mol. Genet. 20, 5000–5011 (2011).

18. Lauc, G. et al. Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers. PLoS Genet. 9, e1003225 (2013).

19. Shen, X. et al. Multivariate discovery and replication of five novel loci associated with Immunoglobulin G N- glycosylation. Nat. Commun. 8, 447 (2017).