Exome sequencing reveals aggregates of rare variants in glycosyltransferase and other genes influencing immunoglobulin G and transferrin glycosylation

It is often difficult to be certain which genes underlie the effects seen in association studies. However, variants that disrupt the protein, such as predicted loss of function (pLoF) and missense variants, provide a shortcut to identify genes with a clear biological link to the phenotype of interest. Glycosylation is one of the most common post-translationalmodifications of proteins, and an important biomarker of both disease and its progression. Here, we utilised the power of genetic isolates, gene-based aggregation tests and intermediate phenotypes to assess the effect of rare (MAF<5%) pLoF and missense variants from whole exome sequencing on the N-glycome of plasma transferrin (N=1907) and immunoglobulin G (N=4912), and their effect on diseases. We identified significant gene-based associations for transferrin glycosylation at 5 genes (p<8.06×10−8) and for IgG glycan traits at 4 genes (p<1.19×10−7). Associations in three of these genes ( FUT8, MGAT3 and RFXAP ) are driven by multiple rare variants simultaneously contributing to protein glycosylation. Association at ST6GAL1 , with a 300-fold up-drifted variant in the Orkney Islands, was detectable by a single-point exome-wide association analysis. Glycome-associated aggregate associations are located in genes already known to have a biological link to protein glycosylation ( FUT6, FUT8 for transferrin; FUT8, MGAT3 and ST6GAL1 for IgG) but also in genes which have not been previously reported (e.g. RFXAP for IgG). To assess the potential impact of rare variants associated with glycosylation on other traits, we queried public repositories of gene-based tests, discovering a potential connection between transferrin glycosylation, MSR1 , galectin-3, insulin-like growth factor 1 and diabetes. However, the exact mechanism behind these connections requires further elucidation. ### Competing Interest Statement P.R.H.J.T. is an employee of BioAge Labs, Inc. G.T. and A.R.S. are full-time employees of Regeneron Genetics Center and receive salary, stock and stock options as compensation. G.L. is the founder and owner of Genos Ltd, a private research organisation that specialises in the high-throughput glycomic analysis and has several patents in this field. A.F.-H., I.T.-A., F.V., and T.P. are employees of Genos Ltd. L.K. is an employee of Humanity Inc., a company developing direct-to-consumer measures of biological ageing. All other authors declare no competing interests. ### Funding Statement We thank Dr Nicola Pirastu for his help and advice regarding the statistical methods. The Orkney Complex Disease Study (ORCADES) was supported by the Chief Scientist Office of the Scottish Government (CZB/4/276 and CZB/4/710), the Royal Society, the MRC Human Genetics Unit, Arthritis Research UK and the European Union framework program 6 EUROSPAN project (contract number LSHG-CT-2006-018947). ORCADES DNA extractions and genotyping were performed at the Genetics Core of the Clinical Research Facility, University of Edinburgh. We would like to acknowledge the invaluable contributions of the research nurses in Orkney, the administrative team in Edinburgh, and the people of Orkney. The CROATIA-Korcula study was funded by grants from the MRC (United Kingdom), European Commission Framework 6 project EUROSPAN (contract number LSHG-CT-2006-018947), Croatian Science Foundation (grant 8875) and the Republic of Croatia Ministry of Science, Education and Sports (216-1080315-0302). Genotyping was performed in the Genetics Core of the Clinical Research Facility, University of Edinburgh. We would like to acknowledge all the staff of several institutions in Croatia that supported the CROATIA-Korcula fieldwork, including, but not limited to, the University of Split and Zagreb Medical Schools, Institute for Anthropological Research in Zagreb, and the Croatian Institute for Public Health in Split. The Viking Health Study-Shetland (VIKING) was supported by the MRC Human Genetics Unit quinquennial programme grant "QTL in Health and Disease". DNA extractions and genotyping were performed at the Edinburgh Clinical Research Facility, University of Edinburgh. We would like to acknowledge the invaluable contributions of the research nurses in Shetland, the administrative team in Edinburgh and the people of Shetland. We acknowledge support from the European Union's Horizon 2020 research and innovation programme IMforFUTURE (A.L. and A.F.-H.: H2020-MSCA-ITN/721815); the RCUK Innovation Fellowship from the National Productivity Investment Fund (L.K.: MR/R026408/1) and the MRC Human Genetics Unit programme grant, "QTL in Health and Disease" (J.F.W. and C.H.: MC\_UU\_00007/10). Finally, this research has been conducted using data from the UK Biobank Resource (under application 26041, 48511 and 19655). For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All studies were approved by local research ethics committees and all participants have given written informed consent. The ORCADES study was approved by the NHS Orkney Research Ethics Committee and the North of Scotland REC. The CROATIA-Korcula study was approved by the Ethics Committee of the Medical School, University of Split (approval ID: 2181-198-03-04/10-11-0008). The VIKING study was approved by the South East Scotland Research Ethics Committee, NHS Lothian (reference: 12/SS/0151). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes There is neither Research Ethics Committee approval, nor consent from individual participants, to permit open release of the individual level research data underlying this study. The datasets generated and analysed during the current study are therefore not publicly available. Instead, the research data and/or DNA samples are available from accessQTL@ed.ac.uk on reasonable request, following approval by the QTL Data Access Committee and in line with the consent given by participants. Each approved project is subject to a data or materials transfer agreement (D/MTA) or commercial contract. The UK Biobank genotypic data used in this study were approved under application 19655, 48511 and 19655 are available to qualified researchers via the UK Biobank data access process. The expression data used for the analyses described in this manuscript were obtained from the GTEx Portal on 25/11/2022. Genebass (https://app.genebass.org/) and AstraZeneca PheWAS portal (https://azphewas.com/) were accessed on 06/12/2022.