Wright, Alan

Hastie, Nicholas

Huffman, Jennifer Elizabeth

Medical Research Council (MRC)

2015-04-13T15:14:20Z

2015-04-13T15:14:20Z

2014-11-28

The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high performance liquid chromatography (HPLC) method for high-throughput glycan analysis, genome-wide association studies (GWAS) of 33 directly measured and 13 derived N-glycan features were performed in 3533 individuals from four European isolated populations. Polymorphisms at six loci were found to show genome-wide significant association with plasma concentrations of N-glycans. Several of these gene products have well characterised roles in glycosylation, however, SLC9A9 and HNF1A were two of the novel findings. Subsequent work performed by collaborators found HNF1A to be a “master regulator” of genes involved in the fucosylation of plasma N-glycans. Additionally, this work led to the discovery that N-glycans could act as biomarkers to discriminate HNF1A-MODY from type 1 and type 2 diabetes mellitus (T1D, T2D) patients. After the success of the total plasma N-glycan GWAS, it was thought that stronger and more biologically interpretable associations may be found from the investigation of N-glycans isolated from a single protein. Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic networks that govern IgG glycosylation, N-linked IgG glycans were quantitated using ultra performance liquid chromatography (UPLC) in 2247 individuals from the same four European populations from the previous study. GWAS of the 77 N-glycan measures identified 15 loci with a p-value<5x10-08. Four loci contained genes encoding glycosyltransferases, while the remaining loci contained genes that have not previously been implicated in protein glycosylation. However, most have been associated with autoimmune and inflammatory conditions and/or hematological cancers. Several high-throughput methods for the analysis of N-glycans have been developed in the past few years but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. To this end, four of these methods were compared, UPLC, multiplexed capillary gel electrophoresis (xCGE), and two mass spectrometric (MS) methods, for quantitative profiling of N-glycosylation of plasma IgG in a subset of 1201 individuals recruited from two of the cohorts used in the previous GWAS studies. A “minimal” dataset was compiled of N-glycan structures able to be measured by all four methods. To evaluate their accuracy, correlations were calculated for each structure in the minimal dataset. Additionally, GWAS was performed to test if the same associations would be observed across methodologies. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and xCGE, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should aid in the selection of the most appropriate method for future studies. This work shows that it is possible to identify new loci that control glycosylation of plasma proteins using GWAS and the potential of N-glycans for biomarker development. It also provides some guidelines for methodology selection for future studies of N-glycans.

http://hdl.handle.net/1842/10038

en

The University of Edinburgh

Lauc G, Essafi A, Huffman JE, Hayward C, Knežević A, et al. (2010) Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1α as a master regulator of plasma protein fucosylation. PLoS Genet 6: e1001256. URL: http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1001256

Huffman JE, Knezevic A, Vitart V, Kattla J, Adamczyk B, et al. (2011) 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. URL: http://hmg.oxfordjournals.org/content/20/24/5000.long

Thanabalasingham G, Huffman JE, Kattla JJ, Novokmet M, Rudan I, et al. (2013) Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile. Diabetes 62: 1329-1337. URL: http://diabetes.diabetesjournals.org/content/62/4/1329.long

Lauc G, Huffman JE, Pučić M, Zgaga L, Adamczyk B, et al. (2013) Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers. PLoS Genet 9: e1003225. URL: http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1003225

Huffman JE, Pučić-Baković M, Klarić L, Hennig R, Selman MH, et al. (2014) Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research. Mol Cell Proteomics. 13(6):1598:610. URL: http://www.mcponline.org/content/13/6/1598.long

Attribution-NonCommercial-ShareAlike 4.0 International

http://creativecommons.org/licenses/by-nc-sa/4.0/

genomewide association studies

glycan

single nucleotide polymorphism

Genetic analysis of protein N-Glycosylation

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy