Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 × 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.

Robustness testing of the high throughput HPLC-based analysis of plasma N-glycans.

BACKGROUND: Plasma glycan analysis using high throughput HPLC-based 96 well platform became a standard procedure for analyzing a large pool of samples for studies comprising thousands of observed individuals. An analytical method which is used to obtain such a huge amount of data should be well characterized and all potentially critical steps should be known. METHODS: Robustness of the high throughput method was tested by Plackett Burman two level, 11-factor, 12 experiment screening design. It provides valuable information about the few most important factors on which further optimization should be focused. According to a long-term laboratory experience, eleven potentially critical factors were chosen for initial screening. Response variable was calculated as coefficient of variance between area % of each peak in each reaction and the area % obtained after performing the procedure according to the laboratory standard operating procedure. RESULTS: Six out of 16, by HPLC separated, glycan groups revealed significant changes according to changes in factor levels. As expected due to their structural and chemical differences, glycan groups did not display uniform response to 11 factors, but effect estimates for six significant glycan groups showed the same direction regarding high and low factor levels. GENERAL SIGNIFICANCE: Screening experiment provided quality data which resolved the questions about optimal conditions and robustness of the high throughput glycan analysis. Fraction factorial design used in this study enabled us to test a great deal of critical steps in time, labor and money saving manner. This article is part of a Special Issue entitled Glycoproteomics.

Epigenetic modulation of the HeLa cell membrane N-glycome.

BACKGROUND: Epigenetic changes play a role in all major events during tumorigenesis and changes in glycan structures are hallmarks of virtually every cancer. Also, proper N-glycosylation of membrane receptors is important in cell to cell and cell-environment communication. To study how modulation of epigenetic information can affect N-glycan expression we analyzed effects of epigenetic inhibitors on HeLa cell membrane N-glycome. METHODS: HeLa cells were treated with DNA methylation (zebularin and 5-aza-2-deoxycytidine) and histone deacetylation (trichostatin A and Na-butyrate) inhibitors. The effects on HeLa cell membrane N-glycome were analyzed by hydrophilic interaction high performance liquid chromatography (HILIC). RESULTS: Each of the four epigenetic inhibitors induced changes in the expression of HeLa cell membrane N-glycans that were seen either as an increase or a decrease of individual glycans in the total N-glycome. Compared to DNA methylation inhibitors, histone deacetylation inhibitors showed more moderate changes, probably due to their higher gene target selectivity. CONCLUSIONS: The results clearly show that composition of HeLa cell membrane N-glycome can be specifically altered by epigenetic inhibitors. GENERAL SIGNIFICANCE: Glycans on the cell membrane are essential elements of tumor cell's metastatic potential and are also an entry point for nearly all pathogenic microorganisms. Since epigenetic inhibitors used in this work are registered drugs, our results provide a new line of research in the application of these drugs as anticancer and antimicrobial agents. This article is part of a Special Issue entitled Glycoproteomics.

Association of medication with the human plasma N-glycome.

Glycosylation is highly variable depending on many environmental factors. Using our fully quantitative high-throughput normal phase hydrophilic interaction liquid chromatography platform we have identified glycosylation changes associated with medication in the plasma N-glycome from three different population cohorts: ORCADES from the Orkney Islands in Scotland and CROATIA-Vis and CROATIA-Korcula from the Croatian islands of Vis and Korcula. Associations between glycosylation and the use of hormones (oral contraceptives, hormone replacement therapy), nonsteroidal anti-inflammatory drugs (aspirin and other NSAIDs), oral steroids (prednisolone) and steroid inhalers (beclomethasone) were investigated. Significant differences associated with usage of oral contraceptives were found with increased core-fucosylated biantennary glycans. Decreases in core-fucosylated biantennary glycans, core-fucosylated triantennary glycans with outer-arm fucose, and high mannosylated glycans were associated with the use of anti-inflammatory drugs. All of the changes in glycosylation were independent of blood group status. In conclusion, hormones and anti-inflammatory medication were associated with changes in glycosylation, possibly as a result of the modulatory effect of these drugs on the inflammatory response. In general, cancer is associated with inflammation, and many glycoproteins in the plasma are acute phase related to the host response. These preliminary data indicate the importance of correcting the levels of glycans used as biomarkers for the effects of medication.

Changes in plasma and IgG N-glycome during childhood and adolescence.

Despite the importance of protein glycosylation in all physiological and pathological processes and their potential as diagnostic markers and drug targets, the glycome of children is still unexplored. We analyzed N-linked plasma and IgG glycomes in 170 children and adolescents between 6 and 18 years of age. The results showed large biological variability at the population level as well as a large number of associations between different glycans and age. The plasma N-glycome of younger children was found to contain a larger proportion of large complex glycan structures (r = -0.71 for tetrasialylated glycans; r = -0.41 for trisialylated glycans) as well as an increase in disialylated biantennary structures (r = 0.55) with age. Core fucosylation and the level of agalactosylated plasma and IgG glycans decreased while digalactosylated glycans increased with age. This pattern of age-dependent changes in children differs from changes reported in adult population in both, direction and the intensity of changes. Also, sex differences are much smaller in children than in adults and are present mainly during puberty. These important observations should be accounted for when glycan-based diagnostic tests or therapeutics are being developed or evaluated.

Mutations in HNF1A result in marked alterations of plasma glycan profile.

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥ 0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.

Epigenetic silencing of HNF1A associates with changes in the composition of the human plasma N-glycome.

Protein glycosylation is a ubiquitous modification that affects the structure and function of proteins. Our recent genome wide association study identified transcription factor HNF1A as an important regulator of plasma protein glycosylation. To evaluate the potential impact of epigenetic regulation of HNF1A on protein glycosylation we analyzed CpG methylation in 810 individuals. The association between methylation of four CpG sites and the composition of plasma and IgG glycomes was analyzed. Several statistically significant associations were observed between HNF1A methylation and plasma glycans, while there were no significant associations with IgG glycans. The most consistent association with HNF1A methylation was observed with the increase in the proportion of highly branched glycans in the plasma N-glycome. The hypothesis that inactivation of HNF1A promotes branching of glycans was supported by the analysis of plasma N-glycomes in 61 patients with inactivating mutations in HNF1A, where the increase in plasma glycan branching was also observed. This study represents the first demonstration of epigenetic regulation of plasma glycome composition, suggesting a potential mechanism by which epigenetic deregulation of the glycome may contribute to disease development.