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

Abstract

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.

Key words:

• protein glycosylation
• plasma glycome
• HNF1A
• CpG methylation
• epigenetics

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Author Contributions

V.Z. and G.L. designed the study and led writing of the manuscript; H.C., C.H., A.F.W., I.R., K.O., M.I.M. and Z.H. designed the study and helped with data interpretation and writing of the manuscript; T.H., M.N., C.C., A.K., M.P. and O.G. performed laboratory analysis and checked the manuscript for intellectual contact; J.H. and O.P. performed statistical analysis and checked the manuscript for intellectual contact.

Acknowledgments

This work was supported by the Croatian Ministry of Science, Education and Sport grant #309-0061194-2023 (to G.L.) and #119-1191196-1224 (to V.Z.), by the Croatian Science Foundation grant #04-47 to I.R., by grants from la Ligue Nationale (Française) Contre le Cancer (France) and the Association pour la Recherche sur le Cancer (ARC, France) to ZH, by the European Commission grants EuroGlycoArrays, GlycoBioM, HighGlycan and CEED3, and by AUF PSCI Grant. Plasma and DNA samples from the inhabitants of Korcula island were collected with support of the FP6 EuroSpan project, the Royal Society, and the Chief Scientist Office of the Scottish Government.

Figures and Tables

Figure 1 Distribution of CpG methylation at four sites in HNF1A in a population. Central box represents the methylation values (the percentage of methylated cytosines at a specific site) from the lower to upper quartile (25 to 75 percentile). The middle line represents the median. The vertical line extends from the minimum to the maximum value, excluding “outside” and “far out” values which are displayed as separate points.

Figure 2 CpG methylation and expression of HNF1A in different cell lines. CpG methylation and expression of HNF1A were quantified in seven cell lines. Expression levels are normalized to expression in Huh7 cell line. Methylation levels are shown as average percentage of methylation on four analyzed CPG sites.

Figure 3 Structures of glycans separated by HP LC-HILIC analysis of the plasma glycome. Individual glycan structures in each HP LC peak were reported previously in reference 22, and are given using Oxford notation.26

Figure 4 Highly branched N-glycans on plasma proteins (GP15 and GP16) are significantly increased in individuals with HNF1A-MODY subtype of diabetes. Central box represents the values from the lower to upper quartile (25–75th percentile). The middle line represents the median. The horizontal line extends from the minimum to the maximum value, excluding “outside” and “far out” values which are displayed as separate points. For GP15 p = 1.3E-14 and for GP16 p = 1.2E-3.

Table 1 Associations between HNF1A methylation and plasma N-glycans