Analysis of O-glycoforms of the IgA1 hinge region by sequential deglycosylation.

A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine with ß1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O-glycans play a key role in IgAN pathogenesis. The clustered and variable O-glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O-glycoform analysis. We combined an automated quantitative analysis of the HR O-glycopeptide profiles with sequential deglycosylation to remove all but Gd O-glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O-glycans were detected; nine glycopeptides carried up to three Gd O-glycans. Sites with Gd O-glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T236, followed by S230, T233, T228, and S232. The new workflow for quantitative profiling of IgA1 HR O-glycoforms with site-specific resolution will enable identification of pathogenic IgA1 HR O-glycoforms in IgAN.

Genome-wide DNA methylation profile of early-onset endometrial cancer: its correlation with genetic aberrations and comparison with late-onset endometrial cancer.

The present study was performed to clarify the significance of DNA methylation alterations during endometrial carcinogenesis. Genome-wide DNA methylation analysis and targeted sequencing of tumor-related genes were performed using the Infinium MethylationEPIC BeadChip and the Ion AmpliSeq Cancer Hotspot Panel v2, respectively, for 31 samples of normal control endometrial tissue from patients without endometrial cancer and 81 samples of endometrial cancer tissue. Principal component analysis revealed that tumor samples had a DNA methylation profile distinct from that of control samples. Gene Ontology enrichment analysis revealed significant differences of DNA methylation at 1034 CpG sites between early-onset endometrioid endometrial cancer (EE) tissue (patients aged ≤40 years) and late-onset endometrioid endometrial cancer (LE) tissue, which were accumulated among 'transcriptional factors'. Mutations of the CTNNB1 gene or DNA methylation alterations of genes participating in Wnt signaling were frequent in EEs, whereas genetic and epigenetic alterations of fibroblast growth factor signaling genes were observed in LEs. Unsupervised hierarchical clustering grouped EE samples in Cluster EA (n = 22) and samples in Cluster EB (n = 12). Clinicopathologically less aggressive tumors tended to be accumulated in Cluster EB, and DNA methylation levels of 18 genes including HOXA9, HOXD10 and SOX11 were associated with differences in such aggressiveness between the two clusters. We identified 11 marker CpG sites that discriminated EB samples from EA samples with 100% sensitivity and specificity. These data indicate that genetically and epigenetically different pathways may participate in the development of EEs and LEs, and that DNA methylation profiling may help predict tumors that are less aggressive and amenable to fertility preservation treatment.