Aberrant DNA methylation is responsible for the epigenetic silencing of genes associated with tumourigenesis and progression of cancer. In this study, we assessed the methylation status of eight genes in 49 snap-frozen primary breast tumours. Epigenetic alterations of 8 genes were analysed with methylation-specific polymerase chain reaction (MS-PCR) (DCR1, DAPK1, RASSF1A and DCR2) or methylation-sensitive high-resolution melting analysis (MS-HRM) (APC, MGMT, GSTP1 and PTEN). MS-HRM performance was validated by bisulfite pyrosequencing regarding the methylation levels of MGMT. Promoter methylation was observed in APC 54.34%, 40.4% DCR1, 37.5% DAPK1, 33.3% RASSF1A, 22.44% MGMT, 16.6% GSTP1, 6% PTEN and 0% DCR2 promoters, respectively. Interestingly, 37 out of 49 cases (75.5%) displayed aberrant promoter methylation in at least one gene. An association of MGMT promoter methylation with age and tumour grade was recorded. Moreover, a correlation with advanced T-category was elicited for GSTP1, RASSF1 and DAPK1 promoter methylation. Finally, concurrent methylation of several genes showed a marginal statistical relationship with N-category. We conclude that APC, DCR1, DAPK1 and RASSF1A promoter methylation represents a common event in breast cancer tumourigenesis. Our results suggest that GSTP1, RASSF1, DAPK1 and MGMT may be implicated in the acquisition of a more aggressive phenotype in breast cancer.

Folic acid is an essential component of 1‐carbon metabolism, which generates methyl groups for DNA methylation. Disruption of genomic imprinting leads to biallelic expression which may affect disease susceptibility possibly reflected in high levels of S ‐adenosyl‐homocysteine (SAH) and low levels of S ‐adenosyl‐methionine (SAM). We investigated the association between folic acid supplementation during pregnancy and loss of imprinting (LOI) of IGF2 and H19 genes in placentas and cord blood of 90 mother–child dyads in association with the methylenetetrahydrofolate reductase ( MTHFR ) genotype. Pyrosequencing was used to evaluate deviation from monoallelic expression among 47 placentas heterozygous for H19 and 37 placentas and cord blood tissues heterozygous for IGF2 and H19 methylation levels of 48 placentas. We detected relaxation of imprinting (ROI) and LOI of H19 in placentas not associated with differences in methylation levels of the H19ICR. Placentas retained monoallelic allele‐specific gene expression of IGF2 , but 32.4% of cord blood samples displayed LOI of IGF2 and 10.8% showed ROI. High SAH levels were significantly associated with low H19 methylation. An interesting positive association between SAM/SAH ratio and high H19 methylation levels was detected among infants with low B ₁₂ levels. Our data suggest profound differences in regulation of imprinting in placenta and cord blood; a lack of correlation of the methylome, transcriptome, and proteome; and a complex regulatory feedback network between free methyl groups and genomic imprinting at birth. —Tserga, A., Binder, A. M., Michels, K. B. Impact of folic acid intake during pregnancy on genomic imprinting of IGF2 / H19 and 1‐carbon metabolism. FASEB J. 31, 5149–5158 (2017). www.fasebj.org

Defective complement activation has been associated with various types of kidney disease. This led to the hypothesis that specific urine complement fragments may be associated with kidney disease etiologies, and disease progression may be reflected by changes in these complement fragments. We investigated the occurrence of complement fragments in urine, their association with kidney function and disease etiology in 16,027 subjects, using mass spectrometry based peptidomics data from the Human Urinary Proteome/Peptidome Database. Twenty-three different urinary peptides originating from complement proteins C3, C4 and factor B (CFB) could be identified. Most C3-derived peptides showed inverse association with estimated glomerular filtration rate (eGFR), while the majority of peptides derived from CFB demonstrated positive association with eGFR. Several peptides derived from the complement proteins C3, C4 and CFB were found significantly associated with specific kidney disease etiologies. These peptides may depict disease-specific complement activation and could serve as non-invasive biomarkers to support development of complement interventions through assessing complement activity for patients’ stratification and monitoring of drug impact. Further investigation of these complement peptides may provide additional insight into disease pathophysiology and could possibly guide therapeutic decisions, especially when targeting complement factors.

Diabetic kidney disease (DKD) is characterized by histological changes including fibrosis and inflammation. Evidence supports that DKD is mediated by the innate immune system and more specifically by the complement system. Using Ins2Akita T1D diabetic mice, we studied the connection between the complement cascade, inflammation, and fibrosis in early DKD. Data were extracted from a previously published quantitative-mass-spectrometry-based proteomics analysis of kidney glomeruli of 2 (early DKD) and 4 months (moderately advanced DKD)-old Ins2Akita mice and their controls A Spearman rho correlation analysis of complement- versus inflammation- and fibrosis-related protein expression was performed. A cross-omics validation of the correlation analyses’ results was performed using public-domain transcriptomics datasets (Nephroseq). Tissue sections from 43 patients with DKD were analyzed using immunofluorescence. Among the differentially expressed proteins, the complement cascade proteins C3, C4B, and IGHM were significantly increased in both early and later stages of DKD. Inflammation-related proteins were mainly upregulated in early DKD, and fibrotic proteins were induced in moderately advanced stages of DKD. The abundance of complement proteins with fibrosis- and inflammation-related proteins was mostly positively correlated in early stages of DKD. This was confirmed in seven additional human and mouse transcriptomics DKD datasets. Moreover, C3 and IGHM mRNA levels were found to be negatively correlated with the estimated glomerular filtration rate (range for C3 rs = −0.58 to −0.842 and range for IGHM rs = −0.6 to −0.74) in these datasets. Immunohistology of human kidney biopsies revealed that C3, C1q, and IGM proteins were induced in patients with DKD and were correlated with fibrosis and inflammation. Our study shows for the first time the potential activation of the complement cascade associated with inflammation-mediated kidney fibrosis in the Ins2Akita T1D mouse model. Our findings could provide new perspectives for the treatment of early DKD as well as support the use of Ins2Akita T1D in pre-clinical studies.