Leukocyte Counts Based on DNA Methylation at Individual Cytosines.

BACKGROUND: White blood cell counts are routinely measured with automated hematology analyzers, by flow cytometry, or by manual counting. Here, we introduce an alternative approach based on DNA methylation (DNAm) at individual CG dinucleotides (CpGs). METHODS: We identified candidate CpGs that were nonmethylated in specific leukocyte subsets. DNAm levels (ranging from 0% to 100%) were analyzed by pyrosequencing and implemented into deconvolution algorithms to determine the relative composition of leukocytes. For absolute quantification of cell numbers, samples were supplemented with a nonmethylated reference DNA. RESULTS: Conventional blood counts correlated with DNAm at individual CpGs for granulocytes (r = -0.91), lymphocytes (r = -0.91), monocytes (r = -0.74), natural killer (NK) cells (r = -0.30), T cells (r = -0.73), CD4+ T cells (r = -0.41), CD8+ T cells (r = -0.88), and B cells (r = -0.66). Combination of these DNAm measurements into the "Epi-Blood-Count" provided similar precision as conventional methods in various independent validation sets. The method was also applicable to blood samples that were stored at 4 °C for 7 days or at -20 °C for 3 months. Furthermore, absolute cell numbers could be determined in frozen blood samples upon addition of a reference DNA, and the results correlated with measurements of automated analyzers in fresh aliquots (r = 0.84). CONCLUSIONS: White blood cell counts can be reliably determined by site-specific DNAm analysis. This approach is applicable to very small blood volumes and frozen samples, and it allows for more standardized and cost-effective analysis in clinical application.

The Hsp90 machinery facilitates the transport of diphtheria toxin into human cells.

Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.

Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures.

Aging is reflected by highly reproducible DNA methylation (DNAm) changes that open new perspectives for estimation of chronological age in legal medicine. DNA can be harvested non-invasively from cells at the inside of a person's cheek using buccal swabs - but these specimens resemble heterogeneous mixtures of buccal epithelial cells and leukocytes with different epigenetic makeup. In this study, we have trained an age predictor based on three age-associated CpG sites (associated with the genesPDE4C, ASPA, and ITGA2B) for swab samples to reach a mean absolute deviation (MAD) between predicted and chronological age of 4.3 years in a training set and of 7.03 years in a validation set. Subsequently, the composition of buccal epithelial cells versus leukocytes was estimated by two additional CpGs (associated with the genes CD6 and SERPINB5). Results of this "Buccal-Cell-Signature" correlated with cell counts in cytological stains (R2 = 0.94). Combination of cell type-specific and age-associated CpGs into one multivariate model enabled age predictions with MADs of 5.09 years and 5.12 years in two independent validation sets. Our results demonstrate that the cellular composition in buccal swab samples can be determined by DNAm at two cell type-specific CpGs to improve epigenetic age predictions.

DNA methylation in PRDM8 is indicative for dyskeratosis congenita.

Dyskeratosis congenita (DKC) is associated with impaired telomere maintenance and with clinical features of premature aging. In this study, we analysed global DNA methylation (DNAm) profiles of DKC patients. Age-associated DNAm changes were not generally accelerated in DKC, but there were significant differences to DNAm patterns of healthy controls, particularly in CpG sites related to an internal promoter region of PR domain containing 8 (PRDM8). Notably, the same genomic region was also hypermethylated in aplastic anemia (AA) - another bone marrow failure syndrome. Site-specific analysis of DNAm level in PRDM8 with pyrosequencing and MassARRAY validated aberrant hypermethylation in 11 DKC patients and 27 AA patients. Telomere length, measured by flow-FISH, did not directly correlate with DNAm in PRDM8. Therefore the two methods may be complementary to also identify patients with still normal telomere length. In conclusion, blood of DKC patients reveals aberrant DNAm patterns, albeit age-associated DNAm patterns are not generally accelerated. Aberrant hypermethylation is particularly observed in PRDM8 and this may support identification and classification of bone marrow failure syndromes.