As a contribution to the discussion about the possible effects of ethnicity/ancestry on age estimation based on DNA methylation (DNAm) patterns, we directly compared age-associated DNAm in German and Japanese donors in one laboratory under identical conditions. DNAm was analyzed by pyrosequencing for 22 CpG sites (CpGs) in the genes PDE4C, RPA2, ELOVL2, DDO, and EDARADD in buccal mucosa samples from German and Japanese donors (N = 368 and N = 89, respectively).Twenty of these CpGs revealed a very high correlation with age and were subsequently tested for differences between German and Japanese donors aged between 10 and 65 years (N = 287 and N = 83, respectively). ANCOVA was performed by testing the Japanese samples against age- and sex-matched German subsamples (N = 83 each; extracted 500 times from the German total sample). The median p values suggest a strong evidence for significant differences (p < 0.05) at least for two CpGs (EDARADD, CpG 2, and PDE4C, CpG 2) and no differences for 11 CpGs (p > 0.3).Age prediction models based on DNAm data from all 20 CpGs from German training data did not reveal relevant differences between the Japanese test samples and German subsamples. Obviously, the high number of included “robust CpGs” prevented relevant effects of differences in DNAm at two CpGs.Nevertheless, the presented data demonstrates the need for further research regarding the impact of confounding factors on DNAm in the context of ethnicity/ancestry to ensure a high quality of age estimation. One approach may be the search for “robust” CpG markers—which requires the targeted investigation of different populations, at best by collaborative research with coordinated research strategies.
A summary of HD and non -HD cases is shown in Table 1. Kidney weights were markedly lower in HD cases than in non-HD cases. MaterialsStandards of o -, m-, and p-cresol were purchased from Wako Pure Chemical Industries (Tokyo, Japan). The deuterated internal standard (IS), p -cresol-d8, was obtained from C/D/N Isotopes Inc. (Quebec, Canada). NaCl, MgSO4, ethyl acetate, acetic anhydride, and acetyl chloride were also obtained from Wako. BSTFA + TMCS (99 : 1, v/v, 100 µL) were purchased from Supelco (Bellefonte, USA). n-Propyl acetate, methanol, pyridine, and heptane were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Concentrated sulfuric acid was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The standard of creatinine was purchased from Katayama Chemical, Inc. (Osaka, Japan). The deuterated internal standard (IS), creatinine -d3, was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). Amicon ! ! Ultra -4 centrifugal filter units were purchased from Merck KGaA (Darmstadt, Germany). Analysis of p-cresol Preparation of case samplesBlood, urine, and organ samples were prepared according to the following methods. A 0.1 -mL aliquot of sample blood and 0.9 mL of distilled water were mixed. A 0.01 -mL aliquot of sample urine and 0.99 mL of distilled water were mixed. A 0.1-g sample of organ tissue was homogenized in a bead crusher with 1 mL of distilled water. Pretreatment before extractionAn outline of the procedure used is showed in Figure 1. "Free p-cresol" is non-protein-bound and unconjugated p-cresol (F p-cresol). "Conjugated p -cresol" is non-protein-binding conjugated p-cresol (C p-cresol). "Total p -cresol" includes free p -cresol, protein-bound conjugated p-cresol, and conjugated p-cresol.
: The erythematous patches and vesicles that are observed in coma patients, usually from an overdose of medication, are known as coma blisters. However, it is unknown whether the degenerated sweat gland is a necrosis or apoptosis. We immunohistochemically examined such skin lesions to investigate the characteristics and pathogenesis of the coma blister. Skin lesions were obtained from a forensic autopsy case, a woman in her thirties, of caffeine intoxication. Those lesions were observed in the left femoral, the lower left thigh, and the right knee. Histologically, the skin lesions showed that the keratinocytes had necrosed and the epidermis was thin in some areas. Eccrine sweat gland degeneration was observed. Obvious inflammatory cell infiltrations were not detected. Immunohistochemically, we stained each skin lesion against CD3, CD8, CD45RO, cytokeratin, 70 kD heat shock protein, ubiquitin, 150 kD oxygen regulated protein, and caspase-cleaved keratin 18 neo-epitope M30. They were also stained with an in situ apoptosis detection kit. Degenerated sweat glands featured CD45RO and M30 immunoreactivity. Immunohistochemical staining for CD45RO, CK-L, and M30 might be useful to observe sweat gland degeneration in the coma blister. Therefore, the apoptosis might be related to coma blisters and sweat gland degenerations. J. Med. Invest. 60 : 256-261, August, 2013
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