DNA methylation-based age estimation and quantification of the degradation levels of bisulfite-converted DNA.

DNA methylation modifications are known to influence epigenetic phenomena and have been a focus of forensic science research for some time. Degraded DNA after bisulfite treatment is widely used in DNA methylation analysis. In this study, we analyzed methylation levels at 12 CpG sites of four selected genomic regions by pyrosequencing after bisulfite treatment. DNA was extracted from buccal swab samples collected from 102 Japanese individuals who were 21-77 years old. We also developed a simple method to quantify the degradation levels of bisulfite-converted DNA by real-time PCR, and evaluated the effect of DNA degradation on age estimation. We found that the methylation levels and chronological ages were highly correlated in the four selected regions, and the mean absolute deviation (MAD) between chronological and estimated ages was low at 3.88 years. These results indicated that pyrosequencing analysis at the 12 CpGs was useful for age estimation in the Japanese population. To develop a sensitive quantification method, we analyzed the amplification efficiency of short and long fragments from 10 regions by real-time PCR. The amplification efficiency was highest for CCDC102B, and the degradation levels of bisulfite-converted DNA for the 102 samples were categorized as moderately or heavily degraded. For the younger age groups (20-49 years), the MADs were lower for moderately degraded DNA than they were for heavily degraded DNA. This finding indicates that degradation levels affected the accuracy of age estimation in most of the samples; the exception was the samples from the 50-77 years age group.

Age prediction by methylation analysis of small amounts of DNA using locked nucleic acids.

Age prediction based on methylation analysis has been reported in many populations, with 10 ng or more of DNA usually required for each determination. In this study, we designed thermostable locked nucleic acid (LNA) primers by replacing a small number of DNA bases in standard DNA primers with LNAs. We evaluated these primer sets by single-base extension analysis using 10, 5, or 2 ng of DNA that would be less than template DNA used in standard methylation testing, and determined sensitivity and accuracy. We analyzed EDARADD, SST, and KLF14 genes, targeting one CpG site in each gene. Melting temperature values of most LNA primers were 4°C higher than those of DNA primers. The intensities of signals from the EDARADD and SST genes were significantly improved by the LNA primers, by 3.3 times and 1.4 times, respectively, compared with the DNA primers using 2 ng of DNA. Coefficient of variation (CV) analysis was used to assess the accuracy of the determined methylation levels. CVs were increased using small amounts of DNA, but lower CVs were detected using LNA primers. We also showed high accuracy of age prediction for 51 individuals using LNA primers. The lowest mean absolute deviation was obtained using 10 ng of DNA and was 3.88 years with the LNA primers. Thermostable PCR primers were simply designed, and the LNAs improved the sensitivity and accuracy of methylation analysis for 10 ng or less of DNA.

Multidirectional analysis for a colchicine poisoning case revealed detail cause of death and its mechanism.

The appearance of Meadow saffron (Colchicum autumnale), which contains colchicine, closely resembles Alpine leek (Allium victorialis), a popular edible wild vegetable in Northern Japan. This often results in the accidental ingestion of Meadow saffron and acute colchicine poisoning deaths. Here, we report on a case of acute colchicine poisoning death caused by the accidental ingestion of Meadow saffron. A man in his 70 s had been given wild vegetables from his neighborhood, which were then cooked and eaten by himself and his wife. Several hours later, they suffered from abdominal pain, vomiting, and diarrhea. They immediately went to the hospital and received routine treatment. While his wife made a full recovery, he died at home two days after consumption of the vegetables. A forensic autopsy was conducted five days after ingestion of the Meadow saffron and a lethal concentration (21.5 ng/mL) of colchicine in the peripheral blood sample was detected by liquid chromatography-tandem mass spectrometry. Distribution of colchicine in body fluids, tissues and gastrointestinal contents was also investigated. Some of the plants he had eaten were identified as Alpine leek or Meadow saffron by genetic analysis of his stomach contents. Histopathological examination showed apoptotic cells and cell cycle arrest at the metaphase in the intestinal crypts and testis. In addition, we detected high concentrations of endotoxins and tumor necrosis factor-α in his blood, indicating that intestinal mucosal injury induced by colchicine poisoning had allowed endotoxins to invade the body, causing death by endotoxin shock.

Discrimination of haplotype in mitochondrial DNA mixtures using LNA-mediated PCR clamping.

Locked nucleic acid (LNA) has been widely used for various genetic analyses, and has many benefits, in terms of the specificity or sensitivity of amplification, because LNA-containing primers/probes form more stable duplexes with template DNA than probes lacking LNA. Here, we developed a new method for discriminating HV1 haplotypes from mitochondrial DNA (mtDNA) mixtures by applying PCR clamping using LNA. PCR clamping is based on the selective inhibition of amplification using LNA-containing probes, which can discriminate single-nucleotide differences. Before designing probes, we selected 171 sequences with single-nucleotide variations from the HV1 region, and evaluated the specificity of LNA-containing probes for them by predicting Tm values. The differences of Tm between mismatched and exactly matched probe-template duplexes depended markedly on the type of LNA nucleotides for discriminating single-nucleotide differences, and the cytosine LNA nucleotide at the site of variations in the probes was most effective to discriminate these differences. For mixture analysis, each probe targeted one or two variations (16209C, 16217C, 16257A/16261T, 16297C/16298C, 16304C, 16362C, or 16362T) that are particularly common in the Japanese population, and seven designed probes completely inhibited the amplification of exactly matched templates. We prepared mixed samples by mixing DNA from two individuals at a ratio of 1:9, 1:4, 1:1, 4:1, or 9:1, and then performed Sanger sequencing analysis after PCR clamping with each probe. Our method distinguished each haplotype at lower ratios from two-person mixtures, and enabled sensitive detection at 12 pg of total DNA including 600 copies of mtDNA. Moreover, we analyzed three-person mixtures with representative sequences, and detected the minor haplotype of one individual present at a rate of 10% by adding two selected probes. The ability to discriminate haplotypes in mixed samples by using LNA-mediated PCR clamping indicates the potential value of mtDNA analysis in criminal investigations.

Assessment of DNA degradation of buccal cells under humid conditions and DNA repair by DOP-PCR using locked nucleic acids.

We analyzed the degradation level of DNA from buccal cells under humid conditions using quantitative PCR analysis. Gauze samples with buccal cells were incubated for up to 12 months under three different conditions (25 °C/dry, 25 °C/humid, or 40 °C/humid). The degradation was evaluated based on two degradation ratios (129:41 and 305:41 bp). DNA degraded slowly under the 25 °C/humid condition, and significant differences in the two degradation ratios were detected between 25 °C/dry and 25 °C/humid conditions after 12 months. Moreover, the degradation rapidly progressed under the 40 °C/humid condition, and the two degradation ratios in this condition were much lower than those from 25 °C/dry and 25 °C/humid conditions after a short incubation period (3 months). To evaluate the effect of DNA repair on low-copy degraded DNA, degenerate oligonucleotide-primed PCR (DOP-PCR) was performed before short tandem repeats (STR) genotyping. As a standard DOP-PCR, we used a 22-base primer with 10 degenerate sequences (5'-CTCGAGNNNNNNNNNNATGTGG-3'), and additionally designed DOP-PCR primers with 2, 4, 6, or 8 locked nucleic acids (LNAs). When slightly degraded DNA (305:41-bp ratio = 0.60) was used, DOP-PCR significantly increased the fluorescent intensity and success rate of genotyping using Identifiler and Globalfiler kits. In particular, the reaction with four LNAs produced the highest value. However, such benefits were not observed in the analysis of moderately degraded DNA (305:41-bp ratio = 0.13). Although the recovery rates of STR profiles by DOP-PCR were dependent on the degradation level of low-copy DNA, the effectiveness of DOP-PCR highlights the potential of LNA for degenerate sequences.

Paraquat toxicity is attenuated by 4-phenylbutyrate-induced phosphorylation of ERK2 via PI3K in A549 cells.

Paraquat (PQ) is a widely used herbicide in the world despite being highly toxic to humans. PQ causes fatal damage to multiple organs, especially the lungs. While oxidative stress is the main toxic mechanism of PQ, there is no established standard therapy for PQ poisoning. In this study, we investigated the cytoprotective effect of 4-phenylbutyrate (4PBA) on PQ toxicity in human lung adenocarcinoma A549 cells. Phosphorylation levels of major survival signaling kinases Akt and ERK, as well as expression levels of antioxidant enzymes catalase and superoxide dismutase 2 (SOD2) were examined. The cytoprotective mechanism of 4PBA against PQ was compared with the antioxidant reagent trolox. We demonstrated that both 4PBA and trolox attenuated PQ toxicity, but their mechanisms were different. 4PBA increased ERK2 phosphorylation levels, which could be inhibited by the PI3K inhibitor LY294002. The cytoprotective effect of 4PBA was also inhibited by LY294002. Catalase expression levels were increased by 4PBA, although this increase was not inhibited by LY294002. 4PBA did not increase SOD2 expression. Trolox did not affect phosphorylation of Akt or ERK, or the expression of antioxidant enzymes. These results suggest that 4PBA attenuated PQ cytotoxicity by ERK2 activation via PI3K. Our study may provide new findings for understanding the molecular mechanism underlying cytoprotection by 4PBA, as well as new therapeutic targets for PQ poisoning.

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.

Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids.

Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5-1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5-9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10(-22), and their combined use can provide robust information for Japanese forensics.

Brain stem hemorrhage due to cerebral amyloid angiopathy: the autopsy of a patient with Alzheimer's disease at a young age.

We report findings from an autopsy of a male in his 40s who died of a brain stem hemorrhage associated with cerebral amyloid angiopathy (CAA), senile plaques (SPs) and neurofibrillary tangles (NFTs), which are histopathological changes associated with Alzheimer's disease (AD). Our immunohistochemical study demonstrated amyloid ß (Aß) deposition in the small cerebral arteries and SPs. Although hypertension (178/132 mmHg) was detected, the subject was not treated accordingly. CAA coupled with hypertension might have caused the intracerebral hemorrhage (ICH).

Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

HRD1 levels increased by zonisamide prevented cell death and caspase-3 activation caused by endoplasmic reticulum stress in SH-SY5Y cells.

Zonisamide, which is commonly prescribed at high doses (200-400 mg/day) for the treatment of partial seizures, has recently been used at a low dose (25 mg/day) for improving parkinsonian syndrome. However, the molecular mechanisms that underlie the antiparkinsonian effects of zonisamide have not been clarified. Here we show that low micromolar concentrations of zonisamide prevented cleavage of caspase-3 and cell death in human dopaminergic SH-SY5Y neuroblastoma cells that were subjected to endoplasmic reticulum stress induced by tunicamycin or 6-hydroxydopamine. Hypodense zonisamide increased the expression levels of SEL1L, which is known to stabilize the ubiquitin ligase HRD1. Indeed, upregulation of HRD1 protein was observed. Thus, the results of this study strongly suggest that low concentrations of zonisamide inhibit neuronal cell death by increasing HRD1 protein levels in patients with Parkinson's disease. Consequently, in addition to the treatment of Parkinson's disease, the therapeutic potential of zonisamide should be considered for the treatment of several neurodegenerative disorders with pathophysiological mechanisms involving endoplasmic reticulum stress.