Detection of deletion/insertion polymorphism profiles from single human hair shafts.

BACKGROUND: Hair is a frequently encountered biological evidence in personal identification. The amount of nuclear DNA that can be extracted from a single strand of rootless hair is most limited, making the detection of short tandem repeat (STR) polymorphisms difficult. To overcome these limitations, deletion/insertion polymorphisms (DIP) as a new type of genetic marker have shown their benefits in detecting low-copy-number DNA. The Investigator DIPplex kit contains 30 biallelic autosomal DIP and amelogenin. The analysis of DIPs combines the advantages of both STR and single nucleotide polymorphism analyses. Thus, this study aimed to detect the DIP distribution of individual hair shafts from individuals. METHODS AND RESULTS: DNA was extracted from the shaft of fresh, aged, and shed hair. After DNA was evaluated, the DIP profiles were detected by capillary electrophoresis. The results indicated that the amount of DNA extracted from hair roots was much higher than that from the hair shafts in the same individual for all samples. The degradation index values of DNA from the aged hair shafts were highest. It is classified to be "mildly degraded." Compared with their hair roots, the full DIP profiles were detected for fresh hair, 70% for aged hair, and 92% for shed hair. Contrarily, except for fresh hair shafts, only three STR loci of the aged and shed strands of hair could be genotyped using AmpFlSTR MiniFiler PCR Amplification Kit. CONCLUSIONS: These results indicate that the detection of DIP profile is an effective tool for personal identification from hair shafts, including aged hair.

A Case-Control Study of the APELA Gene and Hypertensive Disorders of Pregnancy.

Hypertensive disorders of pregnancy (HDPs) are believed to comprise a group of multifactorial genetic diseases. Recently, it was reported that APELA-knockout mice exhibited HDP-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. The aim of the present study is to determine the associations between HDPs and single-nucleotide variants or haplotypes in the human APELA gene through a case-control study. The subjects were 196 pregnant women with HDPs and a control group of 254 women without HDPs. Six single-nucleotide variants (rs2068792, rs13120303, rs4541465, rs13152225, rs78639146, and rs67448487) were selected from the APELA gene region. Although there were no significant differences for each single-nucleotide polymorphism in the case-control study, the frequency of the T-A haplotypes rs4541465-rs67448487 was significantly higher in the HDP group, especially in those with gestational hypertension, than in the control group. The results suggest that the APELA gene may be a disease-susceptibility gene for HDP.

Genotyping DNA isolated from UV irradiated human bloodstains using whole genome amplification.

Analysis of DNA polymorphisms are the primary technique used for personal identification in forensic cases. However, DNA samples collected as evidence from crime scenes are usually degraded by environmental, physical, and chemical factors, which may interfere with the PCR analysis and, consequently, personal identification. Whole genome amplification (WGA) is a useful method to amplify genomic DNA from samples containing low quantity and poor quality of DNA, and it approach that shows promise to overcome the limited small fragments based upon random fragmentation by universal priming sites. In this study, we describe the use of WGA to genotype 15 short tandem repeat (STR) loci from dried blood samples irradiated with different types of ultraviolet (UV) light (UVA, UVB, and UVC). The result showed that UVC was the most damaging to DNA, followed by UVB and UVA. Samples exposed to UVA could be genotyped for all STR loci with or without WGA. For UVB and UVC irradiated blood samples, a greater number of STR loci could be genotyped after WGA. Although it hard to amplified a few higher molecular weight alleles, overall, the WGA method was useful in genotyping template DNA of poor quality but low quantity.

Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method.

Human nail is an important forensic material for parental testing and individual identification in large-scale disasters. Detection of STR polymorphism from hard tissues generally requires DNA purification, which is technically complicated and time consuming. In the present study, we attempted to detect STR polymorphisms from untreated human nail samples by direct PCR amplification method using the primer mixture supplied with the GenePrint® SilverSTR® III System or the AmpFℓSTR® Identifiler® PCR Amplification Kit, and Tks Gflex DNA polymerase known to be effective for amplification from crude samples. A nail fragment measuring approximately 1.5 mm in breadth and 0.5 mm in length was placed directly into a PCR tube, and various PCR conditions were tested. The PCR products were analyzed by denaturing acrylamide gel electrophoresis or CE. Multiple STR polymorphisms were detected successfully. This method that detects STR polymorphisms not only from fresh human fingernails, but also from old nail fragments stored at room temperature for up to 10 years is expected to become a novel DNA analytical method in forensic medicine and genetic studies.

DNA repair and STR PCR amplification from damaged DNA of human bloodstains.

Detection and identification of DNA structure from aged and damaged biological materials such as bloodstain are important for human genetic study and individual identification. However, after a long period of storage, the DNA structure of biological samples is degraded to various degrees depending on several factors including environmental condition. In this study, human bloodstains that have been stored at room temperature for one to 39 years were used to represent damaged biological samples. The numbers of apurinic/apyrimidinic sites (AP sites) were investigated by the DNA Damage Quantification Kit to evaluate the lesions in DNA structure. The damaged DNA from the stored human bloodstains was repaired using seven DNA repair enzymes. As DNA genetic marker, short tandem repeat (STR) genotypes were amplified using the non-repaired and repaired DNA preparations from the stored bloodstains. The results indicated that the number of AP sites increased as the storage time increased. While only 2 to 6 STR loci were detected in the damaged DNA of bloodstains stored for over 30 years, after DNA repair all the genotypes in the STR system could be analyzed even from bloodstains that had been stored for the longest period.

Genetic analysis of twelve X-chromosomal STRs in Japanese and Chinese populations.

We investigated the genetic markers of twelve X-STR loci in 670 healthy, unrelated Japanese (438 men and 232 women) from Tokyo and 488 Chinese (263 men and 225 women) from Shenyang, using the Investigator Argus X-12 kit. Allele and haplotype analyses of twelve X-STRs clustered into four linkage groups indicated that they are highly informative for forensic applications in Japanese and Chinese populations. Hardy-Weinberg equilibrium tests demonstrated no significant deviations in the two populations. Among the four closely linked X-STR trios, some haplotype unique to Japanese or Chinese population were detected. Haplotype diversity for each linkage group ranged from 0.9861 to 0.9968, showing high values in each of the study populations. The genetic distances between populations based on the 12 X-STR loci and the phylogenetic tree revealed long genetic distances between Asian and Caucasian populations and between Asian and African population (Moroccan). These results suggest that the twelve X-STR loci will contribute to forensic casework in Japanese and Chinese populations.

Molecularly imprinted solid-phase extraction for the selective determination of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs in human whole blood by gas chromatography-mass spectrometry.

A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 µL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry.

Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry.

Dextromethorphan was extracted from human plasma samples (100 µL) using MonoTip C(18) tips, which are packed with C(18)-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200 µL of distilled water and 50 µL of 1 mol/L glycine-sodium hydroxide buffer (pH 10). The mixture was extracted to the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.5 µm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320 ng/mL of plasma, and the limit of detection was 1.25 ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug.

Evaluation and SNP typing of DNA from ultraviolet-irradiated human bloodstains using TaqMan assay.

When detecting DNA profiles from forensic materials, it is pivotal to know the extent of degradation and which DNA marker can be genotyped. Ultraviolet (UV) is one of the common external factors that causes DNA damage, through which, an attempt to reveal cardinal genetic information can be made. In this study, after irradiation with three different UV wavelengths, UV-damaged DNA in the bloodstains was analyzed with long and short TaqMan assays using real-time PCR. In addition, both short tandem repeat (STR) profiles and single nucleotide polymorphisms (SNPs) from the damaged DNA at different stages of UV exposure were also assessed. With increasing in UV irradiation cycles, there was a delay of the amplification curves accompanied with a decrease in the DNA amounts collected. Despite the amplification of STR genotype was not altered after 75 cycles of UVC irradiation, all 12 SNP loci could still be detected. Furthermore, a short-assay line was detected in the absence of an amplification of the evaluation curve. The results indicate that, although the DNA template might not be useful and suitable for analysis of STR profile, this approach is of some values in detecting SNPs.

Direct and rapid PCR amplification using digested tissues for the diagnosis of drowning.

We developed a direct and rapid method for the diagnosis of death by drowning by PCR amplification of phytoplankton DNA using human tissues. The primers were designed based on the DNA sequence of the 16S ribosomal RNA gene (16S rDNA) of Cyanobacterium. Samples of lung, liver and kidney tissues were collected from 53 autopsied individuals diagnosed as death by drowning. Without DNA extraction, the tissue fragments were incubated directly in a digest buffer developed in this study, for 20 min. Using 1 microL of the tissue digest solution in PCR, the 16S rDNA was successfully amplified. The specific 16S rDNA fragment was identified from the standard picoplankton Euglena gracilis, the tissues of bodies died from drowning and water samples from the drowning scenes. On the other hand, no PCR products were found in the tissues of individuals who died from causes other than drowning. Various quantities of tissue weighing 1, 5, 10, 20 and 30 mg were tested, and the PCR amplification detected the specific 16S rDNA fragment from all the quantities of tissue tested. This method was found to be more reliable, sensitive, specific and rapid when compared to the conventional diagnosis of death by drowning using the diatom test by acid digestion method.

Automated on-line in-tube solid-phase microextraction coupled with HPLC/MS/MS for the determination of butyrophenone derivatives in human plasma.

This paper describes a fully automated on-line method combining in-tube solid-phase microextraction (SPME) in which sample clean-up and enrichment are conducted through an open tubular fused-silica capillary column and high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) detection for the determination of six butyrophenone derivatives (moperone, floropipamide, haloperidol, spiroperidol, bromperidol, and pimozide) in human plasma samples. The six butyrophenones were extracted by repeatedly aspirating and dispensing plasma sample solutions on a DB-17 capillary column (60 cm x 0.32 mm i.d., film thickness 0.25 microm). The analytes retained on the inner surface of the capillary column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. Extraction efficiencies ranged from 12.7% to 31.8% for moperone, spiroperidol, and pimozide, and from 1.08% to 4.86% for floropipamide, haloperidol, and bromperidol. The regression equations for all compounds showed excellent linearity, ranging from 0.05 to 50 ng/0.1 mL of plasma, except for moperone and spiroperidol (0.01 to 50 ng/0.1 mL). The limits of detection and quantification in plasma for each drug were 0.03-0.2 and 0.1-0.5 ng/mL, respectively. The intra- and inter-day coefficients of variation for all compounds in plasma were not greater than 13.7%.

Real-time PCR assay for the detection of picoplankton DNA distribution in the tissues of drowned rabbits.

The detection of plankton DNA is one of the important methods for the diagnosis of drowning from postmortem tissues. This study investigated the quantities of picoplankton (Cyanobacteria) DNA in the lung, liver, kidney tissues and blood of drowned and non-drowned rabbits, and the sensitivity of detection of picoplankton DNA by polymerase chain reaction (PCR) detect for the diagnosis of death from drowning. For this purpose, the DNA of the 16S ribosomal RNA gene of picoplankton was quantitatively assayed from the tissues of drowned and non-drowned rabbits immersed in water after death. Each of the liver, kidney and lung tissues and blood were obtained from drowned and non-drowned rabbits. Picoplankton DNA in the tissues was extracted using the DNeasy® Blood & Tissue kit to determine the yield of picoplankton DNA from each tissue. TaqMan real-time PCR was performed for quantitative analysis of picoplankton DNA. Target DNA was detected in the liver, kidney and lung samples obtained from the drowned rabbits, while no picoplankton DNA was detected in the non-drowned rabbit tissues (except in lung samples). The results verified that direct PCR for the detection of picoplankton DNA is useful for the diagnosis of drowning. Although we observed seasonal changes in the quantity of picoplankton in river water, we were able to detect DNA from various organs of drowned bodies during the season when picoplankton were not the most abundant.

An autopsy case of miliary tuberculosis in a young adult.

A 23-year-old woman who had worked as a hostess at a nightclub was found dead in her house. The cause of death was diagnosed as miliary tuberculosis from the findings of medico-legal autopsy. Recently, tuberculosis (TB) has re-emerged as a health problem due to recurrence in the aged, and infections among health care workers and young adults like the present case. Currently, the common source of TB transmission is recurrence in the aged, but global migration, difficulty to achieve permanent immunity by BCG vaccination, immunodeficiency such as HIV infection, and drug abuse and/or sexual intercourse are also thought to be associated with tuberculosis in young adults. Forensic pathologists should be aware of such connections with TB, and should take care not to become mediators of TB infections.

A new method for quantitative determination of dimemorfan in human plasma using monolithic silica solid-phase extraction tips.

Dimemorfan was extracted from human plasma samples (100 µL) using MonoTip C(18) tips, which were packed with a C(18)-bonded monolithic silica gel attached to the inside of the tip. The samples, which contained dimemorfan and trimeprazine as an internal standard (IS), were mixed with 300 µL of distilled water and 50 µL of 1M glycine-sodium hydroxide buffer (pH 10). The mixture was extracted onto the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.25 µm) gave adequate separation of the dimemorfan, IS, and impurities. The recoveries of dimemorfan and the IS spiked into plasma were ≥83%. The regression equation for dimemorfan showed excellent linearity from 0.25 to 32.0 ng/100 µL of plasma, and the limit of detection was 0.125 ng/100 µL of plasma. The maximum intra-day and inter-day relative standard deviations were 13%, while accuracy ranged from 88% to 105%. Dimemorfan was stable for at least 12 h at 4°C, 4 weeks at -80°C, and three freeze-thaw cycles in plasma. This new method is expected to have application as a pretreatment for the rapid, simple, and quantitative determination of dimemorfan in plasma samples.

Quantitative determination of phenothiazine derivatives in human plasma using monolithic silica solid-phase extraction tips and gas chromatography-mass spectrometry.

Solid-phase extraction (SPE) using micropipette tips is a useful technique to prepare samples prior to mass spectrometry. However, most commercial SPE tips have loading capacities that are insufficient for quantitative determination. In this paper, we describe a rapid method for quantitative microanalysis of five phenothiazine derivatives, chlorpromazine, levomepromazine, promazine, promethazine and trimeprazine, using a recently introduced C(18) monolithic silica SPE tip, the MonoTip C(18), for extraction from human plasma. The drugs could be extracted within 5 min from 0.1-mL plasma samples, eluted with methanol, and the eluate injected directly into a gas chromatograph prior to mass spectrometry analysis. Only 0.7 mL of solvent was required for each step of the extraction process. The recoveries of the five phenothiazines spiked into plasma were 91-95% and the limits of quantification for each drug were between 0.25 and 2.0 ng/0.1 mL. The maximum intra- and inter-day coefficient of variation was 11%. The validated method was successfully used to quantify the plasma concentration of levemepromazine in a human subject after oral administration of the drug. This new method is expected to have wide applications as a pretreatment for the rapid, quantitative determination of drug concentrations in plasma samples.

Genetic polymorphisms of eight X-chromosomal STR loci in the population of Japanese.

The genetic polymorphisms of eight X-chromosomal short tandem repeats (STR) DXS10135, DXS8378, DXS7132, DXS10074, HPRTB, DXS10101, DXS10134 and DXS7423 were analyzed in a sample of 492 unrelated males (283) and females (209) from the Japanese population. Multiplex PCR amplification was performed using the Mentype Argus X-8 PCR amplification kit. The haplotype frequencies within the four linkage groups were studied for the 283 examined Japanese males. Allele frequencies of eight X-STR loci were calculated separately for males and females, and exact tests demonstrated no significant deviations from Hardy-Weinberg equilibrium. Several microvariant and rare alleles were observed, and forensic efficiency parameters were calculated. The combined powers of discrimination of the loci in men and women were 0.999995 and 0.9999999999988, respectively.

A case of personal identification due to detection of rare DNA types from seminal stain.

Following a rape incident in an apartment in Japan, we were requested to perform a DNA analysis on a body fluid stain left on a bath towel to determine whether it could be attributed to the suspect. The acid phosphatase and prostatic-specific antigen tests confirmed it to be a seminal stain. Based on the DNA analysis by autosomal and Y-chromosome short tandem repeat (STR) systems, no inconsistency was found with the profile of the suspect with African ancestry. In this case, allele 21 of DYS390 at the Y-STR locus was examined, as it is reported to have a distinctly lower frequency in the Japanese population. Furthermore, the haplotype combinations of Y-STR at the DYS389I, DYS389II and DYS390 loci are powerful for personal identification, as these have not yet been found in the Japanese population.