Aya Matsusue

Population genetics of 17 Y-chromosomal STR loci in Japanese

Haplotypes and allele frequencies of 17 Y-chromosomal short tandem repeat (Y-STR) markers were examined using the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems) in a population sample of 1166 Japanese male volunteers in 6 prefectures: Miyagi, Yamagata, Osaka, Tottori, Fukuoka, and Okinawa. A total of 1058 haplotypes were observed from 1166 males, and the most common haplotype detected in 12 males had a frequency of 1.03% and the discrimination capacity was 0.907. The R(ST) analysis showed statistically significant differences between Okinawa and the other subpopulations.

ADAM33 genetic polymorphisms and risk of atopic dermatitis among Japanese children.

OBJECTIVES ADAM33, a disintegrin and metalloproteinase 33, gene has been identified as an asthma susceptibility gene. The relationship between single nucleotide polymorphisms (SNPs) in ADAM33 and atopic dermatitis (AD) in Japanese children was examined using case-control design. METHODS Seven SNPs of ADAM33 (rs2853209, rs2787094, rs2280091, rs2280090, rs628977, rs597980, and rs528557) were analyzed in 140 AD cases and 258 controls aged 3 years. RESULTS Only rs2853209 (T>A) was significantly associated with AD risk. Sex-adjusted odds ratio (OR) for the AA versus the TT genotype was 0.55 (95% confidence interval (CI), 0.30-0.997). Consistent with the results of genotyping analysis, a haplotype carrying rs2853209 A allele was significantly associated with decreased risk of AD compared to all the other haplotypes combined (OR=0.26, 95% CI=0.08-0.69). CONCLUSION This is the first study to provide evidence for an association of the ADAM33 polymorphism with AD risk but the strength of this evidence is limited by our small sample size.

High throughput chiral analysis of urinary amphetamines by GC-MS using a short narrow-bore capillary column

We report very rapid and simultaneous chiral analysis of urinary amphetamine-type stimulants (ATSs), including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyethylamphetamine, using gas chromatography-mass spectrometry (GC-MS) with a simple procedure. A urine sample containing ATSs was subjected to extractive derivatization on a diatomaceous earth tube with trifluoroacetyl-l-prolyl chloride in a single step. The concentrated sample was analyzed by GC-MS, using a short narrow-bore capillary column (10 m × 0.1 mm i.d.) in split injection mode. All chiral isomers of the ATSs targeted in this study were chromatographically distinguishable within 5 min. By our method, ATSs in urine could be measured in the concentration range of 20–1000 ng/ml with coefficients of variation of less than 9%. Our method will be very useful for chiral analysis of ATSs in routine forensic toxicology investigations, because of its simplicity and rapidity.

Application of the drowning index to actual drowning cases

The drowning index (DI) was devised to diagnose drowning deaths, and is the weight ratio of the lungs and pleural effusion to the spleen. Among drowning (94 cases), mechanical asphyxia (47 cases), and acute cardiac (42 cases) deaths, within 2 weeks postmortem we compared six markers, the weight of each lung, pleural effusion weight, total weight of the lungs and pleural effusion, spleen weight, heart weight, and the DI. Statistical analysis revealed that the total weight was heavier, while spleen weight was lighter, and the DI was significantly larger in the drowning group (p<0.05). We examined the relation between the postmortem time and these markers. We divided 94 drowning cases into three groups according to the postmortem duration, group A (0-3 days; 43 cases), group B (3-7; 29 cases), and group C (7-14; 22 cases). The cut-off point of the DI was analyzed using the receiver operating characteristic (ROC) curve. As a result, the DI cut-off point was 14.1 in cases within two postmortem weeks. Drowning is still a difficult autopsy diagnosis, but in our experience, DI is a valuable indicator.

Molecular basis of complement factor I (CFI) polymorphism: one of two polymorphic suballeles responsible for CFI A is Japanese-specific

AbstractIsoelectric focusing has revealed that human complement factor I (CFI) is controlled by two polymorphic alleles, CFI*A and CFI*B, and a few rare variant alleles. In this study the molecular basis of the CFI polymorphism was investigated in 174 Japanese. The CFI*A was divided into two suballeles, CFI*As (R201S) and CFI*Ah (R406H). CFI*Aj, a rare variant allele originating from CFI*Ah, had an additional mutation (R502L). The distribution of these three mutations and two registered SNPs was investigated in a total of 2,471 individuals in 20 populations from various areas, and six haplotypes were observed. Haplotype H3, which is characterized by CFI*As, was found only in Far East populations: the frequencies were about 0.03 in the main island of Japan and lower than 0.01 in Okinawa and Korea. Haplotype H5, characterized by CFI*Ah, prevailed almost exclusively in East Asians and was observed at the highest frequencies in southern Chinese Han and Thais. CFI*Ah must have arisen in a southeastern part of Asia and thereafter have spread to neighboring populations.

Combination of a short middle-bore capillary column with a thicker stationary phase and a short narrow-bore separation column with a thinner stationary phase for the rapid screening of non-volatile drugs by gas chromatography–mass spectrometry

Dear Editor, An increase in requests for drug analysis has led to techniques that can analyze many samples in a short amount of time. For the purpose of enhancing the efficiency of analysis and increasing sample throughput, we looked at increasing the analysis speed of gas chromatography–mass spectroscopy (GC–MS), currently the most widely used technique for forensic drug analysis. The fast GC–MS techniques and the difficulty of their practical implementation have been reviewed in the literature [1]. Our group reported high-throughput chiral analysis of urinary amphetamines by fast GC–MS [2]. It seems very easy to establish a fast GC–MS method with a short narrow-bore capillary GC column and fast temperature programming, if using a modern GC–MS instrument that enables the rapid scanning of mass spectra and powerful pumping to maintain vacuum against the relatively large gas inflow into the ionization chamber. There are short narrow-bore capillary GC columns for fast GC–MS in the presence [3–5] and absence [2, 6, 7] of retention gaps, which are sometimes useful for obtaining narrow peak widths and symmetrical peak shapes. The retention gap is usually a short and deactivated capillary column without stationary phase. In this communication, we have tried to create a drug screening method by fast GC–MS using a short middle-bore capillary column with a thicker stationary phase in place of the inactive retention gap and a short narrow-bore capillary column. Our results showed that more than 20 drugs in whole blood could be screened within 4.1 min of GC–MS analysis. In this study, we mainly selected 30 psychotropic drugs [8, 9] as target compounds for analysis. Pure powder standards of fenfluramine-HCl, imipramine, diphenidolHCl, clomipramine-HCl, diazepam, chlorpromazine-HCl, nordiazepam, and trazodone were purchased from Sigma Aldrich (St. Louis, MO, USA); diphenhydramine-salicylate and nortriptyline-HBr from Wako (Osaka, Japan); and ethenzamide from Nacalai Tesque (Kyoto, Japan). Other drugs in the powder form were obtained from the corresponding pharmaceutical companies. Lidocaine-d10 was purchased from C/D/N Isotopes (Québec, Canada); diazepam-d5 and nordiazepam-d5 (both 1 mg/ml methanol solution) from Cerilliant (Round Rock, TX, USA). Caffeine-d3 (7-methyl-d3-theophylline) and 7-ethyltheophylline were synthesized in our laboratory. Other common reagents were of the highest purity commercially available. Blank whole blood was obtained from a healthy volunteer under permission; it was stored at -20 C until use. Methanolic stock solutions were made of the powder standards at a concentration of 1.0 mg/ml. A working stock solution of the 30 drug standards listed in Table 1 was made up in n-propyl acetate at a concentration of 20 lg/ml. Calibrators were prepared by adding enough of the standard working stock solution to 1 g of blank blood to yield the final concentrations of 0.10, 0.25, 0.50, 1.0, and 5.0 lg/ml in 16 9 125-mm screw cap tubes. A working internal standard (IS) solution containing caffeine-d3 (100 lg/ml), This article is for the special issue TIAFT2012 edited by Osamu Suzuki.

Distinct breakpoints in two cases with deletion in the Yp11.2 region in Japanese population

The amelogenin gene on the Y chromosome (AMELY) is a homolog of the X chromosome amelogenin gene (AMELX), and the marker is employed for sexing in forensic casework. Deletion of the sequences in the Yp11.2 region containing the AMELY locus has been found in males from various ethnic populations. Two cases of AMELY null males found in the Japanese population had different Y haplogroups and deletion mapping. Proximal and distal breakpoints of a sample of haplogroup D2* were located in TSPYA and TSPYB arrays, respectively, suggesting that the deletion mechanism was non-allelic homologous recombination (NAHR). On the other hand, a sample of haplogroup O3a3c* had the distal breakpoint in the TSPYB array and the proximal breakpoint at position 7.94 Mb, not in the TSPYA array. The likely deletion mechanism is non-homologous end-joining. High-resolution STS mapping in the TSPYB array showed the distal breakpoints differed according to the haplogroups. The deletion length was estimated as 3.1–3.7 Mb and 1.6–1.7 Mb for the sample of haplogroup D2* and O3a3c*, respectively. These deletion events should have occurred independently.

Solid-phase microextraction for amphetamines in solid tissues: washing the homogenates with ethyl ether enables their measurements by GC-MS after heptafluorobutyryl derivatization

which the crude homogenate supernatant is washed with diethyl ether. D-Amphetamine (D-AMP), D-methamphetamine (DMAMP), L-AMP-d3 (IS-1), L-MAMP-d6 (IS-2), heptafl uorobutyryl chloride (HFB-Cl), and other reagents used in this study were the same as described in our previous reports [1,5]. Chicken liver was purchased from a local meat store for use in constructing a calibration curve. In an actual autopsy case, the liver, kidney, spleen, lung, brain, and blood were collected from a cadaver of a 29-year-old woman for analysis of amphetamines. The procedure established for analysis of amphetamines in solid tissue samples was as follows. Solid tissue (1.5 g) was incubated at 70°C for 2 h after the addition of 100 ng each of the two ISs and 5 ml of 0.1 M hydrochloric acid (this incubation under acid conditions softened the tissue and made it more suitable for homogenization), and then homogenized with a Polytron homogenizer. The crude supernatant fraction was obtained after centrifuging at 3000 rpm for 25 min. Two milliliters of the fraction was washed with 10 ml diethyl ether by shaking vigorously in a test tube or glassstoppered centrifuge tube. After standing for a few minutes, 1.0 ml of the aqueous phase was placed in a headspace vial, to which 0.5 g of sodium chloride, 0.2 ml of 1 M sodium hydroxide solution, and 0.01 ml of HFBCl were added. The septum-capped vial was shaken gently and set on a tray of an automated sampler for headspace-SPME (AOC-5000 Auto Injector, Shimadzu, Kyoto, Japan) followed by GC-MS analysis. The GC-MS analysis was performed on a QP-5000 instrument (Shimadzu) operated by GCMS-Solution (operation and data analysis system) using an Rtx-5 capillary column (10 m × 0.18 mm i.d., fi lm thickness 0.20 μm; Restek, Bellefonte, PA, USA). The GC oven Received: 1 October 2008 / Accepted: 17 October 2008 / Published online: 21 January 2009

GC-PCI-MS/MS and LC-ESI-MS/MS databases for the detection of 104 psychotropic compounds (synthetic cannabinoids, synthetic cathinones, phenethylamine derivatives).

Designer psychotropic compounds continue to be a major problem in Japan and all around the world. Electron impact mass spectrometry (GC-EI-MS) and liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data on these compounds have been widely reported. In this report, we present a detection method that has been rarely utilized to analyze these types of compounds, gas chromatography with positive chemical ionization and tandem mass spectrometry (GC-PCI-MS/MS). We report on the development of GC-PCI-MS/MS and LC-ESI-MS/MS databases of 104 psychotropic compounds, including 32 cannabinoid derivatives, 29 cathinone derivatives, 34 phenethylamine derivatives, and several other designer compounds. Using this database, we were able to detect 5 psychotropic compounds in an actual forensic autopsy case. If GC-PCI-MS/MS is used together with the more established methods of GC-EI-MS and LC-ESI-MS/MS, we believe the forensic toxicology community could be better prepared to deal with the challenges of these ever-changing compounds.

Development of a preparation method to produce a single sample that can be applied to both LC-MS/MS and GC-MS for the screening of postmortem specimens

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC-MS and LC-MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC-MS and LC-MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratorys daily routine for screening many different compounds from postmortem samples.