Masato Nakatome

Simultaneous detection of multiple STR loci on sex chromosomes for forensic testing of sex and identity

The forensic usefulness of X and Y chromosomal STR loci has recently been demonstrated. One quadruplex-PCR, using 2 X- and 2 Y-STRs (STRX1/HPRTB and DYS390/ DYS393), and 2 duplex-PCRs, each using an X- and a Y-STR (ARA/DYS390 and ARA/DYS393), and detection of PCR products by using an automated DNA sequencer are reported herein. This approach allows us to determine not only the sex of the donor of a sample, but also the X- and/or Y-STR genotypes of the sample. A male biological specimen yields 4 amplified products in quadruplex-PCR and 2 amplified fragments in duplex-PCRs, whereas a female biological specimen yields only 2 amplified fragments of X-STR in quadruplex-PCR and one fragment, also of X-STR, in duplex-PCRs. Our study thus provides useful information for many activities in forensic practice, such as identity testing, paternity testing, especially of deficiency cases, compilation of population data, and sex determination of a biological sample from a single PCR.

Detection of cardiomyocyte apoptosis in forensic autopsy cases

Abstract The purpose of the present study was to determine reliable parameters for the detection of apoptotic cells for use as a diagnostic marker during the early stage of acute myocardial infarction (AMI) in forensic autopsy cases. Myocardial tissues taken from forensic autopsy cases were examined by immunohistochemical and molecular-biological methods using the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labelling (TUNEL) and the DNA laddering methods. In cases of AMI with a time period between 2 h from onset to death and 20 h post-mortem time, the nuclei of cardiomyocytes were stained positive with the TUNEL method and DNA fragmentation of myocardial cells was detected by agarose gel electrophoresis. Similar findings were obtained in cases of carbon monoxide (CO) intoxication. However, no apoptotic cells were found in other cases such as methamphetamine (MAP) intoxication, tetrodotoxin intoxication, alcohol intoxication, asphyxia, head injury, heart injury or myocarditis. These findings suggested that it would be possible to apply TUNEL-positive cells as a diagnostic marker during the early stages of AMI.

Retrospective review of Japanese sudden unexpected death in infancy: The importance of metabolic autopsy and expanded newborn screening

Sudden unexpected death in infancy is defined as sudden unexpected death occurring before 12 months of age. The common causes of sudden unexpected death in infancy are infection, cardiovascular anomaly, child abuse, and metabolic disorders. However, the many potential inherited metabolic disorders are difficult to diagnose at autopsy and may therefore be underdiagnosed as a cause of sudden unexpected death in infancy. In the present study we retrospectively reviewed 30 Japanese sudden unexpected death in infancy cases encountered between 2006 and 2009 at our institute. With postmortem blood acylcarnitine analysis and histological examination of the liver, we found two cases of long-chain fatty acid oxidation defects. Molecular analysis revealed that the one patient had a compound heterozygote for a novel mutation (p.L644S) and a disease-causing mutation (p.F383Y) in the carnitine palmitoyltransferase 2 gene. Furthermore, retrospective acylcarnitine analysis of the newborn screening card of this patient was consistent with carnitine palmitoyltransferase II deficiency. Metabolic autopsy and expanded newborn screening would be helpful for forensic scientists and pediatricians to diagnose fatty acid oxidation disorders and prevent sudden unexpected death in infancy.

Association between the GST genetic polymorphisms and methamphetamine abusers in the Japanese population

Glutathione S-transferase (GST) plays a major role in the detoxification of many compounds by conjugation with glutathione. GSTM1 and T1, which are important members of the GST multigene family, are polymorphic in humans. Complete deletion of the gene results in the null genotype and loss of function. However, it is not clear whether deletion of this gene is associated with a vulnerability to methamphetamine (MAP) abuse. To clarify the potential role and mechanisms of genetic polymorphisms of GSTM1 and T1 in susceptibility to MAP abuse in the Japanese population, we investigated GSTM1 and T1 polymorphisms in subjects with diagnosed MAP-related disorders and in control groups. The risk of MAP abuse associated with GSTM1 null genotype was significantly higher only in females than in subjects with the GSTM1 genotype. GSTM1 and GSTT1 null genotype combined conferred increased risk for MAP abuse compared with GSTM1 and GSTT1 genotype combined. In conclusion, we found that GSTM1 gene deletion may contribute to a vulnerability to MAP abuse in female subjects. Moreover, we identified an association between GSTM1 and GSTT1 null genotype combined and risk of MAP abuse in the Japanese population.

Rapid and clear detection of ABO genotypes by simultaneous PCR-RFLP method.

We reported a new approach of ABO genotyping by a polymerase chain reaction and restriction fragment length polymorphism method. Instead of amplifying the loci containing the positions of nucleotides 258 and 700 of cDNA of the A transferase separately, we successfully amplified these 2 loci together in one reaction mixture using 2 sets of primers. The amplified DNA products were digested at the same time with restriction enzymes Kpn I and Alu I. The digested DNA products were then separated by electrophoresis on polyacrylamide gel. In addition, we evaluated the influence of various amplification parameters (concentration of template DNA, primers, Taq DNA polymerase, MgCl2, and number of cycles). In particular, high Mg2+ concentration (3.5 mM) made effective amplification of this locus without producing any unspecific band. By using that optimized condition for PCR, together with a simultaneous approach, our study proved to be time saving, more economic, and convenient in interpreting the results.

Methylation analysis of circadian clock gene promoters in forensic autopsy specimens

DNA methylation in gene promoter regions influences gene expression. Circadian clock genes play an important role in the formation of a biological clock and aberrant methylation of these genes contributes to several disorders. In this study, we examined forensic autopsy specimens to determine whether DNA methylation status in the promoter regions of nine circadian clock genes (Per1, Per2, Per3, Cry1, Cry2, Bmal1, Clock, Tim, and Ck1e) is related to a change in acquired diathesis and/or causes of death. Methylation-specific PCR and direct sequencing methods revealed that the promoters of Per1, Cry2, Bmal1, Clock, and Ck1e were unmethylated in all the forensic autopsy specimens, while the promoters of Per2, Per3, Cry1, and Tim were partially methylated. Methylation status varied between individuals and between tissues in the same patient. A detailed analysis of methylation patterns in the Cry1 promoter region revealed that the patterns also varied between individuals and the Cry1 promoter had highly methylated patterns in two cases that had been exposed to methamphetamine. These results suggest that the methylation status of clock gene promoters varies between individuals. Methamphetamine use may influence methylation in the Cry1 gene promoter region and disturb circadian rhythmicity.

Simultaneous determination of flunitrazepam and 7-aminoflunitrazepam in human serum by ion trap gas chromatography–tandem mass spectrometry

A method for the determination of flunitrazepam (FNZ) and 7-aminoflunitrazepam (7-AFNZ) in human serum was developed with ion trap gas chromatography (GC)-tandem mass spectrometry. The 7-AFNZ was derivatizated with 50 microl trifluoroacetic anhydride (TFAA), 60 degrees C-20 min. EI mass spectra and tandem mass spectra of FNZ and 7-AFNZ-TFA were m/z 238, 239, 266, 286, 294, 312, 313(M(+)), m/z 350, 351, 360, 378, 379(M(+)), m/z 238, 239, 240 (precursor ion m/z 286, collision energy 1.5 V), and m/z 239, 254, 264, 336 (precursor ion m/z 351, collision energy 1.8 V), respectively. The detection limits of full scan EI mass spectrometry and tandem mass spectrometry for FNZ and 7-AFNZ in human serum were ca. 200 ng/ml, 60 ng/ml, 15 ng/ml and 1 ng/ml, respectively.

Detection of D1S80 (pMCT118) locus polymorphism using semi-nested polymerase chain reaction in skeletal remains

We evaluated the usefulness of a semi-nested polymerase chain reaction (PCR) method for detecting D1S80 (pMCT118) locus polymorphisms of DNA extracted from old skeletal remains. The semi-nested PCR has been applied to the amplification of D1S80 nucleic acid sequences. For amplification of the locus D1S80, a pair of oligonucleotide primers have been used widely as described by Kasai et al. We have designed another set of primers for semi-nested PCR. This method resulted in D1S80-VNTR detection from low-titered DNA isolated from old skeletal remains. The first and second step PCR achieved amplification from as little as 10 ng and 10 pg of template DNA, respectively. Specificity and sensitivity of the amplification products was markedly improved by semi-nested PCR. In DNA extracted from biological samples, this method took about 5 hours to amplify the target DNA and 3 hours for electrophoretic separation. We demonstrated that this semi-nested PCR method was superior in sensitivity to conventional 1-step standard amplification for VNTR typing of the D1S80 locus.

Variants of the melanocortin 1 receptor gene (MC1R) and P gene as indicators of the population origin of an individual

The population origin of an individual is often requested to be determined from specimens left at a crime scene for identifying a suspect and individual identity. The melanocortin 1 receptor gene (MC1R) and P gene are associated with human pigmentation. Although several studies have reported that these genes are highly polymorphic in human populations, it is unclear if the allele variants can be used to determine the population origin of an individual. We aimed to determine the ethnic origin of an individual by using single nucleotide polymorphisms (SNPs). Eighteen SNPs in the MC1R gene and P genes were genotyped in 52 individuals by the direct sequencing method, and 4 SNPs (MC1R gene: R163Q and P gene: IVS5 + 1001, IVS13 + 113, and H615R) were selected on the basis of differences in frequencies. Subsequently, we genotyped these four SNPs in 422 volunteers from six ethnically defined populations using a polymerase chain reaction-based assay. The results revealed that the allele variants were present with high frequencies in Asian populations but were low in European and African populations. On the basis of these results, we defined a specific combination of a genotype (R163Q) and a diplotype group (IVS5 + 1001, IVS13 + 113, and H615R). This study indicates that the specific combination of a genotype and a diplotype group would be effective in estimating the population origin of an individual from a list of population groups.

Diplotype analysis of the human cardiac sodium channel regulatory region in Japanese cases of sudden death by unknown causes

Inherited mutations in the human cardiac sodium channel (SCN5A) gene cause arrhythmogenic diseases such as tachyarrhythmia and bradyarrhythmia. Moreover, mutation subsets in the coding region impair SCN5A function, potentially leading to sudden cardiac death (SCD). In the present study, we performed diplotype analysis of the regulatory region of the SCN5A gene in Japanese people who died suddenly because of an unknown cause (sudden death group; n=70) and controls (n=112). There were no significant differences at six polymorphic loci between the groups. However, 38 diplotypes of 6-nucleotide polymorphism variants were identified. One of these diplotypes-Dip.D (CTG-TC/CCG-TC)-occurred significantly more frequently in the sudden death group than in the controls (p<0.01, OR=5.18, 95% CI: 1.38-19.45). Dip.D has two variants (T-1062C and T-847G), and while it is unclear whether these directly affect mRNA expression, a common polymorphism in this region modulates SCN5A expression in vitro. Our results thus suggest that the transcription of the SCN5A Dip.D variant may be associated with arrhythmogenic diseases that can induce sudden death.