DNA microarray analysis of hypothermia-exposed murine lungs for identification of forensic biomarkers.

We used DNA microarray technology to analyze the pulmonary transcriptome of mice killed by hypothermia. This analysis identified significant differential regulation of 4094 genes; specifically, 1699 genes were upregulated, and 2395 were downregulated in response to hypothermia. The gene encoding cathelicidin antimicrobial peptide was the most upregulated gene, and that encoding BAI1-associated protein 2-like 1 was the most downregulated. Gene-set analysis identified significant hypothermia-induced variations in 101 pathways, and we discovered that pathways related to immunity are involved in the pulmonary pathogenesis of hypothermia. The present findings demonstrate some of the acute pulmonary responses to hypothermia and indicate several pulmonary genes as candidate forensic biomarkers of hypothermia. Furthermore, the present findings suggest that host defense is induced in hypothermic lungs. The present microarray data may facilitate the development of protein analyses for human forensics by immunohistochemistry, western blotting and enzyme-linked immunosorbent assay and may be beneficial in clinical research of hypothermia.

A quantitative morphological analysis of three-dimensional CT coxal bone images of contemporary Japanese using homologous models for sex and age estimation.

Sexual dimorphisms and age-dependent morphological features of the human coxal bone were quantitatively analyzed using homologous models created from three-dimensional (3D) computed tomography images of the pelvis (male: 514 samples, female: 388 samples, age 16-100). Bilateral average coxal images of each sex and age decade were generated separately through principle component analyses (PCA). By measuring average point-to-point distances of 8472 corresponding points (average corresponding point differences [ACPDs]) between each homologous coxal image and the average images, the sex of more than 93% of the samples was correctly assigned. Some principal components (PCs) detected in PCA of the homologous models of the samples correlated fairly well with age and are affected by features of the curvature of the iliac crest, the arcuate line and the greater sciatic notch. Moreover, separate PCA using the average images of each age decade successfully detected the first PCs, which were strongly correlated with age. However, neither multiple regression analysis using PCs related to age nor comparison of ACPDs with the average images of each age decade could produce accurate results for age decade assignment of unknown (blind) samples. Therefore, more detailed analysis of age-dependent morphological features would be necessary for actual age estimation. In addition, some laterality or left and right shape difference of the coxal bone images was also elucidated, and was more significant in females. Analysis of 3D structures using homologous models and PCA appears to be a potential technique to detect subsistent morphological changes of bones.

Pediatric autopsy case of asphyxia due to salmon egg (ikura) aspiration.

Here we report an autopsy case of asphyxia due to aspiration of a salmon egg (ikura) into the airway. The patient was a 19-month-old girl. During breakfast, she put salmon eggs into her mouth, and began to walk. She slipped, fell down, and collapsed. She was pronounced dead following 2 h of resuscitation. The body was autopsied 28 h after death. The gastric contents consisted of rice, orange sections, and white salmon eggs. The lungs were deeply congested and over-inflated. In the right lung, areas of atelectasis in the upper and middle lobes were seen. A yellow salmon egg (8 mm in diameter) was found in the trachea. Although fish eggs are consumed throughout the world, reports of this sort are limited. The aspiration of fish eggs is under-acknowledged and underreported. The importance of preventive measures needs to be emphasized to parents and caregivers.

The global distribution of the p.R1193Q polymorphism in the SCN5A gene.

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations.

Four cases of orbital hyperdensity identified by postmortem computed tomography.

Postmortem computed tomography (PMCT) has become a common examination method in the field of forensic medicine. Head computed tomography provides information of the orbit and eyes, and forensic pathologists may come across abnormal intraocular findings of cadavers upon PMCT. Here, we present four cases in which we identified orbital hyperdensity by PMCT. The first case showed calcified senile scleral plaques (CSSP), whereas the second case showed foreign bodies in the palpebral fissure, which resembled CSSP upon PMCT. The third case showed signs of silicone oil injection in the eye, while the fourth case showed bilateral phthisis bulbi. In the first case, the presence of CSSP was found to be helpful for age estimation, whereas the findings of cases 3 and 4 aided in the personal identification of the subjects. As demonstrated by these cases, intraocular PMCT findings may provide highly useful information, and correct interpretation of the intraocular PMCT findings by forensic pathologists is hence crucial.

DNA microarray analysis of the mouse adrenal gland for the detection of hypothermia biomarkers: potential usefulness for forensic investigation.

We analyzed the adrenal gland transcriptome of mice killed by hypothermia using DNA microarray technology. A total of 4051 significantly expressed genes were identified; 2015 genes were upregulated and 2036 were downregulated.The FBJ osteosarcoma oncogene was the most upregulated,whereas stearoyl coenzyme A desaturase 3 was the most downregulated. Validation by quantitative polymerase chain reaction revealed that results obtained by both methods were consistent. In the gene set analysis, significant variations were found in nine pathways, and we suggest that transforming growth factor ß and tumor necrosis factor α would be involved in the pathogenesis of hypothermia. Gene functional category analysis demonstrated the most overexpressed categories in upregulated and downregulated genes were cellular process in biological process, binding in molecular function, and cell and cell part in cellular component. The present study demonstrated acute adrenal responses in hypothermia, and we suggest that understanding adrenal mRNA expression would be useful for hypothermia diagnosis. Furthermore, the present microarray data may also facilitate development of immunohistochemical analysis of human cases. In forensic practice, the combination of macroscopic and microscopic observations with molecular biological analyses would be conducive to more accurate diagnosis of hypothermia. Although this study is aimed at forensic practice, the present data regarding more than 20,000 genes of the adrenal gland would be beneficial to inform future clinical hypothermia research. From the viewpoint of adrenal gene activity, they could contribute to elucidating the pathophysiology of hypothermia.

Three dimensional surface analyses of pubic symphyseal faces of contemporary Japanese reconstructed with 3D digitized scanner.

Three dimensional pubic bone images were analyzed to quantify some age-dependent morphological changes of the symphyseal faces of contemporary Japanese residents. The images were synthesized from 145 bone specimens with 3D measuring device. Phases of Suchey-Brooks system were determined on the 3D pubic symphyseal images without discrepancy from those carried out on the real bones because of the high fidelity. Subsequently, mean curvatures of the pubic symphyseal faces to examine concavo-convex condition of the surfaces were analyzed on the 3D images. Average values of absolute mean curvatures of phase 1 and 2 groups were higher than those of phase 3-6 ones, whereas the values were approximately constant over phase 3 presumably reflecting the inactivation of pubic faces over phase 3. Ratio of the concave areas increased gradually with progressing phase or age classes, although convex areas were predominant in every phase.

A hypervariable STR polymorphism in the CFI gene: mutation rate and no linkage disequilibrium with FGA.

A hypervariable short tandem repeat (STR) polymorphism in intron 7 of the human complement factor I gene (CFI) was investigated to estimate the mutation rate in Japanese samples and to test linkage disequilibrium (LD) with an STR in the fibrinogen alpha chain gene (FGA). The expected heterozygosity and the mutation rate of CFI were estimated to be 0.917 and 0.002, respectively. No LD was observed between CFI and FGA. CFI is a useful supplementary marker for forensic science.

Distribution of Aconitum alkaloids in autopsy cases of aconite poisoning.

Aconite is a well-known toxic-plant containing Aconitum alkaloids such as aconitines, benzoylaconines, and aconins. We describe here the distribution of Aconitum alkaloids detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS) in three autopsy cases of suicide by aconite poisoning. Case 1: a male in his fifties had eaten aconite leaves. The concentrations of jesaconitine in cardiac blood, urine, and kidney were 12.1 ng/ml, 993.0 ng/ml, and 114.2 ng/g, respectively. Case 2: a female in her fifties had eaten aconite root. The aconite root in the stomach included a high level of mesaconitine. The concentrations of mesaconitine in cardiac blood, liver, and kidney were 69.1 ng/ml, 960.9 ng/g, and 776.9 ng/g, respectively. Case 3: a male in his sixties had drunk liquor in which aconite root had been soaked. The concentrations of mesaconitine and aconitine in cardiac blood were 259.5 and 228.5 ng/ml, respectively. The Aconitum alkaloid levels were very high in the liver. The absorption of ethanol and Aconitum alkaloids might have been increased because of his having undergone total gastrectomy. In all three cases, the Aconitum alkaloid levels were high in the liver and kidney and low in the heart and cerebrum. The level in the cerebrum was lower than that in blood. Data on the distribution of the Aconitum alkaloids in the body in cases of aconite poisoning is useful to elucidate various actions of aconite alkaloids.

Hypothalamic transcript profiling in hypothermia using SuperSAGE.

Understanding of molecular mechanisms underlying hypothermia is of primary importance in devising strategies to diagnose hypothermia. We investigated the hypothalamic transriptome in hypothermia. For transcriptomic analyses, SuperSAGE, an improved method of serial analysis of gene expression, was used. Totally, 62,208 and 54,084 tags were collected from the hypothalami of normal and hypothermia, respectively. And 367 transcripts were differentially expressed at a statistically significant level. That is, 157 and 210 transcripts among them were expressed at a higher level in normal and hypothermic hypothalami. Results obtained by SuperSAGE and quantitative PCR were consistent in 6 selected genes, although levels of differences detected by the 2 methods were not exactly the same. mRNA expressions in the hypothalamus were considered to be useful for hypothermic diagnosis. Various methods have been applied for gene expression analyses and biomarker detections. However in forensic pathology, SuperSAGE would be a promising method, especially in gene discoveries and transcriptomic analyses.

Analyses of sexual dimorphism of reconstructed pelvic computed tomography images of contemporary Japanese using curvature of the greater sciatic notch, pubic arch and greater pelvis.

Three-dimensional pelvic images were reconstructed from multi-slice CT data of contemporary Japanese (males: 124; females: 104, 25-92 years old), and curvature analysis to examine sexual dimorphism was carried out in the great sciatic notch (GSN), the pubic arch and the greater pelvis in the images. Reconstructed pelvic CT images were visualized fairly well and anatomical landmarks were easily recognizable. When calculating the radii (curvature radii) of the best-fit circles for the spline curve lines set along the edges of the GSNs and of the pubic arches, sexes from these regions were correctly identified in 89.1% (males: 93.8%; females: 83.7%) and 94.7% (males: 97.3%; females: 91.8%) of cases, respectively, by setting an appropriate cut-off value. Furthermore, sexing was possible even in deeper regions of the GSN which are relatively resistant to postmortem damage. Curvature radii of the best-fit spheres of greater pelves showed no significant difference between sexes. However, curvature of the best-fit sphere for the left iliac fossa was significantly larger than that of the right one (p<10(-24)) in males, and the ratios were >1.0 in 88% of all male specimens analyzed. Meanwhile, no significant difference was observed among female samples. Although some left-sided dominancy has been reported in 2-dimensional measurements of the human pelvis, this 3-dimensional laterality in males was much more significant, and is a potential index of sex difference.

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.

Y-chromosomal microsatellite mutation rates in a population sample from northwestern Germany.

To estimate Y-chromosomal short tandem repeat (Y-STR) mutation rates, 15 loci (i.e., DYS19, DYS389 I/II, DYS390, and DYS393; DYS437, DYS438, DYS439, and DYS385; DYS391, DYS392, YCA II, and DXYS156) were analyzed in a sample of 1,029 father/son pairs from Westphalia, northwestern Germany. Among 15,435 meiotic allele transfers, 32 mutations were observed; thus, the mutation rate across all 15 Y-STR loci was 2.1 x 10(-3) per locus (95% C.I.: 1.5-3.0 x 10(-3)). With the exception of a three-repeat mutation at DYS385, all remaining mutations were single repeat mutations. Repeat losses were more frequent than gains (20:12), and the mutation rate appeared to increase with age. The Y haplogroups that were detected in the individuals showing a mutation reflect the haplogroup distribution in the Westphalian population. Additionally, the correlation of surnames and haplotypes was tested: Only 49 surnames occurred more than once, and only two men with the same rare surname shared the same haplotype. All other men with identical surnames carried different haplotypes.

Parentally imprinted allele typing at a short tandem repeat locus in intron 1a of imprinted gene KCNQ1.

A short tandem repeat (STR) in the intron 1a of paternally imprinted gene, KCNQ1, is evaluated as a new probe for use in parentally imprinting allele (PIA) typing. This typing can determine the inheritance of one allele from father by the methylation difference. Allelic and genotypic frequencies of the STR were determined using samples from 175 unrelated Japanese and 170 unrelated Germans. The polymorphism information contents were 0.652 and 0.634 for the Japanese and the Germans, respectively, indicating usefulness in individual identification. This method was applied to five Japanese families consisting of 19 individuals. Genomic DNA was digested by methylation-sensitive restriction endonucleases, HhaI and HapII, followed by PCR amplification using two-step sandwich primer sets and the products were analyzed on polyacrylamide gel electrophoresis. For all of the families, each child's paternal allele given by PIA typing corresponded to one of the two alleles from father, not the two from mother, that were determined by the STR genotyping. The results demonstrate that this STR probe is feasible for use in PIA typing and that its typing method can contribute to paternity testing.

Applicability of the parentally imprinted allele (PIA) typing of a VNTR upstream the H19 gene to forensic samples of different tissues.

The parentally imprinted allele (PIA) typing that we have recently developed determines parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene. The usefulness of this typing was demonstrated by its application to blood samples in paternity cases. However, its applicability to other tissue DNA remains to be tested. DNA samples from fifteen different postmortem tissues such as cerebrum, skeletal muscle and skin were examined, all of which were obtained from three autopsy cases 2-11h after death. DNA was digested with a methylation-sensitive HhaI enzyme and diluted solutions of the digests were subjected to the first PCR amplification, providing amplification of only the paternal H19 methylated allele. Subsequent VNTR typing was carried out for the amplified products to determine which allele was of paternal origin. No tissue-dependent difference was observed and all the samples examined, though degraded, were successfully used for determining the paternal allele. These results substantiate the usefulness of PIA typing in forensic examinations. Its application to two identity cases, a burned male body and a male body with adipocere formation, was also shown.

Novel paternity testing by distinguishing parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene.

Conventional PCR-based genotyping is useful for forensic testing but cannot be used to determine parental origins of alleles in DNA specimens. Here we describe a novel method of combined conventional genotyping and PIA typing (parentally imprinted allele typing) at a minisatellite region upstream from the H19 locus. The PIA typing uses two sets of primers and DNA digested with methylation-sensitive Hha I enzyme. The first amplification produces only the methylated fragment of paternal H19 allele, and the second detects polymorphism in the minisatellite. Hence, this distinguishes paternal and maternal alleles by difference in the DNA methylation. Furthermore, the polymorphism in this polymorphic locus was examined using 199 unrelated Japanese and 171 unrelated Germans, their polymorphism information content being 0.671 and 0.705, respectively. Feasibility of this typing is demonstrated for six families, and the usefulness is shown by application to paternity testing.