An optimal skeletal element for DNA testing: Evaluation of DNA quantity and quality from various bone types in routine forensic practice.

For human identification, the quality and quantity of DNA must be sufficient for amplification and analysis. When DNA extraction from bone tissues and teeth is required, the optimal skeletal elements should be selected as samples for DNA extraction because DNA yield differs among elements. Recently, some studies have reported that a high quantity of high-quality DNA can be extracted from the small cancellous bones of the hands and feet. In this study, we evaluated the effectiveness of small cancellous bones in the human identification of skeletal remains in routine forensic genetic casework. Cancellous bones [phalanges, (meta)carpal bones, and (meta)tarsal bones)] and the cortical bones (femur and petrous bones) and teeth, which have generally been recommended as samples, were collected from the same individuals that needed identifying using DNA analysis in our laboratory. The quantity of DNA from small cancellous bones tended to be higher than that from cortical bones, and the quality from the former was as high as that from the latter. This study showed that in routine forensic casework, the small cancellous bones of the hands and feet should be actively selected as samples for DNA testing.

RMplex reveals population differences in RM Y-STR mutation rates and provides improved father-son differentiation in Japanese.

Rapidly mutating Y chromosomal short tandem repeat markers (RM Y-STRs) -characterized by at least one mutation per 100 generations- are suitable for differentiating both related and unrelated males. The recently introduced multiplex method RMplex allows for the efficient analysis of 30 Y-STRs with increased mutation rates, including all 26 currently known RM Y-STRs. While currently available RM Y-STR mutation rates were established mostly from European individuals, here we applied RMplex to DNA samples of 178 genetically confirmed father-son pairs from East Asia. For several Y-STRs, we found significantly higher mutation rates in Japanese compared to previous estimates. The consequent father-son differentiation rate based on RMplex was significantly higher (52%) in Japanese than previously reported for Europeans (42%), and much higher than with Yfiler Plus in both sample sets (14% and 13%, respectively). Further analysis suggests that the higher mutation and relative differentiation rates in Japanese can in part be explained by on average longer Y-STR alleles relative to Europeans. Moreover, we show that the most striking difference, which was found in DYS712, could be linked to a Y-SNP haplogroup (O1b2-P49) that is common in Japanese and rare in other populations. We encourage the forensic Y-STR community to generate more RMplex data from more population samples of sufficiently large sample size in combination with Y-SNP data to further investigate population effects on mutation and relative differentiation rates. Until more RMplex data from more populations become available, caution shall be placed when applying RM Y-STR mutation rate estimates established in one population, such as Europeans, to forensic casework involving male suspects of paternal origin from other populations, such as non-Europeans.

Mutation analysis for 25 Y-STR markers in Japanese population.

In this study, we analyzed DNA samples from 213 Japanese father son pairs with 25 Y-chromosome short tandem repeat (Y-STR) (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS456, DYS390, DYS438, DYS392, DYS518, DYS570, DYS437, DYS385, DYS449, DYS393, DYS439, DYS481, DYF387S1, and DYS533) markers using the Yfiler™ Plus PCR amplification kit. We calculated Y-STR mutation rates for each locus to evaluate the efficacy of the 25 Y-STR markers for paternity testing and forensic identification using samples from male relatives. Six rapidly mutating Y-STR markers (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1), previously reported to have high mutation rates (>1.0 × 10-2), are included in the 25 Y-STR markers, but our findings revealed that the mutation rates for all Y-STR markers except for DYS576 and DYS458 were lower than 1.0 × 10-2. Therefore, the use of these 25 Y-STR markers may be useful for forensic identification in the Japanese population.

Wild-type ATTR amyloidosis may be associated with unexpected death among the elderly.

Wild-type ATTR amyloidosis (ATTR-wt) is characterized by the accumulation of amyloid in the heart, leading to fatal heart failure and arrhythmia. In this study, we investigated the amyloid deposits in the heart from 556 forensic autopsy cases over 60 years of age. The prevalence of ATTR-wt was 5.8% (32 of the 556), with the prevalence increasing as a function of age. We identified an ATTR-wt-specific morbidity rate of 12.3% for patients over 80 years of age, while the prevalence among individuals over 90 years of age was 34.9%. In none of these 32 cases had a clinical diagnosis of ATTR-wt been made. In 29 of the 32 cases found to be ATTR-wt positive, an obvious extraneous cause of death was identified and included burning, drowning, hypothermia, suicide, and traffic accident. On the other hand, heart failure due to ATTR-wt was confirmed as the cause of death in 3 of the 32 cases. It is suggested that ATTR-wt may be associated with unexpected death among the elderly.

High-Frequency Heating Extraction Method for Sensitive Drug Analysis in Human Nails.

Background: A simple, sensitive, and rapid extraction method based on high-frequency (H-F) heating was developed for drug analysis in human nails. Methods: A human nail was placed in a glass tube with an extraction solvent (methanol and 0.1% formic acid; 7:3, v/v), and a ferromagnetic alloy (pyrofoil) was wrapped in a spiral around the glass tube. Then, the glass tube was placed in a Curie point pyrolyzer, and a H-F alternating voltage (600 kHz) was applied. The sample and extraction solvent were heated at the Curie temperature for 3 min. Different Curie temperatures were applied by changing the pyrofoil (160 °C, 170 °C, 220 °C, and 255 °C). Results: The caffeine in the nail was effectively and rapidly extracted into the extraction solvent with the pyrofoil at 220 °C. The peak area obtained for the caffeine using liquid chromatography mass spectrometry (LC-MS/MS) was five times that of what was obtained after conventional ultrasonic irradiation extraction. Because the extraction uses high-pressure and high-temperature conditions in a test tube, the drugs that were strongly incorporated in nails could be extracted into the solvent. The amount of caffeine extracted was independent of the size of the pieces in the sample. Conclusions: Therefore, the sensitive determination of target drugs in nails is possible with rapid (20 min, including H-F extraction for 3 min) and simple sample preparation. The developed method was applied to a nail from a patient with hypertension.

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA.

Six multiplex PCR systems using single-base extension reactions to analyze 46 mitochondrial DNA (mtDNA)-coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.

Analysis of 30 insertion-deletion polymorphisms in the Japanese population using the Investigator DIPplex® kit.

Allele frequencies and forensic parameters for 30 insertion-deletion polymorphisms (INDELs) were investigated in a sample of 251 unrelated Japanese individuals using the Investigator DIPplex® kit (QIAGEN). The frequency distributions showed no deviations from Hardy-Weinberg equilibrium expectations. The combined powers of discrimination and match probability for the 30 INDELs were 0.9999999998 and 2.67×10(-11), respectively. To assess the effectiveness of the kit in typing degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed; most of 30 INDELs and amelogenin were typed successfully. We concluded that the kit offers considerable potential for personal identification from degraded DNA samples due to the small amplicon length and high degree of polymorphisms.

Molecular analysis of potassium ion channel genes in sudden death cases among patients administered psychotropic drug therapy: are polymorphisms in LQT genes a potential risk factor?

Psychotropic drugs can pose the risk of acquired long QT syndrome (LQTS). Unexpected autopsy-negative sudden death in patients taking psychotropic drugs may be associated with prolonged QT intervals and life-threatening arrhythmias. We analyzed genes that encode for cardiac ion channels and potentially associated with LQTS, examining specifically the potassium channel genes KCNQ1 and KCNH2 in 10 cases of sudden death involving patients administered psychotropic medication in which autopsy findings identified no clear cause of death. We amplified and sequenced all exons of KCNQ1 and KCNH2, identifying G643S, missense polymorphism in KCNQ1, in 6 of the 10 cases. A study analysis indicated that only 11% of 381 healthy Japanese individuals carry this polymorphism. Reports of previous functional analyses indicate that the G643S polymorphism in the KCNQ1 potassium channel protein causes mild I(Ks) channel dysfunction. Our present study suggests that administering psychotropic drug therapy to individuals carrying the G643S polymorphism may heighten the risk of prolonged QT intervals and life-threatening arrhythmias. Thus, screening for the G643S polymorphism before prescribing psychotropic drugs may help reduce the risk of unexpected sudden death.

Analysis of Y chromosome haplogroups in Japanese population using short amplicons and its application in forensic analysis.

We designed three mini multiplex PCR systems using single-base extension reactions to identify Japanese Y chromosome haplogroups. We selected a group of 22 Y chromosome single nucleotide polymorphisms (SNPs) from the haplogroups most commonly reported in East Asia. To make the systems more useful in analyzing degraded DNA samples, we designed primers to render amplicons of ≤ 150 bp. Applying these systems, we classified the Japanese population into major haplogroups and confirmed the applicability of these systems in forensic DNA analysis.

Trial investigation of post-mortem non-invasive transnasal endoscopy.

We performed a trial investigation of transnasal endoscopy for post-mortem examinations to assess its efficacy in superficial post-mortem examinations. Transnasal endoscopy proved capable of permitting detailed visual inspections of the respiratory and the upper gastrointestinal tract, equal to direct viewing, on an LCD digital display. In 18 (40.9%) of 44 cases, findings obtained by post-mortem transnasal endoscopy (PMTNE) provided valuable clues regarding cause of death. The cases examined included seven deaths by fire, four by hypothermia, four by asphyxia (three involving death by mechanical asphyxia, the other by choking), two by drowning and one case of lung cancer. In two cases, PMTNE also led to informative findings not directly related to the cause of death. Under Japan's current systems, we are required to diagnose the cause of death for over 80% of all the unnatural death cases based solely on superficial post-mortem examinations, in the absence of an autopsy. Introducing PMTNE to superficial post-mortem examinations will undoubtedly provide much more information on the cause of death than relying solely on superficial post-mortem examinations.

Development of multiplex assay with 16 SNPs on X chromosome for degraded samples.

We selected 16 new X chromosomal SNPs (rs4827155, rs471205, rs7884160, rs16982419, rs985251, rs3813932, rs6630351, rs4132871, rs5966270, rs7471388, rs6641116, rs6521038, rs5990560, rs5959408, rs414960, and rs3006142) and developed the two X chromosomal SNPs Octaplex systems using multiplex single base extension reactions. To make the systems more useful for analyzing degraded DNA samples, we designed primers to render amplicons of 100 bp or shorter (shorter PCR products). Statistical analyses of the 16 SNPs indicated a high usefulness for the Japanese forensic practice. In addition, results of tests on degraded DNA confirm the usefulness of this technique in such samples.

Unusual intracranial stab wounds inflicted with metal tent stakes for a case involving a family murder suicide.

This article presents a highly unusual homicide involving intracranial stab wounds. Of three members of a family killed by intracranial stab wounds apparently inflicted with metal tent stakes, two also showed signs of wounds inflicted during an apparent struggle with the assailant. A wooden mallet appears to be the implement use to drive the metal stakes into the cranial cavity. In all victims, toxicological analysis indicated the presence of brotizolam at concentrations ranging from 30 to 50ngml(-1). The one victim who showed no signs of wounds incurred during a defensive struggle was found to have blood alcohol levels of 2.87mgml(-1). The assailant, another family member with a history of major psychiatric disorders, apparently committed suicide by drowning following the attacks.

Unusual body posture after death leading to signs of strangulation.

This report concerns a case in which the decedent was found in a highly unusual posture, one resembling a yoga backbend pose. Several findings in this case pointed to death by strangulation, including facial congestion, extensive petechiae of the conjunctivae and mucosal membranes of the oral cavity, and congestion and hemorrhaging in the lingual root, laryngopharynx, and cervical lymph nodes. However, no hemorrhaging of the cervical skin or soft tissue was observed. The cause of death was ultimately identified as pulmonary thromboembolism caused by an embolus originating from a deep leg vein thrombosis. If the pulmonary thromboembolism had not been detected, the cause of death may have been misidentified as strangulation.

Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method.

An experimental DNA extraction kit (new kit) was recently developed to extract DNA from degraded skeletal remains without the need for powdering the samples. We compared the utility of the new kit with the conventional phenol/chloroform method using real-time quantitative PCR and multiplex STR analysis. The new kit yielded large amounts of DNA from a compact bone fragment compared with the conventional phenol/chloroform method. We were able to extract sufficient DNA for STR analysis from 75% (3 of 4) and 60% (3 of 5) of the un-powdered tooth and bone samples, respectively, using the new kit. We were able to perform mini-STR analysis of the remaining samples using DNA extracted with the new kit. Furthermore, we successfully performed mitochondrial DNA sequencing of every sample. The new kit simplifies the DNA extraction procedure as it does not require powdering samples. Decreasing the number of procedural steps in DNA extraction will be beneficial in controlling DNA contamination in laboratories. Our results suggest that the new kit may be used for the simple, simultaneous extraction of DNA from multiple samples.

Restriction enzyme analysis of PCR products.

Progress in single nucleotide polymorphism (SNP) detection technologies has provided information for SNP-based studies, such as identification of candidate genes for the complex genetic diseases, pharmacogenetic analysis, drug development, population genetics, evolutionary studies, and forensic investigations. SNP detection is performed by many methods, including hybridization, allele-specific polymerase chain reaction (PCR), primer extension, oligonucleotide ligation, direct DNA sequencing, and endonuclease cleavage. Each of these methods has its specific advantages and disadvantages. Here we introduce the PCR-restriction fragment length polymorphism (RFLP) method, which has certain advantages over many other techniques used for analysis of SNPs. The PCR-RFLP method allows very rapid, simple, and inexpensive detection of point mutations within the sequences of PCR products. The mutation is discriminated by the specific restriction endonuclease and is identified by gel electrophoresis followed by staining with ethidium bromide. This convenient and simple method is useful in a small basic research study.

An unusual suicide case of the combination of asphyxia.

A 52-year-old man died of a combination of suffocation by adherent tape wrapped around the head to cover the airway and ligature strangulation by an electrical cord. An autopsy could not conclusively determine whether it was a case of suicide or homicide. Further investigation, including investigations of the scene, the decedent's background, and the testimony of those close to the victim, helped determine the manner of death. Suicide because of a combination of several forms of asphyxia has rarely been reported in the past. Therefore, this is a very rare and unusual suicide case.

Allele frequencies for 15 STR loci in Tibetan populations from Nepal.

Samples from 105 unrelated healthy Sherpa in Namche Bazaar and 111 unrelated non-Sherpa in Kathmandu valley from Nepal were used to obtain allele frequency data for 15 short tandem repeat (STR) loci (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX and vWA) included in the AmpFLSTR Identifiler kit. No deviations from Hardy-Weinberg equilibrium were observed, but only after applying a Bonferroni correction in the case of D5S818 in the Sherpa population and D7S820 in the Kathmandu population. Genetic parameters of forensic interest were calculated and genetic differentiation between the two populations tested.

Highly sensitive HLA-DNA typing from formalin-fixed and paraffin-embedded tissue samples.

Nucleotide sequences have been determined for more than 1700 different alleles at the core of the human leukocyte antigen (HLA) system. The highly polymorphic character of these genes affects adaptive immune response and is also useful for forensic applications. HLA typing from formalin-fixed and paraffin-embedded tissue provides abundant useful information for both clinical settings and forensic investigations. This study, which investigated the potential use of DNA from formalin-fixed and paraffin-embedded tissue samples in an HLA PCR sequence-specific primer and probe (SPP) system, showed that tissue fixed in formalin for less than 3 days and embedded in paraffin can serve as a useful source of DNA for PCR-SPP typing kits.

Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination.

Validation studies were carried out with the commercially available HLA typing kit using a PCR-SPP (sequence-specific primer and probe) technique. This technique has made it possible to type class I (HLA-A and -B) and class II (HLA-DRB1 and -DQB1) alleles at low-resolution level with total 10 ng of template DNA, in addition to amplify directly from various forms of blood samples without DNA isolation procedure. Experimental examinations with bloodstains smeared on cotton cloth that were a week to 3 months old, bloodstains on gauze stored for 18 years, and buccal cells revealed that this HLA-SPP typing kit is a sensitive and reliable method for forensic investigations.

Correlation between glial fibrillary acidic protein-positive astrocytes and age in the human hippocampus.

The hippocampus is one of the areas most vulnerable to histopathological changes, and such changes may yield useful information in forensic medicine. We found that glial fibrillary acidic protein (GFAP)-positive astrocytes are frequently found in the hippocampus of consecutive series of forensic brains, distributed predominantly in the hippocampal CA4 and hippocampal sulcus (HS) regions. The present study counted GFAP-positive astrocytes in these regions and investigated associations with age, cause of death and postmortem time. Significant correlations were found between age and number of GFAP-positive astrocytes in both CA4 and HS regions. Number of GFAP-positive astrocytes increases in an age-dependent manner, but no correlations were noted between number of GFAP-positive astrocytes and postmortem time and cause of death. Number of GFAP-positive astrocytes in the hippocampus may provide useful information for age estimation.