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.

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.

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.

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.

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.

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.