A panel of 130 autosomal single-nucleotide polymorphisms for ancestry assignment in five Asian populations and in Caucasians.

Ancestry informative single-nucleotide polymorphism (AISNP) panels for differentiating between East and Southeast Asian populations are scarce. This study aimed to identify AISNPs for ancestry assignment of five East and Southeast Asian populations, and Caucasians. We analyzed 145 autosomal SNPs of the 627 DNA samples from individuals of six populations (234 Taiwanese Han, 91 Filipinos, 79 Indonesians, 60 Thais, 71 Vietnamese, and 92 Caucasians) using arrays. The multiple logistic regression model and a multi-tier approach were used for ancestry classification. We observed that 130 AISNPs were effective for classifying the ethnic origins with fair accuracy. Among the 130 AISNPs, 122 were useful for stratification between these five Asian populations and 64 were effective for differentiating between Caucasians and these Asian populations. For differentiation between Caucasians and Asians, an accuracy rate of 100% was achieved in these 627 subjects with 50 optimal AISNPs among the 64 effective SNPs. For classification of the five Asian populations, the accuracy rates of ancestry inference using 20 to 57 SNPs for each of the two Asian populations ranged from 74.1% to 100%. Another 14 degraded DNA samples with incomplete profiling were analyzed, and the ancestry of 12 (85.7%) of those subjects was accurately assigned. We developed a 130-AISNP panel for ethnic origin differentiation between the five East and Southeast Asian populations and Caucasians. This AISNP set may be helpful for individual ancestral assignment of these populations in forensic casework.

Genotyping of 75 SNPs using arrays for individual identification in five population groups.

Single nucleotide polymorphism (SNP) typing offers promise to forensic genetics. Various strategies and panels for analyzing SNP markers for individual identification have been published. However, the best panels with fewer identity SNPs for all major population groups are still under discussion. This study aimed to find more autosomal SNPs with high heterozygosity for individual identification among Asian populations. Ninety-six autosomal SNPs of 502 DNA samples from unrelated individuals of five population groups (208 Taiwanese Han, 83 Filipinos, 62 Thais, 69 Indonesians, and 80 individuals with European, Near Eastern, or South Asian ancestry) were analyzed using arrays in an initial screening, and 75 SNPs (group A, 46 newly selected SNPs; groups B, 29 SNPs based on a previous SNP panel) were selected for further statistical analyses. Some SNPs with high heterozygosity from Asian populations were identified. The combined random match probability of the best 40 and 45 SNPs was between 3.16 × 10(-17) and 7.75 × 10(-17) and between 2.33 × 10(-19) and 7.00 × 10(-19), respectively, in all five populations. These loci offer comparable power to short tandem repeats (STRs) for routine forensic profiling. In this study, we demonstrated the population genetic characteristics and forensic parameters of 75 SNPs with high heterozygosity from five population groups. This SNPs panel can provide valuable genotypic information and can be helpful in forensic casework for individual identification among these populations.

Diatomological investigation in sphenoid sinus fluid and lung tissue from cases of suspected drowning.

We report on the presence, distribution and numbers of diatoms within specific organs as a result of drowning in fresh, treated and seawater. Specimens of sphenoid sinus fluid and lung tissue from 100 cases of suspected drowning and 20 cases where death was by natural causes, to act as a control, were examined for the presence of diatoms. In the 100 cases where the deceased was suspected to have drowned, 94 were confirmed as a death by drowning after autopsy with the other six being reported as death by another cause. No diatoms were found in cases confirmed as death by causes unrelated to drowning, with the exception of possible contamination via open wounds and through decomposition. In 94 cases, where all fatalities were confirmed as death by drowning, there were 81 cases in which diatoms were detected in samples taken from the sphenoid sinus fluid and/or lung tissue. No, or only few, diatoms were observed from the samples where the deceased drowned in treated waters such as spa or swimming pools. A significantly higher number of diatoms were detected in the sphenoid sinus fluid and lung tissue of confirmed drowning cases in fresh water compared to seawater. More diatoms were observed in sphenoid sinus fluid compared to lung tissue regardless of the water in which the deceased drowned. This study illustrates the potential use of diatom screening using both sphenoid sinus fluid and lung tissue to determine the cause of death in suspected cases of drowning. This report also highlights specific variables that need to be considered prior to such as conclusion being reached.

The risk of false inclusion of a relative in parentage testing - an in silico population study.

AIM: To investigate the potential of false inclusion of a close genetic relative in paternity testing by using computer generated families. METHODS: 10000 computer-simulated families over three generations were generated based on genotypes using 15 short tandem repeat loci. These data were used in assessing the probability of inclusion or exclusion of paternity when the father is actually a sibling, grandparent, uncle, half sibling, cousin, or a random male. Further, we considered a duo case where the mother's DNA type was not available and a trio case including the mother's profile. RESULTS: The data showed that the duo scenario had the highest and lowest false inclusion rates when considering a sibling (19.03 ± 0.77%) and a cousin (0.51 ± 0.14%) as the father, respectively; and the rate when considering a random male was much lower (0.04 ± 0.04%). The situation altered slightly with a trio case where the highest rate (0.56 ± 0.15%) occurred when a paternal uncle was considered as the father, and the lowest rate (0.03 ± 0.03%) occurred when a cousin was considered as the father. We also report on the distribution of the numbers for non-conformity (non-matching loci) where the father is a close genetic relative. CONCLUSIONS: The results highlight the risk of false inclusion in parentage testing. These data provide a valuable reference when incorporating either a mutation in the father's DNA type or if a close relative is included as being the father; particularly when there are varying numbers of non-matching loci.

Evaluating the performance of whole genome amplification for use in low template DNA typing.

We report on the performance of two whole genome amplification methods, GenomiPhi™ amplification and modified-improved primer extension preamplification (mIPEP), when analysing low template DNA samples. Template as low as 10 pg treated with mIPEP generated more than 1 ng of DNA that could be used in STR typing. Initial templates of 100-10 pg, when treated with mIPEP, generated an increase in alleles compared with control samples. Partial profiles using the AmpFℓSTR(®) Identifiler™ Kit were produced from this suboptimal DNA template, with 70% of the possible alleles (21.7 ± 2.1 in 32 alleles) recorded, using the mIPEP amplified products with an initial template of 100 pg. Allelic imbalance decreased with samples treated with whole genome amplification method (WGA) compared with those without this initial treatment. Further methods for improvement were also analysed including altering the condition of electrokinetic injection, and the successful DNA typing rate was increased to about 80%. This report illustrates the potential use and limitations of WGA for low template samples.

A novel strategy for sibship determination in trio sibling model.

AIM: To use a virtually simulated population, generated from published allele frequencies based on 15 short tandem repeats (STR), to evaluate the efficacy of trio sibship testing and sibling assignment for forensic purposes. METHODS: Virtual populations were generated using 15 STR loci to create a large number of related and unrelated genotypes (10,000 trio combinations). Using these virtual populations, the probability of related and unrelated profiles can be compared to determine the chance of inclusions of being siblings if they are true siblings and the chance of inclusion if they are unrelated. Two specific relationships were tested - two reference siblings were compared to a third true sibling (3S trio, sibling trio) and two reference siblings were compared to an unrelated individual (2S1U trio, non-sibling trio). RESULTS: When the likelihood ratio was greater than 1, 99.87% of siblings in the 3S trio population were considered as siblings (sensitivity); 99.88% of non-siblings in the 2S1U trio population were considered as non-siblings (specificity); 99.9% of both populations were identified correctly as siblings and non-siblings; and the accuracy of the test was 99.88%. CONCLUSIONS: The high sensitivity and specificity figures when using two known siblings compared to a putative sibling are significantly greater than when using only one known relative. The data also support the use of increasing number of loci allowing for greater confidence in genetic identification. The system established in this study could be used as the model for evaluating and simulating the cases with multiple relatives.

The strategies to DVI challenges in Typhoon Morakot.

Small village populations in which there is a high amount of kinship can cause complications in cases of disaster victim identification. This problem was highlighted by the loss of life after Typhoon Morakot struck Taiwan where over 500 people from small isolated communities lost their lives. Most of the victims were buried by landslides in the remote mountainous areas of southern Taiwan. Only 146 pieces of human remains were recovered after searching for 4 months. Most of the human remains were received for examination as severely damaged fragments prevented possible identification by morphological features. DNA testing using the traditional duo parent/child or sibling screening by STR data opens the possibility of including not only the actual victim but also false positives. Variable likelihood ratios were obtained when comparing DNA types from human remains to those from potential relatives; however, with the DNA typing of numerous members of the same living family, multiple matches to potential families were avoided. Of the 146 samples obtained and collapsed to 130 victims, they were linked to 124 individuals resulting in their identification when compared to a pool of 588 potential relatives. Six of the human remains could not be linked to any living relative and remain unknown.

Polymorphism of 17 Y-STR loci in Taiwan population.

Haplotypes and allele frequencies of 17 Y-STRs included in the AmpFlSTR((R)) Yfilertrade mark kit (DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y GATA H4) were examined in a population sample of 200 unrelated Taiwanese males living in Taiwan.

Urinary excretion of alpha-hydroxytriazolam following a single dose of halcion.

As an approved medicinal product and reportedly an abused substance that have been associated with death and "considered to be a factor...of impaired driving, sexual assault, and other violent crimes", triazolam is controlled at the same level (Level III) as flunitrazepam in Taiwan. Alleged misuses of this substance have been associated with case specimens submitted to this laboratory. A sample preparation (with and without enzymatic hydrolysis) and gas chromatography-mass spectrometry protocols were evaluated and applied to the analysis of free and total alpha-hydroxytriazolam (the main metabolite of triazolam) in urine. Ions designated for TMS-derivatized alpha-hydroxytriazolam and alpha-hydroxytriazolam-d4 are m/z 415, 417, and 430 and 419, 421, and 434, respectively. The overall protocol achieved the following results when applied to the analysis of 2-mL drug-free urine specimens fortified with 10-200 ng/mL alpha-hydroxytriazolam: recovery, 95%; interday and intraday precision ranges, 1.50-3.52% and 0.93-4.71%, respectively; linearity, r2 > 0.99; and limits of detection and quantitation, 0.05 and 0.1 ng/mL, respectively. This protocol was applied to the analysis of case specimens and urine samples collected from two patients (A and B) taking one oral dose of Halcion (0.25 mg triazolam). Excretion profiles of free and total alpha-hydroxytriazolam show that free alpha-hydroxytriazolam is detectable, but at very low levels (< 5 ng/mL). Peak excretion of total alpha-hydroxytriazolam occurs at approximately 5-10 h following the drug intake. Total alpha-hydroxytriazolam is excreted at detectable levels approximately 2-35 h following an oral dose of 0.25 mg triazolam. Total free and conjugated alpha-hydroxytriazolam excreted by A and B are 0.61% and 31.6%; and 0.36% and 57.2% of the dose, respectively.

Improved screen and confirmation test of 7-aminoflunitrazepam in urine specimens for monitoring flunitrazepam (Rohypnol) exposure.

Confirmed and alleged misuses of flunitrazepam (FM2, Rohypnol) have brought about serious interest in the development of an analytical methodology that can be effectively used for preliminary screen and confirmatory test of FM2 (or its metabolites) in urine specimens under high-volume settings. Reported methods do not serve this need well for the following reasons: (1) common benzodiazepine (BZ) immunoassays (IAs) have broad cross-reactivities toward widely prescribed BZs (and their metabolites) and are therefore likely to generate an unacceptable number of false positives and (2) because FM2 is typically used at low doses (1-4 mg), IAs with low cross-reactivities toward FM2 (and its metabolites) are likely to generate false-negative results. In this current study, a familiar and effective two-step IA/gas chromatography-mass spectrometry (GC-MS) approach is successfully developed and applied to clinical specimens. Cross-reacting characteristics of the following BZ IAs toward various BZs (and their metabolites) are evaluated focusing on their effectiveness in serving as the preliminary test reagent in a two-step testing protocol: TDx, Beckman, CEDIA, Roche Cobas Integra, Emit II Plus, and Cozart ELISA. Although other IAs show broad cross-reactivities toward various BZs and their metabolites, diazepam is the only non-FM2 derived compound that exhibits noticeable cross-reactivity toward Cozart ELISA reagent. Cross-reactivity data and data derived from studies conducted on a limited number of clinical specimens demonstrate that, when used to monitor FM2 exposure in a large population group (including those exposed to other BZs), Cozart ELISA has the potential of being as effective as (or better than) those currently used in various workplace drug-testing programs for monitoring respectively targeted drugs. Data derived from this study further suggest that 50 ng/mL apparent 7-aminoflunitrazepam (Cozart ELISA) and 30 ng/mL free 7-aminoflunitrazepam (GC-MS) are potentially effective preliminary test and confirmation test cut-offs. To maximize efficiency, it is further suggested that urine specimens are first diluted by a factor of 5 for the preliminary test in which a 10-ng/mL 7-aminoflunitrazepam standard is used as the assay's cut-off standard.