Y chromosome analysis for common surnames in the Japanese male population.

For many years of Japan's long history, Japanese surnames have been handed down patrilineally. This study investigated relations between major surnames and Y chromosomal polymorphism among the Japanese male population. To analyze genetic phylogeny in namesakes, the Y-single nucleotide polymorphism (SNP) plus Y-short tandem repeat (STR) approach was employed. A haplogroup based on SNPs and haplotypes at 17 STR loci were typed in 567 unrelated volunteers recruited in Kanagawa, Japan. Samples covered 27 common surnames such as Satoh and Suzuki, each name having 10-55 bearers. Significant difference was found for SNP haplogroup compositions and a multidimensional scaling plot using STR haplotypes in several surname groups. By contrast, these common surnames displayed wide diversity with phylogenetic networks, suggesting that no genetic drift event has occurred in their history. In all, 22 descent clusters were found, as judgcriteria ed by ad hoc of groups within five mutational steps in the 15 STR loci with the same haplogroup. The times of the most recent common ancestor ranged from 279 to over 2577 years. According to the approximate millennium span of Japanese surname history, descent criteria are expected to be reasonable for grouping within four step-neighbors. High heterogeneity of common surnames resembles that observed for England and Spain, but not for Ireland. Our results highlight that common Japanese surnames consist of descent clusters and many singletons, reflecting a mixture of long-term bearers and short-term bearers among the population. The genetic study of this population revealed characteristic features of Japanese surnames.

Population Genetics in Malaysia and Japanese Populations Using Power Plex Y23 System.

Population flow between Southeast Asian countries and Japan continues to gather pace. Accordingly, the number of foreigners involved in incidents in Japan has markedly increased, which means that forensic dentistry is now increasingly being faced with the need to analyze DNA from persons of non-Japanese extraction. The DNA test currently used for personal identification mainly utilizes short tandem repeats (STRs) on autosomal chromosomes and the Y-chromosome. This test was developed for use in personal identification, not for distinguishing among races; nonetheless, the standard method for personal identification is often used because the procedure has been established. To determine the degree to which racial differences can be distinguished by standard DNA analysis, 23 STRs located on the Y chromosome were investigated in 218 Malay and 426 Japanese males. The frequencies of each STR were calculated in the two populations. The difference in the power of discrimination between the Malay and Japanese populations ranged from a minimum of 0.01 to a maximum of 0.27; the difference in polymorphic information content ranged from 0.01 (minimum) to 0.23 (maximum). No major differences were noted in the polymorphisms in these two Mongoloid populations, but the distributions of the 17 STRs differed significantly. Short tandem repeat types demonstrating a likelihood of racial differences were identified in 14 of the STRs. Race-specific STR types were identified in 10 STRs. These results suggest that the likelihood of Malay or Japanese genetic background can be judged based on Y-chromosome STR test results.

Evaluation of Y chromosomal SNP haplogrouping in the HID-Ion AmpliSeq™ Identity Panel.

The Y chromosomal haplogroup determined from single nucleotide polymorphism (SNP) combinations is a valuable genetic marker to study ancestral male lineage and ethical distribution. Next-generation sequencing has been developed for widely diverse genetics fields. For this study, we demonstrate 34 Y-SNP typing employing the Ion PGM™ system to perform haplogrouping. DNA libraries were constructed using the HID-Ion AmpliSeq™ Identity Panel. Emulsion PCR was performed, then DNA sequences were analyzed on the Ion 314 and 316 Chip Kit v2. Some difficulties became apparent during the analytic processes. No-call was reported at rs2032599 and M479 in six samples, in which the least coverage was observed at M479. A minor misreading occurred at rs2032631 and M479. A real time PCR experiment using other pairs of oligonucleotide primers showed that these events might result from the flanking sequence. Finally, Y haplogroup was determined completely for 81 unrelated males including Japanese (n=59) and Malay (n=22) subjects. The allelic divergence differed between the two populations. In comparison with the conventional Sanger method, next-generation sequencing provides a comprehensive SNP analysis with convenient procedures, but further system improvement is necessary.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Effects of using the GlobalFiler™ multiplex system on parent-child analyses of cases with single locus inconsistency.

Parent-child analyses sometimes reveal inconsistency of shared alleles at only one locus. This is conventionally called "single locus exclusion", which results from mutational events and the presence of null alleles. Here, in parent-child analyses of the Japanese population, we detected exclusions by using the GlobalFiler™ system comprising 21 short tandem repeat loci. One- or two-step mutations resulting from strand slippage causing gain or loss were observed in seven of 221 parent-child transmissions. The incidences of single locus inconsistency of alleles were 5.88×10(-2) and 8.40×10(-3) for paternal and maternal relationships, respectively. With calculation using a set of 15 loci in the Identifiler® multiplex system, the combined likelihood ratio (CLR) values were limited to less than 100 in all five cases accompanied by single inconsistency. The addition of six loci recovered the CLR values to over 10,000 in three cases. Application of this advanced system may increase the detected occurrence of mutational events, but it should be beneficial for inference in parent-child analyses, particularly in cases accompanied by genetic inconsistency.

Population Genetics of Identifiler System in Malaysia.

Short tandem repeat (STR) polymorphisms were investigated in 341 unrelated Malay individuals (218 males and 123 females) living in or around Kuala Lumpur by using a forensic analysts kit. The following STRs were targeted: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA. The purpose of this study was to elucidate population genetics in Malaysia and calculate statistical parameters for forensic and anthropological research. Data on these STRs in the target population were obtained and subjected to statistical analysis. Accordance with the Hardy-Weinberg equilibrium was proven for all the loci targeted. The combined power of discrimination was greater than 0.9999999999, indicating that this multiplex system is an excellent tool for forensic casework. The allele frequency in the data were weighed against that in four other local populations (Chinese, Iranian, Belgian, and African). The average coefficient of correlation was strongest in the order of Africa (0.092522), Belgium (0.264822), Iran (0.404363), and China (0.706661). These results are consistent with what is known about the anthropological history of and prehistoric human migration in the Malay region. We believe that these data offer a valuable anthropological resource, being applicable to the statistical evaluation of DNA evidence in human identification, as well as the determination of ethnicity in healthy populations.

Assignment of Y-chromosomal SNPs found in Japanese population to Y-chromosomal haplogroup tree.

The relationship between Y-chromosome single-nucleotide polymorphisms (SNPs) registered in the Japanese SNP (JSNP) database (http://snp.ims.u-tokyo.ac.jp) and Y-binary haplogroup lineages was investigated to identify new Y-chromosomal binary haplogroup markers and further refine Y-chromosomal haplogroup classification in the Japanese population. We used SNPs for which it was possible to construct primers to make Y-specific PCR product sizes small enough to obtain amplification products even from degraded DNA, as this would allow their use not only in genetic but also in archeological and forensic studies. The genotype of 35 JSNP markers were determined, of which 14 were assigned to appropriate positions on the Y-chromosomal haplogroup tree, together with 5 additional new non-JSNP markers. These markers defined 14 new branches (C3/64562+13, C3/2613-27, D2a1b/006841*, D2a1b/119166-11A, D2a/022456*, D2a/119166-11A, D2a/119167rec/119167-40rec*, D2a/75888-GC, O3a3c/075888-9T/10T*, O3a3c/075888-9T/9T, O3a3/8425+6, O3a3/119166-13A*, O3a3/008002 and O3a4/037852) and 21 new internal markers on the 2008 Y-chromosome haplogroup tree. These results will provide useful information for Y-chromosomal polymorphic studies of East Asian populations, particularly those in and around Japan, in the fields of anthropology, genetics and forensics.

Multiplex PCR for 18 X-chromosomal STRs in Japanese population.

X-chromosomal STR (X-STR) polymorphisms are particularly useful in complex cases of kinship testing involving inheritance through female subjects. An X-chromosomal multiplex amplifying 18 STRs in one single PCR reaction was developed and optimized in this study. The multiplex system included the DXS7424, GATA172D05, HPRTB, DXS8377, GATA31E08, DXS6810, DXS7423, DXS981, DXS6795, DXS6803, DXS6789, DXS6800, DXS6809, DXS7133, DXS7132, DXS9902, DXS101 and DXS6807 loci, which are distributed over the whole X-chromosome. It was designed as a potential first option in determining recombination within the whole X chromosome in kinship testing. Allele frequencies were obtained from samples from 378 male and 175 female Japanese individuals, all unrelated. The sizes of the amplified products ranged from 82 to 297 bp. The combined power of discrimination of the 18 loci was 0.999999999999997 in females and 0.9999999992 in males. A case is presented in which this system allowed considerable efficacy in reaching a solution. The present multiplex system amplified the largest number of loci among the X-STR multiplex systems tested, indicating its potential in personal identification and determining kinship.

Deletion polymorphism at chromosome 3q26.1 and oral squamous cell carcinoma.

Several recent studies have investigated DNA instability in malignancies including deletions and duplications of part of the chromosome using array-based comparative genomic hybridization (CGH) analysis. Using the same approach on oral squamous cell carcinoma (OSCC) tissue samples, we found a frequent deletion at chromosome 3q26.1 in OSCC patients; this polymorphism showed a gene frequency of 0.293-0.368 in healthy volunteers (n=60) and 0.129-0.195 in OSCC patients (n=54). Detailed analysis around the polymorphic region revealed the deletion breakage point. A significant association of gene frequency for the deletion polymorphism between healthy volunteers and patients implicated genetic factors related to this polymorphism in the development of OSCC. Currently, no gene is predicted to lie within the 3,606-kbp region around the polymorphism. Thus, although a single-gene model could not explain the occurrence of OSCC, we believe that examining this polymorphism could be useful in identifying risk factors for OSCC.

Genetic study of 12 X-STRs in Malay population living in and around Kuala Lumpur using Investigator Argus X-12 kit.

We investigated 12 X-chromosomal short tandem repeat (STR) polymorphisms in 283 unrelated Malay individuals (160 males and 123 females) living in and around Kuala Lumpur using the Investigator Argus X-12 kit. Heterozygosity among the present 12 X-STRs showed a distribution of from 55.3 to 93.5 %. The diversity values of the haplotypes constructed using four closely linked groups were all higher than 0.9865. A comparison of allelic frequency in each system and haplotype variation indicated that the nature of these X-STRs in the Malay population differed from that in East Asian, European, or African populations. Several microvariant alleles found in the Malay population were characterized and compared with known sequence data. The present data may be helpful in forensic casework such as personal identification and kinship testing in the Malay population in Malaysia.

New device for collecting intra-oral findings of unknown body.

In dental identification, the collection of intra-oral images is extremely important. We propose the Dental Watch(®) as a new device for collecting intra-oral findings in situations where sufficient jaw-opening or adequate lighting cannot be obtained in such cases of dead bodies within a day after death or burned bodies encountered in mass disaster. This device is an improved home video camera for taking intra-oral images. It is lightweight and cordless, allowing it to be operated with one hand, and an audio function allows comments to be made and recorded on obtaining findings at the same time as images are taken. In addition, this device allows images of the entire oral cavity to be taken comparatively easily, even when only a minimal degree of jaw movement is available. This device is extremely useful in situations where a single dentist inspector must obtain findings and make an accurate and detailed Dental Chart.

Population genetic study of six closely linked groups of X-STRs in a Japanese population.

X chromosome STR (X-STR) polymorphisms are a useful tool in the fields of human population genetics and personal identification and are quite informative in the investigation of complex kinship or deficiency cases, especially where it is necessary to determine relationships with second-generation offspring in which the same X chromosome may have been inherited. We investigated eight X-STR systems using the Mentype Argus X-8 kit and further developed decaplex PCR for the DXS10148, DXS10161, DXS10160, DXS10159, DXS10079, DXS10075, DXS6799, DXS10102, DXS10106, and DXS10146 loci with the aim of constructing closely linked groups on the X chromosome. The studied population comprised 569 Japanese individuals (390 males and 179 females). Heterozygosity among the present 18 X-STRs showed a distribution of from 54.2% to 90.5%. We constructed six closely linked groups, each comprising three to five X-STRs: DXS10148- DXS10135-DXS8378, DXS10161- DXS10160-DXS10159, DXS7132-DXS10079-DXS10074-DXS10075-DXS981, DXS6809-DXS6789-DXS6799, DXS10102-HPRTB-DXS10101-DXS10106, and DXS8377-DXS10146-DXS10134-DXS7423. The forensic utility of these groups as haplotypes was then evaluated. Haplotype diversity values showed a distribution of from 0.9699 to 0.9959. Analysis of the present closely linked haplotypes will contribute to solving complex kinship cases involving X chromosome inheritance.

Sixteen X-chromosomal STRs in two octaplex PCRs in Japanese population and development of 15-locus multiplex PCR system.

X-chromosome short tandem repeat (STR) polymorphisms are a useful tool in the fields of human population genetics and personal identification and are indispensable in investigating complex kinship or deficiency cases in circumstances where information on mtDNA or Y-chromosome polymorphisms is unavailable. The purpose of this study was to construct a multiplex polymerase chain reaction (PCR) system capable of analyzing a large number of X-STR loci and establish a 16-X-STR database in the Japanese population We developed two octaplex X-STR systems, one including the DXS7424, GATA172D05, HPRTB, DXS8377, GATA31E08, DXS9895, DXS7423, and DXS981 loci and the other the DXS6803, DXS6789, DXS6800, DXS6809, DXS7133, DXS7132, DXS101, and DXS6807 loci, and conducted a population study in 512 Japanese individuals comprising 339 men and 173 women. A 16-locus multiplex system produced unwanted PCR products due to mixture of the DXS9895 primer with the primers of two other loci. However, a 15-locus multiplex system exclusive of the DXS9895 locus did not. The 15-locus multiplex system amplified the largest number of loci among the X-STR multiplex systems used and afforded a power of discrimination of 0.99999999999997 in women and 0.999999997 in men.

Development of vertically adjustable electrically-powered forensic dental examination table.

Dental evidence for post-mortem identification is often examined on a dissecting table through unavailability of an identification table. In mass disasters, however, this is often done on the ground, floor, or in a simple coffin through lack of adequate facilities. We have designed a vertically adjustable, electrically-powered forensic odontological table. Adjustable from a height of 200-1015 mm, compact and light, it offers portability, work efficiency and prevention of infection. Head position components are angle-adjustable and removable from the main body of the table. This table offers the following advantages: (1) Adjustable height enables improved comfort for each type of work according to examiner's height. This new table allows for the optimum posture for each examiner by easy adjustment to a comfortable height; (2) The detachable head component makes taking X-ray photos easy without having to rearrange the position of remains on the table; (3) Contamination of examiner's clothes or personal effects is prevented by virtue of minimally sized top board. Portability and cost performance are enhanced by miniaturization and, most importantly, the integrity and dignity of the remains are improved and preserved. This table is very useful for personal dental identification.

Collection of intraoral findings in corpse with small-scale color dental scanner system.

Together with X-ray radiography and the description in the dental chart (odontogram), the collection of intraoral images is extremely important in dental identification. Recently, thanks to advances in digital devices for taking images in the oral cavity, problems with developing images and images being lost due to scanning errors have been minimized. However, in corpses where postmortem rigidity has firmly set in and burned bodies where the jaw has to be forced open, it is difficult to open the jaw enough to allow images to be taken. In addition, collection of intraoral images requires skill. Our goal was to determine the efficacy of a newly developed, small-scale color dental scanner in collecting intraoral images. The results showed that it was comparatively easy to obtain an entire image of the oral cavity with even a minimum degree of jaw opening. This should enable even a non-expert to perform oral image collection.

[Personal identification using information from cranio-facial region].

Much of Forensic Odontology is concerned with personal identification, through examination of cranio-facial region. This paper describes several studies in which we worked with materials derived from cranio-facial region. The following topics are addressed : (1) Human saliva contains proteins specific to salivary glands, proteins which are highly polymorphic compared with those found in other body fluids. In particular, six genes for proline-rich proteins coded many proteins found in human saliva, and we found several of them. At least five kinds of cystatin are secreted in saliva. We constructed recombinant polymorphic proteins, cystatin SAl and SA2. Using these proteins, we compared effects of amino acid mutation on protease inhibitor activity, and demonstrated a novel function for type-2 cystatin cytokine-inducing activity. (2) Among autosomal STR loci, we identified the D12S67 locus as highly polymorphic, with a heterozygosity of 95%, by investigating differences in nucleotide repeat units. Highly polymorphic autosomal STR loci offer an effective forensic tool under certain conditions, in addition to multiplex PCR, and therefore merit further study in forensic practice. (3) Although digitalization is prevalent in photography, analog images are preferable in certain circumstances as they offer better resolution. (4) Usually, information on mtDNA polymorphisms from HV1 and HV2 in the control region is used in forensic practice. However, information from the coding region considerably increases the discrimination power of mtDNA polymorphisms. It is important to increase the volume of coding region information available with regard to mtDNA polymorphisms for future forensic practice. (5) Y-STR polymorphisms are closely associated with binary haplogroups, and it is possible to estimate a binary haplogroup from an STR haplotype. (6) Mitochondrial DNA and Y-chromosomal polymorphisms can be used to determine geographic origin in individuals from East Asia, something that was considered difficult in the past. (7) A Dental Scan was constructed for the preparation of dental records. It offers a superior method to the taking of pictures as is done in standard forensic odontology.

Proposal for internet-based Digital Dental Chart for personal dental identification in forensics.

A dental chart is very useful as a standard source of evidence in the personal identification of bodies. However, the kind of dental chart available will often vary as a number of types of odontogram have been developed where the visual representation of dental conditions has relied on hand-drawn representation. We propose the Digital Dental Chart (DDC) as a new style of dental chart, especially for open investigations aimed at establishing the identity of unknown bodies. Each DDC is constructed using actual oral digital images and dental data, and is easy to upload onto an Internet website. The DDC is a more useful forensic resource than the standard types of dental chart in current use as it has several advantages, among which are its ability to carry a large volume of information and reproduce dental conditions clearly and in detail on a cost-effective basis.

Murine monoclonal antibody which can distinguish cystatins SA1 and SA2.

To develop a diagnostic trial enabling the selective examination for a target cystatin in human body fluids, we attempted to prepare monoclonal antibodies against human cystatin SA1 (originally cystatin SA) and its variant form (cystatin SA2). BALB/c mice were immunized with recombinant (r-) cystatins SA1 and SA2. Two monoclonal antibodies designated Cys3F11 and Cys2E5 were selected. By ELISA analyses, the Cys2E5 was shown to react with r-cystatin SA2 but also somewhat with r-cystatin SA1 (22% cross-reactivity) and with plasma cystatin C (18% cross-reactivity), indicating a high specificity for cystatin SA2. The Cys3F11 reacted not only with r-cystatin SA1 but also with r-cystatin SA2 (89% cross-reactivity) and plasma cystatin C (47% cross-reactivity). This finding was further emphasized by immunoblotting of human submandibular-sublingual saliva samples. ELISA additivity test suggests that the two monoclonal antibodies bind to distinct epitopes. In conclusion, we have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.

Polymorphism of LPL Locus in Japanese and comparison of PCR amplification efficiency from degraded DNA between LPL locus and the D21S11.

The short tandem repeat (STR) polymorphism of the lipoprotein lipase (LPL) locus was amplified by PCR and analyzed using denaturing polyacrylamide gel electrophoresis followed by silver staining. Among 158 DNA samples from the Japanese population, six alleles were observed. When the sequences of the allelic products were compared, each allelic segment contained 7 and 9-13 TTTA tetranucleotide repeat motifs. Genotypic distribution met Hardy-Weinberg expectations, and included heterozygosity was 48.8%. Most of the Japanese genotypes allele 10. When PCR amplification efficiency for the LPL locus from degraded DNA was compared with that for the D21S11 locus in terms of amplification size, increase in amplification size showed a considerable influence on amplification efficiency, producing inaccurate amplification, such as unbalanced amplification, or amplification of non-target PCR products. These results suggest that reduction in amplification size increases the accuracy and efficiency of PCR amplification from highly degraded DNA.

Identification of unknown body using DNA analysis and dental characteristics in chest X-ray photograph.

An unknown skeletonized body was identified by DNA analysis and dental information. The body had already been cremated when a candidate for the unknown body was proposed. Therefore, for DNA analysis we used teeth that had been kept for a long time after use for serological examination. We also used a chest X-ray photograph of the candidate and photographs of dentition, as well as dental X-ray photographs taken when the unknown body was found. Because DNA obtained from teeth was highly degraded, we amplified three PCR fragments to determine the 766 bp mitochondrial DNA (mtDNA) sequence including HV1 and HV2. Polymorphism of the ABO locus was also analyzed using small PCR fragments. Although the isolated DNA was contaminated, probably with DNA from a different individual, DNA polymorphisms of mtDNA and the ABO locus could be analyzed. We discuss the reliability of our conclusions from the point of view of the necessity of constructing an accurate mtDNA database. Although a dentist who had treated the teeth of the unknown body could not be found, a chest X-ray photograph for medical diagnosis was very useful in comparing dental characteristics, as it included an image of the frontal part of the lower jaw and upper teeth.