Rieko Uchihi

Deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese

The deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs is described. HLA-DQA1 genotypes could be determined from single plucked hair roots. However, it was not easy to type HLA-DQA1 with hair shaft portions. Increase in the specimens of hair shaft portions (over 10 cm in length) to get sufficient DNA caused inhibition of polymerase chain reaction (PCR). Synthetic melanin as well as the one extracted from hairs inhibited the PCR of the genomic DNA template when added to the PCR reaction at the concentrations over than 15 ng/100 microL. Therefore, typability of hair shaft portions seems to depend on the delicate balance of the concentrations of DNA and the contaminated melanin in the final DNA extracts.

Mutations in 14 Y-STR loci among Japanese father-son haplotypes.

In the present study 161 Japanese father/son haplotype transfers in 147 pedigrees were analyzed at 14 Y-STRs with two multiplex PCR-based typing systems. Five isolated single repeat mutations were identified at the DYS389I, DYS439, Y-GATA-H4, DYS389II and DYS391 loci, and a pedigree showing triple alleles at the DYS385 locus (a duplicate locus) without allelic discrepancy between the father and son was also observed. The overall mutation rate estimated across the 14 Y-STRs in the Japanese population was 0.22%/locus/meiosis (95% C.I. 0.09–0.51%). This rate was not significantly different (p>0.05) from those of autosomal STRs and Y-STRs in other populations, including German, Austrian, Polish and Norwegian populations. Furthermore, 138 haplotypes were identified in 147 pedigrees with a haplotype diversity value of 0.9983. Therefore, a combination of the two systems should permit effective analysis with sufficient discriminatory power.

Frequency of HLA-DQA1 alleles in the Japanese population

One of the HLA class II genes, HLA-DQA1, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQA1 gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQA1 genotype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. The HLA-DQA1 genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.

ALLELE DISTRIBUTION AT NINE STR LOCI-D3S1358, VWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 AND D7S820-IN THE JAPANESE POPULATION BY MULTIPLEX PCR AND CAPILLARY ELECTROPHORESIS

Nine tetranucleotide short tandem repeat (STR) loci, D3S1358, vWA, FGA TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820, were analyzed in the Japanese population with a newly released kit for personal identification using multiplex PCR with fluorescent-labeled primers following capillary electrophoresis. The observed heterozygosities were 0.67, 0.77, 0.82, 0.61, 0.62, 0.73, 0.78, 0.81 and 0.74, respectively, and the combined discrimination power of the nineplex was 0.9999999991. None of the nine loci deviated from Hardy-Weinberg equilibrium expectations using the chi-square test, homozygosity test, likelihood ratio test and exact test after the grouping of the alleles. The nine STR loci allele frequencies were significantly different from those of other ethnic populations.

Purification of nuclear DNA from single hair shafts for DNA analysis in forensic sciences

The typing of nuclear DNA from hair shafts has often been unsuccessful to date. We tried to type one of the nuclear DNA loci, HLA-DQA1, from hair shafts, using an efficient cetyl-trimethyl ammonium bromide (CTAB) precipitation for DNA purification and a sensitive semi-nested PCR. After thorough washing with ethanol and water, hair shafts were digested by proteinase K in the presence of dithiothreitol, followed by a purification step including CTAB-DNA precipitation. The specific region of HLA-DQA1 gene was amplified by the semi-nested PCR, and the amplified products were cloned and sequenced. The HLA-DQA1 genotype was determined by comparing the sequence to the known sequence of each allele. All genotypes of HLA-DQA1 were successfully typed with hair shafts from six known heterozygotes, although one of them showed the predominant appearance of one allele. For correct typing, a template DNA equivalent to a hair shaft of 5 or 10 cm in length was necessary. Without the CTAB-DNA precipitation step, DNA extract from such hair shafts inevitably contains enough melanin to inhibit PCR. The present results suggest that hair shafts can be used for the typing of nuclear DNA loci.

Phylogenetic relationship of the populations within and around Japan using 105 short tandem repeat polymorphic loci

We have analyzed 105 autosomal polymorphic short tandem repeat (STR) loci for nine East and South-eastern Asian populations (two Japanese, five Han Chinese, Thai, and Burmese populations) and a Caucasian population using a multiplex PCR typing system. All the STR loci are genomewide tetranucleotide repeat markers of which the total number of observed alleles and the observed heterozygosity were 756 and 0.743, respectively, for Japanese populations. Phylogenetic analysis for these allele frequency data suggested that the Japanese populations are more closely related with southern Chinese populations than central and/or northern ones. STRUCTURE program analysis revealed the almost clearly divided and accountable population structure at K=2–6, that the two Japanese populations always formed one group separated from the other populations and never belong to different groups at K≥3. Furthermore, our new allele frequency data for 91 loci were analyzed with those for 52 worldwide populations published by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian populations with large population size (six Han Chinese, three Japanese, two Southeast Asia) formed one distinct cluster and are closer to each other than other ethnic minorities in east and Southeast Asia. This pattern may be the caviar of comparing populations with greatly differing population sizes when STR loci were analyzed.

Maternal Identification from Skeletal Remains of an Infant Kept by the Alleged Mother for 16 Years with DNA Typing

This is a case study concerning maternal identification by DNA typing at various loci. An infant skeleton was found in the alleged mothers apartment after it was kept for 16 years. We obtained the skeletal remains as well as saliva stains from the alleged mother. DNA typing was conducted for three loci in the HLA class II region (HLA-DQA1, -DPB1, and DRB1), five loci with the AmpliType PM kit (LDLR, GYPA, HBGG, D7S8, and GC), five STR loci (LPL, vWA, F13B, TH01, and TPOX) and D-loop region in mtDNA for maternal identification. Sex determination was accomplished using fluorescent DNA capillary electrophoresis typing. Approximately 5 ng of human DNA was recovered from 1 g of femur bone retrieved from the infant skeletal remains. The probability of two unrelated Japanese sharing the same genotypes was estimated as 7.2 x 10(-11). The combined probability of exclusion that an individual is not the mother was also calculated at 0.998. We therefore conclude that the skeleton is from a female infant, and that there is no inconsistency in the claim that the infant was a daughter of the alleged mother.

A powerful, novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations

A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)‐Japanese and 187 Okinawa‐Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy‐Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu‐Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu‐Japanese were almost the same as in the Okinawa‐Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.

Applications of Minisatellite Variant Repeat (MVR) Mapping for Maternal Identification from Remains of an Infant and Placenta

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to a practical forensic case of an infant and placenta found in an incinerator. They were thought to be left for a few days postmortem, and the infant was severely burnt when found. DNA was extracted from the infantile muscle and maternal placental hematoma. MVR-PCR analysis as well as other common DNA typing (D1S80, HLA-DQA1) were performed on both DNA samples. Both MVR diploid codes were matched although some extra faint bands in the ladder were observed from the maternal placental sample, which probably indicated superimposing of an allele derived only from the mother, and not the infant. In order to detect the original maternal alleles, three flanking polymorphic sites were typed and allele-specific MVR-PCR was performed. Finally, one maternal allele not inherited by the infant and two alleles from the infant were typed. Two alleles suggested the infant and/or mother was Japanese. The two diploid codes including one possibly from the mother were deduced and compared with other codes in the databases for evaluating the discriminating power.

Potential Forensic Applications of Minisatellite Variant Repeat (MVR) Mapping Using the Polymerase Chain Reaction (PCR) at D1S8

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to samples from various human tissues. All DNA samples obtained from an individuals organs at autopsy consistently gave the same digital diploid codes. Even 1 ng of genomic DNA was sufficient to obtain authentic diploid MVR coding ladders. MVR-PCR could be reliably applied to DNA isolated from bloodstains, saliva stains, seminal stains and plucked hair roots, and should become a powerful tool for individual identification in forensic investigations.