Kiyoshi Minaguchi

Sequence polymorphisms of the mitochondrial DNA control region and phylogenetic analysis of mtDNA lineages in the Japanese population

Abstract.Sequence polymorphisms of the hypervariable mitochondrial DNA (mtDNA) regions HVI and HVII, and coding region polymorphisms were investigated in 211 unrelated individuals from the Japanese population. Sequence comparison of the HVI and HVII regions led to the identification of 169 mitochondrial haplotypes defined by 147 variable positions. Among them 145 types were observed in only 1 individual; the other 24 types were shared by 2 or more individuals. The gene diversity was estimated at 0.9961, and the probability of two randomly selected individuals from the population having identical mtDNA types was 0.86%. We also established phylogenetic haplogroups in the Japanese population based on the coding and control region polymorphisms and compared the haplotypes with those in other Japanese, Korean and Chinese populations. As a result, three new subhaplogroups, G4a, G4b, and N9b, and several haplotypes specific for the Japanese and Korean populations were identified. The present database can be used not only for personal identification but also as an aid for geographic or phenotype (race) estimation in forensic casework in Japan.

Y‐chromosomal Binary Haplogroups in the Japanese Population and their Relationship to 16 Y‐STR Polymorphisms

We investigated Y chromosomal binary and STR polymorphisms in 263 unrelated male individuals from the Japanese population and further examined the relationships between the two separate types of data. Using 47 biallelic markers we distinguished 20 haplogroups, four of which (D2b1/‐022457, O3/‐002611*, O3/‐LINE1 del, and O3/‐021354*) were newly defined in this study. Most haplogroups in the Japanese population are found in one of the three major clades, C, D, or O. Among these, two major lineages, D2b and O2b, account for 66% of Japanese Y chromosomes. Haplotype diversity of binary markers was calculated at 86.3%. The addition of 16 Y‐STR markers increased the number of haplotypes to 225, yielding a haplotype diversity of 99.40%. A comparison of binary haplogroups and Y‐STR type revealed a close association between certain binary haplogroups and Y‐STR allelic or conformational differences, such as those at the DXYS156Y, DYS390m, DYS392, DYS437, DYS438 and DYS388 loci. Based on our data on the relationships between binary and STR polymorphisms, we estimated the binary haplogroups of individuals from STR haplotypes and frequencies of binary haplogroups in other Japanese, Korean and Taiwanese Han populations. The present data will enable researchers to connect data from binary haplogrouping in anthropological studies and Y‐STR typing in forensic studies in East Asian populations, especially those in and around Japan.

Cytokine-Inducing Activity of Family 2 Cystatins

Abstract Cystatins are physiological cysteine proteinase inhibitors. Here we report a novel function for some family 2 cystatins that is not related to these activities. The release of interleukin-6 (IL-6) and interleukin-8 (IL-8) by the gingival fibroblasts and that of IL-6 by murine splenocytes were measured using ELISA systems specific for these cytokine molecules. Family 2 cystatins, including cystatins C, SA1, SA2, S, and egg white cystatin, upregulated the IL-6 production by two human gingival fibroblast cell lines or murine splenocytes and also the IL-8 production by gingival fibroblasts at physiological concentrations. After complete saturation with papain, those family 2 cystatins still upregulated IL-6 production, suggesting that the papain- inhibitory site was not involved in the cytokine-inducing activity.

Phylogenetic classification of Japanese mtDNA assisted by complete mitochondrial DNA sequences

We investigated control and coding region polymorphisms in mitochondrial DNA (mtDNA) in 100 unrelated individuals from a Japanese population and determined the basal phylogenetic haplogroup lineages in all samples under updated information. Many of the basal phylogenetic haplogroup lineages assigned on East Asian mtDNA haplogroups corresponded to those previously established. However, new haplogroup lineages such as M7a2a, M7a2b, M7a2*, M7c1b, M11b2*, G2b*, D4c1b1a, D4g2b, A4*, A9, N9b*, B4d1, B4d2, and F1e were identified and established by complete sequencing. Although sequence comparison of the 1.15-kb control region identified 84 mitochondrial haplotypes, examination of coding region polymorphisms increased the total number of haplotypes to 91. Determination of the basal haplogroup lineages increased the discrimination power of mtDNA polymorphisms for personal identification and their usefulness in determining geographic origin in forensic casework in Japanese and other East Asian populations.

Salivary cystatins induce interleukin-6 expression via cell surface molecules in human gingival fibroblasts

Recently, family 2 cystatins have been demonstrated to upregulate interleukin-6 (IL-6) production by human gingival fibroblasts [Biol. Chem. 381 (2000) 1143]. To elucidate the mechanism of the IL-6 inducing activity of cystatins, we tested NF-kappa B activation with salivary cystatins SA1 and SA2-stimulated human gingival fibroblast whole cell lysates. The IL-6 production by human fibroblasts in response to these cystatins was inhibited by tyrosine kinase inhibitors and an inhibitor of NF-kappa B activation. The IL-6 inducing activity of the cystatins was depressed by the anti-CD58 monoclonal antibody. These findings supply evidence that cystatins SA1 and SA2 adhere to human fibroblasts and that the event results in tyrosine phosphorylation and upregulation of the release of IL-6 mediated enhancement of NF-kappa B activity.

MtDNA control region sequence polymorphisms and phylogenetic analysis of Malay population living in or around Kuala Lumpur in Malaysia

Control region polymorphisms in the mitochondrial DNA of 124 unrelated individuals from the Malay population living in or around Kuala Lumpur in Malaysia were investigated and phylogenetic haplogroup lineages were determined. The intergenic COII/tRNALys 9-bp deletion, 3010 and 5178 mutations, and several coding region polymorphisms were examined to discriminate some phylogenetic haplogroups. Sequence comparison of the control regions led to the identification of 117 mitochondrial haplotypes, in which 103 types were observed in only one individual and the other nine types were shared by more than two individuals. Gene diversity was estimated to be 0.997. Phylogenetic haplogroup determination revealed that the gene pool of the modern Malay population in Malaysia consisted mainly of southeast Asian, east Asian, unidentified and unique, and aboriginal southeast-specific haplogroups. These results suggest a multi-original nature for the modern Malay population. The present database may help not only in personal identification but also in determining geographic origin in forensic casework in Malaysian, Southeast Asian and East Asian populations.

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.

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.

Cystatin SA, a cysteine proteinase inhibitor, induces interferon-γ expression in CD4-positive T cells

Abstract Recently, it has been demonstrated that family 2 cystatins upregulate interleukin-6 production by human gingival fibroblasts. In the present study, we investigated the effects of cystatin SA on cytokine production by helper T cells. Human CD4-positive T cells were cultured with phytohemagglutinin in the presence or absence of 0.1 M recombinant cystatin SA1 or SA2. When the amounts of interleukin-4 (IL-4) and interferonγ (IFNγ) were analyzed in an ELISA system after stimulation with either cystatin, no significantly increased levels of IL-4 were detected. However, the amounts of IFNγ were significantly increased after stimulation with the cystatins. Our results suggest that salivary family 2 cystatins are involved in immune responses through the cytokine network.

Genetic polymorphisms of the CST2 locus coding for cystatin SA

A new genetic polymorphism of cystatin SA has been identified in human submandibular-sublingual saliva by means of basic gel electrophoresis and immunoblotting with anti-cystatin S. Two proteins, SA1 and SA2, are given by two alleles of CST2, viz., CST2*1 and CST*2. Inheritance is controlled by two codominant alleles at an autosomal locus. This hypothesis is supported by studies of 16 families 32 children. Gene frequencies for CST2*1 and CST2*2 are 0.935 and 0.065, respectively (n = 341). Eighteen amino acids determined among 20 N-terminal residues of cystatin SA2 are identical with the sequence encoded by CST2. Three forms of cystatin S (mono-phosphorylated cystatin S, di-phosphorylated cystatin S, and non-phosphorelated cystatin S) are present in the 341 saliva samples tested.