HLA genes and haplotypes in Ryukyuans suggest recent gene flow to the Okinawa Islands

Human Biology, Jun 1999 by Hatta, Yoko, Ohashi, Jun, Imanishi, Tadashi, Kamiyama, Haruo, Et al

Abstract Polymorphism of HLA genes was investigated in a population sample of Ryukyuans living on the main island of Okinawa (n = 197), in the southwestern islands of Japan. Serological typing was applied to class I loci (HLA-A, -B, and -C) and to HLA-DRB1; nucleotide sequence-level typing was performed using PCR microtiter plate hybridization and PCR single-strand conformation polymorphism methods. Ryukyuans showed a higher frequency of DRBI *0405 and lower frequencies of DRB1*1502 and DRB1*1302 compared with Hondo Japanese living on main islands. Principal components and phylogenetic analyses of 12 East Asian populations, including Ryukyuans, were performed based on the allele frequencies of HLA-A, -B, and -DRB1. In the principal components analysis 3 Japanese populations (Ryukyuans, Hondo Japanese, and Ainu) formed a cluster and showed the highest affinity to 2 Korean populations. In the phylogenetic tree Ryukyuans and Ainu were neighbors, but the genetic distance between them was larger than the distances between Ryukyuans and Hondo Japanese and between Ryukyuans and Korean populations. The geographic cline of the predominant haplotype in Ryukyuans, A*24-B*54-DRB1*0405, suggests that an ancestral population possessing A*24-B*54-DRB1*0405 moved into the Okinawa Islands after the divergence of Ryukyuans from the Ainu. Such a recent gene flow, probably from South China to the Okinawa Islands, is considered the major cause of difference in genetic characteristics between Ryukyuans and the Ainu.

Numerous attempts have been made to clarify the evolution of modern Japanese. Three major hypotheses-the admixture model, the continuity model, and the transition model-have been evaluated in various fields of anthropology, including morphology and genetics (Suzuki 1969; Yamaguchi 1982; Dodo and Ishida 1990; Omoto 1992). Although the development of modern Japanese is still a matter of controversy, the admixture model is more reasonable than the other 2 models, according to recent publications in both morphology and genetics (Hanihara 1991; Nei 1995; Horai et al.1996; Omoto and Saitou 1997). Thus modern Japanese are considered derived from 2 major groups with different ancestries: a preagricultural indigenous people (Jomon) and post-Neolithic migrants from East Asia (Yayoi).

In the admixture model Ryukyuans living in the Okinawa Islands, the southwestern islands of Japan, are considered relatively pure descendants of the Jomon and of the Ainu living on Hokkaido, the northernmost island. On the other hand, Hondo Japanese living on main islands are thought to have been dramatically influenced by migrants from East Asia by way of the Korean peninsula after the Yayoi era. However, thus far, little attention has been given to the genetic structure of the Ryukyuans and possible gene flow to Hondo Japanese through paths other than the Korean peninsula.

In addition, few reports on these issues have considered HLA (human leukocyte antigen) genes and haplotypes. There are 2 remarkably advantageous features of the HLA system in the genetic analysis of human populations. First, the distribution of alleles reflects well the genetic relationships among human populations because of both the large number of alleles at each locus and the occurrence of population-specific variants. Second, particular HLA haplotypes characterize the migrations and dispersals of populations, because strong linkage disequilibria among HLA loci has resulted from close proximity of HLA genes on the short arm of chromosome 6. Therefore HLA data are powerful tools in anthropological studies (Serjeantson 1989; Tokunaga et al. 1996).

In the present study we investigated the polymorphisms of the HLA-A, -B, -C, and -DRB1 loci in a population sample of Ryukyuans. We recently described unique characteristics of HLA gene polymorphisms in the Ainu (Bannai et al. 1996; Bannai et al., unpublished data, 1998). We found that the Ryukyuans are significantly different from the Ainu in a comparison of genetic affinities between Ryukyuans and other East Asian populations based on HLA genes and haplotypes. Furthermore, the present findings suggest recent gene flow, probably from South China to the Okinawa Islands.

Materials and Methods

Sample. One hundred ninety-seven peripheral blood samples were obtained from unrelated healthy adults living on the main island of Okinawa (Ryukyu), in the southwestern islands of Japan.

Serological Typing of HLA Class I Antigens. Peripheral lymphocytes were prepared by Ficoll-Hypaque centrifugation. Typing for HLA class I (A, B, and C) antigens was performed by the standard National Institutes of Health microlymphocytotoxicity technique with local qualified antisera.

Allele Typing of HLA-DRB1. Genomic DNA was isolated from peripheral lymphocytes using a commercial kit according to the manufacturer's instructions (QIAamp, Qiagen, Hilden, Germany).

For the HLA-DRB1 locus the polymerase chain reaction microtiter plate hybridization (PCR-MPH) method was used (Kawai et al. 1994, 1996). In brief, after PCR amplification of the second exon of the HLA-DRB1 gene with biotinylated primers, the amplified DNA fragments are hybridized with immobilized specific probes on microtiter plate wells; then the amplified fragments bound to the probes are detected colorimetrically using streptavidin conjugated peroxidase and a substrate of 2,2'-azino-di(3-ethylbenzthiazoline-- 6-sulfonic acid) (ABTS). Low-resolution (serology-level) typing was performed using generic PCR primers and 16 different probes.