Genetic Profile of Two Tibetan Populations from China by Analysis of 15 STR Loci

Ying, Binwu

Abstract

Allele frequency data for the STR systems D3S1358, TH01, D21S11, D18S51, PENTAE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PENTAD, VWA, D8S1179, TPOX, and FGA were determined in two population samples of unrelated, healthy Tibetan individuals. All loci met Hardy-Weinberg expectations, and there was no evidence of association of alleles among the 15 loci. These findings suggest that these STR loci could be particularly powerful tools in forensic medicine and could provide the necessary fundamental population genetic data for the reconstruction of recent human evolutionary history.

KEY WORDS: TIBETANS, CHINA, LHASA TIBETAN GROUP, KANGBA TIBETAN GROUR DNA TYPING, SHORT TANDEM REPEAT, FORENSICS, POPULATION GENETICS, D3S1358, TH01, D21S11, D18S51, PENTAE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PENTAD, VWA, D8S1179, TPOX, FGA.

China is a huge multi-ethnic country with 56 identified ethnic groups. Among these groups are the Han, who have a population of 1.1 billion, the largest ethnic group in the world. Each of the 55 ethnic minorities have populations ranging from 1,000 to 16 million. The origin and development of an ethnic group and its blood ties with other groups should be synthetically studied from the point of view of archeology, history, anthropology, linguistics, genetics, and other sciences.

The Tibetan ethnic group is one of the largest ethnic groups in China. The Tibetans number more than 1 million. Tibetan is a general term for people who live in the Himalayan Mountains and speak languages under the Tibetan branch of the Tibeto-Burman subfamily of the Sino-Tibetan family (Matisoff 1991). Tibetans are an important ethnic group in China and have their own customs and habits, costume and ornaments, beliefs, religion, and language.

Ancient Chinese archeological findings have been increasing gradually but are still fragmentary and have many missing links. Previous research is not complete or may be incorrect. Many ethnic groups had no written language in the past, and thus there are no written records about the prehistoric activities of human beings. Many anthropological characteristics (such as skin color and status) are strongly influenced by environmental factors. Populations with different origins evolved in the same direction under similar environments. The characteristics of cultural anthropology, such as customs and habits, costume and ornaments, beliefs and religion, as well as language, can easily change rapidly through conquests, living together, or contact with neighboring ethnic groups (DuandXiao 1997).

People who speak Central and Northern Tibetan are mainly distributed in China (Tibet, Sichuan, and Yunnan), whereas Southern Tibetan speakers live in die southern Himalayan region, for example, in India, Bhutan, and Nepal. The Lhasa Tibetan group and the Kangba Tibetans belong to the Northern Tibetan speaking population. They have similar customs and habits, costume and ornaments, beliefs, religion, and language, but the original relation between the two Tibetan populations is still unclear.

The greatest advantage of genetics markers is their stability, although the nucleotides in genetic material (DNA) change at a defined rate and a small part of the mutations are under the selection of environment, with most of them being neutral, but the DNA is transmitted from generation to generation reliably. Therefore the genetic study of the origin and development of ethnic groups as well as the kinship between them has special significance.

Tibetan populations are distinctive, representative, and forensically relevant population groups in China; therefore our aims in this study were to report and examine allele and genotype frequencies for 15 STR loci in two Tibetan population samples and to determine whether the genotype frequencies conform to Hardy-Weinberg equilibrium. In addition, we calculated forensically relevant parameters to ascertain the utility of the 15 STR loci as genetic markers for the Tibetan population in Chinese forensic casework.

Materials and Methods

Population Sample. Whole blood was obtained by venipuncture into vacutainer tubes containing EDTA. One of the two Tibetan study populations consisted of a Tibetan group in Lhasa and a healthy unrelated Chinese Tibetan population living in Lhasa City in the Tibet region (China). The number of chromosomes analyzed was 126. The other Tibetan group was the Kangba Tibetan population, who live in Sichuan (China); the chromosome number for this group was 105.

DNA Extraction. The DNA was extracted from whole blood using the Chelex-100 extraction method (Walsh et al. 1991), and the DNA concentration was determined by the ethidium bromide dot quantization method (Sambrook et al. 1989).

Typing. Individual DNA samples were amplified for 16 STR loci (including amelogenin) by PCR using the Powerplex 16 kit (Promega Corporation, Madison, Wisconsin) under the conditions recommended by the manufacturer in a reaction volume of 12.5 µl in a PE 9600 thermal cycler (Perkin-Elmer, Foster City, California).

The PCR product of 1 µl was mixed with 0.3 µl internal LlZ size standard (GS-500 LlZ, Applied Biosystems, Foster City, California) and 8.7 µl Hi-Di formamide (Applied Biosystems), and electrophoresed in an ABI Prism1 3100 Genetic Analyzer (Applied Biosystems) using the recommended protocol from the kit. The results were analyzed using the Data Collection (version 1.1), Gene-Scan (version 3.7), and Genotyper (version 3.6) software packages (Applied Biosystems).

Analyses of Data. Evaluation of Hardy-Weinberg expectations was carried out using the chi-square test, and Hardy-Weinberg equilibrium was assessed by means of an X^sup 2^ test; the power of discrimination, the probability of paternity exclusion, polymorphic information content, and observed heterozygosity (H^sub 0^) were calculated using Tereba’s (1999) statistical analysis package, version 1.2.

Results and Discussion

The distributions of observed allele frequencies for the 15 loci (D3S1358, THOl, D21S11, D18S51, PENTAE, D5S818, D13S317, D7S820, D16S539, CSFlPO, PENTAD, VWA, D8S1179, TPOX, and FGA) and the results of the different analytical procedures for testing the correspondence of the genotype frequencies with Hardy-Weinberg expectations in the two Tibetan study populations are summarized in Tables 1 and 2, respectively. These two tables show the forensic value of the analyzed systems expressed as various statistical parameters.

All loci met Hardy-Weinberg expectations, and there was no evidence of association of alleles among the 15 loci. The forensic efficiency parameters revealed the high forensic efficiency of the 15 STR loci analyzed. The data presented in this study can be used to calculate matching probabilities in forensic casework if Tibetan individuals are considered a source of DNA evidence.

Some forensic values of interest were calculated. The power of discrimination (Grunbaum et al. 1978) was calculated to be 0.791 (TPOX) to 0.979 (PENTAE) in the Kangba Tibetan population and 0.797 (TPOX) to 0.977 (PENTAE) in the Lhasa Tibetan population. The probability of paternity exclusion (Ohno et al. 1982) was calculated to be 0.258 (THOl) to 0.825 (PENTAE) in the Kangba Tibetan population and 0.325 (THOl) to 0.789 (D18S51) in the Lhasa Tibetan population. Other statistical parameters of genetic variability and forensic values of interest, such as heterozygosity, the number of alleles, and polymorphic information content, were also calculated. The genetic values of interest in the two Tibetan study populations are summarized in Tables 1 and 2.

Tables 3-5 show the comparisons of distributions of allele frequencies and genetic data of all 15 STR loci in the Kangba and Lhasa Tibetan populations. There are five common alleles of D3S1358 between the two populations; the differences are the presence of allele 19 in the Kangba population and of allele 20 in the Lhasa population. Eleven common alleles of D21S11 were found in the two populations; the differences are allele 33.3 in the Kangba and allele 23 in. the Lhasa population. The Kangba Tibetan population carries D18S51 allele 23, FGA allele 28, D7S820 allele 7, and PENTAE. The Lhasa Tibetan population carries allele 8 of D5S818, CSF1PO, and PENTAE, and allele 9 of D8S1179. The greatest number of common alleles was 17 common alleles for the PENTAE locus, and the least number of common alleles was 5 common alleles for the D3S1358 locus.

Tibetan is a general term for people who live in the Himalayan Mountains and speak languages in the Tibetan branch of the Tibeto-Burman subfamily of the Sino-Tibetan family (Matisoff 1991). The three main language groups in this branch are the Central, Northern, and Southern. People who speak Central and Northern Tibetan are mainly distributed in China (Tibet, Sichuan, and Yunnan), whereas Southern Tibetan speakers live in the southern Himalayan region, that is, India, Bhutan, and Nepal. Historically, it is well known that Tibetans diverged from northeast Asians, which has been confirmed by genetic studies using classical autosomal markers (Du and Xiao 1997) and microsatellite markers (Chu et al. 1998).

Our study investigated the relation of two Tibetan populations (Lhasa Tibetans and Kangba Tibetans) by means of STR markers. These two populations belong to the Northern Tibetan speakers, and they have the same characteristics of cultural anthropology, such as customs and habits, costume and ornaments, beliefs and religion, as well as language. Some population genetics research had been undertaken in the Tibetan population from China by means of STRs (Du et al. 2005; Hu et al. 2004; Tang et al. 2003; Li et al. 2000; Huang et al. 1998; Ying et al. 2003; Yang et al. 2004; Zhao et al. 2004; Bo et al. 2004; Zhu et al. 2005; Gao et al. 2006). The studies have been conducted using euchromosome STRs, Y-chromosome STRs, and mtDNA. But no research has been carried out in the Kangba and Lhasa Tibetan populations using the 15 STRs analyzed here. Thus we have conducted the first genetic population study of this type in these two Tibetan populations. The results indicate that the Kangba Tibetan population is similar to the Tibetan population in Tibet. This study will contribute to further studies of human evolution in the future.

Despite the small number of Tibetan samples studied, forensic parameters-probability of paternity exclusion, power of discrimination, polymorphic information content, and heterozygosity-are high in both populations. These data point to the conclusion that the 15 STRs used here are valuable STR loci for paternity investigations and genetic population studies in the Tibetan population of China.

Acknowledgments This study was supported by the National Natural Science Foundation of China through grants 30425007, 30370627, and 30670921 and by the China Medical Board of New York through grant 00-722, all awarded to F. Q. Wen.

Received 19 January 2006; revision received 12 October 2006.

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BINWU YING,1,3 TINGTING LIU,2 GUO CHEN,1 ZHIHE ZHAO,2 HONG FAN,1,3 AND FUQIANG WEN1

1 Department of Respiratory Medicine and National Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 61004L, Sichuan, People’s Republic of China.

2 Department of Orthodontics, West China College of Stomatology, Sichuan University, Chengdu 610041, People’s Republic of China.

3 Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, People’s Republic of China.

Human Biology, December 2006, v. 78, no. 6, pp. 757-770.

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