Genetic analysis of 15 mtDNA SNP loci in Chinese Yi ethnic group using SNaPshot minisequencing

    Genetic analysis of 15 mtDNA SNP loci in Chinese Yi ethnic group using SNaPshot minisequencing Chun-Ting Hu, Jiang-Wei Yan, Feng Chen, Qing-Xia Zhang, Hong-Dan Wang, Cai-Yong Yin, Han-Ting Fan, Ling-Li Hu, Chun-Mei Shen, Hao-Tian Meng, Yu-Dang Zhang, Hui Wang, Bo-Feng Zhu PII: DOI: Reference:

S0378-1119(15)01180-4 doi: 10.1016/j.gene.2015.09.071 GENE 40889

To appear in:

Gene

Received date: Revised date: Accepted date:

2 February 2015 8 August 2015 28 September 2015

Please cite this article as: Hu, Chun-Ting, Yan, Jiang-Wei, Chen, Feng, Zhang, Qing-Xia, Wang, Hong-Dan, Yin, Cai-Yong, Fan, Han-Ting, Hu, Ling-Li, Shen, Chun-Mei, Meng, Hao-Tian, Zhang, Yu-Dang, Wang, Hui, Zhu, Bo-Feng, Genetic analysis of 15 mtDNA SNP loci in Chinese Yi ethnic group using SNaPshot minisequencing, Gene (2015), doi: 10.1016/j.gene.2015.09.071

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ACCEPTED MANUSCRIPT Genetic analysis of 15 mtDNA SNP loci in Chinese Yi ethnic group using SNaPshot minisequencing

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Chun-Ting Hu1,#, Jiang-Wei Yan2,#, Feng Chen3,#, Qing-Xia Zhang4, Hong-Dan Wang5,

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Cai-Yong Yin3, Han-Ting Fan3, Ling-Li Hu3, Chun-Mei Shen6, Hao-Tian Meng7, Yu-Dang

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Zhang7, Hui Wang1, Bo-Feng Zhu7

1. Neurology Department of Cadre's Ward, 2nd Affiliated Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi Province, P. R. China

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2. Beijing Institute of Genomics, Chinese Academy of Science, Beijing, P. R. China 3. Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu, P. R. China

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4. Forensic Science Service of the Beijing Public Security Bureau, Beijing, P. R. China 5. Medical Genetics Institute of Henan Province, Henan Provincial People's Hospital, People's Hospital of Zhengzhou University, Zhengzhou, Henan, P. R. China

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6. Blood Center of the Shaanxi Province, Shaanxi, P. R. China

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Xi'an, P. R. China.

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7. Research Center of Stomatology, Stomatological Hospital, Xi'an Jiaotong University, Shaanxi,

Correspondence: Dr., Hui Wang, Neurology Department of Cadre's Ward, 2nd Affiliated Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710004, P. R. China

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E-mail: [email protected]

# These authors contributed equally to this work.

Abstract

SNaPshot minisequencing is a rapid and robust methodology based on a single base extension with a labeled ddNTP. The present study detected 15 selected SNPs in the mitochondrial DNA (mtDNA) control and coding regions by minisequencing methodology using SNaPshot for forensic purpose. The samples were collected from 99 unrelated individuals of the Yi ethnic minority group in Yunnan Province. We have predominantly found high-frequency transitions (91.7%) and a significantly lower frequency of transversions (8.3%). The 152, 489, 8701, 10398 and 16183 loci were highly polymorphic, while the 231, 473 and 581 loci were not polymorphic in the

ACCEPTED MANUSCRIPT studied population. Based on these 15 SNPs, a total of 28 mtDNA haplotypes were defined in 99 individuals with the haplotype diversity of 0.9136. Also, we compared the mtDNA sequences of Yi and other 9 populations worldwide and drew a

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Neighbor-Joining tree based on the shared 12 mtDNA SNP loci, which demonstrated a close relationship between Yi and Bai groups. In conclusion, the analysis of the 15

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selected SNPs increases considerably the discrimination power of mtDNA. Moreover, the SNaPshot minisequencing method could quickly detect mtDNA SNPs, and is economical and sensitive. The set of selected 15 SNPs is highly informative and is

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capable for anthropology genetic analysis.

ethnic group; genetic distance

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Introduction

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Keywords: SNaPshot; mitochondrial DNA; single nucleotide polymorphism; Yi

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Human mitochondrial DNA (mtDNA) present outside of the nucleus is a small, closed circular double-stranded DNA molecule [1]. The complete nucleotide sequence of the

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16,569 bp human mtDNA was first determined in 1981 [2] and then revised in 1999 [3]. The revised mtDNA sequence was named revised Cambridge reference sequence (rCRS). The copy number of mtDNA per mitochondrion ranges from 1 to 15 with the

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average around 4-6, and the copy number of mtDNA per cell is estimated to be about 500 [4,5]. MtDNA is maternally inherited as haplotype-block and thus has a low recombination rate [6]. Moreover, the mtDNA sequence evolution rate is much higher than that of nuclear gene [7,8,9]. Along the history and with the human migration, substantial variations arose and accumulated sequentially in the mtDNA, resulting in the divergence of maternal lineages from different geographical regions. Thus, the analyses of these mtDNA mutations enable us to infer the history of human evolution and patterns of migration. In addition, it is an available genetic marker for the studies of anthropology. In the field of forensic genetics, the above mentioned characteristics have made the mtDNA useful for human identification testing and especially for the analysis of

ACCEPTED MANUSCRIPT highly degraded materials or samples containing infinitesimal nuclear DNA such as skeletal remains and hair shafts [10,11,12,13,14,15]. In the previous studies of restriction fragment length polymorphism (RFLP), many stable loci were obtained

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and could be used to identify different mtDNA haplogroups. Most of the mutations observed in both the control and the coding regions of mtDNA in modern human

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populations have occurred on these preexisting haplogroups [16]. The bottleneck of using RFLP in identification testing is the limited discrimination power, and it can only provide less than sufficient information in evolutionary studies. Recently, in

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order to improve the discrimination power of mtDNA, the interest in point mutation also known as single nucleotide polymorphism (SNP) has increased. The mtDNA

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SNPs can be analyzed in amplicons shorter than 150bp [17], therefore they became available for the analysis in degraded samples which could not be genotyped by STR

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typing.

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Yi group is one of the 56 ethnic groups officially recognized by the People's Republic

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of China. In the 6th Chinese population census in 2010, the population of Yi was 8,714,393 and ranked the 7th among all the ethnic groups in China. The Yis are mainly distributed over the provinces of Yunnan, Sichuan, Guizhou, and the Guangxi

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Zhuang Autonomous Region. There are more than five million Yis residing in Yunnan province (http://www.stats.gov.cn/tjsj/pcsj/rkpc/6rp/indexch.htm). Previously, SNaPshot minisequencing has been utilized to subtype mtDNA and resolve mitochondrial macro-haplogroups by many researchers [18,19,20,21]. Here is the first study of the selection of 15 SNPs from both the control and the coding regions of mtDNA in the Yi ethnic group using SNaPshot minisequencing method. We found that the SNaPshot minisequencing methodology was a convenient, robust, and efficient method for mtDNA SNP typing. Furthermore, the relationship between Yi and other 9 populations was analyzed based on the pair-wise comparison at the 12 shared SNPs (16183/16234/16327/16362/150/152/189/195/199/231/473/489). Materials and methods

ACCEPTED MANUSCRIPT MtDNA-SNP selection A set of 15 mtDNA-SNPs (150, 152,189, 195, 199, 231, 473, 489, 581, 8701, 10398,

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16183, 16234, 16327 and 16362), which are termed by position in the mitochondrial

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genome according to the rCRS, was chosen from previously published works for their

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relatively high polymorphism in Chinese groups [22,23,24]. Samples

Blood samples were taken from 99 unrelated individuals of the Yi ethnic minority in

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Yunnan Province, China. DNA was extracted from 10μl EDTA blood samples using phenol-chloroform method. Informed consents were obtained from all the individuals

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in accordance with the Humane and Ethical Research Principles of Xi'an Jiaotong University Health Science Center, China. And the study was approved by the ethical

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PCR-multiplex

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committee of Xi'an Jiaotong University Health Science Center, China.

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The selected 15 SNPs were separated into two multiplex reactions. Multiplex 1 included a selection of 8 SNPs (150, 152, 199, 489, 8701, 10398, 16183 and 16234). Multiplex 2 contained a set of 7 SNPs (189, 195, 231, 473, 581, 16327 and 16362).

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Primers were designed and selected using Primer Premier 5.0 software (PREMIER Biosoft, California, US), which permitted analysis in multiplex reactions with applicable annealing temperatures and amplicon lengths. We performed both multiplexes in a 20μl reaction volume comprising 1ng of DNA template, 2μl 10  Buffer, 1.5μl MgCl2 (25mM), 1 unit of GoldTM DNA Polymerase (Applied Biosystems, Foster City, California, USA), 2μl dNTP (2mM) and 1μl multiplex primers. Amplification was carried out with a first denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and extension at 70°C for 1.5 min with a final extension at 72°C for 7 min. SNaPshot reaction

ACCEPTED MANUSCRIPT After amplification, a purification process was performed in order to remove primers and unexhausted dNTPs. According to the instruction of Exo-sapIT, 5μl of PCR product was incubated with 2μl of Exo-sapIT for 15 min at 37°C followed by 15 min

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at 80°C to inactivate the enzyme, and then at 4°C until further use. The two minisequencing reactions were performed in a GeneAmp 9700 PCR Thermocycler

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(Applied Biosystems, Foster City, California, USA) and both in 7μl volume. The first reaction consisted of 2μl SNaPshot Multiplex, 2.1μl SNaPshot primer and 2.9μl purified PCR products. The second reaction contained 1.5μl SNaPshot Multiplex,

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2.1μl SNaPshot primer and 3.4μl purified PCR products. The single base extension was performed in 25 cycles under the following conditions: 96°C denaturation for 10s,

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annealing at 50°C for 5s and extension at 60°C for 30s. Here the SNaPshot Multiplex contained the enzyme, the buffer and the 4 ddNTPs labeled with different colors: A

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with green, C with black, G with blue and T with red. Using this strategy, the single

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combined ddNTPs can be easily detected. And we could run together an assay with an A/G polymorphism and a C/T polymorphism even if the primers were of the same

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length.

Primer Premier 5.0 software was used to design the minisequencing primers which

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were modified by the addition of non-homologous tails at the 5'-end so that, by the size of the primer we can distinguish the different SNP sites. All minisequencing primers were purified by HPLC to remove incomplete primer synthesis products. After minisequencing reactions, a purification treatment to get rid of the unincorporated ddNTPs can help to prevent high background signal and nonspecific signal. The 7μl products were mixed with 0.7μl of SAP. Incubation was carried out at 37°C for 60 min, followed by 15min at 80°C for enzyme inactivation and at 4°C until further use. The minisequencing products (1.2μl) were mixed with 10μl formamide and 0.5μl of GeneScan LIZ-120 (Applied Biosystems, Foster City, California, United States). Sample analysis was performed on an ABI 3100 Genetic Analyzer (Applied

ACCEPTED MANUSCRIPT Biosystems, Foster City, California, USA). Electrophoresis parameters were used as follows: sample introduction for 10s at 15kv, electrophoresed for 20min at 15kv and 9μA. GeneScanTM3.7 Software (Applied Biosystems, Foster City, California, USA)

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was used to analyze the resulting data.

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Genetic distance analysis

The nucleotides at the selected mtDNA SNP loci were aligned with rCRS for statistical analysis. Software Arlequin 3.0 [25] and Mega 4.0 [26] were employed to

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analyze the pair-wise genetic distances among Yi, Egyptian [27], Tunisian [28], Bai [5], Guangdong Han [29] and other five Han populations (Liaoning Han, Qingdao

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Han, Wuhan Han, Xinjiang Han and Yunnan Han) [30]. Fst genetic distances were calculated. On this basis, we illustrated a phylogenetic tree using Neighbor-Joining

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Results and discussions

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method.

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Mitochondrial DNA has been used in forensic testing because of its maternal genetic characteristics. The analysis of mtDNA has been proved to have irreplaceable value in forensic testing and population genetics. Traditional mtDNA analysis methods,

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including RFLP and Sanger sequencing, have been widely used to identify mtDNA polymorphic loci in the previous studies [3,5]. However, they have the disadvantages of high labor consumption and high cost. In this study, we employed SNaPshot minisequencing methodology for mtDNA SNP typing. The SNaPshot reaction in which dNTP is absent is based on the single base extension with the labeled ddNTP at the position of the SNP. Each kind of ddNTP is labeled with different fluorescent dyes. We interrogated the different SNPs by the color of the ddNTP and the size of the SNaPshot primers. We successfully identified all the genotypes of the chosen SNPs in the 99 Yi individuals using SNaPshot technology. These SNPs would be useful for forensic science, and this method was found to be convenient, robust and efficient.

ACCEPTED MANUSCRIPT Table 1 shows all the allele frequencies observed in the studied population at the 15 selected sites. SNPs can be categorized as either single nucleotide transitions (C∕T, G∕A) or transversions (C∕A, G∕T, C∕G and A∕T). We found that the most common

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mutation was single nucleotide transitions (91.7%) in the studied population and that the frequency of transversions was significantly lower (8.3%) in the studied

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population. Among the 15 loci, all mutations observed were caused by single nucleotide transition except for the 16183 SNP which was caused by transversion (A/C). Because of the principle of this method, we could not distinguish between

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nucleotide substitution, insertion or deletion. In the present study, there was no polymorphism at 231, 473 and 581 loci, however these loci are all variable in Chinese

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Han population [24]. This was probably due to the unique genetic characteristics of these loci in Yi group. In this study, the relative frequencies of the loci 152, 489, 8701,

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10398, 16183, and 16362 were 0.76, 0.57, 0.54, 0.53, 0.77, and 0.55, respectively. A

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relative frequency between 0.20 and 0.80 is the best for individualizing SNPs [17]. Thus, these loci would be helpful in individual identification for forensic mtDNA

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typing in Yi population. The analysis of these polymorphic loci with the combination of traditional polymorphic sites in the control region would improve the power of

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identification in forensic application. As shown in Table 2, a total of 28 mtDNA haplotypes were found in the 99 samples. The haplotype diversity was 0.9136. The available data of this study could provide the genetic basis for mtDNA sequence analysis involving different ethnic groups and inter-ethnic and within-ethnic molecular evolutionary analysis. To improve the identification power of this system, a new group of SNP loci should be added in consideration of that the 231, 473 and 581 loci showed no polymorphisms in this study. For this purpose, more polymorphic mtDNA loci should be screened in future studies. Based on the variations at the shared 12 SNPs of different populations, we reconstructed the genetic structure of Yi group and other 9 groups. Fst and P values

ACCEPTED MANUSCRIPT are showed in Table 3. The results revealed that no significant differences existed among the six Han groups (Liaoning Han, Qingdao Han, Wuhan Han, Xinjiang Han, Yunnan Han and Guangdong Han). Among the genetic distances of Yi and other 9

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groups, Yi had the nearest distance to Bai group (Fst = 0.07453, p < 0.05) while the farthest to Guangdong Han (Fst = 0.18453, p < 0.05). In the NJ tree (Figure 1), three

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main clusters were present. One consisted of all the six Han groups of China, one cluster included Yi and Bai and the other comprised Tunisian and Egyptian. The phylogenetic tree elucidated the closest relationship between Yi and Bai groups,

(http://en.wikipedia.org/wiki/Yi_people)

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which demonstrated the 2 groups’ similar origin corresponding to historic events and

Loloish

language

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(http://en.wikipedia.org/wiki/Loloish_languages#Lesser-known_languages). Considering the genetic distances matrix in conjunction with the phylogenetic tree,

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the results in this research were roughly in line with the relative geographic locations

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of above-mentioned 10 groups. The results demonstrated that the set of selected SNP loci was highly informative while applied to population genetics and human

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anthropology studies. Concluding remarks

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In summary, by analyzing 15 selected SNP sites in Yi population using SNaPshot method we found that loci 152, 489, 8701, 10398 and 16183 were highly polymorphic and suitable for forensic application and population genetics study. Nucleotide transitions accounted for 91.7% compared to 8.3% of transversions. A total of 28 mtDNA haplotypes were defined based on these 15 SNPs with a haplotype diversity of 0.9136. In conclusion, the analysis of 15 selected SNPs increases considerably the discrimination power of mtDNA in the forensic field. Also, we demonstrated a close relationship between Yi and Bai groups and verified that the loci set was highly informative while applied to population genetics and human anthropology studies. Moreover, the SNaPshot minisequencing method could quickly detect mtDNA SNPs, and is economical, sensitive and automatic.

ACCEPTED MANUSCRIPT Acknowledgments This project was supported by the National Natural Science Foundation of China

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(NSFC, No. 81373248, 81400033), the Science and Technology project of Shaanxi

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Province 2006K13-G1(7), the Science and Technology project of Jiangsu Province

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BK20140902. The authors have declared no conflict of interest.

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Figure legend: Figure 1. The NJ tree reconstructed based on 12 mtDNA SNP loci of 10 populations.

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Table 1. The allele frequencies of 15 mtDNA SNPs loci in Chinese Yi population (n = 99)

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Loci (nt) 150 152 189 195 199 231 473 489 581 8701 10398 16183 16234 16327 16362

Allele

Frequency

C/T T/C

0.86/0.14 0.76/0.24 0.98/0.02 0.96/0.04 0.97/0.03 1 1 0.57/0.43 1 0.54/0.46 0.53/0.47 0.77/0.23 0.92/0.08 0.93/0.07 0.55/0.45

A/G T/C T/C C

C T/C

A A/G G/A A/C C/T

C/T T/C

ACCEPTED MANUSCRIPT Table 2. Twenty-eight mtDNA SNP haplotypic diversity of Chinese Yi population (n = 99)

Polymorphic nucleotide arrangement Number Frequency 1 C/T/A/T/T/C/C/C/A/G/G/A/C/C/C 21 0.21 2 C/C/A/T/T/C/C/T/A/A/A/A/C/C/C 13 0.13 3 C/T/A/T/T/C/C/T/A/A/A/A/C/C/T 10 0.10 4 C/T/A/T/T/C/C/T/A/A/A/C/C/C/T 9 0.09 5 C/T/A/T/T/C/C/T/A/A/G/C/C/C/T 8 0.08 6 T/T/A/T/T/C/C/C/A/G/G/A/C/C/T 4 0.04 7 T/T/A/T/C/C/C/C/A/G/G/A/C/C/T 3 0.03 8 C/T/A/T/T/C/C/C/A/G/G/A/C/T/T 3 0.03 9 C/T/A/T/T/C/C/C/A/G/G/A/T/C/C 3 0.03 10 C/C/A/T/T/C/C/C/A/G/G/A/T/C/C 3 0.03 11 T/T/A/T/T/C/C/T/A/G/A/A/C/C/T 2 0.02 12 T/T/A/T/T/C/C/T/A/A/A/A/C/C/T 2 0.02 13 C/C/A/T/T/C/C/C/A/G/G/A/C/C/C 2 0.02 14 C/C/A/T/T/C/C/T/A/A/A/C/C/C/T 2 0.02 15 T/T/A/C/T/C/C/T/A/G/A/A/C/C/T 1 0.01 16 T/T/A/T/T/C/C/T/A/A/A/C/C/C/T 1 0.01 17 T/C/A/T/T/C/C/T/A/A/A/A/C/C/C 1 0.01 18 C/T/A/C/T/C/C/T/A/A/A/A/C/C/C 1 0.01 19 C/T/A/C/T/C/C/T/A/A/A/A/C/C/T 1 0.01 20 C/T/A/T/T/C/C/C/A/G/G/A/C/T/C 1 0.01 21 C/T/A/T/T/C/C/C/A/G/G/C/C/T/T 1 0.01 22 C/T/A/T/T/C/C/T/A/A/A/C/C/T/T 1 0.01 23 C/T/A/T/T/C/C/T/A/A/G/C/T/C/T 1 0.01 24 C/T/G/T/T/C/C/C/A/G/G/A/C/T/T 1 0.01 25 C/T/G/T/T/C/C/T/A/A/A/A/C/C/T 1 0.01 26 C/C/A/C/T/C/C/C/A/G/G/A/C/C/T 1 0.01 27 C/C/A/T/T/C/C/T/A/A/A/A/C/C/T 1 0.01 28 C/C/A/T/T/C/C/T/A/A/A/A/T/C/T 1 0.01 The arrangement order of haplotype was nt150/152/189/195/199/231/473/489/581/8701/10398/16183/16234/16327/16362.

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Haplotype

ACCEPTED MANUSCRIPT Table 3. Fst and p values among 10 populations based on 12 mtDNA SNP loci.

-

0.000 00

0.029 26

-

0.124 59

0.202 18

Guangd ong Han 0.0000 0 0.0000 0 -

0.00 000 0.00 000

Yunn an Han 0.000 00 0.000 00

0.00 000

0.081 08

Bai

Xinji ang Han 0.000 00 0.000 00

Liaon ing Han 0.000 00 0.000 00

0.000 00

Qing dao Han 0.000 00 0.000 00

Wuh an Han 0.00 000 0.00 000

0.000 00

0.00 000

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Tunis ian

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Egyptia n Tunisia n Guangd ong Han

Egypt ian

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Populat ions

0.000 00

Yi 0.00 000 0.00 000 0.00 000

0.091 0.120 0.1489 0.000 0.000 0.000 0.000 0.00 0.00 33 48 4 00 00 00 00 000 000 Yunnan 0.119 0.164 0.0145 0.13 0.117 0.144 0.126 0.58 0.00 Han 50 17 6 473 12 14 13 559 000 Xinjian 0.139 0.186 0.0518 0.12 0.019 0.981 0.729 0.13 0.00 g Han 45 79 3 933 62 98 73 514 000 Liaonin 0.130 0.175 0.0509 0.12 0.013 -0.01 0.783 0.28 0.00 g Han 06 15 9 613 55 717 78 829 000 Qingda 0.154 0.192 0.0616 0.14 0.013 -0.00 -0.00 0.15 0.00 o Han 53 93 8 561 30 965 865 315 000 Wuhan 0.109 0.150 0.0544 0.12 -0.00 0.011 0.001 0.014 0.00 Han 16 40 1 543 524 16 76 86 000 0.115 0.180 0.1845 0.07 0.165 0.165 0.165 0.179 0.16 Yi 15 43 3 453 82 60 25 36 356 Notes: The data of left bottom represent Fst distance; the data of the right upper represent p value; the Fst data underlined means p > 0.05.

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Abbreviations list mtDNA, mitochondrial DNA; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism; rCRS, revised Cambridge reference sequence.

Highlights SNaPshot minisequencing was applied to type SNPin this study.

ACCEPTED MANUSCRIPT 15 SNPs were typed in 99 unrelated individuals from Chinese Yi group.

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Totally 28 mtDNA haplotypes were defined with the haplotype diversity of 0.9136.