Forensic and Genetic Characterization of mtDNA Lineages of Shin, a Unique Ethnic Group in Pakistan

1Centre for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan 2Faculty of Allied Health Sciences, The University of Lahore, Lahore, Pakistan 3Faculty of Medicine, Universiti Sultan Zainal Abidin, Kuala Terengganu, Malaysia 4Karakoram International University, Gilgit-Baltistan, Pakistan Article Information Received 24 October 2019 Revised 11 February 2020 Accepted 09 March 2020 Available online 03 June 2020


INTRODUCTION
M itochondrial DNA has emerged as one of the most popular genetic markers to investigate the genetic diversity of human populations (Pugach and Stoneking, 2015). In forensic practice, mtDNA analysis functions as a pivotal tool for human identity testing, population genetics, phylogenetics, anthropology, archaeology, human evolution and migration studies (Gupta et al., 2015).
MtDNA typing provides a unique maternal genealogical portrait of a person's genetic code. Its remarkable characteristics, which include a high copy number within the cells, an exclusive maternal inheritance, a high level of variation in its control regions, its size, and a neutral mode of evolution, make it a marker of choice in those circumstantial forensic caseworks where routine nuclear markers are not applicable (Conrad et al., 1968;Legros et al., 2004;Nilsson et al., 2008;Khan, 2013;O'Neill, 2013).
Specifically, due to its significantly high copy number per cell, it has an advantage and provides valuable data in the legal scenarios where only degraded DNA is available (Gupta et al., 2015). Moreover, its absolute maternal inheritance pattern and absenteeism of recombination events allow specific mtDNA sequences to be well reserved in all maternally-related family members of a family (Conrad et al., 1968). This has led to an extraordinary evolutionary consistency of genetic factors across multiple generations through the entire four billion spectrum of years since the birth of Adam and Eve (Zenil, 2017). Consequently, forensic comparisons can be made using a reference sample from multiple generations (Conrad et al., 1968).
All these benefits of mtDNA analysis are employed by forensic scientists for multiple purposes such as recognition of the relics of missing persons in disasters or matching evidential DNA recovered from a crime scene to those available in a database (of, e.g., convicted criminal profiles or the database for probable relatives) (Ziętkiewicz et al., 2012). Hence an overall knowledge of mtDNA profiles of worldwide populations is imperative to take advantage of mtDNA in a plethora of applications O n l i n e

F i r s t A r t i c l e
including forensic genetics and phylogenetic studies (Butler, 2009). Population specificity of mitochondrial genome (mtgenome) is widely reported in literature. MtDNA has been found to be very informative for inference of ethnicity (Prieto et al., 2011, Ladoukakis andZouros, 2017). Through historical perspective of human population migrations, South Asia comes next to Africa in holding heterogeneity and genetic diversity of populaces. Pakistan is situated in the core region of South Asia and probably was inhabited during primitive human movements (Shi et al., 2008). This zone is thus considered as the cradle of multiple civilizations. Currently a number of racial groups and minority units reside in Pakistan (Ayub and Tyler-Smith, 2009).
Gilgit-Baltistan is an important independent territory of Pakistan (Afzal, 2017). It is situated in the northern zone of Pakistan and consists of sight worthy valleys disjointed by some of the globe's highest mountain ranges including Hindu Kush, the Himalayas, Karakoram and the Pamir Mountains. Thus, it embraces a mixture of dynamic cultures and civilizations. Hence it is well-intentioned to study the ethnicity of people residing there (Khan, 2013).
One of the dominant populations of Gilgit-Baltistan is Shin, a Dardic tribe, whose mother tongue is Shina (Radloff, 1992;O'Neill, 2013). Unfortunately, this smaller but significant ethnic group of Pakistan has remained neglected and understudied. Understanding the genetic structure of this population is important, not only from a historic standpoint, but also for effective implementation and interpretation of forensic genetics.
In this regard, the current study was aimed to establish the mitochondrial DNA profiles of the Shin population, residing in Gilgit-Baltistan. The entire mtDNA control region of Shin individuals was sequenced and analyzed (as per recommendations) (Parson et al., 2014). This is the first study to report the mtDNA profiles of the Shin population. The main target of this work was to establish the predominant mtDNA lineages of this population to infer their ethnicity and history of their settlements in Pakistan and to compare them with other relevant races. The outcomes of this study will be useful for generating a genetic database of these areas which may be utilized for multipurpose future forensic implications.

Samples
Blood samples were collected from 79 maternally unrelated Shin individuals, both males and females, living in different regions of Gilgit-Baltistan, Pakistan (Fig.  1). Only individuals who confirmed their Shin origin of at least last three generations on the maternal side were included in the study. Scripted informed consent was taken from all the volunteer participants according to the declarations of Helsinki. Sample collection was performed in different towns and cities of Gilgit-Baltistan to achieve a reliable and complete representation of Shin population. A bioethical clearance certificate was obtained by the Bioethics committee of University of the Punjab. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

DNA extraction, amplification and sequencing
Whole blood samples collected in EDTA vials were subjected to DNA extraction via QIAamp DNA Mini Kit as per manufacturer instructions (Qiagen, Hilden, Germany. Cat No./ID: 51304). The quality and purity of extracted DNA samples were determined and adjusted (Nano Drop TM 1000 Spectrophotometer). The amplification of the desired sequences was done by Polymerase Chain Reaction (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA, USA), using specific primers as mentioned in Table I. PCR cyclic reactions were performed in 50µl of reaction mixture containing a total of 25µl 2x PCR hotstart master mix (abm, Canada, Cat. No. G906), a total of 21µl nuclease free H 2 O (Ambion, ThermoFisher Scientific, USA) a total of 2µl forward (10µM) and reverse primers (10µM) and a 2µl of DNA template. The PCR (30 cycles) was engineered to be; initial denaturation at 94°C for 10 min; second denaturation at 94°C for 30 seconds, annealing O n l i n e

Data analysis
The mtDNA control region forward and reverse sequences were aligned through the sequence analysis tool Geneious (Version 7.0.3, Biomatters Ltd, New Zealand) (Drummond, 2009) and were then compared to the revised Cambridge Reference Sequence (Andrews et al., 1999) using mtDNA profiler (Yang et al., 2013). To ensure high quality, two independence evaluations of the raw data were performed as per recommendations (Parson and Bandelt, 2007). The haplogroup assignments were carried out using previously published data (Metspalu et al., 2004;Behar et al., 2008;van Oven et al., 2011;Elmadawy et al., 2013), and by using Mitotool (www. mitotool.org) (Fan and Yao, 2011)  Indices of forensics and population genetics; e.g., genetic diversity, random match probability and power of discrimination were analyzed as explained previously (Tajima, 1989;Prieto et al., 2011). The current study strictly adhered to the guidelines and recommendations from the International Society for Forensic Genetics (ISFG) (Parson and Bandelt, 2007).

RESULTS AND DISCUSSION
The present study generated population data for the complete mtDNA control region (16,024-576) of 79 subjects from the Shin ethnic group. A total of 75 haplotypes were observed including 72 unique and 3 shared haplotypes. The most frequent haplotype (16129A 16223T 16298C 16327T 16519C 73G 249d 263G 315.1C 315.2C 489C) was found in 3.79 % of the sampled population (Table II). In 1122 positions analyzed, 174 variable sites were found in the mtDNA control region of the Shin population.
MtDNA analysis of the subjects revealed that the Shin population exhibited mtDNA genetic diversity of 0.9996, random match probability of 0.0129 and     power of discrimination of 0.9871, as presented in Table III. We compared the forensic and population genetics parameters including no. of haplotypes, unique haplotypes, genetic diversity, random match probability and power of discrimination of the Shin population with the other reported indigenous populations of Pakistan such as Saraiki, Sindhi, Makrani, Pathan, Kashmiri and Hazara, and found that the Shin population had the highest proportion of unique haplotypes reflecting high population heterogeneity in Shins. The large proportion of unique haplotypes in Shin population also corresponded well with their greatest genetic diversity (0.9996) when compared to other ethnic groups of Pakistan, i.e. Pathan (0.9978), Kashmiri (0.9977) Hazara (0.9945), Sindhi (0.9924), Makrani (0.9905) and Saraiki (0.9570) (Rakha et al., 2011(Rakha et al., , 2016Hayat et al., 2015;Siddiqi et al., 2015;Yasmin et al., 2017) (Table IV).

Haplogroup affiliations
The haplogroups observed in the Shin population showed affiliations with different phylogenetic lineages. They were mainly assigned into three continental groups, namely the West Eurasian (59.41%), South Asian (25.32%) and East and Southeast Asian (15.19%) groups. Thus, a high degree of genetic association with West Eurasian lineage was observed as compared to South Asian and South East Asian lineages. The most frequent haplogroups identified in the Shin population were U7a (5.06%), M30 (5.06%) and H32 (5.06%), carried by 15.19% of the population. The rest of the haplogroups observed in the Shin population were C (3.79%), U5a2a  Table V).
The current study revealed that the majority of the haplogroups of the Shin population indicated affiliation with West Eurasian lineage. A similar pattern was observed in other studies conducted on other Pakistani ethnic groups such as the Pathan, Hazara and Kashmiri, Bugti and Laghari, where maximum frequencies of West Eurassian haplogroups were reported. However, the rest of the Pakistani ethnic groups, such as the Gujjar, Araiyn, Bijrani, Chandio, Ghallu, Khosu, Nasrani, Solangi, Laghari, Lashari, Makrani, Saraiki and Sindhi, represented quite contrasting genetic structure and affiliations (Rakha et al., 2011(Rakha et al., , 2016Hayat et al., 2015;Siddiqi et al., 2015;Yasmin et al., 2017;Bhatti et al., 2017Bhatti et al., , 2018a (Fig. 3). The pronounced prevalence of West Eurasian matrilineal lineages may root back to great historical movements from Europe and Central Asia such as the invasion by the soldiers of Alexander the Great, the Arab and Muslim takeovers, and the era of the British Indian Empire (McElreavey and Quintana-Murci, 2005).

CONCLUSION
To the best of our knowledge, this is the first report regarding a forensic dataset of the Shin population including entire mtDNA control region sequences. The results reveal high genetic diversity and low random match probability, predicting the worth of mtDNA profiles of the Shin population for exploring maternal genetic lineages and routine forensic investigations in Pakistan. The outcomes of this study show the West Eurasian O n l i n e

F i r s t A r t i c l e
Forensic and Genetic Characterization of mtDNA Lineages of Shin haplogroups to be predominant in the Shin population. The data reported in this study will contribute in generation of mtDNA databases in Pakistan and will be beneficial for multipurpose future forensic implications.

O n l i n e F i r s t A r t i c l e
Forensic and Genetic Characterization of mtDNA Lineages of Shin