Genetic Characterization of Wakhi People from Hunza Valley of Pakistan by employing Mitochondrial DNA Control Region

Full Length Research Article

Genetic Characterization of Wakhi People from Hunza Valley of Pakistan by employing Mitochondrial DNA Control Region

Marriam Jaffer1, Muhammad Saqib Shahzad1, Zia Ur Rahman1, Azam Ali2*, Saeeda Kalsoom2, Javed Iqbal Bajwa3, Muhammad Farooq Sabar4, Alamgir Alvi5

Adv. life sci., vol. 9, no. 1, pp. 13-17, May 2022
*Corresponding Author: Azam Ali (Email:
Authors' Affiliations

 1. Faculty of Life Sciences, University of Central Punjab, Lahore – Pakistan
2. Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore – Pakistan
. Faisalabad Medical University, Faisalabad – Pakistan
4. Centre for Applied Molecular Biology (CAMB), The University of Punjab, Lahore – Pakistan
5. Soil and Water Testing Lab Jhelum – Pakistan
 [Date Received: 23/10/2019; Date Revised: 12/11/2021; Date Published: 25/05/2022]

Abstractaa download_button



Background: Study of populations at genetic level marks high importance in terms of determination of population specific patterns. The study and analysis of population specific genetic patterns have wide ranging applications from medicine to forensic science. The study of mtDNA control regions gives the insight of maternal lineages of particular ethnic group and their evolutionary as well emigrational history. 

Methods: This research gives information of mitochondrial DNA haplotypes data of CR (control region) covering the area from 16024bp to 576bp of mitochondrial DNA of Wakhi population of Hunza valley from Pakistan. Samples of 40 unrelated Wakhi from upper Hunza were sequenced and their sequences encapsulating mtDNA control region was compared to rCRSs (revised Cambridge reference sequence) to see maternally inherited DNA variation at genetic level in this population.

Results: The results showed that all forty (40) haplotypes are unique. The haplotypes corresponded to 67.9% West Eurasian haplogroups followed by the Middle East and variety of Asian haplogroups exhibiting admixed maternal genetics of this population. Wakhi population comes with high genetic diversity (0.998) in turn lowest random match probability (0.026) and high power of discrimination (0.974).

Conclusion: This study gives interesting highlights important aspect of uni-parental genetics of Wakhi population and is also a contribution to mtDNA control region data of Pakistani populations for applications in criminal investigations.

Keywords: Forensic Science; Control region; Haplogroup; Genetic diversity 

Introduction6th button-01

The mtDNA control region assists to find out the immigration trail of a population throughout the history along with other related uni parental genetic constitution [1]. Analysis of mtDNA haplogroups has been proven valuable in determining not only the evolutionary account of human populations but also the turns the trick in favor of crime investigators to identify a human profile where conventional STR typing comes with limitation to find a hit [2]. Pakistan (Figure 1) happened to be in the most recurrently followed expedition road, which attracted people from out of Africa and referred as one of the ancient regions where contemporary human being chosen to reside [3,4]. By the language and civilization, Pakistani population is mixture of 16 cultural groups of assorted ancestries [5]. Hunza valley is located in the highest Pakistani northern zone in the Karakoram Mountains [6]. Upper part of Hunza is inhabited by Wakhi people believed to be evolved from prehistoric Iranian people. The Wakhi identify them by their Eastern Iranian language which belongs to Pamirian group. Wakhi language is very different from Tajik language, and it is also very different from other Pamiri languages [7]. No genetic study has ever been carried out to dissect the genetic structure of this population. Therefore, we conducted uniparental marker (mtDNA control region) study to find out the maternally inherited DNA pattern of Wakhi population for the usefulness in criminal investigations and snapshot of evolutionary as well as emigrational track record.

Methods6th button-01

Collection of Samples

3-5 ml of blood samples were collected from 40 maternally unrelated individuals of Wakhi population in Hunza Valley of Pakistan. Sampling was done from different areas of Hunza Valley (Figure 2) with the written and oral consent following the according to the declarations of Helsinki [25]. Approval for sample collection was obtained from ethical committee of (UCP) University of Central Punjab, Lahore.

DNA Extraction, Quantification, Amplification

DNA of all samples was extracted by organic method. The genomic DNA was quantified by the real time PCR (7500 SDS Real-Time PCR System).

Entire mtDNA (1122bp approximately) control region was amplified by utilizing the primer set from ( The primer sequence has been given in detail in (Table 1). Amplification was performed in a volume of 25 µl consisting of 1ng of genomic DNA, 0.4 μM of both forward and reverse primers and adding 0.4 µl Taq Polymerase (Thermo scientific #EP0402). The PCR procedure involves the denaturation at 94°C, annealing at 54°C for 30 seconds following by extension at 72°C for 60 seconds. While final extension process was done at 72°Cfor 60 seconds. After the amplification, PCR products were treated with ExoSAP-IT® (USB, Cleveland, OH, USA) to get rid of unused primers and dNTPs.


Bidirectional sequencing of entire mtDNA control region (covering nucleotide position 16,024–16,569 and 1–576) was performed using the Big Dye Terminator Cycle Sequencing v3.1 Ready Reaction Kit (Applied Biosystems) according to the manufacturer’s instructions.

Data Analysis

All bidirectionally sequenced samples were assessed twice by independent researchers as per commendation of [8] utilizing the Geneious (Version 7.0.3, Biomatters Ltd, New Zealand) as sequencing analysis tool. The eminence of mtDNA data was determined by the MitoTool [9] and mtDNA Profiler [10].

Haplogroup assignment was done by using HaploGrep [11] by taking the PhyloTree [12] Build 17 into consideration. The statistical parameters like Genetic diversity, power of discrimination and random match probability were calculated following the [13, 14] to determine richness of gene pool and in formativeness of mtDNA as marker for forensic applications for this population. 


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All of forty (40) samples were sequenced and compared to rCRSs for detection of haplotypes. After the sequence analysis, all samples were found to be different on genetic level marking the rich gene pool of population. Haplogroup frequency of each individual was calculated. The haplogroup H14a+146 were more frequent (5%) in Wakhi population as compared to other haplogroups. The haplogroup frequency of mitochondrial DNA of Wakhi population is presented in table 2. Wakhi population has an admixed mitochondrial DNA pool. 67.9% West Eurasian haplogroups (H101, H15a1b, H14a+146, H1+16239, H14a+146, H4a1a1a3, H15a1b, H2a1, U4b1a1a1, U4b2, U2b2, U4¢9, k1a13, k1b2, T1a, T2d1b, T, T1a1’3,).Wakhi population also exhibited the haplogroup J: 7.5% the haplogroup having origin of Middle East (J1b1b, JT) and found in Middle East with the percentage as high as 12%. 7.5 % of East African haplogroup (N9a1), 5% South Asian haplogroup (M31a1, M3), 2.5% East Asian haplogroup (A+152+16362), 2.5% of African haplogroup, 2.5% of Southeastern haplogroup (B4), 2.5% of Central Asian haplogroup (C4a1), 2.5% of Southeast Asian haplogroup (R2).  The genetic diversity of Wakhi population was high (0.998) with lower random match probability is (0.026) and in turn high power of discrimination (0.974) indicating rich gene pool (Table 3).

Figures & Tables


Discussion6th button-01

Since the age of mankind, has utilized plants for the This study revealed the haplotype data of hypervariable regions of the control region of mitochondrial DNA in Wakhi population living in upper Hunza (Gojal), Pakistan. Each individual’s mitochondrial DNA control region was sequenced and haplogroup assignment was done. Haplogroups U, T, A, M, H, K, J, L, N, B, C and R were observed in Wakhi population. The haplogroups H, U, T and K were frequently found in samples of this population.

Thirty nine (39) unique haplotypes were observed in Wakhi population, hence this population has a very high genetic diversity (0.998) and lowest random match probability is 0.026. The results of this study have been compared to other populations being studied in Pakistan e.g. especially genetic diversities of  Makrani (0.97) [1], Saraiki (0.96) [2], Pakhtun (0.99) [4], Baluchi including three other ethnic groups from Sindh and Baluchistan province (0.97) [19], Sindhi (0.992) [20], Punjabi (0.963) [15], Kashmiri (0.997) [21], Hazara (0.994) [23], Kho from Northwest region (0.0215) [22] and Gujar from Northwest region of Pakistan (0.922) [24] population (Table 4). This population was found to be the most diverse, genetically, than other so far studied populations from Pakistan. The haplogroup diversity observed in present study is comparable with the haplogroup diversity of Pashton population in which predominant haplogroups were of West Eurasian origin (55.6%) as it is common as far as Wakhi are concerned. In this study of Wakhi population, West Eurasian H haplogroup was found in 8 individuals (20%).  Haplogroup U of West Eurasian nature was second most prominent with 17.5% frequency after H haplogroup. Different subgroups of super haplogroups-H have been observed in various other ethnic groups from Pakistan like Makrani, Pathan and Kashmiri people. The most prominent mtDNA super haplogroup-U, is frequently found in Pathan (17%) and in Baluch (74%) in Pakistan. The haplogroup U2 is considered to be the most ancient lineage of super Haplogroup U.  The haplogroup U2 and other sub haplogroups of haplogroup U are found most frequently in Kashmiri and Kalash people from Pakistan. The haplogroup H is considered to be instigated in Southwest Asia around 20,000 to 25,000 years ago [16]. The haplogroup was also observed among the Tryptillians. The clade was found to be in the DNA of ancient mummies which were discovered in the middle Egypt at the archeological site of Abusir el-Meleq dating from the pre Ptolemaic/ late new kingdom and Ptolemaic interlude [17].

The haplogroup U was discovered from human skeletal remnants discovered in western Siberia which has been marked as most ancient DNA belonging to this clade, was dated to 45000 years ago [18]. Around 11 % of native Europeans bearing this haplogroup in their DNA and haplogroup are categorized as most old prehistoric maternally inherited haplogroup found in the region. Likewise, haplogroup H, it was also found in Egyptian mummies at the archeological site of Abusir el-Meleq dated to the 1st millennium BC [17]. In this context, further studies on this ethnic group could shed light on the interesting genetic aspects which could lead to its possible association with southwest Asian and Europe also emigrational history of this ethnic group to or from these continents.

Author Contributions

Marriam Jafar: Study design

Muhammad Saqib Shahzad: Refinement of study design and supervision

Zia Ur Rahman: Technical support

Azam Ali: Data analysis

Rahat Abdul Rehman: Monitoring of research

Saeeda Kalsoom: Drafting

Javed Iqbal Bajwa: Manuscript scanning

Muhammad Farooq Sabar: Manuscript scanning

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.


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