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The ethnic groups inhabiting Dir and Swat Districts of Khyber Pakhtunkhwa Province, Pakistan are known to exhibit cultural and physical diversity. Genetic diversity however among the people of this region remains largely unknown. A research endeavor based on dental anthropology and molecular phylogenetic was conducted in for elaborating phenetic and molecular affinities among the major ethnic groups of the area. The morphological variants of permanent tooth crown were recorded for phenotypic analyses, whereas mitochondrial DNA (mtDNA) and Y-Chromosoal STRs/SNPs were considered for maternal and paternal variation, respectively, among the individuals and in between Gujar, Kohistani, Tarklani, Utmankheil and Yousafzai tribes. Dental casts and oral swabs were collected from volunteers of all the tribes/ethnic groups. Morphological variants of the permanent tooth crown were scored from maxillary and mandibular dental castes in accordance with the Arizona State University Dental Anthropology System (ASUDAS). Two mitochondrial DNA control segments viz Hypervariable segment I (HVSI), Hypervariable segment II (HVSII), 27 Y-STRs and 331 Y-SNPs were used to explore molecular phylogenetic relationships. Dental casts were obtained from 823 healthy unrelated individuals of the five ethnic groups of the two districts. The casts were analyzed for 14 tooth-trait combinations. The data was then compared with 27 samples encompassing 3,185 prehistoric and living individuals representing ethnic groups of the Hindu Kush-Karakoram highlands and Indus Valley of Pakistan, peninsular India, and Central Asia. Inter-sample affinities were computed with C.A.B. Smith’s pairwise Mean Measures of Divergence (MMD) statistic. Patterning of phenetic affinities were assessed with neighbor-joining cluster analysis (NJ), multidimensional scaling (MDS), and principal coordinate analysis (PCA). The results obtained vary with respect to data reduction technique. Neighbor-joining cluster analysis assort Gujars, Kohistanis and Utmankhels with possessing affinities to the ancient Harappans peop;e of the Indus Valley whereas Yousafzais assorted for having affinities with ethnic groups of the Hindu Kush-Karakoram highlands. The Tarklanis exhibit no close affinities to Gujars, Kohistanis, Utmankheils or Yousafzais. The results of mtDNA generated 126 haplotypes among which, 75 were unique and 51 were shared. The results further revealed that 45% of the individuals possess matrilineages of West Eurasian derivation, 36% of South Asian derivation, 6% of individuals possessed lineages of East Eurasian derivation, while frequencies of lineages of other derivations are of extremely low frequency. The West Eurasian haplogroup R was found 62% of individuals was the most frequent haplogroup, followed by South Asian haplogroup M (32%), East Eurasian haplogroup N (5%), while one individual was found to possess the African haplogroup L. The results of Y-STRs analysis revealed 82 haplotypes in which 75% were unique and 25% were shared, yielding a haplotypic diversity of 0.99. High and statistically significant levels of genetic differentiation were obtained in nine of the 10 pairwise comparisons (FST= 0.148-0.596), the exception being the contrast between Tarklanis and Yousafzais (FST = 0.008). Members of the Utmankheil, also considered Pashtuns tribe, were found to be not closely related to any of the other population samples (FST= 0.445-0.596). The high genetic differentiation was also visible in Y-chromosomal SNPs, showing very little overlap between the five population samples, except for Tarklanis and Yousafzais. When analyzed in a larger continental-scale, it is clear that the paternal lineages of these five ethnic groups fall mostly outside the previously characterized Y-chromosomal gene pools of Indo-Pakistani sub-continent. Our findings presented here contribute towards the understanding of the genetic complexity exhibited by the apparently related ethnic groups residing in the northern parts of Pakistan. It provides a sound baseline for elaborating the historical profile and anthropological standings of Pakistani people for the fastly approaching era of personal genomics and personalized medicine.
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