Eighteen binary polymorphisms and 16 multiallelic, short-tandem-repeat (STR) loci from the nonrecombining portion of the human Y chromosome were typed in 718 male subjects belonging to 12 ethnic groups of Pakistan. These identified 11 stable haplogroups and 503 combination binary marker/STR haplotypes. Haplogroup frequencies were generally similar to those in neighboring geographical areas, and the Pakistani populations speaking a language isolate (the Burushos), a Dravidian language (the Brahui), or a Sino-Tibetan language (the Balti) resembled the Indo-European-speaking majority. Nevertheless, median-joining networks of haplotypes revealed considerable substructuring of Y variation within Pakistan, with many populations showing distinct clusters of haplotypes. These patterns can be accounted for by a common pool of Y lineages, with substantial isolation between populations and drift in the smaller ones. Few comparative genetic or historical data are available for most populations, but the results can be compared with oral traditions about origins. The Y data support the well-established origin of the Parsis in Iran, the suggested descent of the Hazaras from Genghis Khan's army, and the origin of the Negroid Makrani in Africa, but do not support traditions of Tibetan, Syrian, Greek, or Jewish origins for other populations.

ABSTRACT1.33 Mb of sequence from the human Y chromosome was searched for tri-to hexanucleotide microsatellites. Twenty loci containing a stretch of eight or more repeat units with complete repeat sequence homogeneity were found, 18 of which were novel. Six loci (one tri-, four tetra-and one pentanucleotide) were assembled into a single multiplex reaction and their degree of polymorphism was investigated in a sample of 278 males from Pakistan. Diversities of the individual loci ranged from 0.064 to 0.727 in Pakistan, while the haplotype diversity was 0.971. One population, the Hazara, showed particularly low diversity, with predominantly two haplotypes. As the sequence builds up in the databases, direct methods such as this will replace more biased and technically demanding indirect methods for the isolation of microsatellites.

The extreme polymorphism found at some of the loci of the HLA system has made it an invaluable tool for population genetic analyses. In this study the genetic polymorphism of six Pakistani ethnic groups was investigated at the HLA-A, -B, -C, -DRB and DQB1 loci using polymerase chain reaction with sequence specific primers. The groups included in this study are the Baloch, Brahui and Sindhi from the south and the Burusho, Kalash and Pathan from the north of Pakistan. The allele frequencies, three-locus haplotype frequencies for HLA-A, -C, -B and HLA-A, -B, -DRB1 are given. Variation in the allele and haplotype distribution between the six Pakistani ethnic groups was observed. A phylogenetic tree and correspondence analysis based on HLA-A, -B, -C, -DRB1 and -DQB1 allele frequencies revealed the Kalash population to be distinct from the remaining Pakistani populations. The Baloch and Brahui were closely related to one another. The Sindhi were closer to the Pathan and Burusho populations than to the neighboring Baloch and Brahui populations, indicating admixture between the northern and southern populations of Pakistan. A phylogenetic tree and correspondence analysis comparing the Pakistani populations with various other world populations showed that the Pakistani ethnic groups lie within the cluster of Asian Indian populations. The three-locus haplotypes found in the Pakistani populations suggest an influence from Caucasian and Oriental populations.

The frequency of inherited bilateral autosomal recessive non-syndromic hearing loss (ARNSHL) in Pakistan is 1.6/1000 individuals. More than 50% of the families carry mutations in GJB2 while mutations in MYO15A account for about 5% of recessive deafness. In the present study a cohort of 30 ARNSHL families was initially screened for mutations in GJB2 and MYO15A. Homozygosity mapping was performed by employing whole genome single nucleotide polymorphism (SNP) genotyping in the families that did not carry mutations in GJB2 or MYO15A. Mutation analysis was performed for the known ARNSHL genes present in the homozygous regions to determine the causative mutations. This allowed the identification of a causative mutation in all the 30 families including 9 novel mutations, which were identified in 9 different families (GJB2 (c.598G>A, p.Gly200Arg); MYO15A (c.9948G>A, p.Gln3316Gln; c.3866+1G>A; c.8767C>T, p.Arg2923* and c.8222T>C, p.Phe2741Ser), TMC1 (c.362+18A>G), BSND (c.97G>C, p.Val33Leu), TMPRSS3 (c.726C>G, p.Cys242Trp) and MSRB3 (c.20T>G, p.Leu7Arg)). Furthermore, 12 recurrent mutations were detected in 21 other families. The 21 identified mutations included 10 (48%) missense changes, 4 (19%) nonsense mutations, 3 (14%) intronic mutations, 2 (9%) splice site mutations and 2 (9%) frameshift mutations. GJB2 accounted for 53% of the families, while mutations in MYO15A were the second most frequent (13%) cause of ARNSHL in these 30 families. The identification of novel as well as recurrent mutations in the present study increases the spectrum of mutations in known deafness genes which could lead to the identification of novel founder mutations and population specific mutated deafness genes causative of ARNSHL. These results provide detailed genetic information that has potential diagnostic implication in the establishment of cost-efficient allele-specific analysis of frequently occurring variants in combination with other reported mutations in Pakistani populations.

Allelic frequencies of 182 tri- and tetra-autosomal microsatellites were used to examine phylogenetic relationships among 19 extant human populations. In particular, because the languages of the Basques and Hunza Burusho have been suggested to have an ancient relationship, this study sought to explore the genetic relationship between these two major language isolate populations and to compare them with other human populations. The work presented here shows that the microsatellite allelic diversity and the number of unique alleles were highest in sub-Saharan Africans. Neighbor-joining trees based on genetic distances and principal component analyses separated populations from different continents, and are consistent with an African origin for modern humans. For the first time, with biparentally transmitted markers, the microsatellite tree also shows that the San are the first branch of the human tree before the branch leading to all other Africans. In contrast to an earlier study, these results provided no evidence of a genetic relationship among the two language isolate groups. Genetic relationships, as ascertained by these microsatellites, are dictated primarily by geographic proximity rather than by remote linguistic origin, Mantel test, R(0) = 0.484, g = 3.802 (critical g value = 1.645; P = 0.05).

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Inactivation of the p53 gene has been found to be associated with the pathogenesis of several neoplasias. Three biallelic polymorphisms in the p53 gene have been linked to predisposition to the development of various malignancies. These include a 16-bp duplication in intron 3 and BstU I and Msp I restriction fragment length polymorphisms (RFLPs) in exon 4 and intron 6, respectively. The prevalence of these polymorphisms was studied in breast cancer patients and nine major ethnic groups of Pakistan. Differences in allele frequencies for all three polymorphisms were observed among the various ethnic groups and breast cancer patients. The absence of the 16-bp duplication was common among the northern ethnic groups, being highest in the Hazara (0.90). The Msp I A1 allele frequency in the southern Makrani population was significantly higher in comparison with the other ethnic groups. In the cancer patients, the absence of the 16-bp duplication in combination with the BstU I Pro and absence of Msp I restriction site were the most frequent. In these patients, ten substitution mutations were found in the p53 gene, seven of which have been reported previously for breast cancer. The remaining three mutations have been found in other malignancies, but not in carcinoma of the breast.

Three populations from northern Pakistan, the Burusho, Kalash, and Pathan, claim descent from soldiers left behind by Alexander the Great after his invasion of the Indo-Pak subcontinent. In order to investigate their genetic relationships, we analyzed nine Alu insertion polymorphisms and 113 autosomal microsatellites in the extant Pakistani and Greek populations. Principal component, phylogenetic, and structure analyses show that the Kalash are genetically distinct, and that the Burusho and Pathan populations are genetically close to each other and the Greek population. Admixture estimates suggest a small Greek contribution to the genetic pool of the Burusho and Pathan and demonstrate that these two northern Pakistani populations share a common Indo-European gene pool that probably predates Alexander's invasion. The genetically isolated Kalash population may represent the genetic pool of ancestral Eurasian populations of Central Asia or early Indo-European nomadic pastoral tribes that became sequestered in the valleys of the Hindu Kush Mountains.