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Asthma, a chronic inflammatory disorder of the lungs and airways, typically results from a combination of multiple environmental and genetic factors. Human leucocyte antigen (HLA) region on chromosome 6p21 encodes the most highly polymorphic loci in the human genome, encoding genes with central roles in the immune function where HLA loci are strongly associated with various immune-mediated diseases such as autoimmunity, allergies and infection. The alleles of HLA class II genes such as DRB1 and DQB1 are the key genetic markers in the development of asthma and have been extensively studied in different ethnicities of the world population. However, the genetic screening of HLA class II alleles and haplotypes in Pakistani asthmatics has not been studied so far. The aim of the present study was to screen the HLA class II DRB1 and DQB1 alleles in asthma cases and controls in a Pakistani population. Seven hundred and two healthy controls and asthma patients were genotyped for HLA class II by sequence-specific polymerase chain reaction assays. The HLA-DRB1 and HLA-DQB1 allele and haplotype frequencies were calculated, and their risk or protective association with asthma was determined. Two-locus haplotypes of DRB1 and DQB1 alleles were imputed using Arlequin version 3.1 software. The signals of association with asthma were stronger at the DQB1 locus as compared to DRB1. HLA DQB1*03:03:02 (odds ratio [OR] = 2.42, 95% confidence interval [CI] = 1.34-4.25) was significantly associated with an increased risk of asthma, as was the haplotype comprised allele DRB1*07:01-DQB1*03:03:02 (OR = 2.40, 95% CI = 1.25-4.62). In contrast, DQB1*06 (OR = 0.39, 95% CI = 0.22-0.70) and DQB1*06:02 (OR = 0.27, 95% CI = 0.10-0.71) emerged as protective alleles for asthma. Our data concludes that the HLA DQB1*03:03:02 allele was a risk allele for asthma, whereas two DQB1 alleles, DQB1*06 and DQB1*06:02, were associated with asthma protection. Our findings highlight a prominent role for HLA-DQB1 alleles in asthma pathogenesis in studied Pakistani cases. More studies, especially with a larger study cohort are needed to confirm the utility of HLA DQB1*03:03:02 as a predictive marker.

Aim: 5,10-MTHFR-single nucleotide polymorphisms are important for normal functioning of the enzyme that plays a key role in DNA synthesis, folate metabolism and methylation reactions. Methodology & results: Male infertility association of C665T and A1298C polymorphisms was explored, this topic is still debatable. Infertile men (232) and controls (114) were genotyped and statistically analyzed. Comparison of patients (6180) and controls (5744) of Caucasian populations was performed by meta-analysis. Pooled results showed A1298C minor allele and homozygous genotype to be of a significantly higher frequency in the low-income group. Increase in per capita income has shown an increasing trend in the minor allele frequency in various world populations, potentially due to dietry-folate compensation. Conclusion: A1298C seems more relevant marker than C665T for infertility association in Caucasian populations and may be addressed by improving dietary folate.

CYP2D6 belongs to a family of Cytochrome P450 and is involved in metabolism of a number of commonly prescribed drugs. This study was designed to identify *4 allelic frequency of CYP2D6 in Pakistani population. The ethno-geographic variations in the CYP2D6 alleles are responsible for varied expression of this enzyme and thus influence the metabolic rate and efficacy of prescribed drugs. In total, 976 volunteers belonging to 16 different ethnic groups of Pakistan were screened for CYP2D6*4 polymorphism. The *4 allele was detected in all the ethnic groups with varied frequency ranging from 3.73%-13.64% and an overall average of 7.22% in different ethnic groups of the population. Maximum frequency was detected in northern Pakistani population including Meo (13.64%), Punjabi (11.96%) and Pathan (10.42%). Low frequency (4%) of*4 polymorphism was observed in Kalash and Makrani groups, whereas an intermediate frequency (5-9%) was observed in all the other ethnic groups. The data indicates that despite ethnic diversity poor metabolizers in Pakistani population are expected to carry CYP2D6*4 allele at a relatively higher frequency than most other Asian populations. (Word count = 186).

Interaction of environmental and genetic elements plays a vital role in the pathogenesis of aplastic anemia (AA). Glutathione S-transferase (GST) is a key detoxifying enzyme. Absence or low levels of this enzyme may genetically predispose individuals to AA. GST genes GSTM1 and GSTT1 are polymorphic. The aim of this study was to screen Pakistani AA patients and controls for GSTM1 deletion GSTM0 and GSTT1 deletion GSTT0 and perform meta-analysis using our data and other published data regarding these polymorphisms. DNA samples from 137 patients and 220 controls were screened using multiplex polymerase chain reaction. GSTM0 emerged as susceptible genotype for AA in Pakistan with a percentage frequency of 49.6 % as compared to 30 % in controls with odds ratio (OR) of 2.25, 95 % confidence interval (CI) of 1.4-3.5 and corrected p = 0.006. The meta-analysis showed a significant association between the null genotype GSTT0 and AA in overall analysis with OR of 1.47, 95 % CI of 1.01-2.13 and p value of 0.04 in random effects model. Studies like these could play a role in understanding the underlying path in AA pathogenesis and therefore can help in designing means for prevention, diagnose and treatment.

Item does not contain fulltext With homozygosity mapping we have identified two large homozygous regions on chromosome 3q13.11-q13.31 and chromosome 19p13.3-q31.32 in a large Pakistani family suffering from autosomal recessive nonsyndromic hearing impairment (arNSHI). The region on chromosome 19 overlaps with the previously described deafness loci DFNB15, DFNB72 and DFNB95. Mutations in GIPC3 have been shown to underlie the nonsyndromic hearing impairment linked to these loci. Sequence analysis of all exons and exon-intron boundaries of GIPC3 revealed a homozygous canonical splice site mutation, c.226-1G>T, in GIPC3. This is the first mutation described in GIPC3 that affects splicing. The c.226-1G>T mutation is located in the acceptor splice site of intron 1 and is predicted to affect the normal splicing of exon 2. With a minigene assay it was shown to result in the use of an alternative acceptor site in exon 2, resulting in a frameshift and a premature stop codon. This study expands the mutational spectrum of GIPC3 in arNSHI.

Asthma is a chronic disease due to inflammation of the airways of lungs that is clinically characterized by variable symptoms including wheezing, coughing and shortness of breath. Angiotensin I-converting enzyme (ACE) plays a major role in fibrous tissue formation and is highly expressed in lungs. The main aim of this research work was to study the role of ACE insertion/deletion (I/D) polymorphism, rs4646994, in asthma in Pakistani patients. A total of 854 subjects, including 333 asthma patients and 521 ethnically matched controls, were studied. The ACE (I/D) polymorphism was genotyped using polymerase chain reaction (PCR). Chi-square, Fisher's exact and Hardy-Weinberg equilibrium tests were used to compare groups. Homozygous insertion genotype II (p less than 0.0001, OR=3.38) and insertion allele (I) was significantly more frequent in Pakistani asthmatics than in healthy controls (p=0.0007, OR=1.40). The ID genotype (p less than 0.0001, OR=0.43) and the deletion allele (D) were associated with protection of disease in Pakistani patients (p=0.0007, OR=0.71). These data suggest the involvement of ACE I/D polymorphism in asthma risk in the Pakistani population. This marker may be an important indication in the molecular mechanism of asthma and can become a useful tool in risk assessment and help in designing strategy to combat disease.

Hepatitis C virus (HCV) infection is prevalent throughout the world and interferon (IFN)-based treatments are currently the only therapeutic option. However, depending upon variations in their human leukocyte antigen (HLA), some patients do not respond well to IFN therapy. The current study evaluated the HLA allele and haplotype distribution of 204 HCV-seropositive individuals from Islamabad, Pakistan, who were receiving standard IFN therapy. In this cohort, 150 patients (74 %) showed a sustained virological response to IFN therapy, whereas 54 (26 %) did not. In addition to the HCV patients, 102 unrelated healthy volunteers were used as controls. DNA was isolated from the blood of the patients and controls for HLA-DRB1 and HLA-DQB1 allele typing, whilst plasma was used for HCV detection and genotyping. HLA-DRB1*04 was found to impart a significant protective advantage [Bonferroni-corrected P value (pc)=0.047] against HCV infection. In patients on IFN therapy, HLA-DRB1*11 and -DQB1*0301 (pc=0.044) were found to be associated with viral clearance. In contrast, HLA-DRB1*07 (pc=0.008) individually or in combination with HLA-DQB1*02 was found to be associated with viral persistence. These associations of HLA with HCV persistence or clearance will be beneficial in deciding the therapeutic regimen for Pakistani patients infected with HCV genotype 3a.

Methylenetetrahydrofolate reductase is an important enzyme in metabolism of homocysteine, and a mutation in the gene predisposes individuals to several disorders related to homocysteine levels. Polymorphism at C677T shows marked heterogeneity based upon ethnicity and geographical location. Pakistani population consists of various ethnic groups confined to different regions of the country where congenital and genetic defects are a major health concern. We have analyzed the distribution of C677T alleles in different Pakistani ethnic groups. A cross-sectional study was conducted from all the four provinces of the country with samples representing the 14 different ethnic clans. A questionnaire with details of their ethnic origin was used, and informed consent was obtained. Blood samples from 701 individuals were collected for DNA isolation and subsequent C677T single-nucleotide polymorphism determination. The data were statistically analyzed. The study revealed that genotypic frequency for CC varied from 0.89 (Mohanna) to 0.38 (Hazara), CT from 0.56 (Hazara) to 0.11 (Mohanna), and TT from 0.06 to 0. Our data revealed a varied distribution of C677T mutation. This information could be helpful for designing future public health strategies, as it can be used to predict the prevalence of several disorders associated with genetic predisposition due to methylenetetrahydrofolate reductase C677T alleles.

Contains fulltext : 138088.pdf (Publisher’s version ) (Open Access) 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.

Background Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma and infects about 3% world population. Response to interferon therapy depends upon the genotype of the virus and factors associated with the host. Despite a good response to interferon therapy, a considerable number of genotype 3a infected patients remains unalleviated. Results In total forty-nine patients including twenty-five non-responders (non-SVR) and twenty-four responders (SVR) were recruited. Patients were tested for viral status at different intervals and the isolated RNA was sequenced for the NS5A region in both groups. The comparison of PKRBD of HCV between the SVR and non-SVR patients did not confirm any significant difference in the number of mutations. However, when the sequence downstream to the PKRBD of NS5A was compared, two important statistically significant mutations were observed; at positions 2309 (Ala to Ser) and 2326 (Gly to Ala). These mutations were then analysed for tertiary protein structure and important structural changes were observed. Statistically significant difference was also observed when age groups of patients were compared; younger patients showed better response than the older ones. Conclusions The region between PKRBD and IRRDR may be important for prediction of response to IFN therapy for genotype 3a. ISDR and PKRBD have not shown any involvement in treatment response. Further functional analyses of these findings can help in understanding the involvement of the NS5A region in interferon treatment of HCV-3a infected patients.

Item does not contain fulltext Bjornstad syndrome is an extremely rare condition characterized by pilitorti and nerve deafness. Only few large families have been reported worldwide. Here we describe a large Pakistani family with five affected individuals. The hair fibers of all the patients were twisted around their axis and devoid of any pigment. In addition the patients had a moderate-to-severe degree of hearing impairment. Genotyping with high-density single-nucleotide polymorphism arrays showed homozygosity in two intervals on chromosome 2. Linkage with one of these regions (genomic position 218745685-221025443, hg19) was confirmed. This region encompasses the BCS1L gene. Mutations in this gene have previously been associated with Bjornstad's syndrome. We sequenced the BCS1L gene for identification of the causative mutation in the family. A novel homozygous missense mutation c.901T>A was identified, which segregated with the disease in the family. This mutation results in the amino acid change p.Tyr301Asn and was predicted to be pathogenic by bioinformatics tools.

The Y chromosome microsatellite markers have been extensively used for population genetic studies and in individual identification and paternity testing in forensic medicine. In the present study, we report the data of five male-specific, polymorphic microsatellites in 740 unrelated male individuals from 12 different ethnic groups of Pakistan. The overall diversities of these individual loci in Pakistan ranged from 0.236 to 0.799. The total haplotypes identified were 152, and of these, 70 different haplotypes were present in only single individuals. Two haplotypes were found more frequently, 9_8_17_11_24 (13.5%) and 9_8_17_11_25 (8.6%), showing population-specific clustering in the Mohanna and the Brahui, respectively. An overall haplotype diversity of 0.965 in Pakistan suggested a high power of discrimination for these loci. Few populations, particularly the Mohanna and the Balti, showed lower haplotype diversity values for these loci (0.662 and 0.758, respectively). This set of microsatellite loci reported in the study can be used for population genetics and forensic medicine analysis. This study also demonstrates the importance of studying haplotype distribution pattern in population genetics.

The present study was designed to investigate the dopamine receptor D4 (DRD4) locus variable number of tandem repeat (VNTR) allelic distribution in different Pakistani ethnic groups. DNA samples from nine different ethnic groups of Pakistan were analyzed. Greek and Somali samples were included as representatives of the European and African populations, respectively. Pakistani, Greek, and Somali populations were also compared to the published data on different world populations. The allelic distribution revealed that the four-repeat allele was the most common allele in all the Pakistani ethnic groups as is in different other world populations, followed by the seven- and two-repeat alleles. To study the evolutionary relationship of the Pakistani ethnic groups among themselves and with a few other world populations, multidimensional scaling based on the allelic frequencies of the DRD4 VNTR was obtained. This analysis grouped most of the Pakistani ethnic groups together and closer to the European and Middle Eastern populations, except for the Mohanna from Sindh, who grouped with the African populations. In addition, the Somali and the Greek samples analyzed in this study grouped closer to the previous data obtained on the African and European populations, respectively.

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.

CCR5 is a G-protein-coupled chemokine receptor that is used as a co-factor by macrophage-tropic (M-tropic) isolates of human immunodeficiency virus-1 (HIV-1) to gain entry into host cells. A 32-bp deletion in the CCR5 gene (CCR5-Delta32) leads to the production of an altered gene product that prevents HIV-1 from entering the host cell. This study was carried out to determine prevalence of CCR5-Delta32 allele frequency in a large Pakistani population sample (n = 821) representing 10 ethnic groups. No individual was homozygous for the mutant allele and the frequency of the CCR5-Delta32 allele ranged from 0.62% to 3.57%. The CCR5-Delta32 allele frequency was generally lower in populations from southern Pakistan. The overall frequency of the CCR5-Delta32 allele in Pakistan was 2.31%, which is much lower than that found in European populations and similar to that in the Middle East. This is consistent with the historical records and genetic data that indicate a close genetic affinity among these populations. This study demonstrates that the Pakistani population is highly susceptible to M-tropic isolates of HIV-1 and public health measures need to be enforced with urgency if Pakistan is to avoid an HIV epidemic.

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.

Introduction Rheumatoid arthritis is an autoimmune disease with poorly understood pathophysiology. Genetic components of disease etiology, especially human leukocyte antigen (HLA) associations, are well known. Ethnic differences account for a number of variations in disease association with the HLA locus and there seem to be differences in various studies regarding its genetic predisposition. This study was aimed at determining the contribution of DRB1 and DQB1 components of HLA class II in rheumatoid arthritis in a Pakistani cohort. Method For this study, 110 patients and 120 healthy controls from the same geographical area and matched ethnicity were enrolled. Blood DNA was isolated from all the subjects and HLA alleles were typed following allele specific amplification. Subsequently, haplotypes were generated and allelic and haplotype distribution frequencies were compared among the patients and controls using χ2 and Arlequin software. The data obtained by this analysis were also compared with other reported associations found in the Pakistani population by meta-analysis. Results HLA allelic status was determined among the patients and controls from the same geographical area to account for differences in ethnicity and environmental factors. Significant associations were found for alleles as well as haplotypes among the patients of rheumatoid arthritis. DRB1*10, DQB1*05 and DQB1*602 were found to be associated with disease susceptibility, whereas DRB1*11 and DQB1*02 had protective effect against the disease. Similarly, haplotype DRB1*10-DQB1*05 was associated disease risk, whereas DRB1*07-DQB1*02 and DRB1*11-DQB1*0301 had a protective effect. Conclusion There is a significant DRB1and DQB1 allele and haplotype association with rheumatoid arthritis susceptibility and protection.

HE HUMAN leukocyte antigen (HLA) system isamong the most polymorphic in the human genome.This polymorphism makes it possible to study the geneticrelatedness and ancestral origins of different ethnic groupsbased on their characteristic allele frequencies and ex-tended haplotypes.The HLA data of three northern Pakistani populations—the Burusho, Pathans, and Kalash—are presented here.The Burusho are of special interest because of their lan-guage, Burushaski, is one of few remaining language iso-lates in the world. Not much is known about the origin ofthe Burusho, but according to legend they are descendantsof soldiers of Alexander the Great. The Pathans, collectedfrom the NWFP, are said to be the progeny of the lost tribeof Israel. The Kalash Kafirs are thought to be of Greekorigin having descended from soldiers of Alexander theGreat, though some literature points towards a Slavicorigin.These populations were typed for HLA-A, HLA-B,HLA-C, DRB1, DRB3, DRB4, DRB5, and DQB1 usingPCR-SSP with 144 primer mixes using the method de-scribed by Bunce et al.