Human Genome Project (HGP) revealed the existence of some 25,000 genes in the human genome; however the function of most of these genes, especially their role in human health and disease, remains uncovered. Elucidating the basis of inherited human disease essentially involves linking genomic variation to clinical phenotype. In this context rare monogenic diseases, often referred to as Mendelian disorders, are of substantial interest because identification of their genetic bases provides insights into the physiological role of the underlying protein, disease pathways and potential therapeutic targets. The recent introduction of next generation sequencing (NGS) technologies has enhanced the disease gene discovery process by replacing the laborious positional cloning studies. Of particular importance is exome sequencing whereby a single streamlined laboratory workflow identifies most of the coding genomic variations. This thesis is an effort to explore the genetic basis of alopecia and skeletal dysplasia in Pakistani populace. Alopecia is a rare heterogeneous common form of hereditary hair loss characterised by a sudden patchy hair loss most commonly occurring on the scalp but occasionally extended to the entire surface of the body. Hypotrichosis refers to diffuse thinning of the scalp hair without any gross defect of the hair shaft morphology while woolly hair is the anomalous variant of strongly curled hair. Evidence for the genetic basis of these disorders has been well established, however a phenotype to genotype relationship is still lacking. Skeletal dysplasias encompass a genetically heterogeneous group of more than 250 disorders of bone and cartilage. They are characterized by abnormal pattering, linear growth, differentiation and maintenance of the human skeleton with the resultant uneven short stature, or dwarfism. Progressive pseudorheumatoid dysplasia (PPD) is a rare autosomal recessive disease characterised by axial and peripheral skeletal dysplasia. Patients with PPD present with stiffness and swelling of joints, motor weakness and joint contractures. Just like alopecia, a phenotype to genotype relation is lacking for skeletal dysplasia, mainly due to variation in clinical phenotype. In this study, 10 unrelated consanguineous families (A to J) were ascertained from different areas of Pakistan. Eight of these families (A to H) were segregating alopecia xiv and two families (I and J) were inheriting skeletal dysplasia. Of the 8 alopecia families, seven families (A to G) showed autosomal recessive mode of inheritance while family H inherited alopecia in autosomal dominant fashion. Both family I and J were segregating skeletal dysplasia in autosomal recessive fashion. Following candidate gene approach all the families were investigated for the involvement of reported genes. Families excluded to known loci/genes were subjected to genome wide scan through microsatellite markers. In the absence of a candidate gene families were subjected to exome sequencing. Families A, B and C were linked to P2RY5 gene locus on chromosome 13q14.11– q21.32. Direct sequencing of the gene revealed a novel mutation (c.insT583) in family A, and a previously known mutation (c. 436 G>A) in the rest of the two families (B and C). Families D, E, F and G were linked to LIPH gene locus on chromosome 3q27-q28. Direct sequencing revealed two known mutations; c.659_660delTA in family D and c.280_369dup in family E. Family F revealed a novel nonsense mutation c.778A>T. Family G was linked to LIPH gene locus but no functional variation could be found when the gene was sequenced. Family H was linked to a novel locus on chromosome 16p13.3. Exome sequencing did not show any functional variant in the entire coding portion of the genome. In both families G and H, the possibility of a mutation in the regulatory region cannot be ruled out. Family I was linked to a large region on chromosome 16 through genome wide scan with microsatellite markers. Exome sequencing identified a missense mutation (c.1415C>G) in the conserved part of a novel gene IRX5 which encodes a transcription factor. This gene will be subjected to further functional assays to get an insight into the mechanism underlying skeletal dysplasia. Family J was subjected to exome sequencing exclusion to a candidate gene. A nonsense mutation (c.156 C>A) associated with a missense mutation (c.248 G>A) in WISP3 gene on chromosome 6q21. Exon 3 of WISP3 gene which carries these mutations is suspected to harbour a mutation hotspot. These findings will provide the basis for genetic counselling and carrier screening in families inheriting alopecia and skeletal dysplasia. These results will also help to establish prenatal diagnosis for the disorders under study. The novel findings of this study will serve as a starting point for future investigations towards understanding the pathogenesis of skeletal dysplasia. Publications 1. Tariq, M., Azhar, A., Baig, S., M., Dahl, N. and Klar, J. 2012. A novel mutation in the Lipase H gene in autosomal recessive hypotrichosis and woolly hair. Sci Rep. Epub ahead of print. DOI: 10.1038/srep00730 2. Tariq, M., Azhar, A., Baig, S. M., Dahl, N., Klar, J. 2012. A novel mutation in Lysophosphatidic Acid Receptor 6 gene in autosomal recessive hypotrichosis and evidence for a founder effect. Eur J. Dermatol. 22(4): 464-6. 3. Khan, T. N., Tariq, M., Malik, N. A., Klar, J., Baig, S., M and Dahl, N. 2014. Evidence for autosomal recessive inheritance in SPG3A caused by homozygosity for a novel ATL1 missense mutation. (Accepted: European journal of Human Genetics). 4. Hussain, M. S., Baig, S. M., Neumann, S., Peche, V. S., Nurnberg, G., Tariq, M., Jameel, M., Khan, T. N., Fatima, A., Malik, N. A., Ahmad, I., Altmuller, J., Frommolt, P., Thiele, H., Hohne, W., Yigit, G., Wollnik, B., Neubauer, B. A., Nurnberg, P. Jand Noegel, A. A. 2013. A gene associated with the centrosome during mitosis and is mutated in a large Pakistani family with autosomal recessive primary microcephaly. Hum mol genet. 20;22(25):5199-214 5. Khan, T. N., Klar, J., Nawaz, S., Jameel, M., Tariq, M., Malik, N, A., Baig, S. M. and Dahl, N. 2013. Novel missense mutation in the RSPO4 gene in congenital hyponychia and evidence for a polymorphic initiation codon (p.M1I). BMC Med Genet. Epub ahead of print. DOI: 10.1186/1471-2350-13-120 6. Baig, S.M., Sabih, D., Rahim, K., Azhar, A., Tariq, M., Hussain, M. S., Naqvi, S. M. S., Raja, G. K., Khan, T. N., Jameel, M., Iram, Z., Noor, S., Baig, U.R., Qureshi, J. A., Baig, S. A., Bakhtiar, S. M. 2012. β-Thalsssemia in Pakistan: a pilot program on prenatal diagnosis in Multan. J of Pediatr Hematol Oncol. 34(2):90-2. 7. Nawaz, S., Tariq, M., Ahmad, I., Baig, S. M., Dahl, N., Klar, J. 2012. Non-bullous congenital ichthyosiform erythroderma associated with homozygosity for a novel missense mutation in an ATP binding domain of ABCA12. Eur J. Dermatol. 22(2):178-81. 8. Nawaz, S., Tariq, M., Azhar, A., Rasool, M., Bakhtiar, S. M., Ahmad, I., Rehman, S. U., Jameel, M., Khan, T. N., Baig, S. A., Klar, J., Dahl, N., Baig, S. M. 2011. Report of a recurrent mutation in ARS (component B) gene with severe Mal de Meleda in a large consanguineous Pakistani family. Pak J Med Sci. 27(3):686-9. 9. Nawaz, S., Klar, J., Wajid, M., Aslam, M., Tariq, M., Schuster, J., Baig, S.M. and Dahl N. 2009. WNT10A missense mutation associated with a complete Odonto-Onycho-Dermal Dysplasia syndrome. Eur J Hum Genet. 17:1600-5. 10. Rasool, M., Schuster, J., Aslam, M., Tariq, M., Ahmad, I., Ali, A., Entesarian, M., Dahl, N., Baig, S. M. 2008. A novel missense mutation in the EDA gene associated with X-linked recessive isolated hypodontia. J Hum Genet. 53:894-8.
Abstract By preserving and narrating hadīth, a chain of narrators was started to maintain its authenticity. When the experts of hadīth realized that some unreliable transmitters might try to fabricate Hadīth, this work started more systematically. Even the chain gradually attained such importance that every Muhaddīth was concerned much about it. In the second century of Hijra, when the teaching and learning of hadīth became the standard of honor and great respect, some people devoted their lives to this work. They travelled to many countries of world and obtained the knowledge of Hadīth from prominent scholars of their time. Experts of Asmā-ul-Rijāl awarded them the certificate of holding the highest position of trust and credibility. The chains having such trustworthy transmitters are considered to be of higher rank than others. Among such traditions there is also one "Mālik-an-Nāfi'-an-Ibn-e-Umar". Due to the reliability of its narrators, Imām Bukhāri and many other Muhaddithīn considered it as "golden chain". When some of the Orientalists started raising objections to the Prophetic Hadīth, they criticized the narrators of the Hadīth as well. Especially the narrators who were declared trustworthy and reliable by Muslim scholars. For this, they especially criticized Abu Hūraira from among the companions and Imām Zuhri among the Successors. The chain of hadīth, (Mālik an-Nāfi' an-Ibn e Umar) “golden chain” was also seriously criticized by Joseph Schacht and Juynboll etc. In this article, a comparative study of the viewpoints of the Muhaddithīn and the Orientalists regarding the chain “Mālik an-Nāfi' an-Ibn eUmar” is presented.
In the present study, we analyzed the prevalence of Hepatitis C virus (HCV) infection and its associated risk factors among pregnant women in five selected districts of Khyber Pakhtunkhwa (KP), Pakistan. The prevention of this highly infectious disease that largely remains asymptomatic, and leads to serious damage during pregnancy carries a substantial socio-economic benefit for the health and welfare of this region. The sensitivity and accuracy of the real-time polymerase chain reaction (qPCR) were also evaluated in comparison with the routine diagnostic methods: Immuno chromatographic technique (ICT), and enzyme-linked immunosorbent assay (ELISA). A total of 750 blood samples from five districts of Khyber Pakhtunkhwa namely, Mardan,Kohat, Peshawar, Nowshera, and Charsadda were screened for HCV infection using a combination of three diagnostic procedures: ELISA, ICT, and qPCR. The results showed that out of the 750 blood samples analyzed, the qPCR assay could confirm the presence of HCV RNA only in 10–30% of anti-HCV-positive samples in each district, with an average of 24% in all districts. ELISA was found to be the most sensitive method for detecting anti-HCV antibodies in 70–100% of HCV-positive blood samples in each district, with an average of 92% in all districts. ICT, which detected antibodies in 27–71% of HCV-positive blood samples from different districts with an average of 44%, is clearly less sensitive than ELISA. The overall prevalence rate in the KP, with 48 of the 750 blood samples tested positive for HCV among pregnant women, was estimated to be 6.4%, which falls within the HCV prevalence range of 4.5 to 8%, reported for Pakistan. However, there was a considerable variation in the rate of HCV prevalence at the district level, which are in order of Mardan (9.33%) >Kohat (7.33%) >Peshawar (6.00%) >Nowshera=Charsadda (4.33%). Logistic regression analysis was performed to determine the association of variables of HCV prevalence, like age, education, residential status, travel to abroad, gravidity, dilation and curettage (D&C), blood transfusion, abortion, general and dental surgery, etc., in participants found to be HCV-positive. The logistic regression model was found to be statistically insignificant (χ2 (8) = 8.785, p >0.05). The model explained 49.9% (Nagelkerke R2) of the variance in HCV-positive cases and correctly classified 95.6% of them. People with a marriage duration of 11–20 years were 17.228 times likely to exhibit HCV symptoms than people in other groups of marriage duration. Illiterates were 50.850 times more likely to acquire HCV infection than people with education. Respondents with urban residential status were 5.221 times more likely to be HCV-positive. Gravidity (primigravida) was also found to have a significant impact on HCV prevalence. A history of dental surgery is likely to increase the chances of HCV-positive status by 2.657 times, whereas the use of therapeutic injections is likely to increase the chances of HCV-positive status by 4.474 times. All other variables were found to be non-significant. District wise chi-square analysis showed age, marriage duration, education, residential status,HCV-positive husband, gravidity, dilation and curettage (D&C), blood transfusion, history of abortion, general and dental surgery, ear and nose piercing, awareness about HCV, history of accident, and use of therapeutic injections to be associated with HCV infection to variable extent in different districts. Our findings of a prevalence rate of 6.4% in KP have important health implications for a large number of pregnant women and their families as most of the infected women are ignorant of HCV infection. Since most of them are asymptomatic, they tend to have normal life implying that there exists a considerable potential for the transmission of HCV to the fetus.