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Title of Thesis

Shaheen N. Khan
Institute/University/Department Details
University of Punjab/ National Centre of Excellence in Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
hemoglobin, genomic dna, thalassemia, chorionic villi, p-globin gene, amplification refractory mutation system (arms), single strand conformation polymorphism (sscp)

Thalassemias are the most common autosomal recessive hemoglobin disorders characterized by the reduction or absence of synthesis of the globin chains of the hemoglobin molecule. In the world population this group of genetic diseases causes far more ill health than any other inherited condition. The most important are β-thalassemia , a-thalassemia and HbE-thalassemia. A β-thalassemia heterozygous person can lead a normal life but homozygous β-thalassemia children require continuous blood transfusions and chelation therapy to prevent death from severe anemia.

In Pakistan β-thalassemia is the most common hemoglobin disorder. In a population of nearly 130 million, and with a β-thalassemia carrier frequency of 5.4%, it is estimated that each year more than 4000 children are born with transfusion dependent β-thalassemia . The affected children become a great source of socioeconomic burden on their families as good quality and screened blood increases its cost. In addition chelation therapy is also very expensive. Identification of the genetic defects in the carriers for genetic counseling and provision of prenatal diagnosis can reduce the incidence of β-thalassemia . As co-inheritance of a-thalassemia reduces the severity of the disease, molecular diagnosis can also help in the clinical management of these patients.

β-thalassemia is usually caused by a set of molecular defects specific for each population. In order to start a preventive program it is essential first to identify the β-thalassemia mutation prevalent in the population. Therefore, β-thalassemia was characterized in molecular terms by analyzing 624 β-thalassemia alleles (312 patients) from six ethnic groups belonging to the four provinces of Pakistan. Molecular characterization of 22 β-globin gene defects revealed that 11 of them account for 97% of all the alleles, thus showing. molecular heterogeneity of β-thalassemia in Pakistani population. The four common mutations are: IVS-I-5, G-+C (37.5%), codons 8/9, +G, (20.8%), 619bp deletion (12.0%) and IVS-I-I, G-+T (9.1%). There are differences among the ethnic groups and also between the provinces in the frequencies of different mutations. The IVS-I-5, G-+C mutation is more prevalent in Sindh and Balochistan, while the codons 8/9, +G mutation is more common in Punjab and the North West Frontier Province. The 619 bp deletion is high in Gujratis and Memons residing in the province of Sindh. The three rare ~-thalassemia mutations, which were not previously reported in Asian Indians or in the Pakistani population were characterized and haplotype analysis suggested their independent origin in our population. Parental consanguinity (69.9%) was high and 67.6% B-thalassemia patients were homozygotes while 32.4% were genetic compounds. Making use of the above information a prenatal diagnosis program was initiated at the Centre for Applied Molecular Biology (CAMB).

Common a-thalassemia rearrangements were studied in 406 alleles from the normal random population and in six ethnic groups of Pakistan. Analyses revealed the presence of only _α37 allele with an overall lrequency of 8.4%. Ethnic dif1erellces were statistically significant for Pashtoon vs. Balochi (P= < 0.0005) and Pashtoon vs. Sindhi (P= < 002). 292 thalassemia patients (584 alleles) were also studied to identify rare

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
116.17 KB
2 1 Introduction 1
49.49 KB
3 2 Literature Review 05
194.78 KB
  2.1 Historical perspective 05
  2.2 Definition and classification 05
  2.3 Hemoglobin composition and production during development 06
  2.4 Globin genes 10
  2.5 Polymorphisms in the genomic DNA 13
  2.6 Hemoglobin variants 14
  2.7 Molecular pathology of thalassemias 16
  2.8 Consanguineous marriages 19
  2.9 Prevention of β-thalassemia 20
4 3 Materials a.nd Methods 22
195.51 KB
  3.1 DNA extraction from blood and chorionic villi 22
  3.2 PCR amplification to detect the 619 bp deletion in p-globin gene 23
  3.3 Principle of Allele Specific Oligonucleotide (ASO) probe hybridization 23
  3.4 Prenatal diagnosis for β-thalassemia by chorionic villi 28
  3.5 Principle of Amplification Refractory Mutation System (ARMS) 29
  3.6 Principle of Single Strand Conformation polymorphism (SSCP) analysis 30
  3.7 DNA sequencing 34
  3.8 Haplotype analysis for the rare β-thalassemia mutations 36
  3.9 Screening for the known a-thalassemia deletions by PCR 36
5 4 Results 39
456.49 KB
  4.1 Screening for the β-thalassemia mutations 39
  4.2 Prenatal diagnosis for β-thalassemia 54
  4.3 Screening for the a-thalassemia mutations 60
6 5 Discussion 71
107.78 KB
7 6 References 80
143.33 KB