Date of Award

11-2024

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr Ambrin Fatima

Second Advisor

Dr Afsar Mian

Third Advisor

Dr Irfan Hussain

Department

Biological and Biomedical Sciences

Abstract

Mendelian disorders, also known as monogenic disorders, are caused by alterations in a single gene with minimal or no environmental influence. These alterations, due to their large effect sizes, offer critical insights for the functional annotation of the genome. Although Mendelian disorders are globally rare, they are observed at three times the rate in inbred or consanguineous populations. Narrowing it down to Pakistani population approximately 7 in 1000 children are born with Mendelian disorder. With a consanguinity rate exceeding 70% and a tradition of large family sizes, the Pakistani population provides a valuable resource for studying inherited Mendelian disorders. In this thesis, we investigated two distinct Mendelian conditions, sickle cell disease (SCD) and families with neurodevelopmental conditions using advanced technologies such as whole exome sequencing, prime editing, and cellular reprogramming for diagnosis. Sickle cell disease, a hemoglobin disorder caused by a point mutation in the HBB gene that leads to red blood cell sickling and widespread vascular occlusion. Current therapeutic strategies, including pharmacological agents, hematopoietic stem cell transplantation, and gene-editing techniques like CRISPR-Cas9, can partially alleviate symptoms. To advance gene therapy specially with context of Pakistan, we generated an induced pluripotent stem cell line by reprogramming a fibroblast sample of a patient suffering from SCD. To best of our knowledge, this is the first iPSC line derived from a SCD patient of Pakistani origin. This characterized iPSC line will serve as a valuable tool for therapeutic interventions in SCD and can also be utilized as a resource for drug discovery and other research applications. This thesis also compares the efficiencies and mechanisms of CRISPR/Cas9, base editing and prime editing, highlighting the advantages and superiority prime editing has over for CRISPR Cas 9 in precise gene correction. Through a series of experiments, pegRNA and plasmids (MLH1DN, pemax, and tevopreq) were used to engineer clones for SCD mutation generation as well as mutation correction. The prime editing system was tested by introducing the SCD mutation into HEK293T cells, results were confirmed by Sanger sequencing. The validated pegRNA will then be used to correct the SCD mutation in both HEK293T cells and iPSCs derived from an SCD patient. 15 In the last part of the thesis, we investigated two families with rare neurodevelopmental disorders using whole exome sequencing. In the first family, we did not identify any variant that segregated with the phenotype. However, in the second family, we identified a missense variant, c.2312G>T: p.771G>V in KIAA0319L, a gene responsible for axonal guidance by interacting with RTN4R, which co-segregated with the phenotype of a neurodevelopmental disorder involving motor development. Furthermore, we conducted in silico analyses to assess the conservation and pathogenicity prediction scores of the identified variant. This thesis contributes to the growing body of knowledge on monogenic disorders by combining advanced gene editing techniques with molecular and in silico analyses, with implications for future therapeutic development

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