Date of Award

10-30-2023

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Ambrin Fatima

Second Advisor

Dr. Afsar Ali Mian

Department

Biological and Biomedical Sciences

Abstract

This project aims to investigate the underlying causes of rare neurological disorders, by using cutting-edge next generation sequencing techniques and variety of functional tools to validate the genetic data and further understand the molecular mechanisms. In Family 1, we identified a homozygous mutation (Col12a1: c.3151C>G; p.V1051L) in Col12a1 gene using whole-exome sequencing (WES), which segregates with the Ulrich muscular dystrophy-2 (UCMD-2) in an autosomal recessive inheritance pattern. Subsequently, our primary objective was to elucidate the pathogenicity of this novel variant and its association with the specific disease. To achieve this, we conducted a comprehensive analysis, including quantitative PCR (qPCR), Western blotting, and immunocytochemistry, on patient-derived and healthy control dermal fibroblasts. Our findings revealed a significant reduction in protein levels of Col12a1 in patient’s cells. This decrease in collagen protein levels may impair its interactions with other extracellular matrix mediators leading into UCMD-2 in patients. In conclusion, we add to the clinical and mutation spectrum of Col12a1- related recessive disease raising the total number of autosomal recesssive patients from 30 to 32. In Family 2, we identified two affected sisters who presented with the history of seizures with developmental regression, born to unaffected consanguineous parents. Targeted gene panel sequencing approach encompassing approximately 200 genes associated with epilepsy yielded no relevant variants. We performed WES on both affected siblings and identified compound heterozygous variants in three genes shared between both patients. Only the variations FAT3:c.A4442G:p.Q1481R and FAT3:c.G7438A:p.A2480T were found to segregate with the phenotype. Reverse phenotyping of family revealed drug resistant seizures with motor regression followed by frequent falls secondary to ataxia at the age of 11 years. Patients also developed cognitive impairments with learning difficulties, tremors and dysarthria. Furthermore, we identified six patients from different countries with FAT3 variants through gene matcher with overlapping clinical presentation of our patients. To explore the underlying disease pathomechanisms due to FAT3 variants, we then took patient-derived fibroblasts and reprogrammed them into induced pluripotent stem cells (iPSCs). Interestingly, we were unable to establish a patient-derived iPSC line, which suggest that the identified gene may have posed obstacles during the reprogramming process highlighting the critical role of the FAT3 during developmental processes. To model pathogenicity of individual/heterozygous variants we have reprogrammed and characterized an iPSC line from a healthy control. Our next step involves using this iPSC line to induce a FAT3 knock-out in order to model the disease. To prepare for this, we have already validated our guide RNAs and the CRISPR-Cas9 mediated electroporation system in K562 cells, successfully achieving knock-outs in this cell type. By progressive translational research, using state-of-the-art methodology, this project has identified genetic cause of two extremely rare neurological disorders and shed further light on understanding of pathogenicity of identified variants. Our findings suggest that FAT3 contributes to early development and it is important to understand the pathophysiology of underlying FAT3 deficiency. The study also established the first iPSC line from a healthy donor of Pakistani origin that will be used as resource for biomedical and stem cell research locally to lay the foundation of disease modeling and personalized medicine.

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Last Page

102

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