All ETDs from UAB

Advisory Committee Chair

Deeann Wallis

Advisory Committee Members

Matthew Alexander

Peter Detloff

Scott Wilson

Talene Yacoubian

Document Type

Dissertation

Date of Award

2021

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Advancements in sequencing technology have enabled the widespread utilization of genetic testing, increasing the diagnosis and understanding of genetic disease. Clinicians and researchers are now faced with the difficulty of interpreting genetic results and understanding how genetic variation can impact patient disease phenotype. Herein we utilized three different genes (frataxin, neurofibromin 1, and NTRK2), which result in Friedreich’s Ataxia (FRDA), Neurofibromatosis Type 1 (NF1), and NTRK2-associated phenotypes respectively, to discuss the impacts of genetic variants on molecular phenotype and explore potential therapeutics. To determine how genetic variation of repeat sequences affects different patient tissues, DNA and protein were extracted from FRDA patient samples and somatic mosaicism of pathogenic variants was analyzed through PCR and Western Blot analyses. We observed a tissue-specific pattern of somatic mosaicism and instability of pathogenically expanded trinucleotide repeat sequences with normal length trinucleotide repeats exhibiting no genetic variation among different tissues. The somatic mosaicism and instability observed represents a potential cause of symptom variability among patients with similar disease-causing variants. Additionally, to determine how different missense mutations impact protein activity we overexpressed NF1 cDNAs that were created with patient-specific missense variants in HEK293 cell-lines to assess protein expression and function. We observed unique effects on protein stability and function with some missense variants resulting in functional protein at a reduced protein expression. Other variants exhibited high levels of protein but were incapable of proper function. This highlights the importance of understanding how genotype can contribute to phenotype in disorders which are caused by thousands of unique variants in one gene. Additionally, we demonstrated that specific variants within NTRK2 can result in drastically different functional consequences associated with distinct phenotypes. We utilized an artificial intelligence based software, mediKanren, to predict potential therapeutics to treat the differing functional consequences and were able to partially rescue variant molecular phenotype. This emphasizes the need to determine how different variants within the same gene can result in separate disorders and whether this impacts patient treatment. Overall, these studies highlight the importance of understanding how intragenic mutation heterogeneity can impact patient presentation and that the molecular mechanisms behind these variants can be targeted for personalized treatments through artificial intelligence-based software in precision medicine.

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