3’ UTR Structural Elements in CYP24A1 are Associated with Infantile Hypercalcaemia Type 1

Ball, Nicole (2022) 3’ UTR Structural Elements in CYP24A1 are Associated with Infantile Hypercalcaemia Type 1. Doctoral thesis, University of East Anglia.

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Ribonucleic acids (RNAs) fold into complex structures that are critical for their function and regulation including post-transcriptional modification, localisation, translation and degradation. RNA structure and potential RNA misfolding has scarcely been studied in a clinical setting. Hypomorphic mutations in the cytochrome P450 family 24 subfamily A member 1 (CYP24A1) protein coding region causing inappropriately elevated active vitamin D metabolites have been observed in some cases of idiopathic infantile hypercalcemia and adult-onset nephrolithiasis. It is unclear why a subset of cases present with superficial CYP24A1 mediated hypercalcemia (CMH) but do not exhibit protein-coding mutations in CYP24A1. This thesis presents a combination of biochemical profiling, next generation sequencing, bioinformatics, proteomic and molecular cytogenetic approaches to examine CYP24A1 in a patient cohort with apparent CMH. This work identified several novel single nucleotide variants (SNVs) located in the CYP24A1 3’ untranslated region (UTR). These SNVs led to CYP24A1 messenger RNA (mRNA) misfolding. The mRNA structural abnormalities observed were associated with an over accumulation of an apparently less active CYP24A1 protein. The generation of a CMH cell line, using CRISPR, mimicking patients with CYP24A1 3’ UTR variants causing mRNA structural alterations provided a model system for in vitro investigations into so-called noncanonical CMH. Subsequent single molecule fluorescence in situ hybridisation (smFISH) methods provided CYP24A1 cellular localisation in addition to mRNA abundance in vitro and ex vivo. The important advancements presented in this thesis are valuable to understanding mRNA structure-function relationships and novel CYP24A1 mutations, which affect mRNA translation and protein expression. The findings of this research provide a framework that can be used to better understand the molecular basis of pathogenesis in patients lacking protein coding region abnormalities.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Chris White
Date Deposited: 26 Apr 2023 07:43
Last Modified: 26 Apr 2023 07:43
URI: https://ueaeprints.uea.ac.uk/id/eprint/91882


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