Investigating the effects of a novel ITP patient derived PRKDC mutation in a human B cell line.
File(s)
Author(s)
Al-Mudhafar, Rand
Type
Thesis
Abstract
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by the PRKDC gene) plays a key role in the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. DNA-PKcs is crucial for V(D)J recombination, the NHEJ-dependent process required for diversification of the genes for B and T cell receptors, and therefore for the generation of a functional vertebrate immune system. Four PRKDC heterozygous missense variants have recently been identified in patients with Immune Thrombocytopenia Purpura (ITP) patients, an autoimmune bleeding disorder characterized by immune-mediated platelet destruction. To investigate the potential significance of one PRKDC variant (c.G5174A; p.A1237T) in ITP, I have used PRKDC gene editing in a human B cell line (DG75) to generate a heterozygous A1237T knock-in clone and several heterozygous (PRKDC+/-) and homozygous (PRKDC-/-) knock-out clones. Compared to parental DG75 cells, the A1237T knock-in cells show slower growth and reduced viability, accompanied by an increase in apoptosis and impaired levels of NHEJ. These phenotypes are similar to those of PRKDC-/- cells and clearly more pronounced than those of PRKDC+/- cells which are intermediate between WT and PRKDC-/- cells. These results suggest that the knock-in allele has a dominant-negative effect, and this was confirmed by targeted inactivation of the knock-in allele which resulted in the reversal of the above-mentioned phenotypes. Western botting showed that the knock-in clone had appreciable levels of DNA-PKcs protein and DNA damage-induced kinase activity. Interestingly, however, the kinetics of kinase induction were slightly advanced, which may help to explain the low NHEJ activity...
Version
Open Access
Date Issued
2022-12-23
Date Awarded
01/11/2023
License URL
Advisor
Porter, Andrew Christopher George
Cooper, Nichola
Publisher Department
Department of Immunology and Inflammation
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)