Novel variants of unknown significance cause pathogenesis through distinct mechanisms in spinocerebellar ataxia 14

Kalsbeek, Robin (2022) Novel variants of unknown significance cause pathogenesis through distinct mechanisms in spinocerebellar ataxia 14. Master's Research Project 2, Biomedical Sciences.

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Abstract

Spinocerebellar ataxia type 14 (SCA14) is a dominantly inherited ataxia caused by missense mutations in the PRKCG gene encoding protein kinase c-gamma (PKCγ). Patients with SCA14 suffer from a slowly progressing cerebellar ataxia. Most pathogenic mutations are found in the C1 domain of PKCγ and cause pathogenesis through increased activity, mislocalization, and protein aggregation. In many cases, genetic tests reveal variants of unknown significance in patients (VUSes). Because SCA14 is a late-onset disease with low prevalence, segregation studies are not always possible, leaving these VUSes unclassified and the patients without a diagnosis. In the present study, we provide evidence on the pathogenicity of the PKCγ variants H174P and R239W in the C2 domain. We performed functional studies on these variants with overexpression plasmids encoding the cDNA of these variants, and we used the novel CRISPR/Cas9 technique prime editing to make isogenic cell models in the human neuroblastoma cell line SH-SY5Y. We showed that both PKCγ H174P and R239W cause pathogenesis through distinct mechanisms. Both PKCγ H174P and R239W form aggregates, PKCγ R239W inhibits proteasomal degradation, and PKCγ H174P increases the activity of PKCγ. Furthermore, Ca++ signaling was sufficient to activate PKCγ H174P, and its expression was toxic in SH-SY5Y cells.

Item Type:	Thesis (Master's Research Project 2)
Supervisor name:	Verbeek, D.S.
Degree programme:	Biomedical Sciences
Thesis type:	Master's Research Project 2
Language:	English
Date Deposited:	19 Apr 2022 09:56
Last Modified:	19 Apr 2022 09:56
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/26934

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