Analysis of CDK13-related disorder in a mouse model

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Abbad, Aseel (2024) Analysis of CDK13-related disorder in a mouse model. PhD thesis, University of Nottingham.

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Abstract

CDK13-related disorder, a developmental syndrome associated with intellectual disability, facial dysmorphism, congenital heart defects, brain defects, and growth abnormalities, is a recently identified syndrome caused by mutations in the serine-threonine protein kinase CDK13. CDK13 is thought to play roles in transcription regulation and splicing. To date, 135 patients have been identified with a total of 58 variants. The most common variant is c.2525A > G, p.N842S, affecting 45/135 patients. The literature is lacking information on the downstream targets of CDK13 and its role in development.

To assess the effect of the mutation on development and gene expression, a mouse model carrying the humanized variant c.2525A > G, p.N842S, and a mouse model carrying the Cdk13 deletion allele Cdk13tm1b were used. Embryonic hearts of Cdk13N842S were phenotypically analysed by high resolution episcopic microscopy. Gene expression was measured using real-time qPCR. Growth of mice was assessed prenatally and postnatally. Brain morphology was studied by cryosectioning and cresyl violet staining. Behavioral tests were performed to assess for neurological symptoms. The total proteome and phosphoproteome resulting from a homozygous deletion of Cdk13 were studied in Cdk13tm1b line. All animal procedures were performed in strict accordance with the Animals Scientific Procedures Act 1986 (ASPA).

Mice homozygous for the variant Cdk13N842S were found to be embryonically lethal before E15.5. Embryonic heterozygous hearts were analyzed at E15.5, and 44.4% (12/27) had a structural defect including ventricular septal defects and outflow tract anomalies, and 28.57% (2/7) of P6 neonates had ventricular septal defects. Markers of heart development were selected from RNAseq data previously produced in the lab, and Vegfa was found to be significantly upregulated. In growth assessment, all mice heterozygous for the mutation Cdk13N842S were growth retarded prenatally and postnatally. The gene expression of Igf1 and Igf2 were not significantly changed in E15.5 embryos, nor the splice variants of Igf1. However, phosphoproteomics results from Cdk13tm1b homozygotes suggested GH and IGF1 to be inhibited.

Cdk13N842S adult heterozygotes performed abnormally in two behavioural tests, clasping and four-limb hanging, and were suggested to have an abnormal gait. Histological analysis of the corpus callosum, lateral ventricle, and cerebellum did not reveal any abnormalities, although additional analysis is required. The total proteome/phosphoproteome analysis revealed residual CDK13 protein in Cdk13tm1b homozygotes, but significantly downregulated phosphorylation of CDK13 at an important activation site T817. The phosphoproteome analysis suggested MEF2C as a phospho-protein that is possibly involved in the cardiac, growth, and neurological phenotype.

This data indicates the humanized mutant mouse p.N842S is the best studied model to date to replicate CDK13-related disorder and its associated phenotypes, such as congenital heart defects, growth problems, and neurological abnormalities. Phosphoproteomics analysis revealed interesting possible interactors with CDK13, and Cdk13tm1b homozygotes are valuable in further investigating the possible targets.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Loughna, Siobhan
Brook, David
Keywords: Gene, mutation, syndrome, developmental disorder, mouse model, CDK13, congenital heart defect, high resolution episcopic microscopy, proteomics
Subjects: R Medicine > RB Pathology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 79950
Depositing User: Abbad, Aseel
Date Deposited: 11 Dec 2024 04:40
Last Modified: 11 Dec 2024 04:40
URI: https://eprints.nottingham.ac.uk/id/eprint/79950

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