Diplegic Spastic Cerebral Palsy Related FBXO31 Mutant Reconstruction and Proliferation Analysis in Human Cells

 

Chemie  |  Biochemie  |  Medizin

 

Jasper Rohner, 2006 | Meisterschwanden, AG

 

Diplegic spastic cerebral palsy (DSCP) primarily results from hypoxia or genetic mutations, exemplified by the D334N mutation on the FBXO31 protein within the SCF-E3 Ligase. FBXO31 regulates substrate levels by marking them for proteasomal degradation. The altered binding function of the D334N mutant disrupts substrate equilibriums, causing homeostatic imbalances in cerebellum cells and subsequent nervous system dysfunction. Previous studies noted potential toxicity upon overexpression in human kidney cells. However, the kidneys in probands exhibited unrestricted functionality, challenging this assumption. This study conducted a proliferative analysis of moderately expressed D334N kidney cells, genetically engineered via CRISPR/Cas9. Results revealed uninhibited proliferation compared to the wild-type protein, indicating a presumed tissue-specific functionality of the mutant correlated with FBXO31 expression levels. This manifested toxicity in regions characterized by elevated FBXO31 expression. The tissuespecific impact of FBXO31 wild-type in proliferation-related mechanisms, such as cancerous cell division, further substantiates this hypothesis.

Introduction

Diplegic spastic cerebral palsy (DSCP), arising from physical factors like hypoxia and genetic mutations such as the D334N variant, remains relatively unknown. Despite the D334N mutation’s presence in various tissues, its impact is not uniform. While disrupting homeostasis in cerebellum cells, kidney cells appear unaffected. This study aims to prove the tissue specificity of the mutant’s impact and understand the reasons for its differential effects.

Methods

Human embryonic kidney cells (HEK293T) were CRISPR/Cas9-engineered to introduce the D334N mutant. D334N mutant cells were analyzed in a proliferative assay, comparing them with FBXO31 wild type and a FBXO31 knockout. A comparative growth assay using Next Generation Sequencing (NGS) measured relative cell-type concentrations over time. CRISPResso analyzed sequencing data, and sequencing and editing quality were assessed. A proliferative analysis of the mutant was performed and a detailed interpretation of its tissue-specificity was conducted later on.

Results

Sequencing quality remained satisfactory, showing constant relative D334N cell concentrations over nine days with minor fluctuations. The D334N mutant exhibited no proliferative disadvantage compared to the wild-type cell line, and no toxic attributes within natural expression levels in HEK293T cells were linked to the mutant.

Discussion

The observed cytoneutral effect in HEK293T cells with the D334N mutant confirms its tissue-specificity. mRNA sequencing of FBXO31 in various tissues suggests homeostatic disruption is prevalent in tissues with high protein expression. Alternative degradation pathways could mitigate mutant toxicity in HEK239T cells. Additionally, the rate of cell division may regulate the mutant’s impact through a resulting shortage of Cyclin D1, a substrate increasingly marked by the mutant. Further studies are needed to confirm this assumption.

Conclusions

This study offers insights into DSCP pathology and demonstrates a cytoneutral effect of the D334N mutant in HEK293T cells. It suggests tissue specificity correlated with FBXO31 expression levels. Future research should explore the exact chemical changes in function across different tissues. Investigating the mutant’s potential proliferation-inhibitory effects against rapid cell growth would be valuable. Optimizing DNA extraction processes and sgRNAs could lead to more precise results.

 

 

Würdigung durch den Experten

Prof. Dr. Eric Kübler

Jasper Rohner hatte sich bereits als Schüler um ein Projekt an der ETH Zürich im Bereich Molekularbiologie bemüht und dabei eine Arbeit gefunden, die sich mit der Krankheit Diplegic Spastic Cerebral Palsy beschäftigt. In einem ausserordentlichen Einsatz konnte er sich die relativ neue CRISPR-Methode aneignen, um damit Studien zu einer mutanten Version des FBXO31-Gens anzustellen. Durch Experimente mit Zellkulturen konnte er zeigen, dass das mutante Protein eine gewebsspezifische Toxizität aufweist. Damit legte Jasper Rohner eine Grundlage dafür, die Unterschiede in den Geweben genauer zu untersuchen.

Prädikat:

sehr gut

Sonderpreis von Life Sciences Switzerland (LS2)

 

 

 

Kantonsschule Wohlen
Lehrer: Erich Bühlmann