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Functional Characterization of a Homozygous TRAF6 Mutation Causing NF-κB Signaling Defects and Human Immunodeficiency Disease
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- Author / Creator
- Lewis, Allison
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As many as 80,000 Albertans are affected by rare diseases; with the greatest prevalence in children such diseases dramatically impact the lives of both those suffering and the families caring for them. Severe combined immunodeficiency (SCID) represents a group of inherited disorders characterized by immune system impairment due to dysfunctional lymphocytes. Ectodermal dysplasia with immunodeficiency (EDA-ID) is a rare disorder where patients experience abnormalities of the ectodermal structures in addition to immunodeficiency. EDA-ID is most often caused by mutations in the gene encoding NEMO, a critical regulator of the NF-κB pathway. This study focused on finding the cause of a life-threatening immunodeficiency with ectodermal dysplasia features affecting a child of Cree descent born to consanguineous parents from Alberta. Conventional genetic testing for SCID was unable to provide a diagnosis prompting an in-depth investigation for possibly novel genetic causes. A combined bioinformatics method of homozygosity mapping, whole-exome sequencing, and in silico modelling was used to identify candidate genes according to an autosomal recessive mode of inheritance. Sanger sequencing was used to determine which candidates segregated with the phenotype in the family. A single candidate gene, TRAF6 (NM_145803 c.G269T; [p.C90F]), was identified according to these criteria.
This gene is a component of the NF-κB pathway and required for NEMO activation, and the mutation is harbored in a domain that is critical for protein structure and function. The functional consequences of the TRAF6 C90F variant at the protein level, and on the NF-κB pathway were determined using patient derived cell lines. Western blotting shows that the mutant protein is present at much lower levels in patient cells than WT protein in control cells, and translational repression of patient cells using cycloheximide suggests that the mutation impacts protein stability. Molecular analysis of NF-κB signaling in patient cells by western blotting, a high-content assay for nuclear translocation, and RT-qPCR showed that NF-κB signaling is impaired or abolished in patient cells. Significantly, introduction of WT TRAF6 gene expression in patient cells rescues signaling defects, confirming that the C90F mutation, and not another unidentified signaling component, is underlying the impairment in pathway activation. Elucidating the causative gene of the disorder in this family has had direct implications for the therapeutic choice to treat the deficiency in this child: a bone marrow transplant, as well as on the family through personalized genetic counselling. This work describes a novel phenotype in a family of Cree descent and proposes a molecular basis for the disease in a gene not definitively known to cause human disease. This research provides an example of a translational study using deep sequencing to identify the cause of a genetic disorder with direct implications for the practice of precision genetics. -
- Graduation date
- Fall 2018
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.