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Functional study of ubiquitin C-terminal hydrolase-L1 gene promoter haplotypes

Functional study of ubiquitin C-terminal hydrolase-L1 gene promoter haplotypes
Functional study of ubiquitin C-terminal hydrolase-L1 gene promoter haplotypes
The Ubiquitin Conjugating System (UCS) describes a system in which the 96-amino acid residue Ubiquitin can be selectively covalently linked to intracellular proteins. This endows cells with an indispensable level of regulation to determine protein fate in a wide range of basic cellular events. The abundant, neuron specific Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is intimately involved with the UCS – both in a hydrolase and ligase capacity. Mutations in UCH-L1 have clearly been associated with various neurodegenerative disorders, including Alzheimer’s, Huntington’s and particularly Parkinson’s disease.
The main and unique objective of this study was to identify any common Caucasian sequence variants in UCH-L1’s promoter, and to investigate whether they are associated with neurodegenerative symptoms, and any change in UCH-L1 transcriptional activity.
Seven novel UCH-L1 Single Nucleotide Polymorphisms (SNPs), as well as the C54A documented coding region polymorphism (Ser18Tyr), were identified using both denaturing High Performance Liquid Chromatography (dHPLC) and DNA sequencing analysis. In relation to the translational start site, the novel SNPs elucidated were: A-307G, A-306G, G-234A, A-24G, C-16T, G12A and G21A. Restriction Fragment Length Polymorphism (RFLP) genotyping analysis was then employed within Caucasian DNA sample sets of 31 and 480 individuals, to firstly elucidate the common UCH-L1 promoter haplotypes that exist within the population, and secondly, in an attempt to uncover any association between the polymorphic alleles and general neurodegenerative symptoms - no association was uncovered.
Using pGEM-T Easy as an initial ‘holding vector’, the three common UCH-L1 promoter haplotypes elucidated – AAGAC, GAGGT and AGAAC - were incorporated into a modified pGL3 vector to ascertain transcriptional activity rates. This was done by Luciferase expression analysis, and the results identified the GAGGT promoter haplotype as having a significantly increased transcriptional activity in all human cell lines tested.
It is my contention, that the pronounced increase in transcriptional activity elucidated for the GAGGT UCH-L1 promoter haplotypes, potentially indicates a primary genetic risk factor for sporadic Parkinson’s disease in the Caucasian population – a novel pathogenic model of which is proposed in this thesis. The fact that RFLP genotyping analysis uncovered no association of the promoter polymorphic alleles with more general neurodegenerative symptoms, indicates the need for further studies to be focused more specifically towards Parkinson’s disease.
Sanassy, Shane
68269389-35b4-4c25-a1f6-daed14f7bd8e
Sanassy, Shane
68269389-35b4-4c25-a1f6-daed14f7bd8e
Ye, Shu
132b6474-1927-4f93-80db-2c620a31c1ab
Zhang, Beatrice
621b5241-1c7a-4f2f-ae7c-ebda60f06891

Sanassy, Shane (2007) Functional study of ubiquitin C-terminal hydrolase-L1 gene promoter haplotypes. University of Southampton, School of Medicine, Doctoral Thesis, 296pp.

Record type: Thesis (Doctoral)

Abstract

The Ubiquitin Conjugating System (UCS) describes a system in which the 96-amino acid residue Ubiquitin can be selectively covalently linked to intracellular proteins. This endows cells with an indispensable level of regulation to determine protein fate in a wide range of basic cellular events. The abundant, neuron specific Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is intimately involved with the UCS – both in a hydrolase and ligase capacity. Mutations in UCH-L1 have clearly been associated with various neurodegenerative disorders, including Alzheimer’s, Huntington’s and particularly Parkinson’s disease.
The main and unique objective of this study was to identify any common Caucasian sequence variants in UCH-L1’s promoter, and to investigate whether they are associated with neurodegenerative symptoms, and any change in UCH-L1 transcriptional activity.
Seven novel UCH-L1 Single Nucleotide Polymorphisms (SNPs), as well as the C54A documented coding region polymorphism (Ser18Tyr), were identified using both denaturing High Performance Liquid Chromatography (dHPLC) and DNA sequencing analysis. In relation to the translational start site, the novel SNPs elucidated were: A-307G, A-306G, G-234A, A-24G, C-16T, G12A and G21A. Restriction Fragment Length Polymorphism (RFLP) genotyping analysis was then employed within Caucasian DNA sample sets of 31 and 480 individuals, to firstly elucidate the common UCH-L1 promoter haplotypes that exist within the population, and secondly, in an attempt to uncover any association between the polymorphic alleles and general neurodegenerative symptoms - no association was uncovered.
Using pGEM-T Easy as an initial ‘holding vector’, the three common UCH-L1 promoter haplotypes elucidated – AAGAC, GAGGT and AGAAC - were incorporated into a modified pGL3 vector to ascertain transcriptional activity rates. This was done by Luciferase expression analysis, and the results identified the GAGGT promoter haplotype as having a significantly increased transcriptional activity in all human cell lines tested.
It is my contention, that the pronounced increase in transcriptional activity elucidated for the GAGGT UCH-L1 promoter haplotypes, potentially indicates a primary genetic risk factor for sporadic Parkinson’s disease in the Caucasian population – a novel pathogenic model of which is proposed in this thesis. The fact that RFLP genotyping analysis uncovered no association of the promoter polymorphic alleles with more general neurodegenerative symptoms, indicates the need for further studies to be focused more specifically towards Parkinson’s disease.

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More information

Published date: October 2007
Organisations: University of Southampton

Identifiers

Local EPrints ID: 64912
URI: http://eprints.soton.ac.uk/id/eprint/64912
PURE UUID: 7f16ff2f-2802-4583-8ef0-b6d10c22b6b7

Catalogue record

Date deposited: 28 Jan 2009
Last modified: 13 Mar 2019 20:20

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Contributors

Author: Shane Sanassy
Thesis advisor: Shu Ye
Thesis advisor: Beatrice Zhang

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