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Characterisation of PAPSS2 genetic variants in vitro / Investigating the role of vitamin D in inflammation related muscle loss

Griffin, Aliesha (2011)
M.Res. thesis, University of Birmingham.

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There are two reports in this MRes thesis.

Report One: Dehydroepiandrosterone (DHEA) is the precursor for sex steroids in humans and its inactivation by sulphonation is a key regulator of active DHEA levels. Sulphonation of DHEA is controlled by the enzyme SULT2A1, which requires the presence of 3’-phosphoadenosine-5’-phospho-sulphate (PAPS) for activity. In humans PAPS is produced by two isozymes, PAPSS1 and PAPSS2. Several variants of human PAPSS2 are associated with changes in functional activity and have been described in patients presenting with hyperandrogenism and severe bone phenotypes.

Aims: The aim of this project was to characterise the functional activity of hsPAPSS2a genetic variants E10K, M281L and E332K within the DHEA sulphonation pathway.

Methods: The hsPAPSS2a variants were overexpressed in HEK293 cells in conjunction with hsSULT2A1. The capacity of the hsPAPSS2a variants to generate PAPS was determined by the ability of the co-expressed SULT2A1 to convert 3H-DHEA to 3H-DHEAS. The steroids were extracted and thin layer chromatography was used to determine the amount of DHEA conversion.

Results: The novel hsPAPSS2a E332G variant was observed to have a reduction of 50% in functional activity. The hsPAPSS2a E10K and M281L variants showed no significant changes in the DHEA sulphonation pathway.

Conclusions: The clinical recommendation from this study is that patients presenting with androgen excess should be screened for the hsPAPSS2a E332G mutation.

Report two:
Background: Rheumatoid arthritis (RA) is an autoimmune disease that results in inflammation of the synovial joints, loss of bone mass and muscle loss. Inflammatory
related skeletal muscle wasting increases the risk of falls and fractures and the mortality and morbidity of patients. There is evidence to suggest that low levels of vitamin D also correlate with muscle weakness and loss. This is supported by observations that vitamin D has an important role in skeletal muscle development and function.

Aim: The aim of this project was to determine if inflammatory related muscle atrophy is directly mediated through impaired vitamin D actions or its metabolism.

Methods: Murine C2C12 myoblasts were differentiated over a six day period and treated with TNFα, IL-6 and IL-1β for 24 hours. Alternatively, C2C12 myoblasts were differentiated for six days in media preconditioned with serum from human RA synovial fibroblasts. Quantitative real time RT-PCR was used to determine changes in the mRNA levels of the vitamin D receptor and mRNA for gene involved in vitamin D metabolism.

Results: Treatment of C2C12 muscle cells with proinflammatory or with preconditioned media from synovial fibroblasts did not cause any changes in gene expression of the vitamin D receptor or the genes responsible for vitamin D activation or catabolism.

Conclusion: It is concluded from this study that inflammatory related muscle atrophy is not a direct result of changes in vitamin D actions through its receptor or its metabolism.

Type of Work:M.Res. thesis.
Supervisor(s):Cooper, Mark and Dhir, Vivek
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:School of Clinical and Experimental Medicine
Subjects:RB Pathology
RC Internal medicine
Institution:University of Birmingham
ID Code:2870
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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