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

Griffin, Aliesha (2011). Characterisation of PAPSS2 genetic variants in vitro / Investigating the role of vitamin D in inflammation related muscle loss. University of Birmingham. M.Res.

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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: Thesis (Masters by Research > M.Res.)
Award Type: Masters by Research > M.Res.
College/Faculty: Colleges (2008 onwards) > College of Medical & Dental Sciences
School or Department: School of Clinical and Experimental Medicine
Funders: None/not applicable
Subjects: R Medicine > RB Pathology
R Medicine > RC Internal medicine


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