Calsequestrin is Decreased in the Thyroid Gland of Patients with Graves’ Disease – Further Evidence for a Role of Autoimmunity against this Protein in Graves’ Ophthalmopathy.
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Date
2014-08-11
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Abstract
Background: The pathogenesis of Graves’ ophthalmopathy (GO) and the mechanism
for its link to thyroid autoimmunity is poorly understood. Our present research focuses on
the role of the skeletal muscle calcium binding protein calsequestrin (CASQ1). Earlier
studies showed that the CASQ1 gene was up-regulated in thyroid tissue from patients
with GO compared to those with Graves’ hyperthyroidism (GH) without eye signs,
however, the protein levels remained the same in both groups, raising the possibility that
the orbital autoimmune reaction begins in the thyroid gland. Here, we measured the
concentration of the CASQ1 protein in normal and Graves’ thyroid tissue, correlating
levels with parameters of the eye signs, CASQ1 antibody levels and the CASQ1 gene
polymorphism rs3838216, shown previously to be a risk factor for ophthalmopathy. Methods: The CASQ1 protein was measured by quantitative Western blotting. Following
electrophoresis, samples were transblotted to polyvinyl difluoride (PVDF) membranes
incubated with a 1:1000 dilution of a rabbit anti-CASQ1 antibody and incubated with an
horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, or anti-mouse
antibodies for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The protein
concentrations were determined from density quantification using the Quantity One 4.4.0
ChemiDoc program and expressed as pmol/mg total protein by reference to CASQ1
standards.
Results: Western blot analysis showed the presence of two forms of CASQ1 in the
thyroid, of 50 and 60 kDa molecular weight respectively. In thyroid tissues, the mean (±
SD) (GO 54.9±86.8 pmol/mg) concentration of the CASQ1 protein (GH 37.1±51.8
pmol/mg) was significantly reduced in patients with Graves’ disease , with or without
ophthalmopathy, compared to normal thyroid tissues from control subjects with multinodular
goitre or thyroid cancer (144.3±162.5 pmol/mg). The difference between GO and
GH was not significant. The decreased CASQ1 protein levels in Graves’ thyroid tissues
correlated with the homozygous genotype of the rs3838216 CASQ1 polymorphism. A
two-fold increase in Levels of CASQ1 protein in toxic nodules compared to Graves’
hyperthyroidism was markedly significant.
Conclusions: Decreased CASQ1 in the thyroid tissues of patients with Graves’ disease
compared to normal thyroid tissues from control subjects may reflect consumption of the
protein in the course of an autoimmune reaction against CASQ1 in the thyroid.
Comparing CASQ1 protein levels in thyroid tissue from five patients with toxic nodular
goitre (74.5±52.8) to controls showed no significance. The relative two fold increase in
CASQ1 levels in toxic nodules compared to Graves’ disease suggests that this is due to
the autoimmune reaction rather than the hyperthyroidism.
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Keywords
Graves’ disease, calsequestrin, thyroid gland, Western blotting, ophthalmopathy, autoantibodies
Citation
Cultrone Daniele, Lahooti Hooshang, Edirimanne Senarath, Delbridge Leigh, Champion Bernard, Wall Jack R. Calsequestrin is Decreased in the Thyroid Gland of Patients with Graves’ Disease – Further Evidence for a Role of Autoimmunity against this Protein in Graves’ Ophthalmopathy. British Journal of Medicine and Medical Research. 2014 Aug; 4(23): 4065-4075.