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Serum copper as a novel biomarker for resistance to thyroid hormone
Jens et al, Biochem. J. (2012) 443, 103-109
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Introduction
TH - development & maintenance of all tissues
Action of TH mediated by TRs
TRα and TRβ – encoded by two genes located on chromosomes 17 & 3 & belongs to family of nuclear receptors
Mediate biological activities of T3 via transcriptional regulation
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Defects in TRβ cause RTH.
Characterized by elevated level of free serum T3 & T4 in the presence of normal and elevated TSH.
Dominant autosomal disease caused by mutations in TRβ gene.
Impaired T3 binding & -ve feedback.
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NEGATIVE FEEDBACK MECHANISM
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Diagnosis of RTH is difficult -: Asymptomatic or euthyroid. Abnormalities in T4 binding proteins Thyrotoxicosis Pituitary adenoma
DIAGNOSIS
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More than 350 families has RTHβ but no one with RTHα.
TRα1R384C mutant mice revealed a TRα1-specific regulation of serum Se.
Alone is not used for RTHVaries with serum TH.
PREVIOUS STUDIES
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AIM
Study the role of serum copper in RTH & its regulation by TH & ceruloplasmin
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Objective 1
Effect of TH on serum copper
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ExperimentMouse strain carrying R384C mutation in TRα1 was
used
Strain crossed to TRβ-deficient mice yielding TRα1 +m TRβ-/- double mutants
Mice were treated with T3 via their drinking water
Spectrometer used to measure trace elements conc.
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Serum Cu is regulated by THInference=
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Objective 2
Investigate the origin of T3-induced increase in serum copper
copper levels in liver, kidney & urine of wild type & TRα +m animals with or without T3.
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13Changes in tissue Cu levels upon TH treatment
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Inference
T3 increases hepatic copper export & improved renal retention
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Objective 3
Investigate the underlying molecular mechanism by analyzing changes in gene expression in liver &
kidney
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ExperimentRNA was isolated
4μg RNA was used for cDNA synthesis
Real-time PCR was performed using HPRT as reference gene
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GENE EXPRESSION STUDY
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Changes in expression of Cu metabolic genes upon TH treatment
GENE EXPRESSION STUDY
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InferenceTH induces expression of two Cu-transport
proteins ATP7A & ATP7B as well as major Cu-transport protein ceruloplasmin
mRNA levels of other Cu-containing enzymes such as SOD1 or Mtt1 & Mtt2 were reduced
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Objective 4
Whether increased mRNA production of ceruloplasmin resulted in higher serum of
this protein
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Experiment 5 μl of mouse serum separated on SDS-
PAGE
Transferred on to nitrocellulose membrane
Ceruloplasmin was detected using specific polyclonal goat antibody & horseradish peroxidase-coupled secondary antibody
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Serum ceruloplasmin levels
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Inference
Increase in ceruloplasmin in serum of wild-type animals upon T3 treatment which was
again slightly more pronounced in TRα1 +m mutants
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Objective 5
Determined Cu levels in patients with RTHβ
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Serum Cu and Se as biomarker for RTHβ
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Inference
Serum Cu concentrations were significantly reduced in RTHβ patients & as expected from
previous studies, serum Se levels were elevated in RTHβ patients
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Summary
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Conclusion
Se/Cu ratio might be helpful during diagnosis of RTH & human RTHα patient might also
present with reduced Se/Cu ratio as in animal model