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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