Serum copper as a novel biomarker for resistance to thyroid hormone

1. Serum copper as a novel biomarker for
resistance to thyroid hormone
Jens et al, Biochem. J. (2012) 443, 103-109
1

2. 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
2

3.  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.
3

4. NEGATIVE FEEDBACK MECHANISM
4

5.  Diagnosis of RTH is difficult -:
 Asymptomatic or euthyroid.
 Abnormalities in T4 binding proteins
 Thyrotoxicosis
 Pituitary adenoma
 DIAGNOSIS
5

6.  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 RTH
 Varies with serum TH.
 PREVIOUS STUDIES
6

7. AIM
Study the role of serum copper in RTH & its
regulation by TH & ceruloplasmin
7

8. Objective 1
Effect of TH on serum copper
8

9. Experiment
 Mouse 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.
9

10. Serum Cu is regulated by TH
10
Inference=

11. Objective 2
Investigate the origin of T3-induced increase
in serum copper
11

12.  copper levels in liver, kidney & urine of wild type &
TRα +m animals with or without T3.
12

13. Changes in tissue Cu levels upon TH treatment 13

14. Inference
T3 increases hepatic copper export & improved
renal retention
14

15. Objective 3
Investigate the underlying molecular mechanism by
analyzing changes in gene expression in liver &
kidney
15

16. Experiment
RNA was isolated
4μg RNA was used for cDNA synthesis
Real-time PCR was performed using HPRT as
reference gene
16

17. 17
GENE EXPRESSION STUDY

18. Changes in expression of Cu metabolic
genes upon TH treatment
18
GENE EXPRESSION STUDY

19. Inference
 TH 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
19

20. Objective 4
Whether increased mRNA production of ceruloplasmin
resulted in higher serum of this protein
20

21. 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
21

22. Serum ceruloplasmin levels
22

23. Inference
Increase in ceruloplasmin in serum of wild-type animals
upon T3 treatment which was again slightly more
pronounced in TRα1 +m mutants
23

24. Objective 5
Determined Cu levels in patients with RTHβ
24

25. Serum Cu and Se as biomarker for RTHβ
25

26. Inference
Serum Cu concentrations were significantly reduced in
RTHβ patients & as expected from previous studies,
serum Se levels were elevated in RTHβ patients
26

27. Summary
27

28. 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
28