THYROID NEWS

 

Section editor:    Daniel Glinoer (M.D.; Ph.D.)  University of Brussels
For THYROID NEWS  articles from 2007, click here                             BACK

 

 April 2008

Contributions by

v Rosalind Brown (Harvard University, Boston, USA)

v Geraldo Medeiros-Neto (The University of Sao Paulo, Brazil)

v Anthony Weetman (The University of Sheffield, UK)

 

Congenital hypothyroidism

 

TOPIC: Congenital central hypothyroidism

Title: Role of the Thyrotropin-Releasing Hormone stimulation test in diagnosis of congenital central hypothyroidism in infants.

Authors: Van Tijn DA, de Vijlder JJM, & Vulsma T.

Reference: Journal of Clinical Endocrinology & Metabolism 93: 410-419, 2008

 
 SUMMARY

Context: A shortage of thyroid hormone during prenatal life and the first years after birth results in a spectrum of neuropsychological disorders, depending on the duration and severity of the deficiency. In the case of congenital hypothyroidism of central origin (CH-C), the majority of patients have multiple pituitary hormone deficiencies (MPHD). This condition poses an additional threat to postnatal central nervous system development, primarily on account of neuroglycopenia due to ACTH/cortisol deficiency with or without additional GH deficiency. Therefore, in CH-C, rapid diagnosis is even more urgent than in congenital hypothyroidism of thyroidal origin.

Objective: In the assessment of hypothalamic-pituitary-thyroid function, the authors considered the pituitary response to intravenous administration of TRH (TRH test) pivotal. They evaluated the usefulness of the TRH test in a cohort of infants with neonatal congenital hypothyroidism screening results indicative of CH-C by analyzing the results within the framework of investigations of the anatomical and functional integrity of the hypothalamo-hypophyseal system.

Design, Setting and Patients: Dutch nationwide prospective study (1994–1996). Patients were included if neonatal CH screening results were indicative of CH-C and patients could be tested within 3 months of birth. Ten male and five female infants with CH-C, detected by neonatal screening, and six infants with false-positive screening results, nonthyroidal illness, or transient hypothyroidism, were included in the study.

Main Outcome Measures: Results of TRH tests, within the framework of extensive endocrinological examinations and cerebral magnetic resonance imaging, were measured.

Results: All patients with type 3 TSH responses to TRH had MPHD, and the majority of patients (67%) with type 2 responses had isolated TSH deficiency.

Conclusions: The TRH test has a pivotal role in the diagnosis of TSH deficiency in young infants. Abnormal TRH test results, especially a type 3 response, urge immediate assessment of integral hypothalamic-pituitary function because the majority of patients have MPHD.

 

COMMENTARY

 

There is no doubt that newborn screening for congenital hypothyroidism (CH), begun over 30 years ago in Quebec and now conducted in most industrialized parts of the world, has been a resounding success. Although there continues to be some controversy as to whether subtle cognitive deficits persist in the most severely affected infants, all agree that the mental retardation due to primary congenital hypothyroidism has been eradicated with the advent of newborn screening and the timely initiation of postnatal therapy. Screening has also permitted the elucidation of the relative prevalence of the various etiologies of this heterogeneous disorder. Screening strategies vary, with some areas of the world (particularly the USA) performing a primary T4/backup TSH method; others (e.g., Europe, Australia, Japan parts of North America employing a primary TSH  program, and a minority measuring both. Each approach has its advantages and limitations but both the primary T4/backup TSH and primary TSH methods appear to be similar in the initial screening goal of identifying babies with ‘classical’ primary congenital hypothyroidism. Whichever approach is employed, rarely babies are missed, due either to limitations in the screening strategy itself or to human error.

 

Since 1995 the Dutch program has initiated a novel multi-step screening strategy that includes, in addition to T4 and TSH, the measurement of thyroxine binding globulin (TBG) in the filter paper specimens with the lowest 5% of T4 values. The T4/TBG ratio is used as an indirect reflection of the free T4, which cannot be measured directly in dried blood spots. The group has shown that, for a relatively small incremental cost, this approach results in increased sensitivity and specificity in the detection of milder cases of primary CH that might otherwise be missed. An additional advantage is the identification of >90% of infants with central congenital hypothyroidism (CH-C), compared with only 30% with primary T4 screening and none with a primary TSH approach. The incidence of CH-C detected on screening was 1 in 21,000 for the years 1994-96 and 1 in 16/000 for the years 1995-2000, much higher than a recent estimate of 1 in 95,933 obtained in the US, and >80% of babies on subsequent testing had multiple pituitary hormone deficiencies. Given the high morbidity and mortality of congenital hypopituitarism, the availability of effective therapy, and an apparent frequency similar to that of  phenylketonuria (1 in 18.000), the disorder for which neonatal screening began, the authors have convincingly argued that the goals of  newborn thyroid screening should be extended to include the detection of babies with CH-C.

 

In the present study, the authors present the results of TRH testing in the original cohort of patients. Of 26 infants who fulfilled the screening criteria suggestive of CH-C, 5 were excluded from further analysis for various reasons and 6 turned out to have false-positive results. All 15 remaining infants had abnormal TSH responses to TRH when compared to adult normative values. Six patients had impaired release of TSH “type 2 response”), and of this group only 4 had had additional pituitary hormonal deficiencies. In contrast, all 9 of the 9 patients whose response was characterized by a delayed peak (“type 3 response”) had additional deficits. Although the authors conclude from these results that the TRH test therefore “has a pivotal role in the diagnosis of TSH deficiency” a statistical analysis was not performed to determine whether this difference was significant. Furthermore, 5/6 of the infants with a type 2 response but only 1/8 patients with a type 3 response had a normal MRI, suggesting that in areas of the world such as the US where TRH is no longer available, brain imaging might provide a suitable alternative.

 

The present study is also at variance with a previous report of 5 infants with congenital hypopituitarism associated with an ectopic posterior pituitary gland (the predominant morphologic abnormality in this study) in which a type 2 response was characteristic of infants with more severe, not less severe hypopituitarism (Brown et al. JCEM 72:12-18, 1991). Clearly more numbers are needed before the utility of TRH testing in identifying infants at risk of multiple pituitary hormone deficiencies can be accepted.

 

Summary and commentary prepared by Rosalind Brown (Related to Chapter 15 of TDM)

 

New insights into Pendred syndrome

 

TOPIC: Pendrin gene mutation induces poor organification, iodine retention & high deiodinase activity

Title: Pendred syndrome in two Galician Families: insights into clinical phenotypes through cellular, genetic and molecular studies.

Authors: Palos F, Garcia-Rendueles ME, Araujo-Vilar D, Obregon MJ, Calvo RM et al.

Reference: Journal of Clinical Endocrinology & Metabolism 93: 267-277, 2008

SUMMARY

Background: Pendred Syndrome is an autosomal recessive disorder characterized by congenital sensorineural hearing loss (enlarged acqueduct, cochlear defects) and variable incidence of goiter with or without hypothyroidism. Mutations in the SLC26A4 (soluble carrier family 26, member 4) gene affects Pendrin, a transmembrane protein expressed in the thyroid apical membrane, inner ear and elsewhere. Pendrin is involved in iodide transport (as well as in chloride, formate and nitrate transport).

Purpose: Two families from Galicia (Spain) were studied. In family A, the proposita had a large goiter and low serum T4 (but normal serum TSH and Free T3). In Family B, affected members showed deafness but not goiter. The objective was to identify the mutations causing Pendred syndrome as well as genetic and cellular mechanisms underlying the phenotypes A and B.

Methods: Pendrin gene (SLC26A4) gene analysis, deiodinase activity in the goitrous tissue, a primary goiter thyrocyte culture and comparison with culture of normal human thyrolytes (western blotting, cofocal microscopy and iodine uptake kinetics).

Results: Proposita A (43 year old deaf woman) had a large multinodular goiter, with normal serum TSH and FT3 but low FT4 attributable to high type 1 and type 2 iodothyronine deiodinase activities in the goiter. She was compound heterozygous for a frameshift mutation at exon 3 (c.279 delT) and a C.578C →T at exon 5. This late mutation results in replacement of c.Thr193Ile. In family B, the propositus (a 26 year old deaf male) bore c.279 delT and a novel mutation c.416-1G →A but no goiter. Thyrocyte cell culture showed truncated pendrin retained intracellularly and high iodine uptake with low efflux leading to iodine retention.

Conclusions: Lack of apical pendrin immunoreactivity was due not only to functional impairment of pendrin but also to defective membrane targeting. Proposita A adapted to poor organification by increasing deiodinase activities in the goiter (avoiding clinical hypothyroidism). Lack of goiter in subjects homozygous for c.416-1G → A was due to incomplete penetrance (some wild type pendrin was generated). Intracellular iodine retention and consequent accumulation confirms the importance of pendrin as an iodide transporter.


 

 

COMMENTARY

In this remarkable article, the authors describe two families with Pendred syndrome from Galicia (Spain). The founder mutation c.279delT (exon 3) causes a frameshift that introduces a stop codon 3 amino acids downstream. The other mutation c.578C → T (exon 5) results in the replacement of a normal threonine with isoleucine at codon 193. The c.416-1G → A located at the acceptor splice site of intron 4 (a novel mutation) replaces a G with an A and had an incomplete penetrance, as some wild type pendrin is translated.

 

Functional studies were conducted in goitrous tissues and normal thyroid specimens. Deiodinase activity was higher in goitrous tissue of patient A, as compared to normal thyroid tissue. MCT8 mRNA expression was also high in this goiter. Although NIS was normally located in the plasma membrane, pendrin was retained outside the Golgi (retained intracellularly). Thus, a high iodine uptake with low iodine efflux led to iodine retention within goitrous follicular cells. Increased D1 & D2 expression and activity were considered to be an adaptative response in order to maintain normal T3 supply, at the expense of T4: hence, the normal serum TSH obseerved in patient A. Moreover, urinary iodine excretion was normal (102 µg/L) indicating a normal nutritional iodine intake. The perchlorate discharge test confirmed the increased intracellular iodine pool with a positive discharge.

Distinct phenotypic clinical appearances are common in large families with Pendred syndrome with or without goiter, with isolated deafness or deafness combined with goiter. The c.279 delT is a founder mutation in Galicia.

In summary, this rather informative and complete study contributes much to our understanding of the patho-physiology of the Pendred syndrome.

 

Summary and commentary prepared by Geraldo Medeiros-Neto (Related to Chapters 2 & 16b of TDM)



Something new with Hashimoto’s encephalopathy

TOPIC: Hashimoto’s encephalopathy

Title: Anti-thyroperoxidase antibodies from patients with Hashimoto’s encephalopathy bind to cerebellar astrocytes.

Authors: Blanchin S, Coffin C, Viader F, Ruf J, Carayon P et al.

Reference: Journal of Neuroimmunology 192: 13-20, 2007

 SUMMARY

Background: Hashimoto’s encephalopathy is a rare and controversial neurological disorder, which is responsive to treatment with corticosteroids and associated with autoimmune thyroid disease. It is currently unclear whether this condition is a direct association between thyroid autoimmunity and a presumptive autoimmune process within the central nervous system, an indirect association or an artefact arising from the frequent occurrence of thyroid autoimmunity in health individuals.

Purpose: In order to test the first of these possibilities, the authors in the present study have attempted to assess the reactivity of thyroid peroxidase autoantibodies from patients with Hashimoto’s encephalopathy with central nervous system tissue.

Methods: The authors obtained sera in cerebrospinal fluid (CSF) from 10 patients with presumptive Hashimoto’s encephalopathy. Control sera were obtained from patients admitted with other neurological conditions, including meningitis, intractable headache, peripheral neuropathy and multiple sclerosis. Two of these coincidentally had thyroid peroxidase antibodies. Further sera were obtained from patients with straightforward Hashimoto’s thyroiditis without encephalopathy. These sera were characterised in detail, and in particular TPO antibodies were sought in the CSF. The main experiments concerned the binding of TPO antibodies to primate central nervous system tissues. Cerebrum, cerebellum and thyroid from rhesus monkeys, as well as cultured human astrocytes were used in standard immunofluorescence assays.

Results: The clinical characteristics of the patients were typical of those with this syndrome. Manifestations included impaired consciousness and a series of other neurological complications but no dementia or behavioural disturbance. EEG analysis on the other hand was abnormal in only 3 of the 10 patients. The patients were variably responsive to steroids. All patients had high levels of TPO antibodies (>50IU/ml) and all those analysed had TPO antibodies in CSF. TPO antibodies declined in most but not all patients after corticosteroid treatment. Indirect immunofluorescence assays were performed with all of the encephalopathy samples, 12 of the Hashimoto’s thyroiditis samples and 4 healthy donor samples. The 10 sera from the encephalopathy patients but none of the Hashimoto’s thyroiditis sera or the healthy sera bound to structures in primate cerebellar tissues. After depletion of the sera on purified human TPO by affinity chromatography, the serum did not bind significantly to either thyroid cells or cerebellar cells, excluding a role for thyroglobulin antibodies in the binding. Four monoclonal antibodies against TPO which were tested bound to primate cerebellar cells. No sera bound to cerebrum.  Further double immunofluorescence assays revealed that the sera appeared to bind to normal human primary astrocyte cultures (GFAP+).

Conclusions: These results indicate that TPO autoreactivity may have a specific role in Hashimoto’s encephalopathy.

 

COMMENTARY

            The association between Hashimoto’s thyroiditis and a specific form of encephalopathy has been hotly disputed, and this paper may have been overlooked by those who concentrate on the endocrine literature. Although there is circumstantial evidence to support a role for thyroid autoimmunity in encephalopathy, detailed reviews of patient cohorts have failed to establish that this is a cause and effect relationship. The present results provide some convincing evidence that TPO antibodies bind to central nervous system structures. The depletion experiments are particularly important in this regard, although of course it remains possible that another cross-reactive autoantibody species, directed against a second autoantigen besides TPO, could be responsible for the encephalopathy. In this regard it is noteworthy that the patients with Hashimoto’s thyroiditis, who had no clinical evidence of encephalopathy, had TPO antibodies which did not bind to cerebellar structures. This clearly suggests that there is an epitopic difference in TPO autoreactivity which determines susceptibility to encephalopathy, although of course the numbers sampled so far are small.

There are parallels to the hypothesis being put forward in this paper. Several neurological disorders such as stiff man syndrome and Lambert-Eaton myasthenic syndrome have been strongly associated with autoantibodies against glutamic acid decarboxylase and calcium channel autoantibodies. Simple experiments could easily be attempted using the autoantibodies from patients with encephalopathy to see if these can induce encephalopathic changes in animal models, and of course it would be fascinating to compare the epitopic recognition of TPO by antibodies from patients with encephalopathy to compare these with straightforward thyroiditis. In addition it would be important to assess the activity of these sera against human rather than primate cerebellum and to understand why encephalopathy can arise from reactivity to only the cerebellar part of the central nervous system. There is clearly much more to be done in this rare but intriguing and clinically important condition.

Summary and commentary prepared by Anthony Weetman (Related to Chapter 8 of TDM)

 

March 2008

Contribution by

v Roy Weiss (The University of Chicago, USA)

 

Non genomic actions of thyroid hormones 

TOPIC: In primary neuron cultures (hippocampus), glutamatergic receptors are affected by thyroid hormones

Title: Non genomic regulation of glutamatergic neurotransmission in hippocampus by thyroid hormones.

Authors: Losi G, Garzon G, & Puia G.

Reference: Neuroscience 151: 155-163, 2008

 SUMMARY

Background: Thyroid hormones (THs) are well known for their genomic effects but, recently, attention has focused also on their non-genomic actions as rapid modulators of membrane receptors. There is substantial evidence also for a complex interaction between the GABAergic system and THs. The hippocampus is a substrate for some forms of learning and memory and sensitive to THs.

 Purpose: Because of the link between THs and brain excitability, the authors investigated the direct non-genomic effect of these hormones on ionotropic glutamate receptors expressed in hippocampal neurons grown in culture and in acutely dissociated hippocampal slices.

 Methods: Primary culture of hippocampal neurons were prepared from newborn (postnatal day (PD) 1) Sprague–Dawley rats. Electrophysiological recordings were performed at room temperature, under voltage clamp in the whole-cell configuration of the patch-clamp technique.

 Results: The authors show that T4 and T3 rapidly decrease N-methyl-D-aspartate (NMDA)-evoked currents in rat hippocampal cultures with potency in the micromolar range. The effect is not mediated by glutamate or glycine binding sites, as an increase in agonist or glycine concentration does not alter TH potencies. Furthermore, THs’ effect on NMDA receptors is independent of voltage and subunit composition. The mechanism of THs’ antagonistic effect does not involve PKC phosphorylation of NMDA receptors since neither blocking nor stimulating PKC changed THs’ modulation. T3, but not T4, inhibits also kainate-evoked currents in hippocampal neurons in culture. In hippocampal pyramidal neurons in slice, T3, but not T4, significantly reduced the frequency of miniature excitatory  postsynaptic currents (mEPSCs) without affecting their  amplitude and decay.

 Conclusions: Thyroid hormones can rapidly affect ionotropic glutamatergic receptors in hippocampal neurons, an effect that could have an important role in their modulation of brain  function in physiological and pathological states.

 

COMMENTARY

      Thyroid hormone (TH) action in the brain is a complex summation of both genomic and non-genomic effects. Major developmental abnormalities of the nervous system are associated with deprivation or excess of TH in the developing brain. Behavioural and neurological effects are seen with perturbations of TH levels in the adult brain. It has been hypothesized by others that the main effects of TH are on the developing brain and likely mediated through genomic effect. However, the effect of TH on neurotransmission in the adult brain is likely to be non-genomic.

The data presented by Losi et al. suggest that TH may play a direct effect via non-genomic pathways on glutamatergic receptors in the hippocampus. Furthermore, these authors demonstrate that T3 (and not T4) is likely to have these non-genomic effects. This raises the question as to the role of exogenous T3 on behaviour and normal brain physiology.

On a somewhat more anecdotal standpoint, it might fuel the fire – slightly – that maybe psychiatrists aren’t so crazy when they give T3 to patients for improvement of psychological or behavioural disturbances.
            Summary and commentary prepared by Roy Weiss (Related to Chapter 3 of TDM)

 

February 2008

 Contributions by
 v Theo Visser (The University of Rotterdam; the Netherlands)

v Carole Spencer (The University of Southern California, USA)

v Samuel Refetoff (The University of Chicago, USA)

 

Role of D3 in the development of the hypothalamus-pituitary-thyroid axis

TOPIC: Thyroid hormone metabolism & action

 

Title: Type 3 deiodinase deficiency results in functional abnormalities at multiple levels of the thyroid axis.

Authors: Hernandez A, Martinez ME, Liao XH, Van Sande J, Refetoff S, Galton VA, & St Germain DL.

 Reference: Endocrinology 148: 5680-5687, 2007

 

SUMMARY

 Background: The type 3 deiodinase (D3) is a selenoenzyme that inactivates thyroid hormones and is highly expressed during development and in the adult central nervous system. The authors have recently observed that mice lacking D3 activity (D3KO mice) develop perinatal thyrotoxicosis, followed in adulthood by a pattern of hormonal levels that is suggestive of central hypothyroidism.

 Results: The authors describe the results of additional studies designed to investigate the regulation of the thyroid axis in this unique animal model. The results demonstrate that the thyroid and pituitary glands of D3KO mice do not respond appropriately to TSH and TRH stimulation, respectively. Furthermore, after induction of severe hypothyroidism by antithyroid drug treatment, the rise in serum TSH in D3KO mice is only 15% of that observed in wild-type mice. In addition, D3KO animals rendered severely hypothyroid fail to show the expected increase in prepro-TRH mRNA in the paraventricular nucleus of the hypothalamus. Finally, treatment with T3 results in a serum T3 level in D3KO mice that is much higher than that in wild-type mice. This is accompanied by significant weight loss and lethality in mutant animals.

 Conclusions: Absence of D3 activity resulted in impaired clearance of T3 and significant defects in the mechanisms regulating the thyroid axis at all levels, the hypothalamus, the pituitary, and the thyroid.

 

COMMENTARY

     Compared with type 1 (D1) and type 2 (D2) deiodinase knockout mice, the phenotype of D3 knockout mice (all generated by the group of Schneider, Hernandez, Galton & St Germain in Lebanon, NH, USA) is much more severe as it is associated with significant mortality and morbidity. Previous studies with these mice have established that D3 gene expression is subjected to imprinting, with predominant expression from the paternal allele and little contribution from the maternal allele.

      Further studies published in the J Clin Invest (116: 476-485, 2007) showed that in the first week of life, serum T3 rises faster in the D3-/- mice than in D3+/+ mice, but decrease subsequently to markedly lower levels in KO than in WT animals. Serum T4 remains very low in the KO animals until weaning and increase thereafter to remain lower than in WT mice. Despite the lower serum T4 and T3 levels in KO mice, serum TSH is not different or only moderately increased compared with WT animals, suggesting a state of central hypothyroidism. D3 is normally expressed in the hypothalamus where it plays a role in the regulation of local T3 levels and, thus, in the negative feedback control of TRH production and secretion. Continuous exposure of TRH neurons to elevated T3 levels during brain development apparently results in the maintenance of an abnormal setting point of the hypothalamus-pituitary thyroid (HPT) axis.

      The authors have further explored the HPT axis in D3 KO vs WT mice, and the results of this study indicate abnormalities at all levels of the HPT axis. Methimazole-induced hypothyroidism results in a much lower up-regulation of hypothalamic TRH expression in D3 KO than in WT mice. It is not excluded that this is explained by still higher hypothalamic T3 levels in the KO vs WT mice. TRH administration induces markedly smaller increases in serum TSH in D3 KO mice than in WT animals, despite the lower thyroid hormone levels in the former than in the latter. Most remarkable, however, are the findings that TSH treatment induces a marked increase in serum T4 in WT animals but no increase at all in KO mice. Therefore, inactivation of D3 is associated with an impaired function of the thyroid gland, indicating that these animals have both central and primary hypothyroidism.

      These studies appear to be related to the findings of central hypothyroidism in the offspring of women with active Graves’ disease during pregnancy (see. Kempers et al., JCEM 88: 5851-5857, 2003). Central hypothyroidism in this clinical context is correlated with exposure of the fetus to high thyroid hormone levels, rather than the presence of maternal autoantibodies. Also in these infants, the state of central hypothyroidism is associated with a loss of thyroid integrity and function (see Kempers et al., JCEM 92: 2984-2991, 2007).

      Further studies in D3 KO mice may help to unravel how a diminished activity of the HPT axis may lead to a functional impairment of the thyroid.

            Summary and commentary prepared by Theo Visser
 (Related to Chapter 3 - Sections 3c & 3d - of TDM)

 

 

Estimation vs measurement of serum free T4 in the pregnant state

 TOPIC: Free T4 measurements during pregnancy

 Title: Tandem mass spectrometry improves the accuracy of free T4 measurements during pregnancy.

 Authors: Kahric-Janicic N, Soldin SJ, Soldin OP, West T, Gu J, & Jonklaas J.

 Reference: Thyroid 17: 303-311, 2007

 

SUMMARY

 

Objective: The aim of the study was to compare a new tandem mass spectrometric (LC/MS/MS) method for measuring free T4 in pregnancy with free T4 measurements made by equilibrium dialysis (ED) and an immunoassay method.

 Subjects & Methods: Ninety eight healthy pregnant women were recruited for an evaluation of free T4 measured by different methods during each trimester of pregnancy, as compared with a group of 29 healthy controls.

 Results: As with other studies, total T4 reached a plateau at 150 percent of non-pregnant values. Only the LC/MS/MS method showed the expected hCG-stimulated rise in free T4 in the first trimester (122% of non pregnant) as compared with immunoassay (95% of non pregnant). Relative to non pregnant, low free T4 immunoassay values were more common in the third trimester (81% of non pregnant) as compared with the LC/MS/MS method (92% of non pregnant). The correlation between the new LC/MS/MS method and the gold standard ED method across pregnancy was good (r = 0.89), but the correlation between the LC/MS/MS method and immunoassay was poor (r = 0.48).

 Conclusion: The LC/MS/MS method is superior to immunoassay for generating trimester-specific free T4 reference values for pregnancy.

 

COMMENTARY

             It is becoming apparent that an adequate supply of maternal T4 is critical for optimal maternal health and fetal development. Unfortunately, non pregnant reference ranges for TSH and T4 do not apply to pregnancy, making assessments of thyroid status during pregnancy somewhat difficult. Specifically, thyroidal stimulation by hCG in early pregnancy results in higher T4 and lower TSH during the first trimester, whereas binding protein changes appear responsible for a lower free T4 as gestation progresses. However, the magnitude and prevalence of low free T4 values in the second and third trimesters appears method-related.

             Most laboratories use automated free T4 immunoassay tests in preference to labor-intensive equilibrium dialysis (ED) reference methods. It is increasingly apparent that immunoassays for free T4 determinations are sensitive to binding proteins and merely ‘estimate’ the free T4 status. In fact, a recent study has shown that free T4 immunoassay values correlate better with total rather free hormone changes (see Fritz et al. in Clin Chem 53:911, 2007).

            This study used LC/MS/MS methodology – the candidate free T4 international reference method. This methodology reported the expected physiological first trimester rise in free T4, whereas the immunoassay method did not. However, this method will not solve the problem of reliable free T4 measurements during pregnancy, because LC/MS/MS equipment is quite expensive (~$ 500,000) and the method necessitates first isolating free from protein-bound T4 by conventional techniques such as ED or ultra-filtration. Fortunately, as with non pregnant patients, TSH is the most sensitive indicator of thyroid status during pregnancy. When free T4 measurements are needed to manage pregnant hyperthyroid patients, a targeting of free T4 to the upper third of the non pregnant immunoassay reference range can compensate for the problem of low free T4 immunoassay values in pregnancy.

            Summary and commentary prepared by Carole Spencer (Related to Chapter 14 of TDM)

 

Local expression of TRß2 & cone photoreceptor differentiation

 TOPIC: Patterning of retinal cone photoreceptor development

 Title: NeuroD1 regulates expression of thyroid hormone receptor ß2 and cone opsins in the developing mouse retina.

 Authors: Liu H, Etter P, Hayes S, Jones I, Nelson B, Hartman B, Forrest D, & Reh T.

 Reference: The Journal of Neuroscience, 28: 749-756, 2008

 

SUMMARY

 Objective: The correct patterning of opsin expression in cone photoreceptors is critical for normal color vision. Thyroid hormone and one of its receptors (TRß2) are important regulators of opsin expression during cone photoreceptor development. Mice have two genes, encoding medium-wavelength (M) and short-wavelength (S) cone opsins. Targeted deletion of TRß2 leads to a uniform expression of S-opsin in all cone photoreceptors and the loss of M-opsin. The control of expression of TRß2 is therefore central to cone differentiation, yet there is little known about its regulation in the retina. Several transcription factors are known to be expressed in developing cones before the onset of TRß2, including those belonging to the basic helix-loop-helix (bHLH) class. Recent analysis of the TRß gene identified cone-specific, intronic control regions containing than E-box consensus site for bHLH.

 Purpose: To determine whether the bHLH transcription factor, NeuroD1, is necessary for sustained expression of TRß2 in immature cone photoreceptors.

 Methods: Mice deficient in NeuroD1 were used to assess the role of this transcription factor in the expression of TRß2 and the resulting effect on opsins during cone photoreceptor development.

 Results: Mice deficient in NeuroD1 develop an opsin phenotype virtually identical with that of TRß2-deficient mice: all cones express S-opsin, and none expresses M-opsin. The introduction of NeuroD1 into embryonic retinal explants from NeuroD1-/- mice restores TRß2 expression. NeuroD1 binds an E-box in the intron control region of the TRß2 gene that mediates cone-specific expression, suggesting that NeuroD1 is a critical contributory factor to the expression of TRß2 in cones.

 Conclusion: These results thus connect the proneural pathway with opsin selection to ensure correct cone patterning during retinal development.

 

COMMENTARY

             This work provides a mechanism for the local expression of the specific TRß2 isoform. However, although NeuroD1 is required for TRß2 expression in the retinal cones, it cannot do so in non permissive cell types in transfection assays. This suggests that the induction process requires the cooperation with other cell-specific factors. The full unravelling of the mechanism regulating cone specific TRß2 gene expression will require the identification of new genes involved in correct cone patterning during retinal development.

            Summary and commentary prepared by Samuel Refetoff (Related to Chapter 3d of TDM)

 

 January 2008

Contributions by
 v Luigi Bartalena (University of Insubria; Varese, Italy)

v                     Bernard Rousset (University of Lyon, France)

v                 Gilbert Vassart (University of Brussels, Belgium)

 Novel somatostatin analogs for treatment of Graves’ orbitopathy

 TOPIC: Graves’ orbitopathy

 Title: Modulation of expression of somatostatin receptor subtypes in Graves’ ophthalmopathy orbits: relevance to novel analogues.

 Authors: Cozma I, Zhang L, Uddin J, Lane C, Rees A, & Ludgate M

 Reference: Am J Physiol Endocrinol Metab 293: E1630-E1635, 2007 (ahead of print)  

 SUMMARY

 Background: Treatment of Graves’orbitopathy (GO) with two somatostatin analogs, octreotide and lanreotide, has been attempted with disappointing results. Octreotide (OCT) and lanreotide mostly bind to somatostatin receptor (SSTR) subtypes 2 & 5. Novel somatostatin analogs with broader affinity for SSTRs might be more effective.

 Purpose: The aim of the study was to investigate SSTR expression ex vivo and to evaluate the effect on preadipocyte proliferation of a novel synthetic somatostatin analog, SOM230, which has a high affinity for a wider range of SSTR subtypes (SSTR 1, 2, 3 & 5).

 Methods: Orbital/adipose tissue was obtained for quantitative PCR studies from 23 GO patients (with inactive disease) and 8 individuals operated on orbital decompression for non-thyroid related ocular diseases. In cultured orbital preadipocytes obtained from 6 additional GO patients (with inactive disease), adipocyte differentiation was obtained by exposure to a peroxisome proliferator-activated receptor- (PPAR) agonist. In this system, the possible effect of TSH receptor (TSHR) activation was evaluated by introducing activating mutant or wild type TSHR into orbital preadipocytes using retroviral vectors. The effect of varying concentrations of OCT and SOM230 on preadipocyte differentiation and proliferation was assessed.

 Results: The expression of SSTR 1 was significantly higher in GO orbital tissue than in control orbital tissue; the expression of SSTR 2 tended to be higher in GO patients, although this did not reach significance; SSTR 3, SSTR 4 and SSTR 5 were expressed at very low levels or were undetectable. TSHR activation did not modulate SSTR expression. In the in vitro model of adipogenesis, SSTR 1 and SSTR 2 were upregulated during adipocyte differentiation; in this system SOM230 produced a greater inhibition of orbital preadipocyte proliferation than octreotide.

 Conclusions: SSTR 1 and, to a lesser extent, SSTR 2 were expressed at a higher level in orbital tissue from GO patients than from controls. Ex vivo analysis of orbital tissues revealed upregulation of SSTR 1 and SSTR 2 in GO patients during adipocyte differentiation. SOM230 reduced preadipocyte proliferation to a greater extent than octreotide.

 

 

COMMENTARY

             Treatment of GO with traditional therapies (intravenous glucocorticoids, orbital radiotherapy) is only partially effective, and patients are eventually unsatisfied with treatment outcome in about one third of the cases. Demonstration of SSTR expression in orbital fibroblasts and lymphocytes derived from GO patients prompted the use of somatostatin analogs (octreotide or lanreotide, mainly interacting with SSTR2 and, to a lesser extent, SSTR 5). After initial positive reports in small and uncontrolled studies, four randomized controlled trials (three using octreotide LAR and one using lanreotide) showed that benefit from such treatment is marginal (if any) in GO. However, it was suggested that novel somatostatin analogs with a wider affinity for SSTR should be investigated and might prove beneficial for the orbitopathy.

             The present study showed a higher level of expression of SSTR 1 and, to a lower extent, SSTR 2 in orbital tissue from GO patients than from controls. This expression appeared to be unrelated to TSHR activation, thus limiting the putative role of TSHR antibodies. SSTR 1 and SSTR 2 were up-regulated during the process of differentiation of predipocyte into mature adipocytes, thus explaining their increased expression measured in GO samples ex vivo. The most interesting finding was the observation that SOM230 exerted a greater inihibitory effect on preadipocyte proliferation than octreotide.

                        These ex vivo and in vitro results suggest that SOM230 might be beneficial (or more beneficial than octreotide or lanreotide) for GO also in vivo. It is likely that the effects might be more favorable if treatment is administered during the early phases of the disease, when the proliferation and/or differentiation of orbital tissue is still in an active ongoing process. However, randomized controlled trials enrolling a sufficiently large number of patients are warranted to establish whether SOM230 may really represent a useful tool in the management of this invalidating disease.
            Summary and commentary prepared by Luigi Bartalena (related to Chapter 12 of TDM)
Full paper obtainable at http://ajpendo.physiology.org/cgi/reprint/full/293/6/E1630           

  

First identification of a mutation in the DUOXA2 gene, in a Chinese patient with CH due to a partial iodine organification defect

 TOPIC: Dyshormonogenesis due to mutations in DUOXA2 gene  

Title: Biallelic inactivation of the dual oxidase maturation factor 2 (DUOXA2) gene as a novel cause of congenital hypothyroidism.

 Authors: Zamproni I, Grasberger H, Cortinovis F, Vigone MC, Chiumello G, Mora S, Onigata K, Fugazzola L, Refetoff S, Persani L, & Weber G.

 Reference: Journal of Clinical Endocrinology & Metabolism (ahead of print) November 27, 2007

 

 SUMMARY

 Background: Dual oxidase 2 (DUOX2) is the catalytic core of the H2O2 generating system which is crucial for iodination of thyroglobulin and thyroid hormone synthesis. DUOX2 deficiency is known to produce congenital hypothyroidism (CH) in humans and mice. It has recently been reported that the expression of active DUOX2 at the apical plasma membrane of thyrocytes requires a protein named DUOXA2, a type of molecular ‘chaperon’ encoded  by a gene located in the DUOX1/DUOX2 intergenic region.

 Purpose: The authors investigated the possibility of thyroid dyshormonogenesis linked to mutations in the DUOXA2 gene.

 Patients: The search for mutation(s) of the DUOXA2 gene was conducted on a series of eleven patients with partial iodine organification defect (PIOD) found to be negative for the other known genetic alterations causing PIOD. These patients had normal or high serum Tg level, no hearing impairment (no pendrin or SLC26A4 mutation), no detectable TPO or DUOX2 mutations.

 Methods: Expression and functional analyses of wild type and mutated form(s) of DUOXA2 performed in HeLa cells.

 Results: Among 11 patients with idiopathic PIOD, 1 chinese girl born to non-consanguineous parents exhibited a homozygous C à G transversion in codon 246, resulting in a nonsense mutation (Y246X). The mutation is expected to lead to the synthesis of a truncated DUOXA2 protein, lacking the last transmembrane domain -helix 5- and the C terminal cytoplasmic domain. Parents and sisters of the proband were heterozygous carriers (C/G) of the mutation and had normal thyroid function parameters. The mutated DUOXA2 gene expressed in HeLa cells yielded a ~30 kDa  glycosylated polypeptide chain instead of the 35-37 kDa native glycoprotein. The 246X mutant protein was N-glycosylated to the same degree as wild type DUOXA2, but its expression was reduced compared to that of normal DUOXA2. Wild-type DUOXA2 showed a dispersed distribution in endoplasmic reticulum, whereas the 246X protein appeared concentrated in foci in the vicinity of the nuclear envelope. Function of the mutated DUOXA2 was studied by DUOX2/DUOXA2 reconstitution experiments in HeLa cells and measurement of H2O2 production. Co-expression of DUOX2 and wild type DUOXA2 in HeLa cells led to expression of a DUOX protein producing H2O2. In contrast, co-expression of DUOX2 and 246X DUOXA2 did not reconstitute DUOX2 activity. Genotyping of unrelated control individuals revealed one heterozygous carrier of the Y246X mutation in 92 Chinese individuals; the mutation was not detected in controls of subjects of Caucasian (178 alleles) or Japanese (82 alleles) ethnicity.

 Conclusions: This article reports the first mutation in DUOXA2 in a patient with CH. Pedigree analysis demonstrate recessive inheritance. Biallelic DUOXA2 mutations are thus a novel genetic event in permanent CH. 

 

 

COMMENTARY

             Molecular studies of DUOX proteins have been hampered for a number of years by the failure to reconstitute active DUOX enzymes in heterologous systems. For unknown reasons, recombinant DUOX was completely retained inside the endoplasmic reticulum (ER) in an immature form. In 2006, Sam Refetoff and coll. provided the answer, by discovering factors (DUOXA1 & DUOXA2) that are specifically involved in the maturation of DUOX1/DUOX2 proteins. The authors found that it was possible to reconstitute a DUOX-based H2O2 generating system in transfected cells, if cells co-expressed a DUOX gene and the respective DUOXA1 or DUOXA2 genes. DUOXA2 allows the exit of folded DUOX2 from ER to the Golgi apparatus. DUOXA2 may be part of a quality control system specific for DUOX2. In the present article, the native form of the DUOX2 maturation factor DUOXA2 was shown to be necessary and sufficient for expression of functional DUOX2 in a mammalian cell line.

             Screening 11 patients with PIOD, the authors were able to find one demonstrative case: a bi-allelic mutation of the DUOXA2 gene in a Chinese patient leading to a complete loss of function of the protein in this patient. The truncated protein appears to be subjected to degradation at the site of synthesis. This study provides evidence for a critical role of DUOXA2 in thyroid hormonogenesis. Search for DUOXA2 mutation in control patients of different ethnic origins led the authors to suggest that homozygosity for the Y246X mutation could be a rather frequent cause of CH in Chinese subjects.

 Summary and commentary prepared by Bernard Rousset (related to Chapter 2 of TDM)
Full paper obtainable at: http://jcem.endojournals.org/cgi/rapidpdf/jc.2007-2020v1

  

Surrounding embryonic tissues (outside the thyroid anlage) play an instructive role in early development of the thyroid gland

 TOPIC: Thyroid development  

Title: Early developmental specification of the thyroid gland depends on han-expressing surrounding tissue and on FGF signals.

 Authors: : Wendl T, Adzic D, Schoenebeck JJ, Scholpp S, Brand M, Yelon D, & Rohr KB.

 Reference: Development 134: 2871-2879, 2007 (doi:10.1242/dev.02872)

 

 SUMMARY

 Background: In all vertebrates, the thyroid gland develops from the ventral floor of the anterior pharyngeal endoderm. Unravelling the molecular mechanisms of thyroid development is expected to help in understanding the mechanisms leading to thyroid dysgenesis, the major cause of  congenital hypothyroidism. Development of the thyroid is comparable between fish and mammals on the molecular level. The thyroid-specific transcriptional programme, including the transcription factors Nkx2.1 (also known as Nk2.1a and Titf1a/TITF1), Pax8 and Hhex, is conserved with respect to expression patterns and function between zebrafish and mouse.

 Purpose: In this study, the authors used zebrafish as a model to investigate the initiation of thyroid development.

 Material & Methods: Zebrafish harbouring mutations in the hands off (han, hand2) and fgf8 genes were used to study the role of these genes in normal thyroid development.

 Results: Zebrafish han mutants displayed severely reduced or absent expression of thyroid-specific developmental genes (titf1, pax8) concomitant with the complete loss of a functional gland. The han locus encodes the bHLH transcription factor Hand2 known to be expressed in cardiac mesoderm, the fin buds and the pharyngeal arches. The hans6 and hanc99 alleles are null and hypomorphic alleles respectively, with hans6 displaying, accordingly, a stronger phenotype. In grafting experiments, the authors showed that han expression in the anterior plate mesoderm or cardiac mesoderm was crucial for thyroid specification. Thus, han gene was required in the tissue surrounding the thyroid for normal gland development. They further showed that FGF signalling was required for thyroid development in zebrafish, and that FGF-coated beads were able to restore thyroid development in hans6 mutants. 

Conclusions: This study provides the first evidence towards understanding the role of surrounding tissue during thyroid specification.

 


 COMMENTARY

             Studies of transcription factors implicated in the regulation of thyroid-specific genes expressed in the adult gland have led to the identification of a limited number of genes involved in normal thyroid development: Nkx2.1 (also known as Nk2.1a and Titf1a/TITF1), Pax8, FoxE1 and Hhex. Despite the identification of mutations in rare cases with thyroid dysgenesis, the vast majority of thyroid developmental defects in human do not show mutations in these genes. In addition, thyroid dysgenesis does not lean itself to gene mapping by classical linkage studies, since most cases are sporadic. Indeed, the disease does not follow mendelian inheritance, nor does it obey to multigenic inheritance since monozygotic twins, as a rule, are discordant.

 Confronted with these problems, groups aiming at understanding the mechanisms leading to thyroid dysgenesis are left with the reasonable option of studying the basic phenomena implicated in early thyroid specification and development. A logical approach has been to attempt identification of novel genes expressed more or less specifically in the thyroid bud. Whereas this endeavour is underway in several laboratories, the study by Wendl et al. demonstrates that genes expressed outside the thyroid anlage, within the surrounding mesenchyme, play an instructive role on the specification of the pharyngeal endoderm. By exploiting loss of function mutations together with grafting, fate mapping and experiments involving soaked beads and FGF signalling inhibitors, they demonstrate unequivocally that the transcription factor gene han, acting upstream of fgf8, is required for induction of cell autonomous development thyroid genes like nxk2.1. With the demonstration of the role of han expression in the anterior lateral plate mesoderm (aLPM), from which the heart later develops, the results of Wendl et al provide a convincing rationale for the close relation between cardiac and thyroid development, and also the observed association between cardiac and thyroid developmental defects.

 As a final comment, it is worth noting that such kind of studies would be extremely difficult, if not impossible, to perform in the mouse. It advertises zebrafish as a fruitful model for exploration of genes/mechanisms involved in thyroid dysgenesi
 Summary and commentary prepared by Gilbert Vassart (related to Chapter 1 of TDM)