Epigenetic developmental programming and intergenerational effects of thyroid hormones.

Mounting evidence is showing that altered signaling through the nuclear hormone receptor superfamily can cause abnormal, long-term epigenetic changes which translate into pathological modifications and susceptibility to disease. These effects seem to be more prominent if the exposure occurs early in life, when transcriptomic profiles are rapidly changing. At this time, the coordination of the complex coordinated processes of cell proliferation and differentiation that characterize mammalian development. Such exposures may also alter the epigenetic information of the germ line, potentially leading to developmental changes and abnormal outcomes in subsequent generations. Thyroid hormone (TH) signaling is mediated by specific nuclear receptors, which have the ability to markedly change chromatin structure and gene transcription, and can also regulate other determinants of epigenetic marks. TH exhibits pleiotropic effects in mammals, and during development, its action is regulated in a highly dynamic manner to suit the rapidly evolving needs of multiple tissues. Their molecular mechanisms of action, timely developmental regulation and broad biological effects place THs in a central position to play a role in the developmental epigenetic programming of adult pathophysiology and, through effects on the germ line, in inter- and trans-generational epigenetic phenomena. These areas of epigenetic research are in their infancy, and studies regarding THs are limited. In the context of their characteristics as epigenetic modifiers and their finely tuned developmental action, here we review some of the observations underscoring the role that altered TH action may play in the developmental programming of adult traits and in the phenotypes of subsequent generations via germ line transmission of altered epigenetic information. Considering the relatively high prevalence of thyroid disease and the ability of some environmental chemicals to disrupt TH action, the epigenetic effects of abnormal levels of TH action may be important contributors to the non-genetic etiology of human disease.

Tafamidis polyneuropathy amelioration requires modest increases in transthyretin stability even though increases in plasma native TTR and decreases in non-native TTR do not predict response.

BACKGROUND: TTR aggregation causes hereditary transthyretin (TTR) polyneuropathy (ATTRv-PN) in individuals with destabilised TTR variants. ATTRv-PN can be treated with ligands that bind TTR and prevent aggregation. One such ligand, tafamidis, is widely approved to treat ATTRv-PN. We explore how TTR stabilisation markers relate to clinical efficacy in 210 ATTRv-PN patients taking tafamidis. METHODS: TTR concentration in patient plasma was measured before and after tafamidis treatment using assays for native or combined native + non-native TTR. TTR tetramer dissociation kinetics, which are slowed by tafamidis binding, were also measured. RESULTS: Native TTR levels increased by 56.8% while combined native + non-native TTR levels increased by 3.1% after 24 months of tafamidis treatment, implying that non-native TTR decreased. Accordingly, the fraction of native TTR increased from 0.54 to 0.71 with tafamidis administration. Changes in native and non-native TTR levels were uncorrelated with clinical response to tafamidis. TTR tetramer dissociation generally slowed to an extent consistent with ∼40% of TTR being tafamidis-bound. Male non-responders had a lower extent of binding. CONCLUSIONS: Native and non-native TTR concentration changes cannot be used as surrogate measures for therapeutic efficacy. Also, successful tafamidis therapy requires only moderate TTR stabilisation. Male patients may benefit from higher tafamidis doses.

Early onset obesity due to a mutation in the human leptin receptor gene.

Summary: Leptin is secreted by adipocytes in response to fat storage and binds to its receptor (LEPR), which is ubiquitously expressed throughout the body. Leptin regulates energy expenditure and is anorexigenic. In this study, we describe the clinical and hormonal findings of three siblings with a personal history of rapid weight gain during the first months of life. They had delayed puberty, high levels of FSH (15.6 ± 3.7 mUI/mL; reference: 1.5-12.4) and LH (12.3 ± 2.2 mUI/mL; reference: 1.7-8.6), normal oestradiol and total testosterone and successful fertility. None of the patients had dyslipidemia, diabetes or thyroid disease. Next-generation sequencing identified a pathogenic homozygous variant c.2357T>C, p.(Leu786Pro) in LEPR. Their parents and children were heterozygous for this mutation. We compared clinical and biochemical findings of homozygous carriers with first-degree heterozygous family members and ten randomly selected patients with adult-onset morbid obesity. Homozygous carriers of the mutation had significantly higher BMI (32.2 ± 1.7 kg/m2 vs 44.5 ± 7.1 kg/m2, P = 0.023) and increased serum levels of leptin (26.3 ± 9.3 ng/mL vs 80 ± 36.4 ng/mL, P = 0.028) than their heterozygous relatives. Compared with the ten patients with adult-onset morbid obesity, serum levels of leptin were not significantly higher in homozygous carriers (53.8 ± 24.1 ng/mL vs 80 ± 36.4 ng/mL, P = 0.149), and thus serum levels of leptin were not a useful discriminative marker of LEPR mutations. We described a rare three-generation family with monogenic obesity due to a mutation in LEPR. Patients with early onset obesity should be considered for genetic screening, as the identification of mutations may allow personalized treatment options (e.g. MC4R-agonists) and targeted successful weight loss. Learning points: The early diagnosis of monogenic forms of obesity can be of great interest since new treatments for these conditions are becoming available. Since BMI and leptin levels in patients with leptin receptor mutations are not significantly different from those found in randomly selected morbid obese patients, a careful medical history is mandatory to suspect this condition. Loss of leptin receptor function has been associated with infertility. However, our patients were able to conceive, emphasizing the need for genetic counselling in affected patients with this condition.

A case report of multiple endocrine neoplasia type 1 and autoimmune disease: Coincidence or correlation?

RATIONALE: Multiple Endocrine Neoplasia type 1 (MEN1) is a familial syndrome that results from the disruption of a tumor suppressor protein called MENIN. Its management is challenging, as MEN1 affects different endocrine tissues and predisposes to both benign and malignant tumors. MENIN-deficient cells have recently been recognized to play a role in triggering autoimmunity. Herein, we present a case of MEN1 with multiple endocrine and autoimmune disorders. PATIENT CONCERNS: A 50 years old female with a 25 years history of complicated nephrolithiasis presented with primary hyperparathyroidism. DIAGNOSES: Over several decades, she was diagnosed with recurrent primary hyperparathyroidism, autoimmune thyroiditis, multinodular goiter, pernicious anemia, metastatic gastric type 1 neuroendocrine tumor, macroprolactinemia, gonadotropin deficiency, mucosa-associated lymphoid tissue lymphoma of the thyroid gland, positive anti-calcium sensor receptor antibodies, and BRCA 1/2-negative invasive breast cancer. The autoimmune regulator gene was sequenced, but no pathogenic variants were found. Next-generation sequencing revealed both a pathogenic MEN1 mutation and a benign CDC73 gene variant. Familial genetic screening revealed a large kindred with multiple carriers of one or both genetic variants (MEN1 = 19; CDC73 = 7). INTERVENTIONS: The patient underwent surgical excision of three parathyroid glands, total thyroidectomy and breast tumorectomy plus tamoxifen, and monthly injections of octreotide. The patient and family members with the MEN1 mutation are under a life-long surveillance program for MEN1 prototypic tumors. OUTCOMES: The patient was stable and alive during a 24-years follow-up period. LESSONS: With the present case, the authors highlight a new interplay between MENIN and the immune system, which may have implications for future targeted life-long surveillance and treatment of MEN1 patients.

Successful long-term use of pioglitazone in Berardinelli-Seip lipodystrophy-associated diabetes.

SUMMARY: Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare autosomal recessive disease, characterized by the absence of subcutaneous adipose tissue, leptin deficiency and severe metabolic complications, such as insulin resistance, diabetes mellitus, and dyslipidemia. The most common mutation occurs in BCSL2 which encodes seipin, a protein involved in adipogenesis. We report a patient with BSCL who was diagnosed with diabetes at 11 years old. He was started on metformin 1000 mg twice daily, which lowered glycated hemoglobin (HbA1c) to less than 7%. Four months later, HbA1c raised above 7.5%, indicating secondary failure to metformin. Therefore, we added the peroxisome proliferator-activated receptor-gamma (PPARG) agonist, pioglitazone. Since then and for the last 5 years his HbA1c has been within the normal range. These findings indicate that pioglitazone should be considered as a valid alternative in the treatment of diabetes in BSCL patients. To the best of our knowledge, this is the first specific report of successful long-term treatment with pioglitazone in a patient with BSCL. LEARNING POINTS: Berardinelli-Seip congenital lipodystrophy (BSCL) is a recessive genetic disorder associated with severe insulin resistance and early onset diabetes, usually around puberty. Failure of oral antidiabetic medication occurs within the first years of treatment in BSCL patients. When failure to achieve metabolic control with metformin occurs, pioglitazone may be a safe option, lowering insulin resistance and improving both the metabolic control and lipodystrophic phenotype. Herein we show that pioglitazone can be a safe and efficient alternative in the long-term treatment of BSCL patients with diabetes.

Increased Hepatic Fat Content in Patients with Resistance to Thyroid Hormone Beta.

Background: Thyroid hormone (TH) has important functions in controlling hepatic lipid metabolism. Individuals with resistance to thyroid hormone beta (RTHß) who harbor mutations in the THRB gene experience loss-of-function of thyroid hormone receptor beta (TRß), which is the predominant TR isoform expressed in the liver. We hypothesized that individuals with RTHß may have increased hepatic steatosis. Methods: Controlled attenuation parameter (CAP) was assessed in individuals harboring the R243Q mutation of the THRB gene (n = 21) and in their wild-type (WT) first-degree relatives (n = 22) using the ultrasound-based transient elastography (TE) device (FibroScan). All participants belonged to the same family, lived on the same small island, and were therefore exposed to similar environmental conditions. CAP measurements and blood samples were obtained after an overnight fast. The observers were blinded to the status of the patients. Results: The hepatic fat content was increased in RTHß individuals compared with their WT relatives (CAP values of 263 ± 21 and 218.7 ± 43 dB/m, respectively, p = 0.007). The CAP values correlated with age and body mass index (BMI) (age: r = 0.55, p = 0.011; BMI: r = 0.51, p = 0.022) in the WT first-degree relatives but not in RTHß individuals, suggesting that the defect in TRß signaling was predominant over the effects of age and obesity. Circulating free fatty acid levels were significantly higher in RTHß individuals (0.29 ± 0.033 vs. 0.17 ± 0.025 mmol/L, p = 0.02). There was no evidence of insulin resistance evaluated by the homeostatic model assessment of insulin resistance in both groups studied. Conclusions: Our findings provide evidence that impairments in intrahepatic TRß signaling due to mutations of the THRB gene can lead to hepatic steatosis, which emphasizes the influence of TH in the liver metabolism of lipids and provides a rationale for the development TRß-selective thyromimetics. Consequently, new molecules with a very high TRß affinity and hepatic selectivity have been developed for the treatment of lipid-associated hepatic disorders, particularly nonalcoholic fatty liver disease.

Physiologic Significance of Epigenetic Regulation of Thyroid Hormone Target Gene Expression.

BACKGROUND: In previous publications, we have reported our findings demonstrating that exposure to high maternal levels of thyroid hormones (TH) has life-long effects on the wild-type (WT, without THRB mutation) progeny of mothers with resistance to thyroid hormone beta (RTHß). The mechanism of this epigenetic effect remains unclear. OBJECTIVES: We reviewed the mechanisms involved in the epigenetic regulation of TH target genes and understand how they may explain the reduced sensitivity to TH in the WT progeny of RTHß mothers. METHODS: The availability of a large, formerly genotyped Azorean population with many individuals harboring the THRB mutation, R243Q, provided us a model to study the influence of fetal exposure to high maternal TH levels. RESULTS: The thyroid-stimulating hormone (TSH) response in WT adults was less suppressible following the administration of L-triiodothyronine (L-T3). This finding suggests reduced sensitivity to TH that is induced by an epigenetic mechanism resulting from exposure to high maternal levels of TH during pregnancy. The persistence of this effect across 3 generations of WT subjects favors transgenerational epigenetic inheritance. Based on preliminary studies in mice, we identified the naturally imprinted gene encoding deiodinase type 3, i.e., DIO3, as a possible mediator of this epigenetic effect through increased inactivation of TH. CONCLUSION: Increased D3 expression and consequently increased T3 degradation appear to be responsible for the reduced sensitivity of the anterior pituitary to administered L-T3. The imprinted DIO3 gene may be a candidate gene that mediates the epigenetic effect induced by exposure to high maternal levels of TH. However, we cannot exclude the role of other TH-responsive genes.

Predictive model of response to tafamidis in hereditary ATTR polyneuropathy.

BACKGROUNDThe hereditary transthyretin (TTR) amyloidoses are a group of diseases for which several disease-modifying treatments are now available. Long-term effectiveness of these therapies is not yet fully known. Moreover, the existence of alternative therapies has resulted in an urgent need to identify patient characteristics that predict response to each therapy.METHODSWe carried out a retrospective cohort study of 210 patients with hereditary TTR amyloidosis treated with the kinetic stabilizer tafamidis (20 mg qd). These patients were followed for a period of 18-66 months, after which they were classified by an expert as responders, partial responders, or nonresponders. Correlations between baseline demographic and clinical characteristics, as well as plasma biomarkers and response to therapy, were investigated.RESULTS34% of patients exhibited an almost complete arrest of disease progression (classified by an expert as responders); 36% had a partial to complete arrest in progression of some but not all disease components (partial responders); whereas the remaining 30% continued progressing despite therapy (nonresponders). We determined that disease severity, sex, and native TTR concentration at the outset of treatment were the most relevant predictors of response to tafamidis. Plasma tafamidis concentration after 12 months of therapy was also a predictor of response for male patients. Using these variables, we built a model to predict responsiveness to tafamidis.CONCLUSIONOur study indicates long-term effectiveness for tafamidis, a kinetic stabilizer approved for the treatment of hereditary TTR amyloidosis. Moreover, we created a predictive model that can be potentially used in the clinical setting to inform patients and clinicians in their therapeutic decisions.

Reduced Sensitivity to Thyroid Hormone as a Transgenerational Epigenetic Marker Transmitted Along the Human Male Line.

Background: Evidence for transgenerational epigenetic inheritance in humans is still controversial, given the requirement to demonstrate persistence of the phenotype across three generations. A previous study showed that exposure of human and mouse embryos to high maternal thyroid hormone (TH) concentrations not only affects the newborns but also subsequently reduces thyrotroph sensitivity to TH during adult life. The current investigation set out to determine if this epigenetic effect is transmitted by humans not exposed in utero to high TH levels to their offspring. Methods: The study took advantage of the high frequency of intrauterine exposure to high TH in the Azorean wild-type population born to healthy mothers with high TH levels because of a heterozygous TH receptor beta gene mutation. Wild-type individuals from F2 (second) and F3 (third) generations were studied, whose parents and grandparents, respectively, were not exposed to high maternal TH levels. Twenty-six individuals belonging to 17 nuclear families were tested for their sensitivity to TH using their thyrotropin (TSH) response to thyrotropin-releasing hormone (TRH) after administration of liothyronine (LT3). Results: Preservation of reduced sensitivity to TH (RSTH) was found in descendants of males but not of females with likewise RSTH. In F2, offspring of fathers but not of mothers exposed to high TH levels had RSTH (TRH-stimulated TSH of 6.39 ± 0.63 vs 1.58 ± 0.41 mIU/L [p < 0.001], respectively, after treatment with LT3). In F3, whose parents nor themselves were exposed to TH excess during their fetal life, descendants of fathers and not mothers had RSTH (TRH-stimulated TSH of 4.60 ± 0.61 vs 1.37 ± 0.23 mIU/L [p < 0.01], respectively, after pretreatment with LT3). Conclusions: Since intrauterine total body and gonadal exposure to elevated TH can potentially affect only the F1 and F2, respectively, the results obtained from F3 confirm a true inheritance of an epigenetic effect, scarcely observed in humans. While the exact mechanism underlying the inheritance of this epigenetic effect remains unknown, it correlates with type 3 deiodinase overexpression demonstrated in pituitary glands of mice born to dams with high TH. This enzyme inactivates TH, and is encoded by an imprinted gene with specific parent of origin expression.

Transient PAX8 Expression in Islets During Pregnancy Correlates With ß-Cell Survival, Revealing a Novel Candidate Gene in Gestational Diabetes Mellitus.

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8 A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 affects islet viability and that PAX8 could be considered as a candidate gene for the study of GDM.

3,5-T2-A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action.

Over the last decades, thyroid hormone metabolites (THMs) received marked attention as it has been demonstrated that they are bioactive compounds. Their concentrations were determined by immunoassay or mass-spectrometry methods. Among those metabolites, 3,5-diiodothyronine (3,5-T2), occurs at low nanomolar concentrations in human serum, but might reach tissue concentrations similar to those of T4 and T3, at least based on data from rodent models. However, the immunoassay-based measurements in human sera revealed remarkable variations depending on antibodies used in the assays and thus need to be interpreted with caution. In clinical experimental approaches in euthyroid volunteers and hypothyroid patients using the immunoassay as the analytical tool no evidence of formation of 3,5-T2 from its putative precursors T4 or T3 was found, nor was any support found for the assumption that 3,5-T2 might represent a direct precursor for serum 3-T1-AM generated by combined deiodination and decarboxylation from 3,5-T2, as previously documented for mouse intestinal mucosa. We hypothesized that lowered endogenous production of 3,5-T2 in patients requiring T4 replacement therapy after thyroidectomy or for treatment of autoimmune thyroid disease, compared to production of 3,5-T2 in individuals with intact thyroid glands might contribute to the discontent seen in a subset of patients with this therapeutic regimen. So far, our observations do not support this assumption. However, the unexpected association between high serum 3,5-T2 and elevated urinary concentrations of metabolites related to coffee consumption requires further studies for an explanation. Elevated 3,5-T2 serum concentrations were found in several situations including impaired renal function, chronic dialysis, sepsis, non-survival in the ICU as well as post-operative atrial fibrillation (POAF) in studies using a monoclonal antibody-based chemoluminescence immunoassay. Pilot analysis of human sera using LC-linear-ion-trap-mass-spectrometry yielded 3,5-T2 concentrations below the limit of quantification in the majority of cases, thus the divergent results of both methods need to be reconciliated by further studies. Although positive anti-steatotic effects have been observed in rodent models, use of 3,5-T2 as a muscle anabolic, slimming or fitness drug, easily obtained without medical prescription, must be advised against, considering its potency in suppressing the HPT axis and causing adverse cardiac side effects. 3,5-T2 escapes regular detection by commercially available clinical routine assays used for thyroid function tests, which may be seriously disrupted in individuals self-administering 3,5-T2 obtained over-the counter or from other sources.

Prenatal Diagnosis of Resistance to Thyroid Hormone and Its Clinical Implications.

Context: Resistance to thyroid hormone-ß (RTH-ß) is an autosomal dominant disorder characterized by reduced sensitivity of target tissues to thyroid hormones (THs). Individuals with RTH-ß have high TH levels usually due to mutations in the TH receptor-ß (THRB) gene. The management of RTH-ß during pregnancy is challenging, as wild-type (WT) fetuses born to RTH-ß mothers have low birth weight and suppressed postnatal thyroid-stimulating hormone (TSH), due to intrauterine exposure to excess TH. Objective: To determine birth weight and postnatal TSH of WT fetuses carried by mothers with RTH-ß whose fT4 levels were maintained below 20% of the upper limit of normal (ULN). Design: Retrospective chart review. Setting: Academic institution in collaboration with off-site hospitals and private practices. Patients: Thirteen women harboring THRB gene mutations were evaluated during 18 pregnancies. Intervention: Prenatal genetic diagnosis by amniocentesis. Women carrying WT fetuses were given the option of treatment with antithyroid medication by their treating physicians with the aim to avoid serum fT4 levels above 20% of the ULN. Results: No significant difference was found in birth weight corrected for gestational age and in serum TSH levels at birth between WT and RTH-ß infants born to RTH-ß mothers. Conclusions: Prenatal diagnosis may play an important role in the management of RTH-ß during pregnancy. Aiming for maternal fT4 levels not above 50% of the ULN in RTH-ß mothers carrying WT fetuses seems to be a prudent approach that prevents the otherwise expected low birth weight and postnatal TSH suppression.

Fetal Exposure to High Maternal Thyroid Hormone Levels Causes Central Resistance to Thyroid Hormone in Adult Humans and Mice.

Context: Fetuses exposed to the high thyroid hormone (TH) levels of mothers with resistance to thyroid hormone beta (RTH-ß), due to mutations in the THRB gene, have low birth weight and suppressed TSH. Objective: Determine if such exposure to high TH levels in embryonic life has a long-term effect into adulthood. Design: Observations in humans with a parallel design on animals to obtain a preliminary information regarding mechanism. Setting: University research centers. Patients or other participants: Humans and mice with no RTH-ß exposed during intrauterine life to high TH levels from mothers who were euthyroid due to RTH-ß. Controls were humans and mice of the same genotype but born to fathers with RTH-ß and mothers without RTH-ß and thus, with normal serum TH levels. Interventions: TSH responses to stimulation with thyrotropin-releasing hormone (TRH) during adult life in humans and male mice before and after treatment with triiodothyronine (T3). We also measured gene expression in anterior pituitaries, hypothalami, and cerebral cortices of mice. Results: Adult humans and mice without RTH-ß, exposed to high maternal TH in utero, showed persistent central resistance to TH, as evidenced by reduced responses of serum TSH to TRH when treated with T3. In mice, anterior pituitary TSH-ß and deiodinase 3 (D3) mRNAs, but not hypothalamic and cerebral cortex D3, were increased. Conclusions: Adult humans and mice without RTH-ß exposed in utero to high maternal TH levels have persistent central resistance to TH. This is likely mediated by the increased expression of D3 in the anterior pituitary, enhancing local T3 degradation.

Transient neonatal diabetes due to a missense mutation (E227K) in the gene encoding the ATP-sensitive potassium channel (KCNJ11).

Neonatal diabetes is a monogenic form of diabetes. Herein, we report on a newborn presenting diabetic ketoacidosis at 17 days of life. A KCNJ11 mutation was identified. In such cases, insulin can be replaced by sulfonylurea with a successful metabolic control, as an example of how molecular diagnosis may influence the clinical management of the disorder.

A new PAX8 mutation causing congenital hypothyroidism in three generations of a family is associated with abnormalities in the urogenital tract.

BACKGROUND: Although thyroid dysgenesis is the most common cause of congenital hypothyroidism (CH), its molecular basis remains largely elusive. Indeed, in only a minority of cases with thyroid dysgenesis (2%-3%) was it possible to identify an underlying genetic defect. The objective of this study was to screen the PAX8 gene and the PAX2 gene in a family with six cases of CH spanning three generations and presenting urogenital malformations. Herein, we report a case series and in vitro characterization of the PAX8 gene mutation. METHODS: Investigations were conducted at a tertiary care referral center. The index case was diagnosed to have congenital hypothyroidism at 7 months of age when he presented with severe impairment of suckling, constipation, and poor development. Treatment with levothyroxine corrected the symptoms and was associated with catch-up growth. His progeny, including two sons, one daughter, and two granddaughters, were affected by CH, and three of them received the diagnosis at neonatal screening. Ultrasound demonstrated normally located thyroid glands with reduced volumes. Five of the six affected family members, including the index case, had urogenital malformations, including incomplete horseshoe kidney, undescended testicles, hydrocele, and ureterocele. Strabismus was found in three out of six affected patients. No other somatic malformations were found. RESULTS: Direct sequencing of the PAX8 gene revealed a new heterozygous mutation (c.74C > G) in all affected individuals. This mutation leads to substitution of proline with arginine at codon 25 (P25R). Fluorescence microscopy showed that P25R is normally located in the nucleus. In transient transfection studies, this mutation causes reduced transcriptional activation ability when using a luciferase reporter construct under the control of a thyroglobulin promoter. This diminished transactivation ability is due to loss of DNA binding capability as shown in electrophoresis mobility shift assay. The sequencing analysis of the PAX2 gene was normal. CONCLUSIONS: We conclude that this novel PAX8 mutation is responsible for a severe form of dominantly inherited CH. The mutation seems to be associated with abnormalities of the urogenital tract.

Serum copper as a novel biomarker for resistance to thyroid hormone.

Thyroid hormone action is mediated by the thyroid hormone receptors TRα1 and TRß. Defects in TRß lead to RTH (resistance to thyroid hormone) ß, a syndrome characterized by high levels of thyroid hormone and non-suppressed TSH (thyroid-stimulating hormone). However, a correct diagnosis of RTHß patients is difficult as the clinical picture varies. A biochemical serum marker indicative of defects in TRß signalling is needed and could simplify the diagnosis of RTHß, in particular the differentiation to TSH-secreting pituitary adenomas, which present with clinically similar symptoms. In the present paper we show that serum copper levels are regulated by thyroid hormone, which stimulates the synthesis and the export of the hepatic copper-transport protein ceruloplasmin into the serum. This is accompanied by a concerted reduction in the mRNA levels of other copper-containing proteins such as metallothioneins 1 and 2 or superoxide dismutase 1. The induction of serum copper is abolished in genetically hyperthyroid mice lacking TRß and human RTHß patients, demonstrating an important role of TRß for this process. Together with a previously reported TRα1 specific regulation of serum selenium, we show that the ratio of serum copper and selenium, which is largely independent of thyroid hormone levels, volume changes or sample degradation, can constitute a valuable novel biomarker for RTHß. Moreover, it could also provide a suitable large-scale screening parameter to identify RTHα patients, which have not been identified to date.

A large family with Carney complex caused by the S147G PRKAR1A mutation shows a unique spectrum of disease including adrenocortical cancer.

CONTEXT: Most tumors in Carney complex (CNC) are benign, including primary pigmented nodular adrenocortical disease (PPNAD), the main endocrine tumor in CNC. Adrenocortical cancer (AC) has never been observed in the syndrome. Herein, we describe a large Azorean family with CNC caused by a point mutation in the PRKAR1A gene coding for type 1-α (RIα) regulatory subunit of the cAMP-dependent protein kinase A, in which the index patient presented with AC. OBJECTIVE: We studied the genotype-phenotype correlation in CNC. DESIGN AND SETTING: We reported on case series and in vitro testing of the PRKAR1A mutation in a tertiary care referral center. PATIENTS: Twenty-two members of a family were investigated for Cushing syndrome and other CNC components; their DNA was sequenced for PRKAR1A mutations. RESULTS: Cushing syndrome due to PPNAD occurred in four patients, including the proposita who presented with AC and three who had Cushing syndrome and/or PPNAD. Lentigines were found in six additional patients who did not have PPNAD. A base substitution (c.439A>G/p.S147G) in PRKAR1A was identified in the proposita, in the three others with PPNAD, in the proposita's twin daughters who had lentigines but no evidence of hypercortisolism, and in five other family members, including one without lentigines or evidence of hypercortisolism. Unlike in other RIα defects, loss of heterozygosity was not observed in AC. The S147G mutation was compared to other expressed PRKAR1A mutations; it led to decreased cAMP and catalytic subunit binding by RIα and increased protein kinase A activity in vitro. CONCLUSIONS: In a large family with CNC, one amino acid substitution caused a spectrum of adrenal disease that ranged from lack of manifestations to cancer. PPNAD and AC were the only manifestations of CNC in these patients, in addition to lentigines. These data have implications for counseling patients with CNC and are significant in documenting the first case of AC in the context of PPNAD.

Autoimmunity in patients with resistance to thyroid hormone.

CONTEXT: Resistance to thyroid hormone (RTH) is an inherited syndrome most often caused by thyroid hormone receptor beta (TRbeta) gene mutations. Given that autoimmune thyroid disease (AITD) is prevalent in the general population, its coexistence with RTH has been presumed coincidental. It was recently proposed that chronic TSH stimulation in RTH may induce an autoimmune response, thereby increasing the chance of their coexistence. OBJECTIVE: The aim was to examine the prevalence of AITD in a large cohort with RTH compared with their unaffected first-degree relatives. SUBJECTS AND METHODS: Among 130 families, 330 individuals with RTH confirmed by the presence of TRbeta gene mutations and 92 unaffected first-degree relatives were tested for thyroglobulin and thyroperoxidase antibodies. The presence of AITD was based on at least one of the two antibodies being positive. Data were analyzed according to genotype, gender, age, and familial association. A large homogeneous family was analyzed separately. RESULTS: Individuals with RTH had an increased likelihood of thyroid autoantibodies (odds ratio = 2.36; P = 0.002). In males, the odds of having AITD were higher in individuals with RTH compared to unaffected first-degree relatives (odds ratio = 2.91; P = 0.042). Although female subjects with RTH had an odds ratio of 1.95 for having thyroid autoantibodies, the difference was not statistically significant (P = 0.097). Antibody prevalence at different ages was not affected by genotype. CONCLUSIONS: Individuals with RTH due to TRbeta gene mutations have an increased likelihood of AITD compared to unaffected relatives, but the prevalence of thyroid autoantibodies with advancing age is not affected by genotype. These novel findings demonstrate that the association between RTH and AITD is not coincidental.