Identification of Mutations in the Thyroxine-Binding Globulin (TBG) Gene in Patients with TBG Deficiency in Korea.

BACKGRUOUND: Thyroxine-binding globulin (TBG) is a major transporter protein for thyroid hormones. The serpin family A member 7 (SERPINA7) gene codes for TBG, and mutations of the SERPINA7 gene result in TBG deficiency. Although more than 40 mutations have been reported in several countries, only a few studies of TBG deficiency and SERPINA7 gene mutation have been performed in Korea. The aim of this study is to review the clinical presentations and laboratory findings of patients with TBG deficiency and to investigate the types of SERPINA7 gene mutation. METHODS: Five unrelated Korean adults with TBG deficiency attending endocrinology clinic underwent SERPINA7 gene sequencing. Four patients harbored a SERPINA7 gene mutation. Serum thyroid hormones, anti-microsomal antibodies, and TBG were measured. Genomic DNA was extracted from whole blood. All exons and intron-exon boundaries of the TBG gene were amplified and sequencing was performed. RESULTS: Two patients were heterozygous females, and the other two were hemizygous males. One heterozygous female had coexisting hypothyroidism. The other heterozygous female was erroneously prescribed levothyroxine at a local clinic. One hemizygous male harbored a novel mutation, p.Phe269Cysfs*18, which caused TBG partial deficiency. Three patients had the p.Leu372Phefs*23 mutation, which is known as TBG-complete deficiency Japan (TBG-CDJ) and was also presented in previous mutation analyses in Korea. CONCLUSION: This study presents four patients diagnosed with TBG deficiency and provides the results of SERPINA7 gene sequencing. One novel mutation, p.Phe269Cysfs*18, causing TBD-partial deficiency and three cases of TBG-CDJ were demonstrated. It is necessary to identify TBG deficiency to prevent improper treatment. Also, sequencing of the SERPINA7 gene would provide valuable information about the TBG variants in Korea.

Thyroxine replacement modifies changes in deiodinase and thyroid hormone transporter expression induced by subclinical hypothyroidism in rats.

PURPOSE: The potential benefits of treating subclinical hypothyroidism (SCH) are unclear and still controversial. Thus, we surgically induced SCH in rats and evaluated the effects of thyroxine (T4) replacement on the gene expression levels of deiodinases and thyroid hormone (TH) transporters in different tissues. METHODS: SCH was induced by hemithyroid electrocauterization. The control animals underwent the same surgical procedure but were not subjected to electrocauterization (sham). After 14 days, half of the SCH animals were treated with T4 (SCH + T4). At the end of the experimental protocol, all of the rats were euthanized, serum hormone concentrations were measured, and RNA analyses were performed on different tissues and organs. RESULTS: Consistent with previous studies, we observed increased TSH levels, normal TH levels, and reduced hypothalamic TRH expression in the SCH group. Additionally, Dio2 mRNA expression was downregulated in the hippocampus and pituitary, and Dio1 was upregulated in the kidney and pituitary of the SCH animals. The changes in Dio3 expression were tissue-specific. Concerning TH transporters, Mct10 expression was upregulated in the pituitary, kidney, hypothalamus, and hippocampus, and Mct8 expression was downregulated in the kidney of the SCH group. Crym expression was upregulated in the kidney and pituitary. Notably, T4 replacement significantly attenuated serum TSH levels and reverted Dio1, Dio2, Mct10, and Crym expression in the pituitary, hippocampus, and kidney to levels that were similar to the sham group. Tissue-specific responses were also observed in the liver and hypothalamus. CONCLUSION: Our results indicate that treatment of SCH should be considered before the appearance of clinical symptoms of hypothyroidism.

Possible mechanism for the polychlorinated bipheny-linduced liver-selective accumulation of thyroxine in rats.

We have previously reported that decrease in level of serum thyroxine T4 by Kanechor 500 (KC500) in rats would occur through the increase in hepatic T4 accumulation rather than the increase in hepatic T4-glucuronyl transferase activity. In the present study, to understand the mechanism underlying the KC500-mediated increase in hepatic T4 accumulation, we examined the relationship between the KC500-mediated changes in hepatic T4 accumulation and the expression levels of mRNAs of hepatic transporters including T4 transporters. [125I]T4 was intravenously injected into KC500-pretreated and control (KC500-untreated) Wistar rats, and [125I]T4 uptake levels of liver parenchymal cells were comparatively examined. The amount of [125I]T4 uptake by hepatic cells increased in a time-dependent manner up to 96 hr after KC500 treatment. Following KC500 treatment, a time-dependent increase in the mRNA level of hepatic T4 influx transporter LAT1 was observed up to 96 hr later, while a significant increase in hepatic T4 influx transporter Oatp2 mRNA occurred only at 96 hr later. No KC500-mediated increases in the mRNAs of other hepatic transporters (Oatp1, Oatp3, Oatp4, Ntcp, LAT2, and Mrp2) were observed at any timepoints, although the mRNA expression of the T4 conjugate(s) efflux transporter Mrp3 significantly increased in a time-dependent manner 24-96 hr following KC500 treatment. The present findings suggest that KC500-mediated increase in hepatic T4 accumulation occurs, at least in part, through the increase in the expression of hepatic T4-transporters, such as LAT1 and Oatp2.

Inherited defects of thyroxine-binding proteins.

Thyroid hormones (TH) are bound to three major serum transport proteins, thyroxine-binding globulin (TBG), transthyretin (TTR) and human serum albumin (HSA). TBG has the strongest affinity for TH, whereas HSA is the most abundant protein in plasma. Individuals harboring genetic variations in TH transport proteins present with altered thyroid function tests, but are clinically euthyroid and do not require treatment. Clinical awareness and early recognition of these conditions are important to prevent unnecessary therapy with possible untoward effects. This review summarizes the gene, molecular structure and properties of these TH transport proteins and provides an overview of their inherited abnormalities, clinical presentation, genetic background and pathophysiologic mechanisms.

A transgenic approach to identify thyroxine transporter-expressing structures in brain development.

Transporters are essential in thyroid hormone metabolism. Thyroxine (T4) is transported by solute carrier organic anion transporter 1c1 (SLCO1C1, OATP14) into the adult brain, where T4 is converted to 3,5,3'-triiodothyronine (T3). In adults, SLCO1C1 expression is found in two brain barrier structures: the blood-brain barrier (BBB) and choroid plexus. However, little is known about how T4 is transported in the developing brain, when the BBB is not yet completely formed. We employed bacterial artificial chromosome recombineering to generate transgenic mice carrying Cre recombinase in the Slco1c1 locus (Slco1c1-Cre mice). In Slco1c1-Cre mice Cre was expressed at the sites that have been previously reported for SLCO1C1 in adults. To trace Cre expression during development, we crossed Slco1c1-Cre transgenic mice with Rosa26 reporter mice. ß-galactosidase staining showed Cre activity in neurones of various brain structures, such as cortical layer 2/3 and the hippocampus, suggesting transient Slco1c1 expression during brain development. At embryonic day15, SLCO1C1 was expressed at the same site as TBR2, a marker of neuronal progenitors. Neurones that express SLCO1C1 during their development could be T4 sensitive. In support of this hypothesis, hypothyroxinaemia induced by propylthiouracil treatment of dams decreased the number of ß-galactosidase-positive neurones in cortical layer 2/3 of newborn Slco1c1-Cre/Rosa26 mice. In conclusion, by generating Slco1c1-Cre transgenic mice, we demonstrated that SLCO1C1 is expressed in the neuronal cell lineage during brain development. Expression of SLCO1C1 may underlie the extraordinary sensitivity of specific neuronal populations to hypothyroxinaemia.

Explanation for the high heat stability of thyroxine binding globulin-Chicago.

OBJECTIVE: Thyroxine binding globulin-Chicago (TBG-Chicago), a variant of TBG with enhanced heat stability, was isolated at the University of Chicago from a 22 year old subject of an African American lineage. High thermodynamic stability in serine proteinase inhibitors (serpin) is the hallmark of reactive center loop (RCL) inserted conformation and this aspect has not been explored in TBG-Chicago, a serpin molecule. It is hypothesised that the high heat stability of TBG-Chicago is due its loop inserted state. METHODS: Recombinant (r) TBG-Chicago and normal (r) TBG were expressed in baculovirus system. Stability of the freshly made proteins was assessed on Native- PAGE by: 1. heating at 65 degrees C/30 min; 2. incubating at 37 degrees C/24 h. Susceptibility of the RCL of both the TBG's to endoproteinase cleavage and the ability to accept synthetic RCL mimetic peptide were assessed before and after incubation at 37 degrees C /24 h (SDS-PAGE/ Native PAGE). RESULTS: It was found that rTBG-Chicago aggregates at 65 degrees C, accepts RCL mimetic peptide and is cleaved by endoproteinase. In contrast, rTBG-Chicago that had been incubated at 37 degrees C/24 h showed enhanced heat stability at 65 degrees C, reduced ability to accept synthetic peptide and decreased susceptibility to endoproteinase cleavage (besides having changed mobility on Native gel). CONCLUSION: The results support the conclusion that freshly isolated TBG-Chicago exists in loop expelled conformation. However at 37 oC, the protein readily converts to a more stable loop inserted conformation and could explain why plasma TBG-Chicago was found to have enhanced heat stability in the 22 year old subject.

Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression.

A survey of the expression of the transthyretin and thyroxine-binding globulin genes in various species during development provides clues as to how the present thyroid hormone distribution network in extracellular compartments developed during vertebrate evolution. Albumin may be the 'oldest' component of the thyroid hormone distribution network as it is found in the plasma of all vertebrates investigated. Subsequent to albumin, transthyretin appeared as the second component in this network during the evolution of vertebrates. The strong expression of transthyretin genes in the liver coincides with the presence of recognition site(s) for liver-enriched transcription factors, such as HNF-3beta (Foxa2), in the transthyretin promoter regions of vertebrates. Finally, the addition of thyroxine-binding globulin to this network occurred at postnatal stages in some marsupials and rodents and in perinatal to adult stages in most eutherians. All vertebrates have defined developmental stages when thyroid hormone-dependent transition from larval to juvenile forms occurs. The inclusion of transthyretin and thyroxine-binding globulin in the thyroid hormone distribution network may be correlated with the increased requirement of thyroid hormones for thyroid hormone-dependent tissue remodeling during these stages and/or increased metabolism in thyroid hormone-target tissues with the acquisition of homeothermy.

A novel variant in Serpina7 gene in a family with thyroxine-binding globulin deficiency.

Thyroxine-binding globulin (TBG) carries approximately 75% of serum T4 and T3. This protein is encoded by serpina7 gene, formerly known as TBG gene, localized on X-chromosome (Xq22.2). A deficiency in TBG is suspected when abnormally low serum total T4 and T3 are encountered in clinically euthyroid subjects in the presence of normal serum TSH. This condition has been associated with different serpina7 gene mutations resulting in amino acid substitutions or truncations in the mature protein. Herein, we report a new serpina7 gene variant in three members of the same family. It results in the replacement of the normal asparagine 233 by isoleucine and, subsequently, in disruption of a glycosylation site. Co-segregation of this new variant with undetectable levels of TBG in the hemizygous man studied and failure to recognize the same variant in 100 alleles at random, made us to consider it as the underlying cause of the TBG deficiency.

Gene expression profiling in rat liver after VMH lesioning.

It has been reported that ventromedial hypothalamic (VMH) lesions induce hepatic cell proliferation and apoptosis and metabolic changes in the body. In the present study, we identified genes of which expression profiles showed significant modulation in rat liver after VMH lesions. Total RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH lesioned rats were investigated using DNA microarray analysis. The results revealed that VMH lesions regulated the genes that were involved in various types of metabolisms and cell proliferations in the liver. Real-time PCR also confirmed that gene expressions of ELOVL6 and SPC24 were upregulated, and that of SERPINA7 was downregulated. VMH lesions may change the expressions of multiple metabolism genes and cell proliferation-related genes in rat liver.

A novel mutation (del 1711 G) in the TBG gene as a cause of complete TBG deficiency.

OBJECTIVE: Inherited thyroxine-binding globulin (TBG) deficiency is caused by mutations in the TBG gene (locus: Xq22.2), which result in defective synthesis or changes in the physical properties or biological function of a protein. DESIGN: We report a novel mutation of the TBG gene causing a complete TBG deficiency in three brothers of Polish origin. DNA was extracted from all of the family members and subjected to sequence analysis. We analyzed the family with a heterozygous mother, a normal father, their three hemizygous affected sons, and their two normal sons. MAIN OUTCOME: Our studies revealed a novel mutation, a single nucleotide deletion (guanine) at position 1711, codon 201 (Asp) in exon 2 (GAC --> AC). This mutation led to a frame shift and premature termination at codon 206, causing a short TBG protein of 205 amino acids (AA) compared to 395 AA of the normal TBG. This new TBG-CD variant was found in the mother and her three affected sons. CONCLUSION: This is a new variant of TBG-CD (TBG-CD-PL Poland) containing 205 AA.

TBG deficiency: description of two novel mutations associated with complete TBG deficiency and review of the literature.

Thyroxine-binding globulin (TBG) is the main thyroid hormone transport protein in serum. Inherited TBG defects lead to a complete (TBG-CD) or a partial (TBG-PD) deficiency and have a diagenic transmission, being clinically fully expressed only in hemizygous males and in homozygous females. In the present study, seven patients from two unrelated families with TBG-CD were studied and two novel TBG mutations were documented. In particular, a T insertion at the 5' donor splice site of exon 0, between nucleotides 2 and 3 at the beginning of intron 1 (g.IVS1+2_3insT) was found in one family and was named TBG-Milano. The other novel mutation is a T deletion at nucleotide 214 of exon 1, which leads to a frameshift at codon 50 with a premature stop codon at position 51 (c.214delT, P50fsX51) and was named TBG-Nikita. According to the X-linked transmission of the defect, females harboring the mutation showed a reduction in TBG levels with normal TSH and total thyroid hormone values at the lower limit of normal. Males harboring either TBG-Milano or TBG-Nikita, showed normal TSH values and low levels of total thyroid hormones and lacked TBG. In conclusion, we report two novel mutations of the TBG gene associated with a complete TBG defect. The first mutation lies at the 5' donor splice site of exon 0 and probably alters the start of translation, while the second is a single nucleotide deletion and leads to a premature stop codon.

Structural mechanism for the carriage and release of thyroxine in the blood.

The hormones that most directly control tissue activities in health and disease are delivered by two noninhibitory members of the serpin family of protease inhibitors, thyroxine-binding globulin (TBG) and corticosteroid-binding globulin. The structure of TBG bound to tetra-iodo thyroxine, solved here at 2.8 A, shows how the thyroxine is carried in a surface pocket on the molecule. This unexpected binding site is confirmed by mutations associated with a loss of hormone binding in both TBG and also homologously in corticosteroid-binding globulin. TBG strikingly differs from other serpins in having the upper half of its main beta-sheet fully opened, so its reactive center peptide loop can readily move in and out of the sheet to give an equilibrated binding and release of thyroxine. The entry of the loop triggers a conformational change, with a linked contraction of the binding pocket and release of the bound thyroxine. The ready reversibility of this change is due to the unique presence in the reactive loop of TBG of a proline that impedes the full and irreversible entry of the loop that occurs in other serpins. Thus, TBG has adapted the serpin inhibitory mechanism to give a reversible flip-flop transition, from a high-affinity to a low-affinity form. The complexity and ready triggering of this conformational mechanism strongly indicates that TBG has evolved to allow a modulated and targeted delivery of thyroxine to the tissues.

C-terminal amino acid alteration rather than late termination causes complete deficiency of thyroxine-binding globulin CD-NeuIsenburg.

CONTEXT: T(4)-binding globulin (TBG) is the main transport protein for T(4) in blood and a member of the superfamily of serine proteinase inhibitors. So far, 14 mutations leading to familial complete TBG deficiency have been reported. Eleven of these are caused by mutations leading to truncation of the molecule, and three are caused by single amino acid substitutions. OBJECTIVE: We report and study the complete deficiency TBG variant found in a patient from NeuIsenburg, Germany (TBG-CDNI). METHODS: Direct DNA sequencing was used to identify the TBG-CDNI mutation in the propositus, which was confirmed by allele-specific amplification. Site-directed mutagenesis and expression in Xenopus oocytes was used to study the secretion defect of TBG-CDNI and several variants by Western blot and T(4)-binding assay. RESULTS: The deletion of two nucleotides in codon 384 (1211_1212delTC) causes a frameshift altering the last 11 residues, introduces a new glycosylation site, and elongates the molecule by seven new amino acids. In contrast to normal TBG, TBG-CDNI was not secreted by Xenopus oocytes. Elongation of normal TBG by seven alanines did not affect its secretion or binding properties. On the other hand, neither disruption of its new glycosylation site nor termination of TBG-CDNI at the normal length repaired its secretion defect. CONCLUSIONS: In this first late termination variant of complete TBG deficiency, alteration of beta-strand 5B, located in the core of the molecule, rather than elongation of the molecule or introduction of a new glycosylation site, suffices to disrupt secretion of TBG-CDNI.

Correction of Hunter syndrome in the MPSII mouse model by AAV2/8-mediated gene delivery.

Mucopolysaccharidosis type II (MPSII; Hunter syndrome) is a lysosomal storage disorder caused by a deficiency in the enzyme iduronate 2-sulfatase (IDS). At present, the therapeutic approaches for MPSII are enzyme replacement therapy and bone marrow transplantation, although these therapies have some limitations. The availability of new AAV serotypes that display tissue-specific tropism and promote sustained expression of transgenes offers the possibility of AAV-mediated gene therapy for the systemic treatment of lysosomal diseases, including MPSII. We have characterized in detail the phenotype of IDS-deficient mice, a model of human MPSII. These mice display a progressive accumulation of glycosaminoglycans (GAGs) in many organs and excessive excretion of these compounds in their urine. Furthermore, they develop skeleton deformities, particularly of the craniofacial bones, and alopecia, they perform poorly in open-field tests and they have a severely compromised walking pattern. In addition, they present neuropathological defects. We have designed an efficient gene therapy approach for the treatment of these MPSII mice. AAV2/8TBG-IDS viral particles were administrated intravenously to adult MPSII mice. The plasma and tissue IDS activities were completely restored in all of the treated mice. This rescue of the enzymatic activity resulted in the full clearance of the accumulated GAGs in all of the tissues analyzed, the normalization of the GAG levels in the urine and the correction of the skeleton malformations. Overall, our findings suggest that this in vivo gene transfer approach has potential for the systemic treatment of patients with Hunter syndrome.

Molecular phylogeny of the carnivora (mammalia): assessing the impact of increased sampling on resolving enigmatic relationships.

This study analyzed 76 species of Carnivora using a concatenated sequence of 6243 bp from six genes (nuclear TR-i-I, TBG, and IRBP; mitochondrial ND2, CYTB, and 12S rRNA), representing the most comprehensive sampling yet undertaken for reconstructing the phylogeny of this clade. Maximum parsimony and Bayesian methods were remarkably congruent in topologies observed and in nodal support measures. We recovered all of the higher level carnivoran clades that had been robustly supported in previous analyses (by analyses of morphological and molecular data), including the monophyly of Caniformia, Feliformia, Arctoidea, Pinnipedia, Musteloidea, Procyonidae + Mustelidae sensu stricto, and a clade of (Hyaenidae + (Herpestidae + Malagasy carnivorans)). All of the traditional "families," with the exception of Viverridae and Mustelidae, were robustly supported as monophyletic groups. We further have determined the relative positions of the major lineages within the Caniformia, which previous studies could not resolve, including the first robust support for the phylogenetic position of marine carnivorans (Pinnipedia) within the Arctoidea (as the sister-group to musteloids [sensu lato], with ursids as their sister group). Within the pinnipeds, Odobenidae (walrus) was more closely allied with otariids (sea lions/fur seals) than with phocids ("true" seals). In addition, we recovered a monophyletic clade of skunks and stink badgers (Mephitidae) and resolved the topology of musteloid interrelationships as: Ailurus (Mephitidae (Procyonidae, Mustelidae [sensu stricto])). This pattern of interrelationships of living caniforms suggests a novel inference that large body size may have been the primitive condition for Arctoidea, with secondary size reduction evolving later in some musteloids. Within Mustelidae, Bayesian analyses are unambiguous in supporting otter monophyly (Lutrinae), and in both MP and Bayesian analyses Martes is paraphyletic with respect to Gulo and Eira, as has been observed in some previous molecular studies. Within Feliformia, we have confirmed that Nandinia is the outgroup to all other extant feliforms, and that the Malagasy Carnivora are a monophyletic clade closely allied with the mongooses (Herpestidae [sensu stricto]). Although the monophyly of each of the three major feliform clades (Viverridae sensu stricto, Felidae, and the clade of Hyaenidae + (Herpestidae + Malagasy carnivorans)) is robust in all of our analyses, the relative phylogenetic positions of these three lineages is not resolvable at present. Our analyses document the monophyly of the "social mongooses," strengthening evidence for a single origin of eusociality within the Herpestidae. For a single caniform node, the position of pinnipeds relative to Ursidae and Musteloidea, parsimony analyses of data for the entire Carnivora did not replicate the robust support observed for both parsimony and Bayesian analyses of the caniform ingroup alone. More detailed analyses and these results demonstrate that outgroup choice can have a considerable effect on the strength of support for a particular topology. Therefore, the use of exemplar taxa as proxies for entire clades with diverse evolutionary histories should be approached with caution. The Bayesian analysis likelihood functions generally were better able to reconstruct phylogenetic relationships (increased resolution and more robust support for various nodes) than parsimony analyses when incompletely sampled taxa were included. Bayesian analyses were not immune, however, to the effects of missing data; lower resolution and support in those analyses likely arise from non-overlap of gene sequence data among less well-sampled taxa. These issues are a concern for similar studies, in which different gene sequences are concatenated in an effort to increase resolving power.

[A family with familial dysalbuminaemic hyperthyroxinaemia].

OBJECTIVE: To report a family of familial dysalbuminaemic hyperthyroxinaemia(FDH). METHODS: Four members, including the female proband, mother, daughter and brother, went through the measurement of thyroid hormone and thyroid-stimulating hormone (TSH). Electrophoretic analysis of the patient's serum proteins was carried out after the patient's serum being incubated with fluorescein isothiocyanate (FITC) labeled thyroxine(T4), The point mutation of Alb gene was determined in all members. RESULTS: The measurements of thyroid hormane and TSH showed that in three members (the proband, her mother and her daughter), the total thyroxine(TT4) serum level was high, the total triiodothyronine(TT3), FT4, FT3 and TSH serum levels were normal. And the enhanced albumin binding of fluorescenced T4 by electrophoresis showed a mutation transition 653 G-->A on DNA coding region of albumin. But in the proband's brother, the thyroid function and the results of electrophoresis of thyroxine-binding protein and determination of albumin gene were normal. CONCLUSION: A family with FDH in China is firstly reported here, a mutation at albumin gene DNA coding region 653G-->A causing enhanced albumin binding of T4 results in high T4 level.