Complete Thyroxine-binding Globulin Deficiency Research Articles

A Novel Mutation (del 1711 G) in theTBGGene as a Cause of Complete TBG Deficiency

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. 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. 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. This is a new variant of TBG-CD (TBG-CD-PL Poland) containing 205 AA.

Two novel mutations leading to familial partial and complete thyroxine-binding globulin deficiency

Two novel mutations leading to familial partial and complete thyroxine-binding globulin deficiency

TGB 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.

Complete Thyroxine-Binding Globulin (TBG) Deficiency in Two Families without Mutations in Coding or Promoter Regions of the TBG Genes: In Vitro Demonstration of Exon Skipping

Inherited thyroxine-binding globulin (TBG) deficiency is caused by mutations in the TBG gene located on the X-chromosome. We now describe two families (K and H) with X-linked complete TBG deficiency without mutations in the coding or promoter regions of the TBG gene. The propositi of both families presented with euthyroid hypothyroxinemia and were found to have undetectable TBG in serum. Affected females had approximately half the normal serum TBG concentration except for one woman who also had undetectable TBG (family H). All four of her children (two boys and two girls) were affected. Affected members of family K had no mutations in any of the five exons or in the minimal promoter region of the TBG gene. However, a G to A substitution, five base pairs downstream from exon 3, was associated to the phenotype of TBG deficiency (TBG-Jackson) and was not present in 100 normal alleles. In contrast to individuals without this mutation, no TBG mRNA could be detected in fibroblasts of the propositus, expressing solely TBG-Jackson. In vitro transcription of genomic DNA containing the mutant intron in an exon trapping system showed that this mutation, reducing the consensus value on the 5′ donor splice site, affects the normal splicing process. The transcript of TBG-Jackson lacks exon 3 and is unstable. The deduced amino acid sequence has a frameshift and an early stop codon at position 325. Affected subject of family H had no mutations in the TBG gene including all exons, all introns, the minimal promoter, and the 3′ untranslated sequence. However, an intragenic A/G polymorphism (125 bp upstream from exon 2) was identified. It allowed us to confirm a cosegregation of the phenotype to the TBG gene and to show that the single female with complete TBG deficiency was homozygous for the polymorphic TBG allele. The cause of TBG deficiency in this family remains unknown.

Gene screening of thyroxine-binding globulin (TBG) deficiencies in the Japanese: only two mutations account for TBG deficiencies in the Japanese.

T4-binding globulin (TBG) is the principal transport protein for thyroid hormone in the circulation. Twelve mutations in the human TBG gene have been reported, and the inheritance of those variant TBGs was shown to be X-chromosome linked. We previously reported a nucleotide deletion at the codon 352 (TBG-CDJ) and a nucleotide substitution at the codon 362 (TBG-PDJ) of the TBG gene in a Japanese male manifesting TBG complete deficiency and partial TBG deficiency, respectively. In this communication we investigate the prevalence of both mutations among 50 unrelated Japanese subjects manifesting complete or partial TBG deficiency from various areas of the Japanese Archipelago. Mutant alleles were identified by amplification of their genomic DNAs by PCR with allele-specific primers. In addition, the presence of a polymorphic mutation in codon 283 (TBG-Poly) in these variants was investigated. All male subjects manifesting complete TBG deficiency (n = 30) and all female subjects manifesting partial TBG deficiency (n = 4) were demonstrated to be hemizygotes and heterozygotes for the mutation of TBG-CDJ, respectively. All male subjects manifesting partial TBG deficiency (n = 16) were shown to have the mutation of TBG-PDJ as hemizygotes. TBG-Poly was consistently absent from these variants. We conclude that only TBG-CDJ and TBG-PDJ may account for complete and partial TBG deficiencies in the Japanese.

Mass screening for complete deficiency of thyroxine-binding globulin in adult Japanese by comprehensive health examination.

The incidence of complete thyroxine-binding globulin (TBG) deficiency (TBG-CD) was determined for a Japanese population from a comprehensive health examination, in which a T3 resin uptake test of the upper 5% (78 subjects from among 1,589 men) was the screening line for TBG-CD. Further analysis of the known mutation in TBG-CD gene of the Japanese population (reported as TBG-CDJ with codon 352 deletion) was performed on 72 subjects, and three were found to have TBG-CDJ, two of whom were siblings. Only those three subjects had a serum TBG concentration of less than 5 mg/l. The six subjects for whom the DNA analysis was not performed, did not have a serum TBG level of less than 5 mg/l. From these findings, the gene frequency of TBG-CDJ was calculated to be 0.13%. The incidence of TBG-CDJ in the total Japanese population is suggested to be 0.09%.

Thyroxine-binding globulin: organization of the gene and variants.

Thyroxine-binding globulin (TBG), the principal thyroid hormone transport protein in human serum, is synthesized by the liver and secreted into the bloodstream as a 54-kD acidic glycoprotein made up of a single polypeptide chain of 395 amino acids and four heterosaccharide units. The carbohydrate chains are important for the correct posttranslational folding, secretion and degradation of the molecule but are not required for hormone binding. TBG, encoded by a single gene copy located on Xq22, consists of five exons spanning 5.5 kbp. An upstream sequence of 218 nucleotides containing a hepatocyte nuclear factor 1 binding motif imparts to the gene a strong liver-specific transcriptional activity. Inherited TBG defects produce three phenotypes based on the level of TBG in serum of affected hemizygotes: complete TBG deficiency (TBG-CD), partial TBG deficiency (TBG-PD) and TBG excess (TBG-E). The molecular basis of the TBG defect has been identified in 12 of 16 known TBG variants. TBG-CD is caused by either premature termination of translation or an amino acid substitution resulting in failure of secretion. Point mutations resulting in single amino acid substitutions are responsible for the alteration of the properties and/or concentration of TBG-PD variants. Gene duplication and triplication has been recently identified in subjects with TBG-E.

Gene screening in Japanese families with complete deficiency of thyroxine‐binding globulin demonstrates that a nucleotide deletion at codon 352 may be a race specific mutation

Thyroxine-binding globulin (TBG) is a serum protein that transports 75% of circulating thyroxine. Eleven naturally occurring mutations in the human TBG gene have been identified, ten of which alter the properties of the molecule. Three of these mutations produce complete deficiency of TBG (TBG-CD) and four are associated with a second mutation in codon 283 (TBG-poly) which is polymorphic in some ethnic groups but, when present alone, does not alter the properties of the TBG molecule. In this communication we investigate whether two unrelated Japanese families with TBG-CD harboured the TBG-CDJ mutation in codon 352 associated with TBG-CD in families residing in more distant locations of the Japanese Islands. In addition we examined the possible association with TBG-poly and its incidence in the Japanese population. Mutant alleles were identified by amplification of genomic DNAs by the polymerase chain reaction, using allele-specific oligonucleotide primers. Eight family members and 25 normal subjects. Serum free thyroxine and TBG concentration were measured by a conventional radioimmunoassay and a more sensitive enzyme immunoassay. Genomic DNAs were extracted from white blood cells and specific mutations at codons 352 and 283 were identified by allele-specific amplification. Three males and three females, whose serum TBG levels were decreased, had mutations at codon 352 as hemizygous and heterozygous, respectively. This mutation was not present in the DNA of any of the related or unrelated subjects with normal TBG concentration. The presence of TBG-poly was demonstrated in only one heterozygous family member and in six out of 30 alleles (20%) in normal unrelated subjects. The frequency of this TBG polymorphism in the Japanese is similar to that of 16% reported in French Canadians. We conclude that TBG-CDJ might be a prevalent cause of complete deficiency of thyroxine-binding globulin in the Japanese and that TBG-poly probably appeared before the divergence of human races.

A truncated thyroxine-binding globulin due to a frameshift mutation is retained within the rough endoplasmic reticulum: a possible mechanism of complete thyroxine-binding globulin deficiency in Japanese.

We have previously reported six unrelated Japanese families having the same mutation in the TBG gene and manifesting complete TBG deficiency (TBG-CDJ). The deficiency consists of a single nucleotide deletion resulting in the production of C-terminal truncation due to a frameshift and premature termination. However, the reason for the failure to detect TBG in the serum of subjects harboring this mutation remains unknown. In this communication we investigated the mechanism of the TBG deficiency associated with TBG-CDJ. The complementary DNAs of TBG-CDJ and normal TBG (TBG-N) were expressed in COS-1 cells. Pulse-chase experiments revealed a complete absence of secretion of TBG-CDJ, whereas TBG-N was already present in the medium at time 0 and was almost entirely secreted by 3 h. In cell lysates, TBG-CDJ had a smaller molecular mass [52 kilodalton (kDa)] than TBG-N (54 kDa) and gradually decreased during the chase. Thus, failure of TBG-CDJ secretion accounts for the complete TBG deficiency. The molecular mass of TBG-CDJ (42 kDa) was also smaller than that of TBG-N (44 kDa) when synthesized in the presence of tunicamycin. These findings are consistent with the premature termination of the TBG-CDJ molecule deduced from the nucleotide sequence analysis. Intracellular TBG-N was resistant to endoglycosidase H but not TBG-CDJ, suggesting the retention of TBG-CDJ within the rough endoplasmic reticulum. Indeed, subcellular fractionation revealed that most of TBG-CDJ was located in the rough endoplasmic reticulum compartment, and TBG-N was distributed in the Golgi fractions. Our results suggested that the lack of intracellular transport of the truncated TBG molecule is the cause for the absence of immunoreactive TBG in the serum of subjects harboring the TBG-CDJ variant.

A truncated thyroxine-binding globulin due to a frameshift mutation is retained within the rough endoplasmic reticulum: a possible mechanism of complete thyroxine-binding globulin deficiency in Japanese

A truncated thyroxine-binding globulin due to a frameshift mutation is retained within the rough endoplasmic reticulum: a possible mechanism of complete thyroxine-binding globulin deficiency in Japanese

Gene screening of 23 Japanese families with complete thyroxine-binding globulin deficiency: identification of a nucleotide deletion at codon 352 as a common cause.

Thyroxine-binding globulin (TBG) is a major thyroid hormone transport protein in human serum. Its complete deficiency (TBG-CD) is one of inherited TBG abnormalities that transmit on X-chromosome. We previously reported a nucleotide deletion at codon 352 of the TBG gene (TBG-CDJ) in Japanese families with TBG-CD. To determine the prevalence of this mutation in Japanese with TBG-CD, 23 affected subjects (19 males and 4 females) belonging to unrelated families living in 4 major islands of Japan were analyzed with regard to the mutation at codon 352. Their genomic DNAs were amplified by the polymerase chain reaction with allele specific primers. Nineteen male and four female subjects were shown to have the mutation as hemizygotes and heterozygotes, respectively. It is concluded that TBG-CDJ may be a common cause of TBG-CD in Japanese and might have appeared in the ancestors of the Japanese after the human race divergence.

Hereditary Complete Thyroxine-Binding Globulin Deficiency: Identification by T3 Resin Uptake Test and DNA Analysis.

Complete thyroxine-binding globulin deficiency (TBG-CD) was uncovered in a subject receiving a comprehensive health examination. The subject had an abnormally high T3 resin uptake. A family study showed that the TBG abnormality had been inherited by X-chromosome linkage. Genetic analysis revealed single nucleotide deletion, common among Japanese with TBG-CD, from the allele specific amplification of the TBG genes of the family.

In vitro expression of thyroxine-binding globulin (TBG) variants. Impaired secretion of TBGPRO-227 but not TBGPRO-113.

Thyroxine-binding globulin (TBG) is a glycoprotein that transports thyroid hormones in blood. Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency. In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes. Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed. TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in thyroxine (T4) binding. TBG-CD5 had altered electrophoretic mobility and did not bind T4. TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly. Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M. The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion.

Complete thyroxine-binding globulin (TBG) deficiency caused by a single nucleotide deletion in the TBG gene

Thyroxine-binding globulin (TBG) is the major thyroid hormone transport protein. Several inherited TBG variants resulting in partial and complete TBG deficiency have been shown to be caused by one or two nucleotide replacements in the coding regions of the TBG gene. Each manifests as a different change in the physical properties and/or biological function of the mature TBG protein. We now report sequencing of the entire coding region and exon-intron junctions of a TBG allele of a subject with inherited complete TBG deficiency, previously classified as TBG-CD6. A single nucleotide deletion was found in the codon for amino acid 165 of the normal TBG molecule. The shift in the reading frame due to this nucleotide deletion results in downstream encoding of two different amino acids followed by an early stop codon. This results in a mature protein of only 167 residues, as compared with the normal 395. Nine members of the TBG-CD6 family were tested for the presence of this mutation by allele-specific amplification. Seven of them were found to be carriers of the defect, with five hemizygous subjects manifesting complete TBG deficiency. The mutant allele correlated with the results of TBG analysis in serum. This defect was not present in affected subjects of any of seven unrelated families with complete TBG deficiency tested.

Nucleotide Deletion Resulting in Frameshift as a Possible Cause of Complete Thyroxine-Binding Globulin Deficiency in Six Japanese Families

Complete T4-binding globulin deficiency (TBG-CD) is inherited in an X-linked fashion. A nucleotide substitution has been shown to cause this hereditary condition in caucasians of French Canadian origin. Heterogeneity in molecular mechanisms for TBG-CD has also been reported. Genomic DNA from a Japanese male exhibiting TBG-CD was subjected to polymerase chain reaction, and the generated DNA fragments were sequenced. A single nucleotide deletion was found in the first base of the codon for amino acid 352 of the common-type TBG molecule. This mutation causes a frameshift in translation and premature termination. Compared with common-type TBG, the mutated polypeptide results in 1) 22 different amino acids on its carboxy-terminus, 2) a 22-amino acid truncation, and 3) the absence of a potential N-linked glycosylation site. These alterations may lead to profound changes in the secondary and tertiary structures of the molecule. To ascertain the presence of this nucleotide deletion in the genomic DNA of affected subjects, a mutated primer was designed which together with the nucleotide deletion produced a new endonuclease restriction site in the polymerase chain reaction fragment. Results revealed the presence of the mutation in genomic DNA of the subject, and his mother was shown to have both mutant and normal alleles. The same mutation was also detected in five other unrelated families carrying TBG-CD. This mutation may be frequent in Japanese subjects with TBG-CD.

Replacement of Leu227by Pro in Thyroxine-Binding Globulin (TBG) Is Associated with Complete TBG Deficiency in Three of Eight Families with This Inherited Defect*

The T4-binding globulin (TBG) gene is a single copy located on the X-chromosome. Previous studies have failed to elucidate the molecular defect in individuals with complete TBG deficiency (TBG-CD). Indeed, no major deletions, insertions, or other rearrangements were observed in the TBG gene of six unrelated males with this defect. To clarify the molecular basis of TBG-CD, we have cloned and sequenced the TBG gene of an affected male (CD5) of French Canadian origin. The sequence of the exons encoding the mature protein, adjacent introns, and the promoter region revealed two nucleotide substitutions: CTA(Leu)----CCA(Pro) at codon 227 and TTG(Leu)----TTT(Phe) at codon 283. The Leu----Phe substitution, a relatively conservative replacement, is a TBG polymorphism present in 16% (3 of 19) of French Canadian males. It has no effect on the serum concentration or properties of the common type TBG (TBG-C). The new Leu----Pro substitution, which is predicted to alter the higher order of TBG structure, is probably responsible for the TBG-CD phenotype of the individual studied and two other families with TBG-CD. It possibly impairs TBG biosynthesis or secretion or perhaps alters TBG structure to such a degree that the molecule is not recognized by antibodies against native or denatured TBG and does not bind T4.

Thyroxine-binding globulin deficiency re-examined.

Seven sera, previously categorised as completely deficient in thyroxine-binding globulin (TBG) by an immunoelectrophoretic technique, were re-examined with a sensitive ELISA method. Only one of the sera was confirmed completely deficient by ELISA. The remaining six contained 0.08-0.19 mg/l. The protein was immunologically identical with 'normal' TBG, could bind to thyroxine and had the correct mobility in reverse-flow electrophoresis. Complete TBG deficiency may therefore be rare.