| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Other Original Articles |
Departments of Medicine (Si.R., Sa.R.) and Pediatrics (Sa.R.) and the J. P. Kennedy Jr. Mental Retardation Research Center (Sa.R.), The University of Chicago, Chicago, Illinois 60637; and Division of Endocrinology (O.E.J.), Department of Medicine, University of Essen, Essen D-45122, Germany
Address all correspondence and requests for reprints to: Samuel Refetoff, M.D., The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: refetoff{at}medicine.bsd.uchicago.edu
Abstract
Inherited T4-binding globulin deficiency is caused by mutations in the T4-binding globulin gene located on the X chromosome. We describe herein three novel mutations in three different families producing complete T4-binding globulin deficiency. The proposita of a family from Harwichport is a female with XO Turner’s syndrome who expressed only the mutant T4-binding globulin allele. Her T4-binding globulin sequence has a 19-nucleotide deletion in the distal portion of exon 4. This causes a frameshift and a premature stop at codon 384 of the mature protein. Structure analysis with the Swiss PDB-Viewer revealed that this mutation removes ß-strand s5B from the core of the T4-binding globulin molecule, leading to a severe folding defect that is likely to prevent synthesis and secretion. The propositi of complete T4-binding globulin deficiency 7 and 8 were 7-month-old and 3-wk-old male infants who were identified because of low serum T4 levels detected during neonatal screening. Sequencing of complete T4-binding globulin deficiency 7 revealed a single nucleotide deletion, a G at position 2690 in exon 3. This leads to an alteration of the amino acid sequence starting at codon 283 and a premature stop at codon 301. Complete T4-binding globulin deficiency 8 also has a deletion of the first nucleotide of exon 4, a G at position 3358. This leads to a frameshift and a premature stop at codon 374. As in the case of complete T4-binding globulin deficiency J, which has also a nucleotide deletion but downstream (position 3421) and a stop at codon 374, these two T4-binding globulin mutants undoubtedly have a defect in intracellular transport and therefore fail to be secreted. This explains the lack of T4-binding globulin in the hemizygous affected subjects.
T4-BINDING GLOBULIN (TBG) is a 54-kDa acidic glycoprotein synthesized by the liver (1). It is composed of a single polypeptide chain of 395 amino acids. TBG is encoded by a single gene copy located on the long arm of the human X chromosome (Xq22.2) (2, 3, 4). With one exception (5), inheritance of TBG abnormalities, including TBG deficiency and TBG excess, follows an X-linked pattern. TBG deficiency can present as partial TBG deficiency or complete deficiency (TBG-CD), depending upon the serum TBG concentration in hemizygotes. The defects are fully manifested in hemizygous males but only partially in heterozygous females (6). However, variable phenotypes in females have been observed in some instances of selective inactivation of the X chromosome carrying the normal TBG gene of the two alleles and in a female with XO Turner’s syndrome (7, 8).
Seven distinct mutations have been identified in subjects with TBG-CD. Six have truncated molecules because of an early stop codon. These are TBG-CDB (Buffalo) (9) with one nucleotide substitution, TBG-CD6 (10), TBG-CDJ (Japan) (11), TBG-CDY (Yonago) (12), and TBG-CDN (Negev) (13), each with one nucleotide deletion, and TBG-CDK (Kankakee) (14), with one nucleotide addition caused by a mutation in the acceptor splice junction. Substitution of the normal Leucine 227 with a Proline, in TBG-CD5 (15), caused failure of secretion of the variant molecule because of aberrant posttranslational processing (16).
We describe herein three novel mutations in the TBG gene causing TBG-CD. The proposita of TBG-Harwichport (TBG-CDH) is a female with XO Turner’s syndrome. Her TBG gene has a 19-nucleotide deletion at the 3' end of exon 4. The other two, TBG-CD7 and TBG-CD8, each have one nucleotide deletion in exon 3 and exon 4, respectively. All three produce truncated TBG molecules.
Materials and Methods
Subjects
The proposita of TBG-CDH (III-3), a phenotypic female, was first seen in 1965 at the age of 13 yr with the chief complaints of retarded growth, primary amenorrhea, and absence of secondary sex characteristics. The details of the patient’s history, physical examination, laboratory test results, and those of members of her family were previously reported (8). In brief, she demonstrated the stigmata of Turner’s syndrome. A buccal smear was negative for Barr bodies and karyotyping revealed 45,XO chromosome pattern consistent with Turner’s syndrome. Laboratory studies in 1965 showed low PBI (an estimate of the serum T4 concentration), normal free T4 index (FT4I) and undetectable TBG. No inhibitor of T4 binding to TBG was found, and there was no evidence of T4 deficiency at the tissue level. The amount of T4 degraded each day was normal. The pedigree and thyroid function test results are shown in Fig. 1.
The affected males of TBG-CD7 (II-1) and TBG-CD8 (III-1) were 7-month-old and 3-wk-old infants, respectively. Both were identified because of low serum T4 levels detected through neonatal screening programs for hypothyroidism in California and in the province of Quebec, respectively. The diagnosis of complete TBG deficiency was subsequently confirmed. Family members were studied. These included the parents of TBG-CD7 and maternal grandfather of TBG-CD8. The study protocols were approved by the institutional review board, and informed consents were obtained from all individuals who participated in the study.
Tests of thyroid function
Serum total T4, total T3, TSH, and TBG concentrations were measured by RIAs. The FT4I was calculated as the product of the serum TT4 concentration and the T4-resin uptake value. TBG capacity was measured as previously described (17). Thyroid peroxidase and Tg autoantibodies were measured by agglutination.
Sequencing of the TBG gene
Genomic DNA was extracted as previously described
(18) from peripheral white blood cells of the propositi of
TBG-CDH, TBG-CD7, and the grandfather of TBG-CD8. Genomic DNA
fragments were amplified by PCR using specific oligonucleotide primers.
All PCRs were performed in a volume of 100 µl with 8 µmol/liter of
each primer and buffer containing 2.5 mmol/liter magnesium, 10
nmol/liter desoxynucleotide triphosphates, and 0.2 U
Taq DNA polymerase. Amplified sequences included the entire
four coding exons with splice junctions, the 5' untranslated exon 0,
and the promoter region of the TBG gene. Primers and PCR conditions
used to amplify exons 2, 3, and 4 were as previously reported
(9). Primers used to amplify exons 1 and 0 with the
promoter region are shown in Table 1
. PCR
conditions were denaturation at 94 C for 1 min, annealing at
temperatures shown in Table 1 for 1 min, and extension at 72 C for 1
min, for a total of 38 cycles. PCR products were sequenced directly
using an automated fluorescence-based cycle sequencer (ABI,
Perkin-Elmer Corp., Foster City, CA). Primers used for
sequencing were those for DNA amplification. Additional internal
primers used for sequencing are listed in Table 1. Of note, nucleotide
numbering is different from that in GenBank (accession number L13470).
Position +1 in this publication is the adenine of the ATG at the
translation start point, and -1 is the preceding nucleotide with
numbering in the 3' to 5' direction. These two nucleotides correspond
to positions 4319 and 4318 in GenBank, respectively, and are
nucleotides +1668 and +1667 published by Hayashi et al.
(3).
|
The crystal structure of TBG is not available but assumed to be
very similar to 
1-proteinase inhibitor and
other members of the superfamily of serine proteinase inhibitors
(serpins) (19). Thus, a model of wild-type and mutant TBGs
was obtained by submitting a homology structure of intact
1-proteinase inhibitor (Entrez Structure: 1PSI
and 1QLP) with the TBG sequence (Entrez Nucleotide: L13470; Entrez
Protein: AAA16067) to the automated protein modeling server SWISS-MODEL
running at the GlaxoWellcome Experimental Research in Geneva,
Switzerland (20). The structure model was modified and
refined using the Swiss-PDB Viewer (GlaxoWellcome Experimental
Research) and POV-Ray programs (POV-Ray Team, Indianapolis, IN).
Results
Thyroid function tests
The pedigree of the three families and thyroid function test
results of their members are shown in Figs. 1, 2, and 3. Those of
the Harwichport family were previously
described in detail and are briefly summarized here (Fig. 1). They show
that the mother (II-4) and half-sister (III-1) of the proposita are
partially TBG deficient, and her half-brother (III-2) and grandfather
(I-2) have the TBG-CD phenotype. The inheritance is consistent with
an X-linked pattern and permitted identification of the maternal origin
of the single X chromosome in the proposita.
|
|
|
). His mother (I-1) demonstrated partial TBG
deficiency with a low serum T4 level of 50
nmol/liter before pregnancy. During the time of the study, she was 5
months pregnant. Her TBG concentration, which should be normally
elevated because of the effect of E, was in the lower range of normal
and her serum T4 was still low at 63 nmol/liter.
Thyroid test results of his father (I-2) were all within the normal
range.
The propositus of TBG-CD8 (III-1) had low serum
T4 and T3 levels but a
normal TSH concentration for age (Fig. 3). His serum TBG was
undetectable. Blood samples for DNA extraction could not be obtained
from him or his parents, but a sample was provided by his affected
maternal grandfather (I-2). At that time, he was being treated with
T4. Despite a high FT4I and suppressed TSH level,
indicating overreplacement, his serum T4 and
T3 concentrations remained low. This suggests
that the grandfather also has TBG deficiency, which was transmitted to
his daughter (II-2) and through her, to the propositus.
TBG sequences and structure analysis
Sequencing of the TBG gene from the Harwichport proposita
(Fig. 4A) revealed a 19-nucleotide
deletion in exon 4, from position 3515 to 3524. This causes a
frameshift, leading to the substitution of two amino acids and a
premature stop (TGA) at codon 384, a deletion of 12 amino acids.
Structure analysis showed that this modification results in removal of
ß-strand s5B from the core of the TBG molecule, implicating a severe
folding defect of the molecule (Fig. 5).
|
|
). This causes an alteration of the amino acid
sequence starting at codon 283 (TTG 
TTT, Leu Phe) and a
frameshift downstream. This leads to a premature stop (TGA) at codon
301, producing a molecule missing approximately one-fifth of the
carboxyl terminus (Fig. 5). This mutation must not be confused with
TBG-P (Poly), a polymorphism without any changes of the properties of
the molecule. The TBG-P has a single amino acid substitution at codon
283 (TTGTTT, LeuPhe) (6).
The TBG-CD8 sequence has a deletion of a guanine at position 3358,
which is the first nucleotide of exon 4 (Fig. 4C). This also leads to a
frameshift and a premature stop (TGA) at codon 374 (Fig. 5).
Discussion
TBG-CD is defined as virtual absence of TBG in serum or a concentration less than 0.5 µg/dl in hemizygotes (XY males or XO females) who express only the mutant allele (6). This level corresponds to 0.03% of the average serum TBG concentration in adults. The prevalence is approximately 1:15,000 newborn males (6). Until now, seven mutations causing TBG-CD have been described, six of which (9, 10, 11, 12, 13, 14) present early stop codons and one has a missense mutation (15).
Herein we describe three novel mutations in three different families causing TBG-CD. All propositi had undetectable TBG concentrations. The proposita of TBG-CDH is an XO female who expressed only the TBG-CD allele. Her TBG sequence has a 19-nucleotide deletion in the distal portion of exon 4, the largest deletion ever described in TBG mutants. This causes a frameshift and early termination of translation at codon 384, lacking 3% of the TBG molecule. Structure analysis of this mutant TBG, which has two substituted amino acids and 12 deleted amino acids at the carboxyl terminus, reveals that this modification essentially removes the ß-strand s5B from the core of the molecule (19). This leads to a severe folding defect, an important step of posttranslational acquisition of the molecule’s tertiary structure before being secreted from liver cells (6). We conclude that this is the cause of TBG-CDH in this family and explains why the deletion of only a small portion of the TBG molecule can cause the TBG-CD phenotype.
TBG-CD7 and CD-8 each have one nucleotide deletion in exon 3 and exon 4, producing premature stops at codons 321 and 374, respectively. Although we did not determine the mechanism responsible for the absence of these mutant TBG molecules in serum, a previous study (11) has been performed with another TBG-CD, TBG-CDJ (Japan). This TBG mutant has a deletion at the first base of the codon for amino acid 352 (exon 4), leading to the premature termination producing a TBG molecule of 373 amino acids. Expression of the cDNA in COS-1 cells revealed complete absence of TBG secretion. Additionally, subcellular fractionation showed that most of TBG-CDJ was located in the rough endoplasmic reticulum compartment, suggesting the lack of intracellular transport of the truncated TBG molecule. It is thus expected that TBG-CD7 and -CD8, which produce TBG molecules of 320 and 373 amino acids, also by a single nucleotide deletion, will have a similar defect of intracellular transport and secretion. This explains the inability to detect TBG in the serum of subjects harboring these mutations.
Acknowledgments
We thank Dr. Hebert Selenkon (Boston, MA), Dr. Jean H. Dussault (Quebec City, Canada), and Dr. B. Sheikholislam (Carmichael, CA) for obtaining blood samples from members of the Harwichport, TBG-CD8, and TBG-CD7 families, respectively. We also thank Dr. Neal H. Scherberg and the technical staff of the Endocrinology Laboratory at the University of Chicago for performing some of the tests of thyroid functions. Special thanks are due to the members of the three families for their consent to participate in this study.
Footnotes
This work was supported in part by NIH Grants RR 00055, DK 15070, and DK 07011; funds from Blum-Kovler and Tivoli Wein Katz; and the Deutsche Forschungsgemeinschaft (DFG Ja 671/1-3; to O.E.J.).
Abbreviations: FT4I, Free T4 index; TBG, T4-binding globulin; TBG-CD, complete TBG deficiency.
Received April 13, 2001.
Accepted June 6, 2001.
References
1-
proteinase inhibitor and thyroxine-binding globulin properties. J
Biol Chem 274:15046–15051This article has been cited by other articles:
 |
|
 |
|
  L. C. Moeller, A. Fingerhut, H. Lahner, H. Grasberger, B. Weimer, J. Happ, K. Mann, and O. E. Janssen C-Terminal Amino Acid Alteration rather than Late Termination Causes Complete Deficiency of Thyroxine-Binding Globulin CD-NeuIsenburg J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3215 - 3218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Fingerhut, S. Reutrakul, S. D. Knuedeler, L. C. Moeller, C. Greenlee, S. Refetoff, and O. E. Janssen Partial Deficiency of Thyroxine-Binding Globulin-Allentown Is Due to a Mutation in the Signal Peptide J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2477 - 2483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Prapunpoj, S. J. Richardson, and G. Schreiber Crocodile transthyretin: structure, function, and evolution Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2002; 283(4): R885 - R896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Reutrakul, A. Dumitrescu, P. E. Macchia, G. W. Moll Jr., H. Vierhapper, and S. Refetoff 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 J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1045 - 1051. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
