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Journal of Clinical Endocrinology and Metabolism
Copyright © 1998 by The Endocrine Society
Vol. 83, No. 10
Printed in U.S.A.
Complete Thyroxine-Binding Globulin (TBG) Deficiency
Produced by a Mutation in Acceptor Splice Site Causing
Frameshift and Early Termination of Translation
(TBG-Kankakee)*†
GISAH A. CARVALHO, ROY E. WEISS,
AND
SAMUEL REFETOFF
Departments of Medicine (G.A.C., R.E.W., S.R.), Pediatrics (S.R.), and the J. P. Kennedy, Jr. Mental
Retardation Research Center (S.R.), The University of Chicago, Chicago, Illinois 60637
ABSTRACT
Fourteen T4-binding globulin (TBG) variants have been identified
at the gene level. They are all located in the coding region of the gene
and 6 produce complete deficiency of TBG (TBG-CD). We now describe
the first mutation in a noncoding region producing TBG-CD. The
proband was treated for over 20 yr with L-T4 because of fatigue
associated with a low concentration of serum total T4. Fifteen family
members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in
serum. Sequencing of the entire coding region and promoter of the
T
4-BINDING
globulin (TBG) is a 54-kDa glycoprotein
synthesized in the liver (1). TBG defects are inherited
as X chromosome-linked traits, compatible with the presence
of a single copy of the TBG gene on the X chromosome (2, 3).
TBG deficiencies are divided into three types depending on
the level of TBG in serum: complete deficiency (TBG-CD),
partial deficiency (TBG-PD), and excess (TBG-E) (1, 2). These
defects are fully manifested in hemizygous males but only
partially in heterozygous females, because dosage compensation for X chromosome-linked genes between males and
females is achieved by random inactivation of one of the X
chromosomes in females (4). Although in most instances
random inactivation produces TBG levels in heterozygous
females that are intermediary between those in affected and
nonaffected males (2), variable phenotypes have been observed in some instances of selective inactivation of the X
chromosome carrying one of the two alleles (5).
Fourteen distinct mutant TBGs have been identified at the
gene level. Six mutations produce TBG-CD: TBG-CD5 (6),
TBG-CD6 (7), TBG-CDJ (Japan) (8), TBG-CDY (Yonago) (9),
TBG-CDB (Buffalo) (10), and TBG-CDBe (Bedouin) (11).
Received May 18, 1998. Accepted June 22, 1998.
Address all correspondence and requests for reprints to: Samuel Refetoff, The University of Chicago, MC3090, 5841 South Maryland Avenue
Chicago, Illinois 60637. E-mail:[email protected].
* This work was supported in part by the National Institutes of Health
Grants DK-02081 and DK-17050 and the Seymour J. Abrams Thyroid
Research Center. Presented in part at the 70th Annual Meeting of the
American Thyroid Association, October 15–19, 1997, Colorado Springs,
Colorado.
† This paper is dedicated to the memory of Dr. Niall O’Meara who
as a fellow in the Section of Endocrinology contributed patients to this
study and whose enthusiasm for endocrinology was an inspiration to all
of us.
TBG gene revealed no abnormality. However, an A to G transition was
found in the acceptor splice junction of intron II that produced a new
HaeIII restriction site cosegregating with the TBG-CD phenotype.
Sequencing exon 1 to exon 3 of TBG complementary DNA reverse
transcribed from messenger RNA of skin fibroblasts from an affected
male, confirmed a shift in the ag acceptor splice site. This results in
the insertion of a G in exon 2 and causes a frameshift and a premature
stop at codon 195. This early termination of translation predicts a
truncated TBG lacking 201 amino acids. (J Clin Endocrinol Metab
83: 3604 –3608, 1998)
TBG-CD5 has a single amino acid substitution causing aberrant posttranslational processing. The other five TBG-CDs
have truncated molecules caused by early termination of
translation caused by a single nucleotide substitution (TBGCDB) or by a frameshift caused by a nucleotide deletion
(TBG-CD6, TBG-CDJ, TBG-CDY, and TBG-CDBe) (Fig. 1).
Until now, no mutations have been identified in noncoding regions of the TBG gene that cause TBG abnormalities.
We describe here the first such mutation in a North American
family of German ancestry with the TBG-CD phenotype.
Sequence analysis of genomic DNA (gDNA) revealed an A
to G transition in the acceptor splice junction of intron II
(ag3gg). Genotyping, using a mutation specific restriction
site, confirmed the presence of this nucleotide substitution in
all affected family members.
Sequencing of TBG complementary DNA (cDNA) transcribed from fibroblast messenger RNA (mRNA) confirmed
a shift in the ag acceptor splice site resulting in the insertion
of a G in exon 2 that produces a frameshift with a premature
stop codon. This premature stop in the transcript predicts a
truncated TBG lacking the C-terminal 201 amino acids.
Materials and Methods
Patients
The proband (II-3), a 55-yr-old woman, was treated for 20 yr with L-T4
for what was presumed to be hypothyroidism based on low total T4 (TT4)
concentration and fatigue. The diagnosis of TBG deficiency was confirmed when 15 family members were studied, some showing low TT4
inherited as an X chromosome-linked trait. Skin biopsies were obtained
from subjects II-3 and II-2 (Fig. 2A) for fibroblast cultures. All subjects
gave informed consent for genetic testing in accordance with the University of Chicago Institutional Review Board.
3604
SPLICE SITE MUTATION OF TBG-CD KANKAKEE
3605
FIG. 1. Mutations causing TBG-CD
(left) and TBG-PD (right) and their location on the gene. Both mutant nucleotide and resulting amino acid change
are shown in bold letters. Y, Yonago; Be,
Bedouin; B, Buffalo; J, Japan; SD, San
Diego; G, Gary; M, Montreal; S, Slow; A,
Aborigine; Cgo, Chicago; and Q, Quebec. For detailed description see Ref. 2.
FIG. 2. Pedigree and genotype of family with TBG-CD Kankakee. A, Pedigree aligned with restriction enzyme
analysis of gDNA samples from respective individuals. B, Genotype analysis.
Replacement of an A to a G at acceptor
splice site in intron II creates a new
recognition site for enzyme HaeIII that
digests amplified fragment reducing its
size from 467 to 411 bp. Affected females are heterozygous; affected and
normal males show only mutant (411
bp) or normal (467 bp) allele, respectively. Mol wt, DNA size marker (Fx,
digested with HaeIII).
Tests of thyroid function
Serum TT4, total T3 (TT3), total reverse T3 (TrT3), TSH, and TBG
concentrations were measured by RIAs. The serum free T4 index (FT4I)
was calculated as the product of the serum TT4 concentration and the
T4-resin uptake value. P values were calculated using the unpaired
Student’s t test.
phenol/guanidine isothiocyanate (Trizol, Gibco BRL, Gaithersburg,
MD). The integrity of the RNA was assessed by formaldehyde-agarose
gel electrophoresis. cDNA was synthesized by RT of very small amounts
of TBG mRNA present in fibroblasts (illegitimate transcription) (12).
Avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI) and oligo-dT primer were used to prepare the first strand of
cDNA. gDNA was isolated from peripheral blood leukocytes (13).
Preparation of RNA, cDNA, and gDNA
Total RNA was extracted from cultured skin fibroblasts and peripheral blood leukocytes from one normal individual, one heterozygous
female, and one affected hemizygous male. RNA was extracted with
Sequencing of TBG gene
gDNA fragments were amplified by PCR using specific oligonucleotide primers (7). Amplified sequences included the entire coding re-
3606
CARVALHO ET AL.
gion, splice junctions, and the promoter region of the TBG gene. Amplified DNA segments were subcloned into M13 bacteriophage (New
England Biolabs, Inc., Beverly, MA) or pGEM T plasmids (Promega
Corp.) and sequenced by the dideoxynucleotide chain termination
method (Sequenase version 2.0, United States Biochemical Corp., Cleveland, OH).
Two consecutive PCRs were needed to amplify a TBG cDNA fragment across exon 2, Oligonucleotide primers for the first PCR were:
59-GCATCTGATCTGTTCACTGAATTTC-39 sense, nucleotide (nt) 339 –
364 and 59-GTTTCAGACCATTGTCCTCT-39 antisense, nt 2798 –2817. A
second, nested PCR, used the following oligonucleotides: 59-CTAATTCAAGACCTCAAGC-39 sense, nt 562–580 and 59-GAAAACTTTGGAACAAAC-39 antisense, nt 2690 –2707. The conditions of the PCR
reactions were as follows: initial denaturation at 94 C for 3 min, followed
by 35 cycles consisting of 94 C for 1 min, 55 C for 1 min, 72 C for 1 min,
and a final extension at 72 C for 15 min. Automated fluorescence-based
cycle sequencing (ABI, Perkin Elmer, Foster City, CA) was performed
directly with the PCR products. The oligonucleotide primers used were
the same as those used in the second PCR.
Genotyping
The replacement of the normal nucleotide A with a G in the acceptor
splice junction of intron II (ag3gg) creates a new restriction site for the
enzyme HaeIII. gDNA from peripheral blood leukocytes was amplified
by PCR using the primers employed to amplify exon 2 from gDNA for
sequencing (7). Amplified gDNA fragments were separated by electrophoresis in 3% agarose gel before and after digestion with HaeIII and
photographed under ultraviolet light after staining with ethidium bromide. The allele containing the wild-type sequence resists the endonuclease and retains its original size of 467 bp, whereas the mutant allele
is digested into two fragments, 411 and 56 bp (Fig. 2B).
JCE & M • 1998
Vol 83 • No 10
phenotype of low total serum iodothyronine levels and low
TBG concentration inherited in a X chromosome-linked pattern (Figs. 2A and 3). The 4 affected males (II-1, II-2, II-4, and
II-9) presented with low TT4 levels of 23.5 6 1.2 nmol/L
(mean 6 sd) compared with those of unaffected subjects of
106.0 6 12.8 nmol/L, P , 0.0005. Serum TT3 was also significantly lower in the affected males (1.0 6 0.1 nmol/L, P ,
0.0005) as compared with the unaffected individuals (2.0 6
0.2). Serum TBG level of all affected males was undetectable,
, 0.5 mg/L (normal range 11–21 mg/L, P , 0.0005). The 6
affected females (I-1, I-2, I-5, II-3, II-6, and II-7) had on the
average low serum TT4 (52.5 6 19.3 nmol/L, P , 0.001) and
TT3 (1.2 6 0.2 nmol/L, P , 0.0005) levels. However, in only
3 of them were values below the lower limit of normal. In
contrast, all affected females had serum TBG concentrations
below the limit of normal (6.8 6 1.9 mg/L). The 5 unaffected
members of the family showed normal levels of total iodothyronines and TBG in serum (Fig. 3). All 15 individuals had
normal serum FT4I and TSH, except for one affected female
with suppressed TSH caused by treatment with L-T4. Serum
FT4I was significantly lower in the affected males, 87.5 6 10.1
(normal range 76 –135, P 5 0.01), as compared with unaffected individuals (110.68 6 8.29) caused by underestimation
of the resin T4 uptake value in samples from the affected
males.
Genetic analysis
Results
Thyroid function tests
The clinical diagnosis of TBG deficiency was confirmed in
10 of 15 family members who showed cosegregation of the
gDNA from an affected male with TBG-CD (II-2) was
sequenced. The sequences of the exons and the promoter
region did not show any abnormality. However, sequences
of the flanking introns revealed an A to G transition in nu-
FIG. 3. Tests of thyroid function in individual members of family. One affected female with suppressed TSH was on L-T4 therapy.
SPLICE SITE MUTATION OF TBG-CD KANKAKEE
cleotide 1671 of the acceptor splice site in intron II (Fig. 4). The
mutation was present in all affected family members as confirmed by genotyping. Affected and normal males showed
only the mutant allele or normal allele, respectively, and
heterozygous females had both normal and mutant allele
(Fig. 2B).
To evaluate the effect of this mutation on the splicing
pattern, it was necessary to determine the sequence of TBG
mRNA. TBG mRNA is transcribed in liver and therefore is
not readily accessible for sampling. We attempted illegitimate amplification of TBG mRNA from peripheral blood
leukocytes, as previously accomplished for the TSH receptor
and other mRNA (14), and failed on several attempts, though
the extracted RNA was of good quality. However, we did
succeed in synthesizing TBG cDNA using RNA extracted
from cultured skin fibroblasts. The sequence of this TBG
cDNA transcribed from fibroblasts of a normal subject was
identical to that transcribed from liver mRNA of an unrelated
person with normal TBG, confirming the same pattern of
splicing in both tissues.
To determine the effect of the mutation on the patient’s
TBG transcripts, RNA from fibroblasts was processed in the
same manner. Direct sequencing of the PCR cDNA product
from an affected male (II-2) revealed a shift of one nucleotide
upstream of the authentic consensus ag acceptor splice site.
Sequencing of cDNA from a heterozygous female (II-3) demonstrated both the mutant and the normal allele. This mutation resulted in the insertion of the G in exon 2 causing a
frameshift with a premature stop at codon 195 (Fig. 5). As a
result of this mutation, the new acceptor splice site better
matches the ideal consensus sequence compared to the wildtype sequence (Fig. 6).
Discussion
Mutations are randomly distributed throughout the TBG
gene. There are no hot spot areas, and there is no correlation
between the degree of TBG deficiency and location of the
mutation (2). Until now, six TBG-CDs have been described,
FIG. 4. Section of a sequencing gel of TBG gene from gDNA displaying A to G transition of nucleotide 1671 in intron II at acceptor splice
site junction. Lower case letters are in intron II; upper case letters are
in exon 2, and substituted nucleotide is in bold and detached.
3607
FIG. 5. Direct sequencing of TBG gene from cDNA showing codons
186 through 189. Normal sequence (common type, TBG-C) is compared with sequence of affected male (hemizygous). Latter sequence
shows insertion of G (in bold) in exon 2 causing a frameshift downstream to amino acid 187 and ultimately a stop codon 195. Altered
codons 188 and 189 are also indicated in bold.
FIG. 6. mutation a3g in nucleotide 1671 (*) creates a new ag acceptor
splice site (four nucleotides shown in bold) in intron II. Compared
with wild-type sequence (also in bold), new acceptor recognition sequence is a better match to consensus sequence. This shifts the ag
splice site resulting in insertion of a G (short arrow) in exon 2 and
causing a frameshift that produces an early stop at codon 195.
all located in the coding region of the gene. Five present early
stop codons (nucleotide deletion or nonsense mutation) and
one has a missense mutation (6 –11).
Herein we describe the first splice junction mutation causing TBG-CD (TBG-CD Kankakee). A novel A3 G transition
in the acceptor splice site of intron II resulted in an one
nucleotide shift in the acceptor splice site, predicting a truncated protein containing only the first 194 of 395 amino acids
in the wild-type molecule. Similar to the other five TBG-CDs
described, this mutation has an early stop codon. Instead of
deletion of a nucleotide in a coding exon, the frameshift is
caused by a single nucleotide insertion in exon 2.
It has been estimated that up to 15% of known single base
pair substitutions causing human genetic disease disrupt the
normal splicing of mRNAs (15). For genes expressed in a
tissue-specific manner, analysis of the consequences of such
defects on mRNA processing has often been hindered by the
inaccessibility of the mRNA transcripts. Ectopic transcript
analysis (illegitimate transcription) is an invaluable tool for
the characterization of defects of mRNA splicing, although
splicing patterns exhibited by normal and ectopic transcripts
might not be identical (16). We found that processed TBG
3608
CARVALHO ET AL.
mRNA transcripts from both expressing (liver) and nonexpressing (fibroblast) cells were indistinguishable at the site of
the mutation. Although illegitimate transcription should,
theoretically, amplify minute amounts of any mRNA present
in all tissues, the amount of mRNA in nonexpressing tissues
depends not only on gene-specific but also tissue-specific
influences (17). This explains why illegitimate amplification
of TBG mRNA from peripheral blood leukocytes failed,
whereas that from skin fibroblasts was successful.
The splice junction mutation causing TBG-CD Kankakee
creates a new acceptor splice site one nucleotide upstream of
the authentic acceptor splice site adding an intronic G to the
downstream exon. This is in agreement with the majority of
acceptor splice site mutations reported (15, 18). Exonic and
intronic recognition sequences have an established role for
splice-site selection, also important are the consensus sequences in the immediate vicinity of the exon-intron borders
(19 –21). A consensus value (CV) can be calculated by comparing the actual nucleotide sequence with the mutation
(CVM) to the ideal consensus sequence (CVN) (22). A perfect
match would have a CV score of 1. The calculated CV for the
four nucleotide (11 to 23) wild-type acceptor splice recognition sequence using the primate nucleotide weight table of
Shapiro and Senapathy (22) is 0.66 compared with the CV of
0.83 for the new sequence resulting from the mutation.
We have, therefore, described a novel mechanism for the
molecular basis of complete TBG deficiency identified by
sequencing the intron exon junction of the TBG gene. Although this mechanism is not uncommon in other genetic
defects, it has not been previously described in TBG defects
of which 15 have been so far identified.
Acknowledgments
We thank Dr. Neal H. Scherberg and the Technical Staff of the Endocrinology Laboratory for performing the tests of thyroid function, and
all family members for their consent to participate in this study.
3.
4.
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21.
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