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Original Studies |
-Subunit, and Variable Circulating Thyrotropin (TSH) Levels as Hallmark of Central Hypothyroidism due to Mutations of the TSHß Gene1
Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore Istituto de Ricovero e Cura a Carattere Scientifico (M.B., P.B.-P.); Istituto Auxologico Italiano Istituto de Ricovero e Cura a Carattere Scientifico (L.P.); and Department of Pediatrics, University of Milan, H. S. Raffaele Istituto de Ricovero e Cura a Carattere Scientifico (M.C.P., G.W., G.C.), 20100 Milan, Italy
Address all correspondence and requests for reprints to: Luca Persani, M.D., Ph.D., Laboratorio di Ricerche Endocrinologiche, Istituto Auxologico Italiano IRCCS, Via Ariosto 13, 20145 Milan, Italy. E-mail: persani{at}auxologico.it
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit were recorded. Despite the fact that
mutant TSHß lacks 60% of the C-terminal amino acid sequence, it
forms with the -subunit a heterodimer with preserved
immunoreactivity in some TSH measurement methods, but the mutant
heterodimer is completely devoid of bioactivity. In conclusion, high
circulating free glycoprotein -subunit levels, variable TSH levels,
and, possibly, hyperplastic pituitary gland are the hallmark of ICH due
to mutations of the TSHß gene. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Since the cloning of the TSHß gene (16, 17), three
different TSHß mutants have been described in several familial
settings of congenital ICH. All of the affected patients had homozygous
mutations in exon 2 or 3 of the TSHß gene. Two mutations located in
the N-terminal part of the subunit (Q12X and G29R) prevented the
assembly of the heterodimer, and the affected patients were
biochemically characterized by undetectable circulating TSH
concentrations (7, 8, 9, 10). On the contrary,
heterodimerization was not completely prevented by a particular
frameshift mutation (C105
) in the TSHß carboxyl-terminal with a
premature stop codon in the "seat-belt region" of TSH
(C105,114X), and the affected patients had variable serum TSH
concentrations depending on the TSH measurement method employed
(11, 12, 13, 14). In fact, in noncompetitive assays the ability of
anti-TSH monoclonal antibodies to bind epitopes still present on the
mutant heterodimer leads to the formation of the "sandwich" and,
therefore, to the measurement of certain amounts of TSH. Recently,
another form of inherited central hypothyroidism has been recognized as
being due to an inactivating mutation of the TRH receptor gene, a
situation accompanied by a mild hypothyroid state and normal basal TSH
and PRL levels that are unresponsive to TRH stimulation
(15).
Recognition of the genetic defect underlying ICH is often difficult due
to the low number of cases reported to date and the consequent lack of
clear-cut clinical and biochemical diagnostic criteria. ICH with
undetectable or low TSH levels is, in general, considered suggestive of
TSHß mutations, in particular when high levels of glycoprotein
hormone -subunit (GSU) are concomitantly recorded (11, 18). Conversely, resistance to TRH due to TRH receptor mutations
is suspected on the basis of blunted responses of both TSH and PRL to
the TRH test (15).
In all of the above patients, the hypothyroid state is not detected at neonatal screening for congenital hypothyroidism, as the only test used in most centers is TSH evaluation on a dry blood spot (19). The diagnosis is, therefore, delayed, and the hypothyroid state is often severe (20). The delayed start of L-T4 replacement therapy may result in slight to severe mental and growth retardation (7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20).
Here, we report a girl with congenital ICH carrying a novel homozygous mutation in the TSH ß-subunit gene. The data collected along with those previously reported allow delineating the diagnostic criteria of such a rare pituitary-thyroid disorder.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Early studies. The propositus is an Egyptian girl, normally
delivered at term after an uneventful gestation. Her birth weight and
length were 2250 g and 46 cm (10th percentile), respectively. She
is the first of four sisters. The parents are consanguineous (first
cousins). She was referred for feeding problems, jaundice, growth
(length, 51 cm), and neuromotor delay at 75 days after delivery.
Physical examination showed umbilical hernia, severe hypotonia, large
posterior fontanel, hoarse cry, dry skin, and epicanthus. Routine
biochemical tests were normal. Thyroid function tests showed severe
hypothyroidism in the presence of normal serum TSH (Table 1
) and absence of antithyroid
autoantibodies, including those against the TSH receptor. TRH
administration was followed by an impaired TSH response (basal, 3.4
mU/L; peak, 4.8 mU/L). Other pituitary functions, including PRL, GH,
and adrenal axes, were normal.
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Follow-up studies. The patient was reevaluated at 21 months
of age during L-T4 therapy (Table 1
).
She was 78 cm in height (third percentile). The compliance with therapy
was judged to be good on the basis of normal circulating levels of free
T4 (FT4) and
FT3. A sella computed tomography scan showed a
completely normalized pituitary size. The GH response to arginine
(basal, 2.0 µg/L; peak, 10.0 µg/L) and basal PRL levels (294 mU/L;
normal, 100–500) were normal. In that period, the patient’s
family moved back to Egypt, and subsequent follow-up became difficult.
The patient was seen again at the age of 7 yr while receiving
L-T4 treatment. Bone age was delayed
(
1 yr), and her growth rate was at the third percentile. An
improvement of the neuromotor delay was noted, and hearing function was
normalized, but her neurological conditions, including language and
visual-spatial abilities, were impaired. Her intelligence quotient
(score, 65) was low. Although FT4 and
FT3 levels were within the normal range, the
L-T4 dose was not adequate for
patient age and body weight (2.3 vs. an adequate regimen of
3–4 µg/kg BW·day) (21). The inadequacy of
substitutive therapy was subsequently confirmed by the finding of
-GSU levels much higher than those found in controls matched for
similar circulating levels of pituitary glycoprotein hormones.
One year later, at the age of 8 yr, the patient was reinvestigated at
our institutes both before and after
L-T4 withdrawal. Serum TSH was
measured by means of several immunometric assays. In addition, free
-GSU and basal and TRH-stimulated PRL levels were tested (Table 1
and Fig. 1). Blood samples for genetic
and biochemical studies were obtained from all family members after
informed consent of the parents was given.
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Immunoreactive TSH was measured using two second generation
methods [enzyme-linked immunosorbent assay (Roche,
Mannheim, Germany): sensitivity, 0.05 mU/L; Delfia (Wallac, Inc. Turku, Finland): sensitivity, 0.03 mU/L] and three third
generation methods [enzymatic chemiluminescent immunoassay
(Roche): sensitivity, 0.007 mU/L; Myria (Bouty, Sesto S.
Giovanni, Italy): sensitivity, 0.002 mU/L; AutoDelfia Ultra
(Wallac, Inc.): sensitivity, 0.005 mU/L]. The TSH
International Reference Preparation 80/558 was the reference
preparation for all of these assays. All methods, except the Delfia
Ultra kit, are two-site immunoassays based on the sandwich technique,
in which two monoclonal antibodies are directed against two separate
antigenic determinants on the TSH heterodimer. One monoclonal antibody
is directed against a specific /ß conformational antigenic site,
and the other is directed against a specific epitope on TSHß. The
Delfia Ultra Kit is based on the use of three monoclonal antibodies
conferring a very high specificity to the TSH assay; the capture
antibody is directed against an /ß conformational epitope of the
TSH heterodimer, whereas the other two monoclonal antibodies are
directed against two different antigenic sites of TSHß subunit
(three-site immunoassay). The cross-reactivity of free -GSU or
TSHß is absent in all of the above TSH immunoassays. Possible
interference (leading to an overestimation of serum TSH values) due to
the presence in the serum of heterophilic antimouse autoantibodies is
prevented by the use of chimeric (mouse-human) antibodies (in the
enzyme-linked immunosorbent assay and the enzymatic chemiluminescent
immunoassay) and/or the addition of mouse serum to the assay
buffer. To exclude methodological interference due to anti-TSH
antibodies or other circulating factors cross-reacting with the assay
monoclonal antibodies, serum dilution (1:2.5, 1:5, and 1:10) and
recovery (8, 16, and 32 mU/L TSH IRP 80/558 were added to the serum
samples) tests were carried out by using both second and third
generation Delfia assays.
Serum FT3 and FT4 levels
were measured by direct back-titration methods, using Delfia technology
(Wallac, Inc.). Serum levels of -GSU were evaluated by
a sensitive and specific two-site immunoradiometric assay (Biocode,
Sclessin, Belgium). Other hormones were evaluated by means of sensitive
and specific commercial kits.
DNA sequencing
Genomic DNA was extracted from peripheral whole blood using NUCLEON BACC2 (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy). The coding region of the TSHß gene was amplified using primers and PCR conditions previously described (12). PCR was performed in 100 µL reaction solution containing 400 ng genomic DNA, 1.5 mmol/L MgCl2, 40 pmol of each primer, and 0.5 U DNA Taq polymerase (Promega Corp., Madison, WI). The purified PCR products were directly sequenced using the Big Dye Terminator kit and the ABI 310 automated sequencer (PE Applied Biosystems, Fosters City, CA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Data collected at the age of 8 yr both before and after
L-T4 substitutive therapy withdrawal
showed normal circulating TSH concentrations in most of the
noncompetitive immunoassays (Table 1). TSH immunoreactivity was
undetectable in two immunoassays, including that showing the highest
specificity (Delfia Ultra). In both second and third generation Delfia
assays, dilution curves of samples collected after
L-T4 withdrawal were parallel to the
standard curve, and a recovery of 100 ± 2% was observed at the
TSH recovery test. Together, these findings show the lack of
methodological interference in the measurement of TSH
concentrations.
After withdrawal of L-T4 for 6 weeks,
the TRH test showed an impaired TSH response in two immunoassays,
whereas serum TSH immunoreactivity was always undetectable in the
remaining two (Fig. 1). Serum FT4 (basal, 2.1
pmol/L; at 120 min, 2.5 pmol/L) and FT3 (basal,
1.5 pmol/L; at 120 min, 1.1 pmol/L) did not increase after TRH
administration. Serum thyroglobulin levels were low both before and
after L-T4 therapy withdrawal. Serum
-GSU levels were extremely elevated and showed an exaggerated
response to TRH after withdrawal of
L-T4 treatment (Table 1 and Fig. 1).
Moreover, the high levels of -GSU recorded during
L-T4 substitutive therapy indicated
that the patient was undertreated, as also documented by the relatively
low FT4 concentrations. Serum PRL was at the
upper limit of the normal range in basal conditions (508 mU/L;
normal, 100–500) and increased normally in response to TRH
injection (PRL peak, 1552 mU/L).
The parents and three sisters of the proband had normal thyroid
function tests and -GSU levels (Fig. 2).
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Direct sequencing of the TSHß gene showed a homozygous C to T
transition in exon 3, at position +205 of the coding sequence
(17) (Fig. 2). The mutation leads to a premature stop at
codon 69, resulting in the synthesis of the mature mutated protein Q49X
(after excision of the signal peptide of 20 amino acids). The mutant
Q49X lacks 60% of the C-terminal tail of the mature protein. The
parents and 2 sisters were heterozygous carriers of the same
substitution (Fig. 2).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-GSU are detected. Normal secretion of the other pituitary
hormones, including PRL, rules out other forms of congenital
hypothyroidism, such as combined pituitary hormone deficiency
(1, 2, 3, 4, 5, 6) and possible mutation of the TRH receptor gene
(15).
TSHß mutations located in the amino-terminal part prevent subunit
assembly (Table 2). The peptide is
truncated after 11 amino acids in the case of the nonsense substitution
(Q12X) reported by Dacou-Voutetakis (9), whereas the
substitution (G29R) found in the Japanese cases (7, 8, 10)
alters the domain called the CAGYC region, which is a highly conserved
sequence among the ß-subunits of glycoprotein hormones (7, 22, 23). Microinjections of this mutant TSHß messenger ribonucleic
acid in Xenopus laevis oocytes lead to the formation of
conformationally altered peptides that could not associate with the
-subunit, indicating that the integrity of the CAGYC region is
required for correct subunit folding and regular dimer formation
(7, 22, 23). As expected, the above mutations are
accompanied by undetectable circulating TSH levels. On the contrary,
mutations in the carboxyl-terminal part of TSHß, such as C105,114X
(11, 12, 13, 14) and Q49X described here, do not prevent formation
of the heterodimer (Table 2). Hence, immunoreactive TSH may be detected
using both competitive and noncompetitive immunometric assays. In fact,
if the mutant TSH possesses the epitopes recognized by the assay
monoclonal antibodies, a sandwich may be formed, and a certain amount
of TSH may be measured. Indeed, the highest levels of TSH were measured
in competitive RIAs using polyclonal antibodies recognizing not only
the heterodimer but also the circulating free TSHß mutant
(14). Interestingly, the mutant (Q49X) heterodimer was not
recognized by highly sensitive and specific TSH methods, such as the
Delfia Ultra Kit, based on the use of three monoclonal antibodies
directed against three different epitopes on the TSH heterodimer. Of
course, possible methodological interference by circulating anti-TSH
antibodies or unknown factors mimicking TSH molecules should be ruled
out by appropriate tests, such as dilution and recovery tests.
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-subunit and maintains the conformation and bioactivity of the
hormone (11, 22). Moreover, different experimental
approaches have shown that the C-terminal part of TSHß (deleted in
the Q49X mutant) contains several domains required for high affinity
TSH receptor binding and signal transduction (22, 25)
(i.e. sequence 88–105 encompassing the seat belt region as
well as sequence 58–69 within the ß-hairpin ßL3 loop). Therefore,
mutations of the TSHß gene represent an additional cause of
discrepancy between immunoreactive and bioactive TSH levels commonly
found in central hypothyroidism (26, 27).
The extremely high circulating values of the common -GSU and their
exaggerated response to TRH or L-T4
withdrawal in the presence of low TSH, FSH, and LH represent a
fundamental marker of ICH due to TSHß gene mutations and indicate a
very active, although deranged, synthetic process within thyrotropes
(7, 11, 18). This is indirectly confirmed by the
hyperplastic pituitary gland found by magnetic resonance imaging at the
time of diagnosis and by the normalization of pituitary size during
L-T4 replacement therapy. These
findings indicate that the assembly of Q49X TSHß and the -subunit
itself is only partially conserved, so that a large amount of the
synthesized -subunit is circulating uncombined. Thus, the
carboxyl-terminal region of TSHß, lost in the Q49X mutant, appears to
be required for optimal stabilization and maintenance of the
heterodimer. Finally, it must be underlined that -GSU has been found
hypersecreted in the few cases of TSHß gene mutations in which it was
measured (Table 2) (11, 18). Moreover, the measurement of
-GSU is very useful in monitoring the adequacy of
L-T4 replacement therapy. In fact,
TSH measurement is not a good parameter to follow-up
L-T4 replacement therapy in patients
with any form of central hypothyroidism (28), and a
decrease in -GSU to an undetectable level was reported to occur in
two patients with G29R mutations during replacement therapy with
thyroid hormone (18). In the present case,
L-T4 undertreatment was revealed by
the finding of elevated -GSU values (Table 1), leading to the
adjustment of the daily L-T4 dose to
a higher level (i.e. 3.0 µg/kg BW·day).
Finally, the dramatic consequences of delayed L-T4 administration due to the missed neonatal diagnosis of congenital central hypothyroidism should promote further efforts aimed at improving neonatal screening programs to prevent cretinism in these cases. This might be achieved by the combined measurement of TSH and T4 levels in the dry blood spot.
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Acknowledgments |
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Footnotes |
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Received September 18, 2000.
Revised December 28, 2000.
Accepted January 8, 2001.
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References |
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