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Divisions of Pediatric Endocrinology (J.-M.V., J.D., G.G., P.E.M.) and Molecular Human Genetic (S.G.), University Children’s Hospital, 3010 Bern, Switzerland; and Dokuz Eylül Faculty of Medicine (A.B., P.C.), 35340 Izmir, Turkey
Address all correspondence and requests for reprints to: Prof. Dr. Primus E. Mullis, Division of Pediatric Endocrinology, University Children’s Hospital, CH-3010 Bern, Switzerland. E-mail: primus.mullis{at}insel.ch
Abstract
We identified a new nonsense mutation of the TSH-ß
subunit gene responsible for a severe isolated TSH deficiency
in two children from the same consanguineous kindred. These affected
children are homozygous for a C-to-T transition at nucleotide 654 of
the TSH-ß subunit gene, leading to the conversion of a
glutamine (CAG) to a premature stop codon
(TAG) in the codon 49 (Q49X). The resulting nascent
peptide does not contain the seat belt region (amino acid residues
88–105), a TSH-ß subunit region crucial for the dimerization with
the 
-subunit, and, hence, the correct secretion of the mature TSH
heterodimer is hampered. Free T3, free T4 as
well as basal TSH levels were extremely low in both affected
individuals and, importantly, TRH stimulations failed to increase serum
TSH, but not PRL, confirming isolated TSH deficiency. Using the new
StyI endonuclease restriction site generated by the
mutation, we confirmed that the affected children were homozygous for
the Q49X TSH-ß mutation whereas their unaffected parents as well as
their unaffected brother were heterozygous. Consequently, this isolated
TSH deficiency follows an autosomal recessive mode of
inheritance.
CENTRAL HYPOTHYROIDISM IS due mainly to acquired lesions (e.g. tumors, traumas) either in the pituitary (secondary hypothyroidism) or the hypothalamus (tertiary hypothyroidism) or both. However, the few congenital cases are principally due to mutations of homeobox genes involved in the pituitary development including HESX1, LHX3, PROP1, and POU1F1 (1, 2, 3, 4). Because more than one pituitary cell type is deficient in these genetic defects, they often lead to variable phenotypes of combined pituitary hormone deficiency (5).
Isolated central hypothyroidism remains a rare disease and is due
mainly to genetic deficit of ß-subunit of TSH (OMIM no. 188540). TSH
is a 28- to 30-kDa glycoprotein synthesized and secreted from
thyrotrophs of the anterior pituitary gland. It is a member of the
glycoprotein hormone family, which includes FSH, LH, and CG. The
glycoprotein hormones are heterodimeric cystine knot proteins
consisting of a common -subunit and a specific ß-subunit that
confer proper biologic effect onto each hormone (6). The
ß-subunit [118 amino acids (aa)] heterodimerizes noncovalently with
the -subunit through a segment termed "seat-belt" (aa 88–105),
because it wraps around the -subunit long loop (7).
Importantly, therefore, all the genetic isolated defects of TSH
described so far involved the chromosome 1-located TSH-ß
gene (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). So far, three allelic variants of the
TSH-ß gene have been described. The first one was
described in three Japanese families probably sharing a common
ancestry, where homozygosity for a G-to-A substitution in codon 29 of
the TSH-ß subunit led to the conversion of a glycine to an arginine
(G29R) (9, 10, 11, 12). The second variant was found in two
related Greek families, where a G-to-T substitution in codon 12
introduced a premature stop codon (GAA
TAA,
glutamic acid 12 to stop codon substitution, E12X) and, consequently,
led to the deletion of the aa residues 12–118 (13).
The last variant was described in four families of different origins, where a 1-bp deletion in codon 105 caused a frameshift mutation. This resulted in cysteine105 to valine substitution and yielded an additional 8-aa nonhomologous peptide extension on the mutant protein (C105V, 114X) (14, 15, 16, 17). Interestingly, one of the two disulfide bridges (aa 19–105 and 88–95) stabilizing the seat belt region was disrupted through this C105V amino acid substitution (18).
Important to stress, these three mutations caused in all subjects a severe form of congenital hypothyroidism if not treated as early as possible. This underscores the importance of a quick and accurate diagnosis, even though central hypothyroidism due to TSH-ß gene mutation is a rare condition.
In the present study, we report a new mutation of the
TSH-ß gene in a consanguineous Turkish family. Sequencing
of the genomic DNA and endonuclease digest revealed homozygosity for a
C-to-T transition at nucleotide 654 of the TSH-ß subunit
gene (19). Thus, glutamine 49 (CAG) was
converted to a premature stop codon (TAG) (Q49X). The
resulting nascent peptide does not contain the seat belt region (aa
88–105), a TSH-ß subunit region crucial for the dimerization with
the -subunit and, hence, important for the correct secretion of the
mature TSH heterodimer. Confirming this hypothesis, serum TSH-ß was
extremely low in the homozygous individuals and even stimulation with
TRH failed to induce a TSH release.
Subjects and Methods
Index case (patient II.1)
The index case is a Turkish girl who was referred to Dokuz Eylul
University, Pediatric Endocrinology and Adolescence Unit, at the age of
73 d because of the history of congenital hypothyroidism in her
brother. She was born through normal spontaneous vaginal delivery at 36
wk gestation with a birth weight of 3100 g. She had no problems
during or after delivery and was exclusively breast-fed. Her 8-yr-old
brother was diagnosed to have congenital hypothyroidism at birth in
another city. Her other 7-yr-old brother is healthy. Her parents were
first-degree relatives. Family histories are otherwise noncontributory.
At referral, she was 4300 g in weight (1–25th percentile), 54 cm
in height (3–10th percentile), and her head circumference was 38 cm
(10–25th percentile). She had no jaundice but had dry skin with some
scaling, macroglossia, and coarse facial features. Anterior fontanelle
was 5 x 2 cm, and posterior fontanelle was 1 x 1 cm. She
presented with an umbilical hernia, and her liver was 2 cm palpable
below the costal margin. Her physical examination was otherwise
unremarkable. On laboratory testing, values were: total
T4, 1.29 nmol/liter (normal range, 57.9–140.3
nmol/liter); total T3, 0.3 nmol/liter (normal
range, 0.92–2.79 nmol/liter); free T4, 1.29
pmol/liter (10.3–19.3 pmol/liter); free T3, 0.15
pmol/liter (3.54–6.45 pmol/liter); TSH, 0.02 mU/liter (0.35–5.5
mU/liter); knee x-ray was consistent with 36 wk gestation. Because of
the very low basal TSH concentration a TRH stimulation test (TRH
Ferring, Protirelin, 200 µg/m2 iv;
Ferring Pharmaceuticals Ltd., Dübendorf,
Switzerland) was performed. As shown in Table 1
, no TSH increase was found, which is in
contrast to the normal response of PRL. An ultrasound of the thyroid
gland revealed a normally positioned, but hypoplastic gland (volume of
0.125 ml, below the 10th percentile) (20). Therefore, the
diagnosis was central hypothyroidism. To rule out multiple pituitary
hormone deficiencies, additional hormone tests revealed a spot GH level
of 11.3 µg/liter (normal) and a morning cortisol level of 645.6
nmol/liter (110.4–618 nmol/liter), and ACTH was 17.8 pmol/liter
(5.5–13.8 pmol/liter). Slightly increased ACTH and cortisol levels at
that time were interpreted as stress induced as further analysis
revealed no abnormalities. She was started on levothyroxine
replacement. During follow-up, she presented a catch-up growth and her
height at the age of 14 months was 77 cm (50–75th percentile).
Developmentally, she sat without support at 7 months of age, she
started walking at the normal age of 12 months, and she said a few
words at 13 months of age.
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The brother of the index case was diagnosed at birth with
congenital hypothyroidism in another city and was started on
levothyroxine replacement in the newborn period. He has been taking his
medication regularly since then and has developed normally ever since.
His records were not available to us, and, therefore, the etiology of
his hypothyroidism was investigated only at age 8 yr after his sister
was diagnosed with central hypothyroidism. After discontinuation of the
levothyroxine replacement therapy for 4 wk, his thyroid function tests
revealed a total T4 of 10.42 nmol/liter (normal
range, 57.9–140.3 nmol/liter), total T3 of 0.58
nmol/liter (normal range, 0.92–2.79 nmol/liter), free
T4 of 2.57 pmol/liter (10.3–19.3 pmol/liter),
free T3 of 0.15 pmol/liter (3.54–6.45
pmol/liter), and TSH of 0.01 mU/liter (0.35–5.5 mU/liter). A
TRH stimulation test (see patient II.1) showed no TSH response at all
with, however, a normal PRL concentration (Table 1
). A fasting morning
cortisol value was 247.7 nmol/liter (110.4–618 nmol/liter), whereas
FSH and LH values were in the prepubertal range, 2.7 IU/liter (1.6–8.0
IU/liter) and 0.3 IU/liter (0.02–0.8 IU/liter), respectively. At the
age of 8 yr, the physical examination was absolutely fine with weight
and height of 26.4 kg (50–75th percentile) and 125 cm (25–50th
percentile), respectively. His thyroid gland was not palpable. The
diagnosis was familial-isolated TSH deficiency, and levothyroxine
replacement therapy was continued.
DNA isolation
For the genetic studies, written informed consent was obtained from both parents. Genomic DNA was isolated from peripheral leukocytes of the affected subjects and relatives, as described previously (21). The concentration of each sample was determined by measuring the optical density of the purified DNA at 260 and 280 nm.
Amplification and sequencing of genomic DNA
The TSH-ß gene was amplified as follows. Using a
5'-sense primer (5'-TGTAAAACGACGGCCAGTCTTTCTGATTTTAACAAATAGG-3')
and a 3'-antisense primer
(5'-CAGGAAACAGCTATGACCCAAGCACATTTAACCAAATTGC-3'), we amplified a 985-bp
sequence encompassing exon 2, intron 2, and exon 3 (Fig. 1). PCR was performed in a total volume
of 50 µl containing 500 ng genomic DNA, 0.2 mM
dNTPs, 10 pmol each of PCR primers, and 1.25 U Amplitaq DNA polymerase
(Perkin-Elmer Corp., Rotkreuz, Switzerland) in 2.5
mM MgCl2, 10
mM Tris-HCl (pH 8.3), and 50
mM KCl for 30 cycles as follows: 94 C for 45 sec,
53 C for 45 sec, and 72 C for 45 sec. After an extra 3-min extension
period at 72 C in the final cycle, PCR products were purified with
QIAquick spin column (QIAGEN AG, Basel, Switzerland).
Direct sequencing of the PCR products was carried out according to the
thermal cycle sequencing protocol (PE Applied Biosystems,
373 DNA Sequencer; Perkin-Elmer, Rotkrenz, Switzerland)
using the same primers as mentioned above. Analysis of the sequences
was performed using the Seqman computer software (Lasergene;
DNASTAR Inc., Madison, WI).
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To confirm the DNA sequencing, all PCR products were digested to completion with the restriction enzyme StyI under the conditions recommended by the commercial suppliers (Roche, Rotkreuz, Switzerland). The C-to-T transition in codon 49 of TSH-ß introduces a StyI restriction site, and, therefore, digest of the amplified product allows us to confirm the presence of an altered allele. Digestion products were run in ethidium bromide-stained 2% (wt/vol) agarose gel and photographed by UV transillumination.
Results
Genetic analysis
Gene amplification by PCR was performed on exons 2 and 3 and the
in-between intron of the TSH-ß gene. As shown in Fig. 1, this part of the gene is encoding the protein. Sequencing was performed
with both sense and antisense primers to avoid any artifact. On
electropherograms the two affected individuals (II.1 and II.2)
presented with a homozygous C-to-T nucleotide change at codon 49,
whereas the unaffected parents (I.1 and I.2) as well as an unaffected
brother (II.3) were heterozygous for the mutation (Fig. 2). The new mutation replaces a glutamine
codon (CAG) by a premature stop codon (TAG) (Q49X). Furthermore, the
nucleotide change generates a new restriction site for StyI
that allowed us to confirm the DNA sequencing results by endonuclease
digest. Moreover, StyI restriction enzyme analysis of
the amplified TSH-ß gene fragment clearly confirmed the
homozygosity of the affected patients as well as the heterozygosity of
the unaffected relatives when compared with the normal controls (Fig. 3). In that way, the autosomal recessive
mode of inheritance of the Q49X TSH-ß gene mutation was
confirmed.
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As described by Matzuk et al. (22) the
TSH-ß subunit (similar to the LH-ß subunit) is intracellulary
degraded when it is expressed in a cell without the -subunit.
Therefore, it is generally assumed that the TSH secretion depends on
the noncovalent association between the TSH- and -ß subunits
(6). Moreover, the correct dimerization between the
TSH- and TSH-ß subunits strongly depends on the integrity of the
ß-subunit seat belt region (aa 88–105) as well as the two disulfide
bridges (C-C 19–105 and 88–95) involved in the stabilization of the
seat belt. For example, patients suffering from the C105V-114X mutation
of the TSH-ß (known to break the 19–105 disulfide bridge) had
extremely low serum TSH levels (14, 15, 16, 17). Altogether, these
data underscore the importance of the TSH-ß seat belt region for the
secretion of a mature TSH heterodimer.
Here, we report two children suffering from isolated TSH deficiency
associated with a nonsense mutation in codon 49 (Q49X) of the
TSH-ß subunit gene (Figs. 2 and 3). The resulting nascent
peptide lacks the seat belt region (aa 88–105). Consequently, the
dimerization with the -subunit may be impaired, and no full active
TSH-dimer is produced. This hypothesis deduced from the genetic study
is confirmed by our hormonal test results, especially the TRH tests,
performed in the two affected individuals (Table 1). Both affected
children presented with normal corticotroph, somatotroph, and
lactotroph functions. However, TRH failed to increase the serum TSH
levels, but not the PRL, confirming the isolated deficiency of TSH
secretion.
In conclusion, we report a new autosomal recessive mutation of the TSH-ß subunit gene caused by a truncated Q49X TSH-ß subunit peptide.
Acknowledgments
We thank Peter Kopp, M.D. (Northwestern University, Chicago, IL), for advice and technical assistance.
Footnotes
This study was supported by The Swiss National Science Foundation (Grant 32-53714.98; to P.E.M.) and by a M.D.-Ph.D. grant from the Swiss Academy of Medical Sciences (Grant 31-54879.98; to J.D.).
1 J.-M.V., J.D., and A.B. contributed equally in the realization of
this work. 
Received April 11, 2001.
Accepted May 29, 2001.
References
Cys at codon 120 (R120C). J Clin Endocrinol
Metab 83:3727–3734This article has been cited by other articles:
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  G. Borck, A. K. Topaloglu, E. Korsch, U. Martine, G. Wildhardt, N. Onenli-Mungan, B. Yuksel, U. Aumann, G. Koch, G. Ozer, et al. Four New Cases of Congenital Secondary Hypothyroidism due to a Splice Site Mutation in the Thyrotropin-{beta} Gene: Phenotypic Variability and Founder Effect J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4136 - 4141. [Abstract] [Full Text] [PDF]
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