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Original Studies |
Department of Pediatric Endocrinology, Vestische Kinderklinik Datteln, University of Witten-Herdecke (B.M.D., W.A.), Datteln; and the Department of Pediatrics, RWTH Aachen (R.W.P.), Aachen, Germany; and the Thyroid Study Unit, University of Chicago (J.P.), Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Beate M. Doeker, M.D., Vestische Kinderklinik, University of Witten-Herdecke, Lloydstrasse 5, D-45711 Datteln, Germany. E-mail: 101.29837{at}germanynet.de
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Abstract |
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Direct sequencing of the entire coding region of the human TSH
ß-subunit gene revealed a homozygous single base pair deletion in
codon 105, resulting in a frame shift with a premature stop at codon
114. The truncated TSHß peptide lacks the terminal five amino acids.
Furthermore, the cysteine in codon 105 that is believed to be important
for the interaction of the TSH ß-subunit with the 
-subunit, is
replaced with a valine (C105V), supporting the theory of a
conformational change in the TSH molecule.
Genotyping confirmed that the proposita was homozygous for this mutation, whereas her unaffected parents, the paternal grandmother, and the maternal grandfather were heterozygous. Thus, isolated TSH deficiency follows an autosomal recessive mode of inheritance in this kindred.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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After the first report in 1971 of familial congenital hypothyroidism due to TSH deficiency (2), several cases, occurring worldwide, have been reported. The gene encoding for the human TSH ß-subunit was identified by Wondisford et al. in 1988 (3). It is located on chromosome 1 (4) and consists of 3 exons encoding for a protein of 118 amino acids. Subsequently, the molecular defect causing TSHß deficiency has been elucidated in some of the previously described cases (5, 6, 7, 8). Three mutations in different populations were found, all located in different areas of the coding region of the TSHß gene. Interestingly, one of them was identified in three unrelated Japanese families (6, 8), suggesting a common genetic background in this population.
However, for western countries no common mutations in the TSHß gene have been identified. Here, we describe an infant of German parents with no known consanguinity who presented with isolated TSH deficiency caused by a homozygous mutation in the TSH ß-subunit gene. Interestingly, the same mutation was previously described in a large Brazilian kindred (7), suggesting a new hot spot area for mutations in the TSH ß-subunit gene, although common ancestry cannot be ruled out.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The proposita, a daughter of unrelated healthy parents of German
ancestry, was delivered at term after an unremarkable gestation through
cesarean section because of facial presentation. Her birth weight was 4
kg, and her length was 50 cm. Routine neonatal screening did not
disclose hypothyroidism. Her development proceeded slowly, and at the
age of 5 months she presented developmental delay. On physical
examination her weight was 5100 g with a length of 56 cm. She had
psychomotor retardation and exhibited typical signs of hypothyroidism
with muscle hypotonia, hoarse cry, enlarged tongue, umbilical hernia,
and an open posterior fontanel. Her thyroid gland was not enlarged, and
ultrasound showed a small thyroid gland of normal density. The serum
thyroglobulin level was normal (33 ng/mL; normal range, 5–50). Bone
age (<3 months) was delayed. An undetectable TSH level of less than
0.04 mU/liter (normal, 0.5–4.0) in combination with undetectable
levels of T3 (<10 ng/dL; normal range, 67–167) and
T4 (<0.4 µg/dL; normal range, 5.2–12.6) in serum led to
the diagnosis of central hypothyroidism. Treatment with 50 µg/day
L-T4 was initiated. Physical and mental
development improved dramatically, and the typical signs of
hypothyroidism disappeared. After iv administration of 100
µg/m2 TRH, the TSH concentration remained undetectable
(<0.04 mU/l), whereas the serum PRL concentration increased from 10.1
to 42 µg/L after 20 min. These results and other endocrine function
tests are shown in Table 1
. Because of
isolated TSH deficiency, DNA was obtained from white blood cells for
molecular analysis. After 6 months of treatment, the patient grew along
the third percentile for height and the third percentile for weight.
She is clinically euthyroid and doing well.
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Serum total T4 and total T3
concentrations were measured by chemiluminescent immunoassays with
sensitivities of 0.4 µg/dL and 10 ng/dL, respectively (Amersham
Buchler, Braunschweig, Germany). TSH levels were determined by three
different methods. Neonatal screening was performed using the
AutoDELFIA neonatal human (h) TSH assay with a sensitivity of 2 µU/mL
(Wallac Oy, Freiburg, Germany). This is a solid phase, two-site
fluoroimmunometric assay based on the direct sandwich technique in
which two monoclonal mouse antibodies are directed against the hTSHß
molecule and partly against the hTSH -subunit. The second TSH assay
(sensitivity, 0.02 mU/L; IRMAmat TSH, Byk-Sangtec Diagnostica,
Dietzenbach, Germany) was a RIA with antimouse antibodies against the
- and ß-subunits of the hTSH molecule. Finally, TSH was measured
with a chemiluminescent immunoassay (Amerlite, Amersham) with mouse
monoclonal anti-TSH and anti-TSH ß-subunit antibodies (sensitivity,
0.04 µU/mL).
LH, FSH (both at baseline and 30, 60, and 120 min after iv administration of 25 µg/m2 LHRH), and cortisol (only baseline) were measured by chemiluminescent immunoassays (Amersham); PRL (Chiron Diagnostics, Fernwald, Germany) and thyroglobulin (Brahms Diagnostica, Berlin, Germany) were determined with immunoluminometric assays. GH was determined with an immunoradiometric assay (Medgenix Diagnostics, Fleurus, Belgium), and IgF1 was measured with a RIA (Nichols Institute Diagnostica, Bad Nauheim, Germany).
Ultrasound of the thyroid gland was performed with an Acuson 128 linear 5.0-megahertz scanner (Acuson, Erlangen, Germany). Bone age was determined from x-ray of the left hand according to the method of Greulich and Pyle (9).
Sequencing of the TSH ß-subunit gene
Genomic DNA was extracted from leukocytes using the Qiagen Blood DNA kit (Qiagen). DNA fragments containing the entire coding region of the TSH ß-subunit gene were amplified by PCR using the primers 5'-GATCATATGCATTGGGATGG-3' and 5'-TGCGTATCCATTGTGCTGAG-3' for exon 2 and 5'-GTCCTGTCACATTATGCTCTC-3' and 5'-GCTTTATTTCAGGCAAGCAC-3' for exon 3. PCR was performed as follows: 50 µL of the reaction solution contained 100 ng genomic DNA, 50 pmol of each primer, 0.5 U DNA polymerase from Thermus brockianus (Primezyme, Biometra, Gottingen, Germany), 200 µmol/L of each deoxy-NTP, and 1.5 mmol/L MgCl2 in 10 mmol/L Tris-HCl and 50 mmol/L KCl, pH 8.8, buffer. PCR was performed on a Mastercycler (Eppendorf, Hamburg, Germany). It consisted of an initial denaturation step at 93 C for 4 min and then 30 cycles of 92 C for 30 s, 55 C for 30 s, and 72 C for 50 s, followed by a final extension step at 72 C for 5 min. The PCR products were resolved by 2% agarose gel electrophoresis and stained with ethidium bromide. For automated sequencing the PCR products were purified with the Qiaquick PCR purification kit (Qiagen) and directly sequenced after amplification with a Taq DyeDeoxy Terminator Cycle Sequencing Kit (ABI Prism, Perkin-Elmer, Warrington, UK) using the same primers and an ABI 373A Sequencer (ABI, Perkin-Elmer).
Genotyping
The deletion of nucleotide (nt) 410 creates a new restriction
site for SnaBI. Therefore, exon 3 was amplified from genomic
DNA of the patient, all available family members, and 50 normal
individuals. The amplified PCR product was then digested with
SnaBI (Promega, Mannheim, Germany), and the products of
digestion were resolved by 3% agarose gel electrophoresis and finally
stained with ethidium bromide. The allele containing the wild-type
TSHß fragment remained undigested (344 bp), whereas the mutant allele
produced 2 fragments of 251 and 93 bp (Fig. 1B
).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). The
resulting protein would lack the five C-terminal amino acids. The
proposita was homozygous for this mutation, as the wild-type nt 410T
was not detected by direct sequencing (Fig. 2), and SnaBI
completely digested the amplified PCR product containing the mutation
(Fig. 1B). Furthermore, genotyping using the SnaBI site
showed that the mutation was inherited in an autosomal recessive
manner. The unaffected father and mother as well as the paternal
grandmother and maternal grandfather were heterozygous carriers of one
mutant allele (Fig. 1B), as the normal allele remained undigested.
Fifty normal individuals did not harbor the mutation in nt 410,
indicating that this mutation occurs in less than 1% of the
population.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit. This so-called disulfide-linked
seatbelt region is extremely important for maintenance of the
conformational and biological activities of the TSH molecule.
Medeiros-Neto et al. demonstrated that the substitution of
the cysteine in codon 105 with a valine and not the absence of the
terminal 13 amino acids is responsible for the impaired bioactivity of
the altered TSH molecule (7), thus explaining our clinical finding of
severe hypothyroidism in the proposita. However, the phenotype in our
patient is slightly different from that of the previously reported
family, in which some affected individuals had low or even normal
baseline serum TSH concentrations. Although harboring the same
mutation, in our case TSH was not detectable even after administration
of TRH. The fact that the affected child was receiving thyroid hormone
replacement with 50 µg L-T4 when the TRH test
was performed still does not explain the absent TSH response. Our
finding, therefore, might indicate that the TSH ß-subunit molecule is
not detectable because the molecule is very unstable or simply not
present in serum. The messenger ribonucleic acid has a premature stop
codon, and it is well known that eukaryotic cells have mechanisms to
degrade such ribonucleic acids (11). If the molecule was synthesized,
the altered interaction with the -subunit might contribute to an
impaired secretion or shortened half-life of the TSH. This hypothesis
could be investigated clinically by repeating the TRH test in the
patient without thyroid hormone substitution, but has not been
performed because of the young age of the proposita. Our findings are
in agreement with the observations of Dacou-Voutetakis et
al. (5) and Hayashizaki et al. (6), who also did not
detect TSH concentrations in serum in the two previously described
families with mutations in the TSH ß-subunit gene.
Investigation of the genetic backgrounds of the two families harboring the same mutation in codon 105 but originating from different populations would elucidate whether other factors might contribute to the conformational structure or stability of the TSH molecule. Furthermore, this might reveal the presence of a common ancestor in both families or identify this region of the gene as an area where mutations are more likely to occur.
Taking into account that the present case of isolated TSH deficiency is inherited in an autosomal recessive manner and that the occurrence of a homozygous mutation in a family that originated from different parts of Germany is very unlikely, we anticipated that the mutant allele might occur more commonly. However, screening of 50 normal individuals did not detect the mutant in any allele, indicating a gene frequency of less than 1%.
Although the frequency of mutations in the TSH ß-subunit gene is not known, it is important to keep in mind that isolated TSH deficiency will not be detected by routine neonatal screening based on TSH measurement. Neonatal screening for low levels of T4 would have picked up this rare condition and, therefore, is an alternative to prevent the delay of treatment.
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Acknowledgments |
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Footnotes |
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Received December 31, 1997.
Accepted January 20, 1998.
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References |
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