Severe congenital hypothyroidism due to a homozygous mutation of the βtsh gene

ABSTRACT Isolated TSH deficiency leading to hypothyroidism seems to be a rare condition, escaping the diagnosis by neonatal screening programs, which are based on the primary determination

of TSH. This is the first report of a case with an autosomal recessive TSH defect caused by a homozygous mutation of the βTSH gene that was diagnosed in the early neonatal period.

Hypothyroidism in the first child of apparently unrelated parents was suspected because of the classical symptoms of congenital hypothyroidism, which were fully expressed already on the 11th

day of life. Routine neonatal TSH-screening on the 4th day of life had been normal, but subsequent determination of serum thyroid hormone levels revealed almost undetectable levels and

thyroid hormone substitution was immediately started. Because there was no indication for other pituitary hormone deficiencies, sequence analysis of the βTSH gene was initiated. A homozygous

T deletion in codon 105 was found resulting in a change of a highly conserved cysteine to valine followed by eight altered amino acids and a premature stop codon due to the frame-shift.

This altered βTSH is a biologically inactive peptide. Because of the early development of severe symptoms, it is possible that this altered TSH suppresses the physiologic constitutive

activity of the unliganded TSH receptor. Rapid molecular diagnosis in this patient clarified the diagnosis without additional endocrine and imaging studies and it is concluded, that symptoms

of hypothyroidism in the neonatal period should result always in an immediate comprehensive work-up of thyroid function including molecular genetic studies irrespective of the screening

result. SIMILAR CONTENT BEING VIEWED BY OTHERS NOVEL HOMOZYGOUS VARIANT IN THE _TPO_ GENE ASSOCIATED WITH CONGENITAL HYPOTHYROIDISM AND MILD-INTELLECTUAL DISABILITY Article Open access 27

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PRIMARY CONGENITAL HYPOTHYROIDISM OVER 24 YEARS IN FINLAND Article Open access 03 June 2022 MAIN Central congenital hypothyroidism caused by various hypothalamic-pituitary defects is

considered to be a rare disorder with an estimated frequency of 1 in 30 000 to 150 000 newborns(1). Isolated TSH deficiency is even rarer and only three studies have described cases, in

which isolated βTSH deficiency was proven by molecular genetic studies(2–4). The clinical diagnosis of hypothyroidism in all of these patients was made after the newborn period and molecular

genetic studies were performed only in later life because these patients belonged to consanguineous kindreds with a multiple occurrence of central hypothyroidism. Although it has been

generally assumed that congenital central hypothyroidism alone may not lead to severe symptoms in the neonatal period and that mental retardation in the patients with various

hypothalamic-pituitary defects is the result of accompanying endocrine, metabolic, and developmental CNS disturbances(5), mental retardation has also been described in some, although not

all, patients with isolated TSH-deficiency. It is possible that even in the complete absence of TSH, like in pituitary aplasia, the considerable physiologic constitutive activity of the

unliganded TSHR of the thyroid cell that distinguishes the TSHR from other glycoprotein receptors(6) conserves the crucial thyroid hormone production, which is necessary to protect the

newborn from the development of severe symptoms. In contrast, binding of a ligand that decreases the constitutive receptor activity, _e.g._ a maternal immunoglobulin(7), and the congenital

loss of normal TSHR properties caused by mutations of the TSHR gene(8) have been shown to result in severe congenital hypothyroidism and mental retardation in some of the cases. We describe

the first case of nonfamilial congenital βTSH deficiency, in which rapid molecular genetic analysis enabled the diagnosis already in the newborn period and made extensive endocrine and

imaging studies unnecessary. Besides this useful application of molecular genetic techniques, this case, by the presence of severe symptoms and hypoplasia of the thyroid gland already in the

neonate, indicates that some forms of isolated TSH deficiency can result in severe congenital hypothyroidism and subsequent mental retardation. PATIENT The patient is the first child of

unrelated parents without any history of thyroid disease in the families of both parents. He was born after 40 wk of an uneventful pregnancy with a birth weight of 3435 g and a length of 51

cm. The mother was not given iodine during pregnancy and no iodine-containing drugs were used perinatally in the mother or the newborn. He was discharged at the 7th day of life from the

maternity ward despite documented hyperbilirubinemia, decreased muscle tone, and feeding problems. At home during the 2nd wk of life, he developed constipation, and feeding became

increasingly difficult because he was very sleepy. The general pediatrician suspected hypothyroidism, contacted the screening laboratory, and asked for the result of the determination of TSH

in dried blood spots of the 4th day of life, which had given a normal result of 1.3 mU/L. The patient was referred to the neonatology unit on the 14th day of life to rule out infectious or

other metabolic diseases. On admission the newborn presented with the typical symptoms of congenital hypothyroidism: prolonged jaundice (bilirubin 172 µmol/L), dry skin, muscular hypotonia,

enlarged tongue, muscular hypotonia, lethargy, enlarged cranial sutures, and wide open anterior and posterior fontanels. METHODS _HORMONE MEASUREMENTS._ Serum levels of growth hormone,

prolactin, IGF-I, and cortisol were measured using specific radioimmunoassays (Pharmacia, Freiburg; IBL, Hamburg, Germany). Serum thyroid hormone, serum TSH, and neonatal TSH in filter-paper

blood were measured by fluoroimmunoassay (Delfia, Wallac, Freiburg, Germany). TSH was also measured by an automated ELISA (Boehringer Mannheim, Mannheim, Germany) using a different βTSH

antibody. _SAMPLE PREPARATION FOR DNA ANALYSIS._ Genomic DNA was prepared from peripheral white blood cells of the patient and family using a DNA extraction kit (QIAamp Blood Kit, Qiagen,

Hilden, Germany). _ANALYSIS OF THE ΒTSH GENE._ The second and third exons of the human βTSH gene were amplified using polymerase chain reaction (PCR) with 0.5 µg of genomic DNA as template

and the following pairs of oligonucleotides: Exon 2: forward 5′ GCA TGA TCA TAT GCA TTG GG 3′, reverse 5′ CCA ATA ACC ATT AGA TTT GGG 3′. Exon 3: forward 5′ GTC CTG TCA CAT TAT GCT CTC 3′,

reverse 5′ GGC AAG CAC ATT TAA CCA AAT TG 3′. Sequencing was performed with BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Weiterstadt, Germany). The sequencing

reactions were analyzed with an automatic sequencer (ABI 373, Applied Biosystems, Foster City, CA). The deletion of a T at codon 105 creates a new restriction site for _Sna_BI in this

fragment. Restriction analysis of exon 3 was performed at 37°C for 2 h and afterward separated on a 2% agarose gel. RESULTS _BIOCHEMICAL FINDINGS._ The determination of thyroid hormone

levels in serum after hospital admission on the 14th day of life revealed an almost undetectable low total T4 of 10.2 nmol/L and T3 level of 0.46 nmol/L, whereas TSH was also low with 0.07

µU/L. The TG level was in the low normal range (17.5 µg/L). Blood glucose concentrations, which were taken every 4 h during the first 24 h after admission, were at normal levels (3.6-4.1

mmol/L) and baseline cortisol, prolactin, and growth hormone levels were in the normal range, ruling out multiple pituitary hormone deficiencies. All other biochemical findings were in the

normal range besides a still elevated free serum bilirubin of 172 µmol/L. There was no indication for an infectious cardiovascular or metabolic disease. Thyroid hormone replacement was

started on the 15th day of life, and a control examination given after 1 wk showed normal results for T4 with 248.4 nmol/L and T3 with 1.9 nmol/L and an unmeasurable TSH of < 0.01 mU/L.

After 1 wk of therapy, the boy had a normal stool frequency, increased muscle tone, and longer periods of being awake. Feeding problems improved resulting in a weight gain of 260 g within

this week. Maternal and paternal thyroid disease was excluded by the determination of TSH: thyroid hormones and thyroid autoantibodies were in the normal range. The results of the

biochemical studies are summarized in Table 1. _ULTRASOUND STUDIES._ Cerebral ultrasound investigation showed normal findings, especially the septum pellucidum, and the corpus callosum

presented normally. Ultrasound examination of the thyroid gland revealed the presence of normal thyroid tissue in the normal position, but the gland was significantly reduced in size (0.6

mL) compared with neonatal reference values(9). Bone age, as assessed by x-ray and ultrasound of the knee and ankle, was significantly retarded being only 35 wk at an age of 43 wk of

gestation. _IDENTIFICATION OF THE ΒTSH MUTATION._ The association of severe symptoms of congenital hypothyroidism, low circulating thyroid hormone concentrations, and a low TSH level

strongly suggested a TSH deficiency as the cause for hypothyroidism. Because no other pituitary hormone deficiencies were detected, a search for mutations in the βTSH gene of the patient was

started immediately. The human βTSH gene is located on chromosome 1p13 and consists of three exons. The first exon is untranslated, the two other exons are encoded for a 118-amino acid

mature βTSH peptide after release of a 20-amino acid signal peptide(10). Amplification of exons 2 and 3 was performed in single fragments. Direct sequencing of both PCR products with an

automated sequencer showed a homozygous single base deletion (T) at codon 105 in exon 3, which resulted in a frameshift. This mutation results in the loss of a highly conserved cysteine

residue at codon 105(11), which is substituted by valine and followed by eight nonhomologous amino acids. The amino acid sequence of the mutated TSH compared with the wildtype sequence is

shown in Figure 1. Due to the premature stop codon, the mutated peptide is five amino acids shorter than the wild type. The deletion of a T in codon 105 creates a new unique restriction site

for _Sna_BI in exon 3 of the βTSH gene. Restriction analysis of PCR products were performed in the patient, all available family members, and 20 control persons (Fig. 2). The patient is

homozygous for the mutation indicated by a complete cut of exon 3 resulting in a 250-bp and a 82-bp fragment. All other family members were heterozygous for the mutation. Therefore one

allele of exon 3 is uncut shown by the 332-bp fragment and the other allele is cut shown by the 250-bp fragment. Exon 3 of the βTSH of a control person was not restricted (see Fig. 2). The

DNA analysis of both parents and grandmothers, who are not related and originate from different parts in Germany, showed that they all are heterozygous for this mutation. Although there was

no familial history of hypothyroidism, the cause for TSH deficiency in this newborn was clarified within 4 wk, and made further investigations, _e.g._ MRI studies and endocrine function

tests, unnecessary. DISCUSSION Autosomal recessively inherited βTSH defects leading to hypothyroidism seem to be a rare disease and only a few familial cases have been reported

previously(2–4). More recently in three of these families, mutations of the βTSH gene have been identified as the cause of TSH deficiency. The first described mutations where found in exon 2

(G29R)(4) and a G to T transversion at nucleotide 94 leading to a premature stop codon resulted in an abolished synthesis of β TSH(2). The mutation of the patient in this study is identical

to that which Medeiros-Neto _et al._(3) described in a Brazilian kindred with four affected offspring. This mutation leads to the production of a biologically inactive TSH. It has been

postulated that the highly conserved cysteine in position 105 together with the cysteine 19 is crucial for the formation of the so-called "seat-belt buckle" that holds the

α-subunit within the βTSH molecule(10). The integrity of the α- and βTSH-subunit conformation is necessary for the interaction with the TSHR of the thyroid cell. _In vitro_ findings have

shown a decreased biologic activity of the C105ΔT βTSH mutant, inasmuch as the cAMP production was reduced compared with the wild type. Furthermore, it was shown that the loss of biologic

activity was solely due to the loss of cysteine and not to the loss of the following amino acids (3). The severe clinical picture with extremely low thyroid hormone concentrations of the

patient in this study, which is in contrast to the usually mild symptoms of pituitary hypothyroidism and is as profound as in patients without functioning thyroid tissue, is astonishing and

would be explainable by a circulating mutated βTSH peptide, which upon interaction with the TSHR decreases the constitutive activity of the unliganded state. However, _in vitro_ expression

studies are limited in their implications for the _in vivo_ situation and extensive expression studies are necessary to prove that the disruption of the TSH conformation has blocking effects

on the TSH receptor. The most frequent cause of congenital pituitary hypothyroidism are developmental defects of the pituitary, which now can be approached also by the analysis of the genes

encoding for pituitary transcription factors, _e.g._ pit-1 or prop-1(12,13). Important conclusions have to be drawn from this study. Molecular genetic studies are a valuable tool to clarify

the diagnosis of pituitary hypothyroidism within a short period of time and to avoid invasive and costly imaging studies and endocrine function tests. Moreover, this is the first case

without a family history of hypothyroidism, in which homozygosity for a mutation of the βTSH gene has been demonstrated and who was already diagnosed and treated in the newborn period.

Unlike previously assumed, inherited βTSH defects can lead to severe neonatal hypothyroidism with the risk of mental retardation solely due to hypothyroidism. Therefore, if symptoms of

hypothyroidism are present in the newborn period that occur despite a normal TSH measurement in newborn screening, an immediate and comprehensive work-up of thyroid function including

molecular genetic studies has to be initiated, because central hypothyroidism is not detectable by TSH-screening. During the revision process of this paper, we were able to identify the

C105ΔT mutation in two additional newborns with severe symptoms of congenital hypothyroidism born in 1998. Therefore this mutation seems to be frequent in patients with severe congenital

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skillful technical assistance. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Paediatric Endocrinology, University Children's Hospital, Charité Campus Virchow Klinikum, Humboldt

University, Berlin, 13353, Germany Heike Biebermann, Klaus-Peter Liesenkötter & Annette Grüters * Neonatalogy, University Children's Hospital, Charité Campus Virchow Klinikum,

Humboldt University, Berlin, 13353, Germany Michael Emeis & Michael Obladen Authors * Heike Biebermann View author publications You can also search for this author inPubMed Google

Scholar * Klaus-Peter Liesenkötter View author publications You can also search for this author inPubMed Google Scholar * Michael Emeis View author publications You can also search for this

author inPubMed Google Scholar * Michael Obladen View author publications You can also search for this author inPubMed Google Scholar * Annette Grüters View author publications You can also

search for this author inPubMed Google Scholar ADDITIONAL INFORMATION This study was supported by grants from the Sonnenfeld-Stiftung, Berlin, Germany. RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Biebermann, H., Liesenkötter, KP., Emeis, M. _et al._ Severe Congenital Hypothyroidism Due to a Homozygous Mutation of the βTSH Gene.

_Pediatr Res_ 46, 170–173 (1999). https://doi.org/10.1203/00006450-199908000-00007 Download citation * Received: 31 July 1998 * Accepted: 26 March 1999 * Issue Date: 01 August 1999 * DOI:

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