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A Novel Mechanism for Isolated Central Hypothyroidism: Inactivating Mutations in the Thyrotropin-Releasing Hormone Receptor Gene¹

Department of Pediatrics and the Research Unit on Biology of Reproduction and Development (R.C., J.T., J.C., G.L., N.M., C.H., C.D., G.V.V.), and Department of Biochemistry (E.D.), Sainte-Justine Hospital, University of Montreal, Montreal, Canada H3T 1C5; and the Medical Research Council Reproductive Biology Unit, Center for Reproductive Biology (E.F., K.A.E.), Edinburgh, United Kingdom

Address all correspondence and requests for reprints to: Dr. Robert Collu, Research Center, Hôpital Sainte-Justine, 3175 côte Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5. ). E-mail: Collu{at}justine.umontreal.ca

	Abstract

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Isolated central hypothyroidism, characterized by insufficient TSH secretion resulting in low levels of thyroid hormones, is a rare disorder. We report a boy in whom isolated central hypothyroidism was diagnosed at 9 yr of age. Complete absence of TSH and PRL responses to TRH led us to speculate that he had an inactivating mutation of the TRH receptor gene. The patients’ genomic DNA was isolated, and the entire coding region of the TRH receptor was amplified by the PCR and sequenced directly. Confirmation of the mutations and haplotyping of the family was performed using restriction enzymes. The biological activity of the wild-type and mutated TRH receptors was verified by evaluating the binding of labeled TRH and stimulation by TRH of total inositol phosphate accumulation in transfected HEK-293 and COS-1 cells. The patient was found to be a compound heterozygote, having inherited a different mutated allele from each of the parents; both mutations were in the 5'-part of the gene. Mutated receptors were unable to bind TRH and to activate total inositol phosphate accumulation. Our report is the first description of naturally occurring inactivating mutations of a G protein-coupled receptor linked to the phospholipase C second messenger pathway. The prevalence and phenotypic spectrum of TRH receptor mutations in isolated central hypothyroidism remain to be established.

	Introduction

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CENTRAL hypothyroidism, characterized by insufficient TSH secretion resulting in low levels of thyroid hormones, is a rare disorder with an estimated frequency of 0.005% in the general population; most cases are associated with other pituitary deficiencies and are due to tumors or infiltrative diseases of the hypothalamic-pituitary area or to pituitary atrophy (1). Isolated central hypothyroidism due to mutations of the TSH ß-subunit has been described (2, 3). On the other hand, inactivating mutations of receptors for hypothalamic hormones can also lead to pituitary hormone deficiencies, as recently reported for the GHRH receptor (4).

We report a patient with central hypothyroidism whose plasma TSH and PRL levels did not rise after the administration of TRH. He was found to be a compound heterozygote, having inherited from each parent a different mutation in the TRH receptor gene. Both mutations resulted in a receptor with reduced or absent biological activity.

	Case Report

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The propositus, the second of three sons born to nonconsanguineous Caucasian parents, was referred for evaluation of short stature at 8.9 yr of age. His clinical history was unremarkable except for poor school performance. On examination, his height was 115.2 cm (-2.6 SD), his weight was 23.0 kg (-0.4 SD), and his heart rate was 64 beats/min without other signs of hypothyroidism. The bone age was 4 yr (-4.1 SD). Plasma T₄ was 4.0 µg/dL (52 nmol/L; normal, 4.5–11.5 µg/dL), and TSH was 1.3 mU/L (normal, 0.1–5.0). A retrospective verification of T₄ and TSH values obtained at neonatal screening revealed that the T₄ value, at 4.9 µg/dL (64 nmol/L), was abnormally low (normal, >6.7 µg/dL), but the TSH concentration was normal (13 mU/L; normal, <15 mU/L). Because of the normal TSH value, the patient had not been recalled. The peak plasma GH reponses to clonidine and levodopa were 31.8 and 15.6 µg/L, respectively (normal, >8). Baseline plasma PRL was 6.2 µg/L (normal, <15), and iv TRH failed to induce a rise in either TSH or PRL levels. On a computerized tomographic scan, the pituitary volume was normal at 175 mm³ (5); on films of the abdomen, taken after the scan, a stippled right femoral epiphysis was noted. A diagnosis of central hypothyroidism of unknown cause was made. The patient was started on T₄ at a daily dose of 50 µg. Normalization of plasma T₄ levels (9.3 µg/dL or 119 nmol/L) was associated with an increase in heart rate (to 100 beats/min) and a slight and transient increase in height velocity (from 4.8 to 7.3 cm/yr). At 10.9 yr, height velocity decreased again to 2.7 cm/yr, although the compliance was good, and the dose of T₄ was increased to 75 µg/day. When the patient was 12.3 yr old, it was decided to repeat the TRH test after 1 month of T₄ withdrawal, to confirm the pituitary unresponsiveness to the peptide, and to compare it to the response of the other family members who had normal thyroid function tests. The patient’s baseline plasma T₄ was 3.3 µg/dL (42 nmol/L), and TSH was 2.2 mU/L; iv TRH failed again to release TSH and PRL, whereas all of the other family members had a normal response to the peptide (Fig. 1). At 11.9 yr, the patient’s overall intellectual quotient (I.Q.) was determined, by the Wechsler Intelligence Scale for Children (revised) (6), to be 92 (verbal, 79; Performance, 109). The testicular volume started to increase at 12.5 yr, with normal pubertal progression thereafter. The two brothers and the parents had no signs or symptoms of hypothyroidism. The overall I.Q. of the elder brother at age 16 yr was 76 (verbal, 74; performance, 81), and that of the younger brother at age 11 yr was 89 (verbal, 86; performance, 93). Although the parents did not undergo formal I.Q. testing, they appeared to be of normal intelligence, but their educational level was low.

View larger version (19K): [in this window] [in a new window]   Figure 1. Plasma TSH and PRL responses to TRH in all family members schematically represented in the pedigree diagram. The figure shows the plasma concentration of TSH (open squares) and PRL (solid squares) before and after the iv administration of TRH. Half-filled rectangles and circle represent heterozygotes for either the paternal mutation (solid filling) or the maternal mutation (cross-hatched filling). The rectangle with half-solid and half-cross-hatched filling represents a compound heterozygote carrying both mutations (the propositus).

	Materials and Methods

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Plasma T₄ and TSH were measured on the AutoDelfia by time-resolved immunofluorometric assays (Wallac, Vaudreuil, Canada). PRL was measured by RIA (Intermedico, Markham, Canada). The TRH test was performed as previously reported (7, 8), using 7 µg/kg (maximum, 200 µg) Relefact (Hoechst-Roussel Canada, Montreal, Canada).

Preparation of genomic DNA and DNA sequencing

Genomic DNA was isolated from peripheral blood cells as previously described (9). For sequencing, the entire coding region of the TRH receptor gene was subdivided into four overlapping segments (no. 1–4 in a 5'-3' direction). DNA was amplified by the PCR as previously described (9), using sets of primers obtained from Dr. Q. H. Dong (Molecular Pathophysiology Branch, NIH/NIDDK, Bethesda, MD) for segments 1, 2, and 4. For segment 3, which contains the intron, customized primers were synthesized and used to amplify the intron-exon boundaries of this part of the TRH receptor gene (10). Direct sequencing was performed as previously described (11) on segments recovered with the Sephaglas BandPrep kit (Pharmacia, Baie d’Urfé, Canada). Segment 1 (nucleotides 1–455) was also used for cloning.

Confirmation of the mutations and haplotyping

The creation of new restriction sites by each of the two mutations was used to confirm the presence of the mutations and to identify family members as carriers of the mutated alleles. PCR was performed under the same conditions as previously described (9), except for the addition of [³²P]deoxy-ATP. After amplification of genomic DNA of each family member, the PCR products were digested with the appropriate enzymes and electrophoresed on a polyacrylamide gel. The gel was dried and exposed overnight to an x-ray film. The appearance of additional digested fragments indicated the presence of a mutation in one of the two alleles.

Construction of wild-type and mutant TRH receptor complementary DNA (cDNA) expression vectors

The full-length, wild-type TRH receptor cDNA was synthesized by reverse transcription of TRH receptor messenger ribonucleic acid extracted from a normal human pituitary gland obtained at autopsy. The cDNA was cloned (9) into pcDNA3 (Invitrogen Corp., San Diego, CA). Segment 1 of the patient’s TRH receptor gene was cloned in pBluescript KS⁺ (Stratagene, La Jolla, CA). More than three clones of each allele were verified by sequencing with Sequenase kit 2.0 (U.S. Biochemical Corp., Cleveland, OH). Mutant TRH receptors (maternally derived, designated M-Stop and paternally derived, designated F-TM3) were constructed by removing the mutated segment 1 and using it to replace the normal segment 1 of the wild-type receptor in pcDNA3. The entire sequence of the cloned receptors was verified and was identical to that of the published TRH receptor (12, 13), except for the expected mutations in the mutant clones.

Functional studies of the TRH receptors in a transient expression system

HEK-293 and COS-1 cells were maintained in DMEM (Sigma Chemical Co., Dorset, UK) containing 10% heat-inactivated FCS at 37 C and 5% carbon dioxide. Monolayer cultures of either HEK-293 or COS-1 cells (1.2 x 10⁶ cells/well) were transiently transfected with either the wild-type or the mutant human TRH receptor cDNAs cloned in pcDNA3 (5 µg/well) using Transfectam (Promega, Southampton, UK). The efficiency of the method of DNA transfection has been verified by cotransfecting the cells with a plasmid containing the gene for ß-galactosidase and assaying the enzyme activity in cell extracts using a commercial kit (Promega).

Radioligand binding assays were performed as previously reported (12). Briefly, cell membranes were prepared 48 h after transfection by lysing the cells on ice for 10 min in 20 mmol/L Tris-HCl (pH 7.2) containing 2 mmol/L MgCl₂, then homogenizing them in a glass homogenizer and spinning them at 14,000 rpm for 20 min at 4 C. Cell membranes were resuspended in assay buffer, and 20–50 µg protein were added per tube. Displacement curves were generated using tritium-labeled [3Me-His²]TRH (New England Nuclear-DuPont, Hertfordshire, UK) and unlabeled [3Me-His²]TRH (Peninsula Laboratories, Merseyside, UK). Samples were incubated at 4 C for 2 h before filtration through a cell harvester. All values are the means of triplicate determinations, and the experiments were performed on at least three independent occasions.

For studies of inositol phosphate accumulation, normal DMEM was replaced with inositol-free DMEM containing myo-[2-³H]inositol (Amersham, Aylesbury, UK) 24 h after transfection. This medium was removed 48 h later, and the cells were incubated for 20 min in phosphate-buffered saline containing 10 mmol/L lithium chloride and various concentrations of TRH. Tritium-labeled total inositol phosphates were then extracted, separated using an anion exchange resin, and assayed by liquid scintillation counting (12).

	Results

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DNA sequencing and haplotyping

Several germ-line mutations were found only in segment 1 of the patient’s DNA. Sequencing of 10 clones of segment 1 further indicated that the mutations were in different alleles, as shown in Fig. 2. In one allele, a cytosine to thymine (C to T) mutation at position 49 resulted in arginine (CGA) being substituted by a premature stop codon (TGA) at peptide position 17. In the other allele, a deletion of nine nucleotides from position 343 to 351 plus a mutation of guanine to adenine (G to A) at position 352 were found. The deletion resulted in the loss of three amino acid residues (Ser¹¹⁵, Ile¹¹⁶, and Thr¹¹⁷) at the cytoplasmic end of the third transmembrane domain of the receptor. The mutation at position 352 resulted in the replacement of alanine (GCC) by threonine (ACC) at peptide position 118 (Fig. 2). The presence of different mutations in each of the patient’s alleles indicated that he is a compound heterozygote. The mutation at position 49 generated a new restriction site for the enzyme NlaIII, whereas the deletion plus mutation at positions 343–352 created a new restriction site for the enzyme RsaI. Family studies applying restriction site enzymatic digestion to the respective parts of the gene traced each of the mutant alleles to either one of the parents (Fig. 3) and provided evidence that the elder brother of the patient had also inherited the mother’s mutant allele. The heterozygous state of each parent and that of the elder brother were confirmed by the presence of one normal allele (Fig. 3). These results indicate that the patient and his elder brother had inherited germ-line mutations. No other mutations were found by direct sequencing of segments 2, 3 and 4 of the patient’s DNA.

View larger version (73K): [in this window] [in a new window]   Figure 2. Sequencing gel showing the mutations in the TRH receptor gene in the propositus. Genomic DNA from peripheral blood leukocytes was separated in fragments 1–4 in a 5' to 3' direction. Fragments 1, 2, and 4 (F1, F2, and F4) were amplified by PCR using primers generously provided by Dr. Q. H. Dong, Molecular Pathophysiology Branch, NIH/NIDDK (Bethesda, MD; F1s, 5'-GAA GAT GGA AAA CGA GAC AGT C-3'; F1as, 5'-AAA GCC CAG ACA AAG ATG ATA ATC-3'; F2s, 5'-CCC ATC AAA GCC CAG TTT CTC TGC-3'; F2as, 5'-TTC CTT GAA GAT ACT GTG CTG TTG-3'; F4s, 5'-ATG GAT GCC CTA CAG GAC TCT A-3'; F4as, 5'-ATT GGC CAT GTT CTC CCT TTT G-3'). The intron-containing fragment 3 was amplified by PCR using custom-synthesized primers (701s, 5'-GGA AAA ATG ATT CAA CCC ATC AGA-3'; int177as, 5'-ATG ATT GTA TTT TGG TTT CGC CAT-3'; int996s, 5'-CTT AAT CCT GCT TCC TGG GAT CAC-3'; 907as, 5'-ATA AAT GCA AAT TCT GCA AAA GAG-3'). In the paternal allele, nucleotides at positions 343–351 were missing, leading to the deletion of Ser115, Ile116, and Thr117 amino acid residues and the mutation GCCACC resulted in the substitution of alanine by threonine at position 118. In the maternal allele, the mutation CGATGA resulted in a premature stop codon instead of arginine at position 17. The substituted nucleotides are outlined in black.

View larger version (88K): [in this window] [in a new window]   Figure 3. Confirmation of the mutations in the TRH receptor gene in family members. Genomic DNA from peripheral leukocytes was amplified by PCR using the sense primer 5'-GGC TCA CCA GGT AGC AGT TTG T-3' and the antisense primer 5'-TGA GGC TGG CAG TAG CTG ATC T-3'. The presence of thymidine at position 49 produces an additional NlaIII restriction site, whereas the deletion of nucleotides 343–351 plus the presence of adenine at position 352 creates an additional NlaI restriction site. The upper gel shows that all family members have either two alleles (father and one son) or one allele (mother and two sons) that are normally digested with NlaIII, creating a 139-bp fragment (Nla 1) and three smaller fragments (not shown). It also shows that the mother and two sons (the propositus and the elder son) all have a mutant allele containing a new restriction site that, when digested with NlaIII, cuts the 139-bp fragment in two DNA fragments of 85 and 54 bp (Nla 2). The lower gel shows that all family members have either two alleles (mother and two sons) or one allele (father and the propositus) that are normally digested with RsaI, creating a fragment of 220 bp and another of 177 bp (RsaI); it also shows that the father and the propositus have a mutant allele containing a new restriction site that, when digested with RsaI, cuts the 177-bp fragment to produce a DNA fragment of 149 bp (Rsa 2) and a smaller fragment (not shown). The pedigree above the gels shows the pattern of inheritance of the mutant TRH receptor alleles. u, Undigested; d, digested.

Figure 4 (left panel) shows radioligand displacement curves with [3Me-His²]TRH and membranes prepared from HEK-293 cells expressing wild-type and mutant human TRH receptors. The wild-type receptor exhibited high affinity binding with an ED₅₀ of approximately 1.6 nmol/L. In contrast, the maternally derived mutant (M-Stop) showed no measurable binding, whereas the paternally derived mutant (F-TM 3) showed a very small amount of binding. Untransfected HEK-293 cells did not demonstrate any binding (results not shown). The dose-response curve of TRH-induced increase in total inositol phosphate production in COS-1 cells expressing either the wild-type or the mutant receptor is also shown in Fig. 4 (right panel). The EC₅₀ value of total inositol phosphate stimulation by TRH for COS-1 cells expressing the wild-type receptor was 2.4 nmol/L. COS-1 cells expressing either one of the two mutant receptors showed no stimulation of total inositol phosphate production in response to increasing concentrations of TRH. Similarly, untransfected or sham-transfected COS-1 cells demonstrated no inositol phosphate response at any of the TRH doses used.

View larger version (15K): [in this window] [in a new window]   Figure 4. Functional studies of the wild-type and the mutant TRH receptors. The left panel shows representative curves of displacement by [3Me-His2]TRH of 3H-labeled [3Me-His2]TRH binding to HEK-293 cell membranes transiently expressing wild-type (WT) and mutant human TRH receptors [mutant M-Stop (maternally derived) and mutant F-TM3 (paternally derived)]. Data points represent the mean (±SE) of triplicate samples. The right panel shows total inositol phosphate production in COS-1 cells transiently expressing wild-type and mutant human TRH receptors after stimulation with TRH. Untransfected COS-1 cells were used as a negative control. Data points represent the mean (±SD) of duplicate samples.

	Discussion

Top Abstract Introduction Case Report Materials and Methods Results Discussion References

Central hypothyroidism, defined as low plasma total and free T₄ concentrations in the presence of normal TSH values, is rare. It may result from either hypothalamic or pituitary lesions and usually occurs in association with other pituitary hormone deficiencies (1). The clinical manifestations of central hypothyroidism are usually mild, especially when it is isolated (1). Indeed, in our patient, the only presenting symptoms were short stature with markedly delayed bone maturation, which shows the exquisite sensitivity of these markers of hypothyroidism in children (18). It is uncertain whether the TRH-R gene defect is causally related to our patient’s cognitive deficiencies, as his two unaffected brothers had similar learning difficulties and subnormal I.Q.; however, we suspect that these problems may be related to the low socioeducational status of the family.

Extrapituitary expression of the TRH receptor has been reported, suggesting that TRH may have nonendocrine functions (19). However, our patient’s phenotype, characterized only by isolated central hypothyroidism, indicates that endogenous TRH does not appear to play a major physiological role outside of the pituitary. The prevalence of TRH receptor mutations in patients with isolated central hypothyroidism and their phenotypic spectrum remains to be determined.

	Footnotes

 
¹ This work was supported by grants from Biopedia, Inc., the Interservice Club Council, and the NIH (DK-42792).

Received November 18, 1996.

Revised January 24, 1997.

Accepted January 27, 1997.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Collu, R. Articles by Van Vliet, G. Search for Related Content PubMed PubMed Citation Articles by Collu, R. Articles by Van Vliet, G.