Novel mutational mechanism in the thyroglobulin gene: imperfect DNA inversion as a cause for hereditary hypothyroidism.

The objective of this study was to perform genetic analysis in three brothers of Turkish origin born from consanguineus parents and affected by congenital hypothyroidism, goiter and low levels of serum TG. The combination of sequencing of DNA, PCR mapping, quantitative real-time PCR, inverse-PCR (I-PCR), multiplex PCR and bioinformatics analysis were used in order to detect TG mutations. We demonstrated that the three affected siblings are homozygous for a DNA inversion of 16,962bp in the TG gene associated with two deleted regions at both sides of the inversion limits. The inversion region includes the first 9bp of exon 48, 1015bp of intron 47, 191bp of exon 47, 1523bp of intron 46, 135bp of exon 46 and the last 14,089bp of intron 45. The proximal deletion corresponds to 27bp of TG intron 45, while the distal deletion spans the last 230bp of TG exon 48 and the first 588bp of intergenic region downstream TG end. The parents were heterozygous carriers of the complex rearrangement. In conclusion, a novel large imperfect DNA inversion within the TG gene was identified by the strategy of I-PCR. This aberration was not detectable by normal sequencing of the exons and exon/intron boundaries. Remarkably, the finding represents the first description of a TG deficiency disease caused by a DNA inversion.

Thyroid cancer recurrence in patients clinically free of disease with undetectable or very low serum thyroglobulin values.

DESIGN: This was a retrospective clinical study. SETTING: The study was conducted at a university-based tertiary cancer hospital. PATIENTS: One hundred seven patients had initial thyroid cancer surgery and subsequent remnant radioiodine ablation. Patients underwent recombinant human TSH (rhTSH)-mediated diagnostic whole-body scan and rhTSH-stimulated thyroglobulin (Tg) measurement before April 2001 if they had no antithyroglobulin antibodies, were clinically free of disease, and had one or more undetectable (≤0.5 ng/ml) or low (0.6-1 ng/ml) basal Tg measurements on levothyroxine. Patients were stratified according to their rhTSH-Tg responses: group 1, Tg 0.5 ng/ml or less (68 patients); group 2, Tg from 0.6 to 2.0 ng/ml (19 patients); and group 3, Tg greater than 2 ng/ml (20 patients). MAIN OUTCOME MEASURES: Tumor recurrence was measured. RESULTS: In group 1, two of 62 patients (3%) with follow-up recurred. In group 2, 63% converted to group 1, whereas two of 19 (11%) converted to group 3 and then recurred. Sixteen of the initial 20 group 3 patients (80%) recurred, including recurrence rates of 69 and 100% for those with an initial rhTSH-Tg greater than 2.0 ng/ml but 5.0 ng/ml or less, and 4.6 ng/ml or greater, respectively. One group 3 patient died of distant metastases. rhTSH-Tg more accurately predicted tumor recurrence than basal Tg. An rhTSH-Tg threshold of 2.5 ng/ml or greater optimally predicted future recurrence with sensitivity, specificity, and negative and positive predictive values of 80, 97, 95, and 84%, respectively. CONCLUSIONS: The prevalence of postablation thyroid cancer recurrence is predicted by the rhTSH-Tg response with an optimal Tg threshold of 2.5 ng/ml. Still, recurrent disease occurs in some patients with an initial rhTSH-Tg of 0.5 ng/ml or less.

Does recombinant human thyrotropin-stimulated positron emission tomography with [18F]fluoro-2-deoxy-D-glucose improve detection of recurrence of well-differentiated thyroid carcinoma in patients with low serum thyroglobulin?

BACKGROUND: Thyrotropin (TSH) stimulates thyrocyte metabolism, glucose transport, and glycolysis. The interest in using recombinant human TSH (rhTSH) stimulation of fluoro-2-deoxy-D-glucose (FDG) with positron emission tomography (PET) has been shown, but mainly for patients with high serum thyroglobulin (Tg) concentration. We evaluated the use of rhTSH-stimulated PET-FDG in patients with low serum Tg concentration. METHODS: Sixty-one PET/computed tomography (CT)-FDG (Biograph Sensation 16; Siemens Medical Solutions, Knoxville, TN) were performed in 44 patients (28 women and 16 men; 51 +/- 16 years) with positive Tg levels, negative or no contributive iodine-131 whole-body scintigraphy results, and no contributive morphological imaging results (ultrasound, magnetic resonance imaging, and CT). Thirty-eight patients had papillary carcinoma and six had follicular thyroid carcinoma. All patients had previously undergone total thyroidectomy and postoperative iodine ablation of thyroid bed remnant tissue. The rhTSH-stimulated PET/CT-FDG (5 MBq/kg) was performed after two 0.9 mg intramuscular doses of rhTSH (Thyrogen; Genzyme) which were administered 48 and 24 hours before imaging, while patients continued levothyroxine (LT(4)). Blood sampling was performed immediately before FDG injection for measurement of serum TSH and Tg concentrations (TSH(1) and Tg(1)) and after 48 hours (TSH(2) and Tg(2)). PET/CT-FDG findings were compared with the Tg: (i) at the initial iodine treatment during T(4) withdrawal (Tg(ini)), (ii) under T(4) (Tg(T4)) within 3 months before the PET/CT-FDG, (iii) with Tg(1), and (iv) with Tg(2). PET/CT-FDG findings were correlated with the findings of histology, iodine-131 whole-body scintigraphy, morphological imaging, or clinical follow-up. RESULTS: The mean Tg(ini) was 785 +/- 2707 microg/L for a TSH of 73 +/- 64 mU/L. The mean Tg(T4) was 7 +/- 15 microg/L (T(4) = 195 +/- 59 microg/day; mean TSH of 0.24 +/- 0.57 mU/L). Among the 44 patients, PET/CT-FDG findings were positive in 20 and negative in 24. Among the 61 PET/CT-FDG, 25 PET/CT-FDG were positive (41%). Among the 25 positive PET, the Tg(T4) values were less than 10 microg/L for 19, including 9 true-positive patients (20% of the 44 patients). There was no difference of PET/CT-FDG results (positive vs. negative) as related to the serum Tg concentrations (p = 0.99 for Tg(ini), p = 0.95 for Tg(T4), p = 0.07 for Tg(1), and p = 0.42 for Tg(2)). No relation was observed with PET/CT-FDG results and initial tumor size (p = 0.52) or node metastasis (p = 0.14). CONCLUSION: In the diagnosis of recurrent disease in patients with differentiated thyroid carcinoma and low Tg level, the sensitivity of rhTSH-stimulated PET/CT-FDG seems to be low and no correlation was observed between PET/CT-FDG findings and Tg level. However, positive PET-FDG results have been found in 9/44 (20%) patients with serum Tg levels lower than 10 microg/L. Therefore, this series shows that a cutoff value of 10 microg/L for the Tg under T(4) is probably not the best criteria to select patient candidates for PET/CT-FDG examination to detect the recurrence of differentiated thyroid carcinoma.

Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C>T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7.

BACKGROUND: Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre. OBJECTIVES: The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied. METHODS: Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed. RESULTS: Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing. CONCLUSIONS: In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.

Pediatric thyroid testing issues.

Thyroid problems are common in children. While serum thyroid function tests lead to an accurate diagnosis in most patients, unique patient situations can produce misleading results. Total T4 measurements can incorrectly suggest hypothyroidism in congenital thyroid binding globulin (TBG) deficiency and hyperthyroidism in TBG excess, as seen in high estrogen states. Free T4 (FT4) measurement techniques involve either physical separation of unbound thyroxine from serum binding proteins or estimation of FT4 levels in the presence of binding proteins. These estimation techniques are susceptible to under- or over-estimation of FT4 levels when binding proteins are low or high. Other complicating factors arise in the setting of prematurity or systemic non-thyroidal illness (NTI), simulating central hypothyroidism. Thyroid stimulating hormone (TSH) levels in children have a wider normal range than in adults and are affected by drugs and NTI. Additionally, heterophile and anti-T4 or anti-TSH antibodies can interfere with accurate T4 or TSH measurement.

Goitrous congenital hypothyroidism in a twin pregnancy causing respiratory obstruction at birth: implications for management.

We report a twin pregnancy complicated by fetal goitrous hypothyroidism secondary to dyshormonogenesis caused by thyroglobulin deficiency. Antenatal treatment with intra-amniotic thyroxine was considered but not performed, given the late gestational age at diagnosis and the multiple nature of the pregnancy. Both twins developed airway obstruction at delivery, requiring intubation and ventilation. We review the literature and describe the practical issues relating to the antenatal assessment and perinatal management of fetal goitre.

Two distinct compound heterozygous constellations (R277X/IVS34-1G>C and R277X/R1511X) in the thyroglobulin (TG) gene in affected individuals of a Brazilian kindred with congenital goiter and defective TG synthesis.

In this study, we have extended our initial molecular studies of a nonconsanguineous family with two affected siblings and one of their nephews with congenital goiter, hypothyroidism, and marked impairment of thyroglobulin synthesis. Genomic DNA sequencing revealed that the index patient (affected nephew) was heterozygous for a single base change of a cytosine to a thymine at nucleotide 886 in exon 7 (886C>T, mother's mutation) in one allele and for a novel guanine to cytosine transversion at position -1 of the splice acceptor site in intron 34 (IVS34-1G>C, father's mutation) in the other allele. The two affected siblings inherited the 886C>T mutation from their mother and a previously reported cytosine to thymine transition at nucleotide 4588 in exon 22 from their father (4588C>T). The 886C>T and 4588C>T substitutions resulted in premature stop codons at amino acids 277 (R277X) and 1511 (R1511X), respectively. In vitro transcription analysis showed that the exon 35 is skipped entirely when the IVS34-1G>C mutation is present, whereas the wild-type allele is correctly spliced. SSCP (exon 7 and 35) and restriction analysis (exon 22) using Taq I indicated that the two affected siblings, the affected nephew, his mother, and his unaffected brother were all heterozygous for the R277X mutation. The two affected siblings, their father, and three unaffected siblings were all heterozygous for the R1511X mutation, whereas the affected nephew and his father were heterozygous for the IVS34-1G>C mutation. Moreover, in this kindred, we have characterized polymorphisms (insertion/deletion, microsatellite, and single nucleotide polymorphism) located within introns 18 and 29 and exon 44 that are associated with the described mutations. Haplotype analysis with these polymorphic markers in two unrelated Brazilian families (present family studied and previously reported family) harboring the R277X mutation suggests a founder effect for the R277X mutation. In conclusion, the affected individuals of this family are either compound heterozygous for R277X/IVS34-1G>C or R277X/R1511X. This observation further supports that thyroglobulin gene mutations display significant intraallelic heterogeneity.

Bocio multinodular gigante por defecto de síntesis de tiroglobulina / Giant multinodular goiter due to thyroglobulin synthesis defect

Los defectos de la síntesis de tiroglobulina son una causa poco frecuente de hipotiroidismo congénito. El diagnóstico neonatal de esta entidad puede escapar a los programas de screening por la existencia de niveles normales de hormonas tiroideas y discretamente elevados de TSH. Se presenta el caso clínico de una niña que consulta a los 14 años por bocio muy voluminoso (grado 3) y disfagia a sólidos. El estudio de la función tiroidea mostró concentraciones muy bajas de tiroglobulina y de T4, valores normales de T3 y elevación moderada de los niveles de TSH, hipercaptación tiroidea y respuesta negativa al test de descarga de perclorato. El screening neonatal para hipotiroidismo congénito fue negativo. Se diagnostica a los 3 años de hipotiroidismo por bocio difuso (grado lb). No recibió tratamiento sustitutivo con hormona tiroidea por decisión familiar, Siguiendo tratamiento naturalista con aminoácido 1-tirosina, zinc y Suplementos vitamínicos. El desarrollo pondoestatural y psicomotor fue normal. En resumen, el desarrollo precoz de bocio en niños con screening neonatal normal y en estado de eutiroidismo debe hacernos sospechar la existencia de una dishormonogenesis, y entre ellas la producida por defecto de la síntesis y secreción de tiroglobulina (AU)

Congenital hypothyroidism with impaired thyroid response to thyrotropin (TSH) and absent circulating thyroglobulin: evidence for a new inactivating mutation of the TSH receptor gene.

Congenital hypothyroidism due to impaired thyroid response to TSH was originally described by Stanbury. A diagnosis of congenital hypothyroidism with thyroid unresponsiveness to TSH is accepted if the patient has congenital hypothyroidism, the thyroid gland is in the normal position in the neck, the size of the thyroid is either normal or atrophic, the serum TSH level is increased, the bioactivity of TSH is intact, and the response of the thyroid gland to TSH stimulation is decreased. In all originally described cases serum thyroglobulin was undetectable. We describe a 22-yr-old female patient who was severely hypothyroid and mentally retarded. Serum T4 and T3 concentrations were below the sensitivity of the methods, with elevated serum TSH levels. Serum thyroglobulin was undetectable. A normally shaped hypoplastic gland located in the appropriate anatomical position in the neck was found at scintiscan. The gland did not respond after administration of bovine TSH in terms of 131I uptake, serum thyroid hormones, and thyroglobulin secretion. A diagnosis of congenital hypothyroidism due to TSH unresponsiveness was formulated. Genetic analysis in the propositus showed a homozygous inactivating mutation of the TSH receptor that had not been previously described. The mutation consisted of the substitution of an isoleucine in place of a highly conserved threonine at position 477 in the first extracellular loop of the receptor (T477I). The brother, one sister of the father (whose DNA was not available), the mother of the propositus, one sister, and the brother were heterozygous for T477I. All the heterozygous persons were unaffected. After transfection in COS-7 cells, the mutant receptor displayed an extremely low expression at cell surface. At variance with cells transfected with the wild-type TSH receptor, cells transfected with the mutant T477I did not show constitutive activity for the adenylyl cyclase pathway. A dramatic reduction in the amount of cAMP accumulation after bovine TSH challenge was observed in cells transfected with the mutant T477I receptor. A structural defect in the mutant TSH receptor protein was probably responsible for the poor routing of the receptor to the cell membrane. This is the first time that a loss of function mutation of the TSH receptor is described in a patient with severe congenital hypothyroidism and absent circulating thyroglobulin due to TSH unresponsiveness and the first time that an inactivating mutation of the TSH receptor is described in the first extracellular loop.

[Immunohistochemical identification of biochemical defect of dyshormonogenetic goiter]. / Defeito bioquímico de bócio disormonogenético identificado através de estudo imuno-histoquímico.

This is a report of two cases of congenital goiter in which it was possible to indicate, through immunohistochemistry method, the disorder in the hormonal synthesis. One of these cases was of a sixteen-year-old woman, with goiter since childhood who used thyroid hormone replacement for ten years. Pathology showed coloid goiter with follicular adenoma. Immunohistochemistry showed strong mainly apical follicular cell thyroglobulin. Thyroglobulin was not found in the coloid. This finding is compatible with a malfunction of thyroglobulin exocitosis. The reaction for thyroid peroxidase was adequate for the age. The other case was a 13 year-old-boy with goiter and thyroid hormone replacement since the age of 1 year. He presented slight mental and growth retardation. Pathology showed coloid goiter with follicular adenoma. Immunohistochemistry study showed a very weak and diffuse reaction for thyroid peroxidase while the reaction for thyroglobulin was adequate. These findings indicate quantitative defect of thyroid peroxidase.

Congenital hypothyroid goiter with deficient thyroglobulin. Identification of an endoplasmic reticulum storage disease with induction of molecular chaperones.

Recent advances in understanding the molecular pathogenesis of congenital hypothyroid goiter in cog/cog mice, have raised important questions concerning the maturation of thyroglobulin (the thyroid prohormone) in certain human kindreds with congenital goiter. We have now examined affected siblings from two unrelated families that synthesize an apparently normally glycosylated, > 300 kD immunoreactive thyroglobulin, yet have a reduced quantity of intraglandular thyroglobulin and that secreted into the circulation. From thyroid tissues of the four patients, light microscopic approaches demonstrated presence of intracellular thyroglobulin despite its absence in thyroid follicle lumina, while electron microscopy indicated abnormal distention of the endoplasmic reticulum (ER). We have confirmed biochemically that most intrathyroidal thyroglobulin fails to reach the (Golgi) compartment where complex carbohydrate modification takes place. Moreover, the disease in the affected patients is associated with massive induction of specific ER molecular chaperones including the hsp90 homolog, GRP94, and the hsp70 homolog, BiP. The data suggest that these patients synthesize a mutant thyroglobulin which is defective for folding/assembly, leading to a markedly reduced ability to export the protein from the ER. Thus, these kindreds suffer from a thyroid ER storage disease, a cell biological defect phenotypically indistinguishable from that found in cog/cog mice.

Congenital human thyroglobulin defect due to low expression of the thyroid-specific transcription factor TTF-1.

TTF-1 and Pax-8 are thyroid-specific transcription factors, from homeo and paired box genes, respectively, that are responsible for thyroid development and for thyroglobulin and thyroperoxidase gene expression. However, TTF-1 and Pax-8 preferentially bind to the thyroglobulin and thyroperoxidase promoters, respectively. Here, we have studied a patient with defective thyroglobulin synthesis. Thyroglobulin mRNA was found at very low levels while the mRNA for thyroperoxidase was found to be more abundant compared with control tissue. The low levels of thyroglobulin mRNA are caused by a transcriptional defect due to the virtual absence of TTF-1 expression as determined by Northern blot analysis, reverse transcriptase-PCR, and electrophoretic mobility shift assays. The level of Pax-8 mRNA was the same in the goiter and in the control thyroid. These results are the first reported evidence of a congenital goiter with a thyroglobulin synthesis defect due to the low expression of the thyroid-specific transcription factor TTF-1. Moreover, these data suggest that TTF-1 and Pax-8 would be differentially regulating thyroglobulin and thyroperoxidase gene transcription.

[Thyroglobulin (Tg) gene and familial Tg synthesis defect].

The thyroglobulin (Tg) gene is a 300-kilobase (kb) single copy gene, containing at least 42 exons, mapped in man to chromosome 8 (8q24) and codes for a glycoprotein with a molecular weight 660,000, which functions as a matrix for the thyroid hormone (T4, T3) and iodothyronine synthesis. Recent progress in genetic technology enables us to study a family case of hereditary goiter with hypothyroidism due to Tg synthesis defect. RT-PCR and subsequent sequencing of the Tg gene revealed a C to G conversion at -3 position of the acceptor splice site in intron 3. This splice site mutation resulted in exon. 4-deleted (major) and exon 3-5-deleted (minor) mRNAs in the goiter thyroid. This defect in this patient indicates the importance of the tyrosine No. 130, coded within the exon 4, in the thyroid hormone formation.

A nonsense mutation causes human hereditary congenital goiter with preferential production of a 171-nucleotide-deleted thyroglobulin ribonucleic acid messenger.

Defective or impaired thyroglobulin (Tg) synthesis usually results in congenital goitrous hypothyroidism, virtual absence of Tg in thyroid tissue, and the presence of an elevated concentration of iodoalbumin. The final result of these abnormalities is a decreased rate of T3 and T4 synthesis. We have previously reported two siblings with this syndrome that was attributable to decreased levels of thyroid tissue Tg mRNA, resulting in decreased translation of a fully mature Tg. Further molecular studies in this family are the subject of this report. The Tg mRNA from normal and goitrous thyroid tissue was first reverse transcribed and divided into five overlapping portions from positions 57-8448, and the resulting cDNAs were amplified by polymerase chain reaction and analyzed by agarose gel electrophoresis. The amplification of nucleotides (nt) 4502-5184 from control thyroid tissue Tg mRNA showed a predominant fragment of 683 basepairs (bp) and a minor fragment of 512 bp. This latter fragment contained a 171-nt deletion that mapped between positions 4567 and 4737 of the Tg mRNA. In contrast, the fragment predominantly present in the congenital goiter was 512 bp. The sequencing of the 683-bp fragment revealed that the responsible mutation is a cytosine to thymine transition, creating a stop codon at position 1510. This results in loss of a TaqI restriction site. The point mutation is, thus, removed from a portion of the transcripts by the preferential accumulation in the goiter of a 171-nt-deleted Tg mRNA. The reading frame is maintained and is potentially fully translatable into a Tg polypeptide chain shorter by 57 residues. The presence of the deleted Tg mRNA in normal thyroid tissue, albeit at a low level, strongly suggests that the deleted mRNA sequence corresponds to a complete exon. Our studies suggest that the shorter, alternatively spliced Tg mRNA predominates in the goitrous tissue and probably has a shorter half-life. This would explain the tissue's low Tg mRNA levels, previously reported. Moreover, translation of the mutated transcript would generate a severely truncated Tg polypeptide with limited ability to generate thyroid hormone, resulting in congenital goitrous hypothyroidism.

[Molecular biology of the genetic defects of synthesis, transport and peripheral action of the thyroid gland hormones]. / Biologia molecular dos defeitos genéticos de síntese, transporte e ação periférica dos hormônios da glândula tireóide.

Recent advances in molecular biology and the use of modern techniques made possible the identification and cloning of DNA portions of the human genome encoding proteins that are important for the synthesis, storage, transport and action of thyroid hormones. These achievements have led to the diagnosis of the basic defects at the DNA level and the understanding of the biochemical disturbance in the genetic disorders of thyroid metabolism. In patients with defective expression of thyroid peroxidase (TPO) the presence of polymorphism (RFLP) associated with Bgl-I segregates with the affected individual with goiter, hypothyroidism and positive perchlorate discharge test. Defects in the thyroglobulin are either quantitative (no Tg is synthetized) or qualitative (an abnormal Tg is expressed). Low messenger RNA codifying for Tg was present in one of the families with virtual absence of Tg in serum and thyroid tissue. In another family absence of sialic acid incorporation into the Tg molecule produced a Tg with structural defect, incapable of T3 and T4 generation. A point mutation in the nucleotide 281 of exon 5 has been claimed as the mutation producing a defective TBG. Finally, the resistance to thyroid hormone action is linked to a mutation in the c-erb-A beta, detected by the presence of a polymorphism EcoRV, that will encode a defective protein or will produce a protein that might block the T3 binding to the receptor.

Low levels of thyroglobulin messenger ribonucleic acid in congenital goitrous hypothyroidism with defective thyroglobulin synthesis.

We characterized the virtual absence of immunoassayable thyroglobulin (Tg) in the serum and thyroid gland of two siblings (MA, JNA) and one nephew (RSS) from a family without inbreeding or familial goiter. Diagnosis of defective Tg gene expression was based on findings of normal PBI and low serum T4, low or normal serum T3, negative perchlorate discharge test, and virtual absence of the serum Tg response to challenge by bovine TSH. This conclusion was confirmed by analysis of proteins in the goiter extracts. Only minute amounts of immunoassayable Tg were detected by RIA (MA, 0.11; JNA, 0.19 mg/g tissue; compared to 70-90 mg/g in normal thyroid tissue). Gel filtration in Sephacryl S300 showed the absence of a normal Tg peak at 280 nm and concentration of label mostly on albumin. A minor intermediate peak of radioactivity was also detected, with the size of, approximately, normal Tg. Sodium dodecyl sulfate-agarose gel electrophoresis indicated the absence of Tg dimer and monomer, and Western blotting and immunoelectrophoresis confirmed this finding. Dot blot quantification of Tg and thyroid peroxidase mRNA indicated decreased hybridization of the patients' mRNA (MA, 44%; JNA, 63%) with phTgM2 (Tg probe) and increased hybridization (MA, 191%; JNA, 182%) with the pM5 (thyroid peroxidase probe) compared with control thyroid tissue. Dot blot analysis of Tg mRNA from the two siblings weakly hybridized with 3' and 5' Tg probes. RNA analysis by means of Northern transfer showed a clear signal of hybridization with Tg probe (phTgM1) in the 8- to 9-kilobase range, corresponding to the normal size Tg mRNA. No major polymorphisms were noted in Southern blotting, using seven restriction endonucleases. We conclude that no gross alteration of the 5' region of Tg gene was present in these patients. Ultrastructural examination of the thyroid tissue indicated that the rough endoplasmic reticulum was not augmented, nor were the cisternae of rough endoplasmic reticulum dilated. The defect observed in these goiters is diminished tissue concentration of Tg mRNA with defective translation. However, small amounts of functionally active Tg could be synthesized, iodinated, and immediately hydrolized, yielding mostly T3, owing to the intense tissue stimulation by TSH.

Normal level of thyroglobulin messenger ribonucleic acid in a human congenital goiter with thyroglobulin deficiency.

Two siblings with congenital goiter were investigated from clinical, biochemical, and molecular biology standpoints. The association of clinical and biological hypothyroidism with undetectable levels of serum thyroglobulin (Tg) and the presence of iodohistidines in the urine suggested the diagnosis of defective Tg gene expression. This conclusion was confirmed by analysis of proteins present in goiter extracts. Only minute amounts of Tg-related material was detected by RIA (0.28 and 0.17 mg/g tissue compared to 80-100 mg/g in normal thyroid tissue), by Sepharose 6B chromatography, and by sucrose density gradient centrifugation. Surprisingly, the goiters contained normal amounts of Tg mRNA. The size of the mRNA and the sequence organization of its first five exons also were normal. We conclude that no gross alteration of structure or transcription of the Tg gene was present in these patients. The results are compatible with a lesion affecting the mRNA sequence (point mutation, splicing error etc.), leading to defective translation or abnormal routing of the translation product through the membrane system of the cell. This latter hypothesis is supported by the extreme distension of the goiter endoplasmic reticulum found on electron microscopy.

[Plasma thyroglobulin in treated hypothyroidism in children]. / La thyroglobuline plasmatique au cours de l'hypothyroïdie traitée chez l'enfant.

In order to appreciate the value of the dosage of thyroglobulin (Tg) in the reappraisal of the classification of hypothyroidism after the onset of substitutive treatment and the supervision of patients treated with L-thyroxin (LT), plasma Tg and FT4 levels were studied in 42 samples from 21 hypothyroid children (ages ranging from 18 months to 16 years) under LT treatment. These patients were divided into 2 groups according to the results of scanning: group I: a thyroid (n = 8) and group II: ectopic or hypoplastic thyroid gland (n = 13). A control group consisted of 60 apparently healthy children of the same ages. Tg was undetectable in 11 samples of 6 children but significant levels (6.8 to 17 ng/ml) were found in 5 samples of 2 children. In group II, Tg could be measured in 20 of 26 samples, the mean level (+/- SEM) not being different from that in the control group (14.32 +/- 2.25 and 18.12 +/- 1.28 ng/ml, respectively). However, in this group, Tg levels seemed to be lower (9.75 +/- 3.94 ng/ml) in samples from patients with LT excess than in euthyroid or hypothyroid patients whose values of Tg were 15.8 +/- 3.36 ng/ml and 16 +/- 3.91 ng/ml, respectively.