A 3' splice site mutation in the thyroglobulin gene responsible for congenital goiter with hypothyroidism.

A case of congenital goiter with defective thyroglobulin synthesis has been studied in molecular terms. The patient is the fifth of a kindred of six, three of which have a goiter. The parents are first cousins. Segregation of thyroglobulin alleles in the family was studied by Southern blotting with a probe revealing a diallelic restriction fragment length polymorphism (RFLP). The results demonstrated that the three affected siblings were homozygous for the RFLP. Northern blotting analysis of the goiter RNA with a thyroglobulin probe suggested that thyroglobulin mRNA size was slightly reduced. Polymerase chain reaction amplification of the 8.5-kb thyroglobulin mRNA as overlapping cDNA fragments demonstrated that a 200-bp segment was missing from the 5' region of the goiter mRNA. Subcloning and sequencing of the cDNA fragments, and of the patient genomic DNA amplified from this region, revealed that exon 4 is missing from the major thyroglobulin transcript in the goiter, and that this aberrant splicing is due to a C to G transversion at position minus 3 in the acceptor splice site of intron 3. The presence in exon 4 of a putative donor tyrosine residue (Tyrosine nr 130) involved in thyroid hormone formation provides a coherent explanation to the hypothyroid status of the patient.

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line.

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.

Effects of naloxone and morphine on the proestrous surge of prolactin and gonadotropins in the rat.

The effects of naloxone hydrochloride and morphine sulfate on the proestrous surge of PRL and gonadotropins (LH and FSH) were investigated in normal cycling Sprague-Dawley rats. Blood samples (0.45-0.50 ml) were withdrawn without anesthesia every 20 min from 1400-2000 h through an atrial cannula implanted the same morning. RIA revealed that a single iv injection of naloxone (0.2 mg/kg) at 1400 h completely suppressed the surge of PRL, and this was reversed by a concomitant injection of morphine (10 mg/kg). Morphine itself did not alter the peak of the PRL surge. Morphine suppressed only the early phase of the LH surge, and this was reversed by naloxone. Naloxone alone did not change the peak of the LH surge but maintained higher levels than controls during the declining phase. The FSH surge was not altered by either morphine or naloxone. These results suggest that endogenous opioid peptides may have a role in regulating the PRL and LH surges during proestrus in the rat.

A novel missense mutation (G2320R) in thyroglobulin causes hypothyroidism in rdw rats.

The rdw rat is a hereditary hypothyroid variant initially derived from the Wistar-Imamichi strain. Proteome analysis by two-dimensional gelelectrophoresis showed that molecular chaperones accumulated in the thyroid glands, suggesting retention of abnormal proteins in the endoplasmic reticulum (ER). Anatomical studies indicated that thyroglobulin (Tg) was not secreted into the follicular lumina, but retained in the dilated ER. Sequencing of the entire Tg complementary DNA from the rdw rat revealed a missense mutation (G2320R) in the acetylcholinesterase-like domain at the 2320th amino acid residue. Carbohydrate residues of the G2320R Tg mutant were of the high-mannose ER type, as shown by sensitivity to the treatment with endoglycosidase H. Molecular chaperones, GRP94, GRP78, and calreticulin, were all accumulated in the rdw rat thyroid glands. Computer analysis of protein secondary structure predicted that the mutation would cause extension of the helix where beta-sheet and turns were formed in the normal Tg. Altered folding of Tg might account for the impaired intracellular transport of Tg and activated premature degradation by the same mechanism as in ER storage diseases.

Preferential renal excretion of iodide derived from thyroxine and triiodothyronine deiodination in man.

Tracer doses of 131I- (Carrier free), 131I-T3 and 131-T4 were administered po to 19 healthy male volunteers at intervals 2 to 8 weeks to study whether or not part of the iodide generated in the kidney from T3 and T4 deiodination may enter the renal tubular lumen and be excreted in the urine without entering the blood stream. U(urine)/T(thyroid) ratios of the radioactivity from these materials were employed as the index of the comparison. U/T ratios were severalfold higher 24 h after 131I-T3 or 131I-T4 administration than after 131I-. The data indicate that the 131I- derived from T3 and T4 metabolism is more readily excreted into urine than 131I- which reaches the kidney as inorganic iodide.

Two novel cysteine substitutions (C1263R and C1995S) of thyroglobulin cause a defect in intracellular transport of thyroglobulin in patients with congenital goiter and the variant type of adenomatous goiter.

We analyzed the thyroglobulin (Tg) gene of 2 unrelated patients with congenital goiter and the Tg gene of 2 siblings with the variant type of adenomatous goiter. The clinical characteristics of the patients with congenital goiter and the variant type of adenomatous goiter were very similar, except for serum Tg levels, which were less than 15 pmol/L in the patients with congenital goiter, but 117-181 pmol/L in the patients with the variant type of adenomatous goiter (normal, 15-50 pmol/L). The tissue content of Tg in the thyroid glands of all 4 patients was reduced at 0.9-3.8% of total protein (normal, 19-40%). The missense mutation C1263R was detected in the 2 unrelated patients with congenital goiter; the pedigree study showed an autosomal recessive pattern of inheritance. In the 2 siblings with the variant type of adenomatous goiter, the missense mutation C1995S was homozygously detected. In the Tg complementary DNA of 110 normal subjects, the allelic frequencies of the C1263R and C1995S mutations were each less than 0.5%. Also in the normal subjects were detected 35 nucleotide polymorphisms, the insertion of 3 nucleotides, and 1 alternative splicing, each of which was not associated with any specific thyroid disease. From these data, the molecular mechanism of the C1263R and C1995S mutations was elucidated. We first analyzed the carbohydrate residues of C1263R Tg and C1995S Tg. Sensitivity to treatment by endoglycosidase H suggests that C1263R Tg and C1995S Tg were retained in the endoplasmic reticulum (ER). Also, the presence of endoglycosidase H-resistant Tg as well as endoglycosidase H-sensitive Tg in the patients with the variant type of adenomatous goiter suggests that a fraction of C1995S Tg was transported to the Golgi and associated with the mildly increased serum Tg levels. Native PAGE and Western blot analysis with anti-Tg antibody showed that C1263R Tg and C1995S Tg form high mol wt aggregates in the ER. Our results suggest that missense mutations that replace cysteine with either arginine or serine cause an abnormal three-dimensional structure of Tg. Such misfolded Tg polypeptides are retained in the ER as high mol wt aggregates.

Polymorphism of the polyalanine tract of thyroid transcription factor-2 gene in patients with thyroid dysgenesis.

OBJECTIVE: One of the thyroid-specific transcription factors, thyroid transcription factor-2 (TTF-2), performs a crucial role in the development of the thyroid gland. We performed genetic analysis of the TITF2 gene (encoding TTF-2) in patients with thyroid dysgenesis. METHODS: By direct sequencing of the PCR products of TITF2, we screened the genomic DNA from 46 patients with thyroid dysgenesis (five had agenesis, six had hypoplasia, 15 had ectopy, and 20 were undetermined). We also studied the transcriptional activities of TITF2 by co-expressing the luciferase gene directed by the human thyroglobulin gene promoter. RESULTS: Human TITF2 consists of a forkhead domain, a polyalanine tract, and unique C-terminal residues. In one of the patients with an ectopic sublingual thyroid, we found a polyalanine tract of 11 alanine residues on one chromosome instead of the 14 alanine residues found in normal controls. In one patient with hypoplasia, the polyalanine tract consisted of 12 heterozygous alanine residues. The reduced polyalanine tracts were not detected in 101 normal individuals. However, the expression study showed that the transcriptional activities of TITF2 with reduced polyalanine-tract lengths were equal to that of TITF2 with an unreduced polyalanine tract. CONCLUSION: These results suggest that the polymorphism of the polyalanine tract of TITF2 is not a frequent cause of developmental defects of the human thyroid gland.

Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease.

To confirm the significance of excretion of annexin V into the urine and the change of urinary annexin V concentration in kidney disease, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two monoclonal antibodies. Urinary annexin V concentration was measured in healthy individuals and patients with kidney and other diseases. Urinary annexin V did not change over a range of pH between 5.0 and 8.0, and was stable during the course of the study for 24 h at room temperature and for 8 days at 4 degrees C. The mean urinary annexin V concentration in 105 normal healthy individuals was 1.5+/-1.5 ng/ml, while that in patients with nephrotic syndrome and systemic lupus erythematosis (SLE) nephritis was 9.3+/-9.1 and 6.6+/-6.7 ng/ml, respectively, and that in IgA nephropathy and chronic renal failure was 2.6+/-2.1 and 1.3+/-0.7 ng/ml, respectively. Annexin level correlated with urinary protein concentration (r=0. 717), but not the serum creatinine concentration, blood urea nitrogen (BUN) and 24-h creatinine clearance. Mean urinary annexin V concentration in patients with ischemic heart disease, hypertension, and diabetes mellitus was 1.4+/-1.0, 1.4+/-1.1, and 1.7+/-1.3 ng/ml, respectively. In one case of relapsing nephrotic syndrome, the urinary annexin V concentration was markedly increased in the early phase after admission and then decreased. This patient later required hemodialysis. These results suggest that a high urinary annexin V concentration may be an indicator of acute renal injury related to the urinary protein level.

Analysis of the promoter of the thyrotropin receptor gene and the entire genomic sequence of thyroid transcription factor-1 in familial congenital hypothyroidism due to thyrotropin unresponsiveness.

We previously reported that our patients with congenital primary hypothyroidism associated with thyrotropin (TSH) unresponsiveness through an autosomal recessive pattern of inheritance did not have mutations in the coding region of the TSH receptor gene. In the current study, we analyzed the promoter of the TSH receptor gene and the entire region of the thyroid transcription factor-1 (TTF-1) gene, including promoter, two exons, and one intron, because expression of the rat TSH receptor gene is reported to be stimulated by the interaction of the promoter of the TSH receptor gene with TTF-1. Screening for mutations was performed by RNase cleavage assay, and the polymerase chain reaction (PCR) products were subsequently sequenced by the automatic sequencer. In the promoter of the TSH receptor gene, a duplication of nucleotides -346 to -330 was detected in one allele, but haplotype analysis of the family demonstrated lack of linkage between the duplication and the TSH unresponsiveness. The same duplication was also observed in some normal subjects. In the TTF-1 gene, we detected a transition (guanine to adenine) in the intron at the minus four position of cryptic 3' splice site in one allele, but absence of linkage suggested that the transition was not responsible for the TSH unresponsiveness. The same transition also was found in some normal subjects. These results suggest that TSH unresponsiveness in our patients is unlikely to be caused by mutations either in the promoter of the TSH receptor gene or in the TTF-1 gene.

[Review of thyroid disease and recent progress in thyroid research].

Major thyroid diseases and recent progress in thyroid research are reviewed, including our clinical experiences and data on genetic analysis. Of the 19,944 patients receiving care in our endocrinology and metabolism department over the past 26 years(from 1974 to 2000), there were 4,471(22.4%) patients with thyroid diseases. Of these patients with thyroid disease, 37.3% had Graves' disease, 24.1% had Hashimoto's thyroiditis, and 22.2% had a benign thyroid tumor. Male-to-female ratio for Graves' disease was 1:3.2. The precise mechanism and genetic or environmental factors underlying the onset and progression of autoimmune thyroid disease need further investigation, although recent thyroid research, especially molecular level studies, has resulted in many new insights. Our genetic analysis of patients and experimental animals with thyroglobulin(Tg) abnormalities indicated the amino acids involved in the surface electric charge were important in maintaining the solid structure of Tg and thyroid hormone synthesis in addition to tyrosine and cysteine. In three patients with hyperthyroid Graves' disease, Hashimoto's thyroiditis or idiopathic hypothyroidism, followed by the author for 8 to 20 years, it was indicated that continued comprehensive care was needed for various episodes, even those arising from non-endocrine conditions, throughout the clinical course, although clinical and laboratory findings showed improvement of the thyroid disease itself.

[Recent advance in the thyroid testing with special reference to the gene analysis].

Thyroglobulin (Tg) is a large (660 kd) homodimeric glycoprotein molecule, encoded by a gene on chromosome 8, that is secreted uniquely by thyroid follicular cells. The steps of mature dimeric Tg synthesis include folding and assembly of nascent Tg with glycosylation, in the endoplasmic reticulum (ER), and dimerization and carbohydrate modification in the Golgi apparatus, followed by incorporation into exocytotic vesicles for export into the lumen of thyroid follicles, after which thyroid peroxidase catalyses iodination of tyrosyl residues and coupling of some of them within the Tg polypeptides to form thyroid hormones (thyroxine and triiodothyronine). Here, we reviewed recent progress in the study of Tg synthesis mechanisms, especially of the function of some molecular chaperones which possibly participate in the Tg synthesis. Our recent findings indicated that Tg mutations C1263R and C1995S caused a defect in intracellular transport of Tg. The thyroid disease caused by Tg gene mutations was considered as a model of the defect in the intracellular transport of de novo synthesized protein (the ER storage disease [ERSD]). ERSDs seen in organs other than the thyroid gland are also briefly reviewed. Gene abnormalities in the other proteins in the thyroid gland, such as thyroid peroxidase, Na/I symporter, TSH receptor, thyroid transcription factor (TTF) 1, TTF 2, and PAX 8, are also discussed.

[Present situation and problems in allergy testing in vitro].

The present situation and problems related to in vitro assays for measurement of specific immunoglobulin E(IgE) are described. A comparison of four commercially available assay kits(CAP, AlaSTAT, MAST and QAS) revealed that of the allergens studied, common ragweed, mugwort and fungus(aspergillus, candida and alternaria) showed low correlation coefficients. The precise reason for this discordance is not clear, however, we speculated that difference in the origin and extraction method of allergens may affect the correlation results. Random between-the-lot-difference was noted: the results for egg white and milk in QAS showed differences of two classes, although the total imprecision expressed in CV(%) was less than 12% when only a single lot was tested. These results suggest that the specific IgE assay may need an improvement in reagents(approval of standard materials and establishment of standard procedures for allergen extraction) and careful daily quality control by the user.

[DNA diagnosis: its induction, problem and future--with special reference to congenital goiter].

PCR technique is a powerful tool for genetic analysis of various diseases including infectious diseases, inherited diseases and malignant tumors. Some of our experiences and problems in gene level diagnosis using mainly the PCR technique newly introduced to our clinical laboratory were discussed. A case of congenital goiter in which the analysis at the gene level was the first demonstration of a mutation associated with the abnormal expression of the thyroglobulin gene in man, is also presented. The direct demonstration from the sputum of the presence of M. tuberculosis DNA by PCR will be a great benefit to the clinician in the diagnosis and treatment of tuberculous infection. At present, it is possible to report the results within 2 days from the submission of samples, but further considerations on the sensitivity and specificity are required. The clinical laboratory will be required to accumulate sufficient knowledge of gene level analysis of various disease conditions and master the techniques.

[Serum thyroglobulin measurement and its clinical significance].

The tissue specific origin of thyroglobulin (Tg) in the blood has made this protein very applicable as a diagnostic marker in various thyroid diseases such as differentiated thyroid cancer (DTC), subacute thyroiditis (ST), destructive thyroiditis, Graves' disease (GD) and congenital thyroid diseases, although the mechanism by which Tg is released and the molecular structure in which it appears in the circulation are not fully understood. This review first describes serum Tg measurements using the immuno-radiometric assay kits available in Japan and the problem of this assay in relation to interference by the autoantibody to Tg. Finally, future directions of serum Tg measurement and its clinical application are considered based on the recent evidence that the molecular form of Tg in the circulation has characteristics that differ for specific thyroid diseases (DTC, ST or GD), and that the detection of the Tg molecule by reverse transcriptase polymerase chain reaction (RT-PCR) is possible from using thyroid cells in peripheral circulation and is effective in diagnosis and monitoring of DTC.

[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.

Diversity of molecular forms of plasma brain natriuretic peptide in heart failure--different proBNP-108 to BNP-32 ratios in atrial and ventricular overload.

OBJECTIVE: Recent studies have shown that plasma levels of brain natriuretic peptide (BNP)-32 and proBNP-108 are increased in heart failure (HF) and that the BNP-32 assay kit in current clinical use cross-reacts with proBNP-108. We investigated why proBNP is increased without processing in HF was investigated. DESIGN, SETTING AND PATIENTS: Plasma BNP-32 and proBNP-108 in normal individuals (n=10) and in patients with atrial fibrillation (AF) (n=18) and HF (n=132) was measured. BNP-32 and proBNP-108 in ventricular and atrial tissue and in pericardial fluid using a specific fluorescent enzyme immunoassay following Sep-Pak C18 (Waters, Milford, Massachusetts, USA) cartridge extraction and gel filtration was also measured. MAIN OUTCOME MEASURES: Levels of both BNP-32 and proBNP-108 were higher in HF than in control or AF (both p<0.01), and the levels of these peptides significantly correlated (r=0.94, p<0.001). The proBNP-108/total BNP (BNP-32+proBNP-108) ratio was widely distributed and lower in HF (0.33 (0.17)) than in control (0.41 (0.06), p<0.05) and AF (0.45 (0.04), p<0.002). The proBNP-108/total BNP ratio was higher in HF with ventricular than in HF with atrial overload (0.45 (0.10) vs 0.20 (0.11), p<0.001). Consistent with this finding, the major molecular form were proBNP-108 and BNP-32 in ventricular (n=6, 0.67 (0.04)) and atrial (n=7, 0.76 (0.05), p<0.0001) tissues, respectively. ProBNP-108 was also the major molecular form of BNP in pericardial fluid (n=8, 0.82 (0.05)). The proBNP-108/total BNP ratio increased and decreased with HF deterioration and improvement, respectively. CONCLUSION: These results suggest that BNP-32 and proBNP-108 is increased in HF and that the proBNP/total BNP ratio increases in association with pathophysiological conditions such as ventricular overload.