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.

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.

[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.

[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.

Localization of annexin V in rat normal kidney and experimental glomerulonephritis.

The localization of annexin V, a calcium binding protein, was immunochemically and immunohistologically studied in experimental rat glomerulonephritis using annexin V polyclonal antibody. Plasma and urinary annexin V levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Urinary annexin V level, which was correlated with urinary L-lactate dehydrogenase activity, N-acetyl-beta-D-glucosaminidase activity and protein level, increased time-dependently after the injection of nephritogenic antigen (bovine glomerular basement membrane), progressively increasing to attain a peak level at 4 weeks of 51.5 +/- 11.3 ng/h. However, plasma annexin V level showed no increase during the study period. Normal kidneys showed strong staining for annexin V in distal tubules, being particularly strong in tubules of the inner stripe of the outer medulla, but could not be detected in proximal tubules. Annexin V was seen in visceral epithelial cells. Bowman's capsule of the glomerulus, the vascular endothelium of arterioles and interlobular arteries, and vascular smooth muscle. In nephritis, the lumen of distal tubules and the luminal cell membrane were deeply stained, with leakage of annexin V being observed from tubular cells. In the present study, renal annexin V was markedly excreted into urine, and its urinary level reflected the severity of damage of renal tissue and the progression of nephritis. These changes of annexin V in the distal tubule and visceral epithelial cells may be of significance in cell injury of the kidney.

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.

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.

Measurement of serum low density lipoprotein-cholesterol in patients with hypertriglycemia.

Low density lipoprotein-cholesterol (LDL-c) concentration measured by a homogeneous enzymatic assay was reported to correlate well with the modified beta-quantification assay, especially in samples with high triglyceride (TG) concentration. In this study, we evaluated a homogeneous enzymatic assay, Cholestest-LDL assay system, in hypertriglycemic patient samples, and found that 56% (9/16) of serum samples with intermediate TG concentrations (2.27-4.52 mmol/L) showed more than 10% discrepancy with concentration by the modified beta-quantification assay. Such serum samples originated from patients with hyperglycemia of type II a (three cases), type II b (two cases), type III (one case), and type IV (six cases). Differential staining of cholesterol and triglyceride after agarose gel electrophoresis revealed that these serum samples contained significant amounts of intermediate fractions between pre-beta- and beta-lipoproteins. Since lipoprotein (a), which migrates between pre-beta- and beta-lipoproteins, is not correlated with the discrepancy, we believe the intermediate fraction consists of intermediate density lipoprotein (IDL) and a chylomicron remnant. A part of IDL and chylomicron remnant, which contain a significant amount of triglyceride, might be measured as LDL-c by the homogeneous enzymatic assay, but not by the modified beta-quantification assay.

[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.

Secretion of TNF-alpha, IL-8 and nitric oxide by macrophages activated with polyanions, and involvement of interferon-gamma in the regulation of cytokine secretion.

When macrophages derived from rat bone marrow were cultured in the presence of polyanions such as acetyl lignin (EP3), sulfonyl lignin (LS) or dextran sulfate (DS), the cells secreted TNF-alpha, IL-8 and nitric oxide (NO). EP3 had a dose-dependent effect on the secretion of TNF-alpha, IL-8 and NO. EP3 significantly affected secretion at concentrations greater than 5 microg/ml. The EP3 effect was at its maximum between concentrations of 50 and 100 microg/ml. LS and DS induced a slight increase in the secretion of cytokines and NO at a concentration of 100 microg/ml. The use of the reverse-transcription polymerase chain reaction (RT-PCR) showed that the increases in cytokine and NO secretion were due to an increase in cytokine mRNAs or NO synthase mRNA. Anti-TNF-alpha antibodies partially inhibited NO secretion by EP3-activated macrophages, although IL-8 secretion was independent of antibody treatment. The secretion of TNF-alpha and NO was also unaffected by the addition of anti-IL-8 antibodies. The addition of interferon-gamma (IFN-gamma) to the culture medium did not alter TNF-alpha and NO secretion by the EP3-activated macrophages, however, IL-8 secretion was increased when a low concentration of IFN-gamma (0.2 U/ml) was added, but was reduced in the presence of a high concentration of IFN-gamma (2000 U/ml). IFN-gamma produced similar effects on cytokine and NO secretion in macrophages activated with lipopolysaccharide (LPS). Therefore, it is concluded that macrophages treated with polyanions secrete cytokines and NO, and that INF-gamma is involved in the regulatory mechanism of cytokine and NO secretion.

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.

[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.

Missense mutation (C1263R) in the thyroglobulin gene causes congenital goiter with mild hypothyroidism by impaired intracellular transport.

Metabolic abnormalities in thyroid hormonogenesis cause congenital goiter. Here we studied a case of mild hypothyroidism caused by a novel missense mutation in the thyroglobulin (TG) gene. A female patient underwent thyroidectomy twice at the age of 27 and 43 years because of gradual enlargement of the thyroid. By RNase cleavage assay and PCR direct sequencing we identified a thymine to cytosine transition at nucleotide 3828 (from the transcription start site) which causes amino acid change from cysteine to arginine at codon 1263. A pedigree study suggested autosomal recessive inheritance due to consanguineous marriage of her parents. Immunohistochemical study suggested impaired intracellular transport of the mutant TG. Sensitivity to endoglycosidase H confirmed that the mutant TG failed to reach the Golgi compartment. Native polyacrylamide gel electrophoresis and Western blot analyses showed that formation of monomers and homodimers was defective with abundant high molecular-weight aggregates which are normally formed transiently after translation. To examine if the mutant TG is functionally defective, we separated thyroid tissue extract on a Biogel A5m column and measured T4 and T3 released from proteins in each fraction by treatment with proteinase K. Although thyroid hormones released per mole of the mutant TG protein did not decrease, those released per mg of total protein decreased. In conclusion, the missense mutation in the TG gene caused congenital goiter with mild hypothyroidism due to an altered protein structure which resulted in defective intracellular processing and premature degradation by "quality control" mechanisms. Although the tissue TG content was greatly reduced, the hypothyroidism was mild with slow progression of the goiter, because the mutant TG was a relatively good substrate for the synthesis of the thyroid hormones.

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.

Sequence analysis of thyroid transcription factor-1 gene reveals absence of mutations in patients with thyroid dysgenesis but presence of polymorphisms in the 5' flanking region and intron.

Congenital hypothyroidism is caused by several mechanisms. The most common cause worldwide is iodine deficiency, but in iodine-sufficient regions thyroid dysgenesis is the most common cause of congenital hypothyroidism. In the present study we analyzed the thyroid transcription factor-1 (TTF-1) gene in patients with congenital hypothyroidism due to thyroid dysgenesis: three patients with athyrosis, five with ectopy, and one with hypoplasia. Genomic DNA was isolated from peripheral leukocytes, and the TTF-1 gene, including a 5' flanking region, two exons and one intron was amplified by polymerase chain reaction (PCR) with 4 pairs of primers. The PCR products were directly sequenced by the Dye Terminator Cycle Sequencing method. We could not find any mutations specific for the thyroid dysgenesis in the 5' flanking region, two exons and one intron in the TTF-1 gene, but two heterozygous nucleotide substitutions were detected in the intron: a G to A transition at nucleotide 469 (G469A) and a C to A transversion at nucleotide 866 (C866A). The same nucleotide changes were detected in some normal subjects. Allelic frequencies of the polymorphisms G469A and C866A were 23% and 10%, respectively. Another normal polymorphism in the 5' flanking region was a G to T transversion at nucleotide -845 from the transcription start site (G-845T). The allelic frequency of the polymorphism G-845T was 28%. We also found 12 polymorphisms in the 5' flanking region, two in the intron and one in the 3' untranslated region. These polymorphisms were detected in 100% chromosomes. These results suggest that congenital hypothyroidism associated with thyroid dysgenesis is unlikely to be caused by mutations in the TTF-1 gene in which, however, were detected normal polymorphisms in the 5' flanking region, intron and 3' untranslated region.

Activation of macrophages by lactoferrin: secretion of TNF-alpha, IL-8 and NO.

When macrophages were cultured with lactoferrin, cytokines such as tumor necrosis factor (TNF-alpha), interleukin 8 (IL-8) and nitric oxide (NO) were secreted. Secretion of TNF-alpha peaked at 6 h of incubation in the presence of lactoferrin and then declined. About 80% of the maximum secretion of IL-8 was observed at 6 h of incubation. The concentration of IL-8 in the culture medium remained almost constant between 24-72 h. In contrast, no significant effect on NO secretion was observed at 6 h, but a significant effect was observed at 24 h and secretion gradually increased between 24-72 h. The effects of lactoferrin on the secretion of TNF-alpha, IL-8 and NO were dose-dependent and lactoferrin had a significant effect on secretion of at concentrations greater than 10 mg/ml. The use of reverse transcription-polymerase chain reaction (RT-PCR) showed that the results obtained were consistent with the cytokine secretion results. It is concluded that lactoferrin activates macrophages which result in the secretion of TNF-alpha, IL-8 and NO.

Secretion of TNF-alpha from macrophages following induction with a lignin derivative.

Macrophages derived from rat bone marrow were treated with macrophage colony stimulating factor (M-CSF) to obtain a sufficient number of cells for the tumor necrosis factor (TNF-alpha) assay. The present study has been designed to investigate whether the production of TNF-alpha, which induces multinucleated giant cell formation, is regulated by polyanions such as lignin derivatives. ELISA for TNF-alpha showed that the polyanion induced TNF-alpha production by macrophages. The secretion of TNF-alpha from the cells reached a maximum at 3-6 h, and then showed a slight decline. Northern blotting of TNF-alpha mRNA showed that the amount of TNF-alpha reached a maximum within 1 h of macrophage culture in the presence of a lignin derivative. On the other hand, TNF-alpha mRNA was undetectable in the control cells. It was concluded that stimuli such as that provided by lignin derivatives increases the amount of TNF-alpha mRNA, which is then followed by translation of TNF-alpha.

[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.

[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.