Genomic organization of the 3' region of the human thyroglobulin gene.

The genomic organization of the 3' end of the human Thyroglobulin (Tg) gene has not previously been characterized. We isolated and characterized seventeen lambda phage clones from a human genomic library that included nucleotides 6263 to 8410 of the Tg mRNA, encompassing the last thirteen 3' exons of the Tg gene. The region contained exons ranging in size from 94 to 222 nucleotides, split by introns of 1 to 64 kb. We estimate a total of 48 exons in the Tg gene. All the intron-exon boundaries were sequenced. We found that the splicing sequences diverged considerably from the 3' and 5' consensus. However, the GT-AG rule was perfectly respected in all the exons. A total of 5788 intronic bases and most of the sequences contained in the 13 exons were analyzed (1846 bases). One sequence variation, TT to CC at positions 8377-8378, was found in the 3' untranslated segment. The three tyrosine residues involved in thyroid hormones synthesis (amino acids 2554, 2568, and 2747) at the carbosyl termini of Tg, are encoded by exons 44, 45, and 48. The knowledge of the precise organization of the Tg gene should help to direct studies of Tg gene mutations in families in which a defect in the synthesis of Tg occurs.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

The 5' region of the human thyroglobulin gene contains members of the Alu family

Repeated sequences were identified in the 5' region of the human Tg gene in introns 4, 5, 10, and 11. Another repeated cluster was located in the 5' flanking sequences, approximately 6 Kb upstream from the first exon. The nucleotide sequence analysis indicated that these repeated sequences are members of the Alu family. The homology between the sequences of the intron 4 and the Alu consensus was 86%. The Alu member studied was oriented in the direction of transcription of the Tg.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Identification of a minor Tg mRNA transcript in RNA from normal and goitrous thyroids.

Polymerase chain reaction (PCR) amplification of nt 4502 to nt 5184 of the thyroglobulin (Tg) mRNA from several patients, with or without elevated serum thyrotropin (TSH), showed a predominant fragment of the expected size (683 bp) and a minor fragment of 512 bp. The sequence of this minor fragment revealed that 171 bp were missing between position 4567 and 4737. It is highly probable that the deleted sequence corresponds to a complete exon, suggesting an alternative splicing as mechanism for the generation of the minor transcript.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Defective thyroglobulin synthesis and secretion causing goiter and hypothyroidism.

The integrity of the Tg structure as a protein is essential for adequate synthesis of thyroid hormone. Also a large supply of iodine and of thyroid hormone is stored into the Tg molecule and available for secretion on demand. Mutations in Tg gene or hyposialylated Tg due to a defective sialyltransferase activity would cause a structurally defective protein and severely impair the functional ability of Tg. In this review we attempt to cover the abnormalities in the synthesis of Tg described in both animals and man. Hereditary congenital goiter with or without hypothyroidism is the phenotypic major clinical finding in these species. Affected animals include sheep, bovine cattle, bongo antelope, goats, and mice. As in man the inheritance mode is autosomal recessive. In most animal studies structurally abnormal Tg is present. The molecular basis for the defective Tg synthesis was attributable to nonsense mutation in exon 9 (Afrikander cattle) and in exon 8 (Dutch goats). In man the Tg defective synthesis has been reported in 89 subjects and frequently more than one sibling is affected in a given generation. Characteristically these patients exhibit hereditary congenital goiter with relatively low Tg levels that do not increase after stimulation with bovine TSH. High PBI concentrations with low serum T4 values indicate the serum presence of iodinated proteins (mainly iodoalbumin). Also iodinated peptides are frequently excreted into the urine. Tissue studies confirm that there is an absent Tg peak at gel filtration, and virtually no immunoassayable Tg is present in the tissue extracts. The molecular basis of these defects have been recently reported in a patient and includes low tissue Tg mRNA probably due to premature degradation of a defective Tg mRNA. The responsible mutation is a cytosine to thymine transition creating a stop codon at position 1510. The point mutation is removed by the preferential accumulation of a 171 nt deleted Tg mRNA. In another subject molecular studies revealed that exon 4 was missing from the major Tg transcript due to a cytosine to guanine transversion at position minus 3 in the acceptor splice site of intron 3. It is anticipated that other mutations responsible for these defects will be identifiable in the near future.Journal ArticleResearch Support, Non-U.S. Gov'tReviewSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Genomic organization of the 5' region of the human thyroglobulin gene

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Identification of a new thyroglobulin variant: a guanine-to-adenine transition resulting in the substitution of arginine 2510 by glutamine.

We analyzed thyroglobulin (Tg) reverse transcription polymerase chain reaction (RT-PCR) products from three congenital goiters and three normal thyroid tissues by Taq I digestion. Tg coding sequences were amplified from position 57 to 8448 in 12 amplification fragments. A Taq I restriction fragment length polymorphism was detected in the most 3' RT-PCR product (nt 7584 through 8448). Data from the sequence showed a G-->A transition (nt 7627) causing the disappearance of the Taq I site in position 7625. It produced the substitution of arginine for a glutamine at position 2510. Afterwards, we established that the glutamine allele is present in normal unrelated individuals, with an allelic frequency of 62%. This Tg variant is thus widely represented in the human population. The available sequence information from rat and bovine Tg showed the presence, in both, of glutamine at position 2510.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Molecular genetics of thyroid diseases.

Over the past 20 years, recombinant deoxyribonucleic acid technology has led to the cloning of three major thyroid specific protein genes (thyroglobulin, thyroperoxidase and thyrotrophin receptor), which happen to be also the main thyroid autoantigens implicated in thyroid diseases. In this context, the impact that molecular genetics has had on the understanding of aetiopathogeny and diagnosis of thyroid diseases is summarized, with special emphasis on a recently discovered genetic mechanism responsible for toxic nodules. One fruitful outcome of the basic research on thyroid-specific gene expression has been the possibility of targeting the expression of a series of genes to the thyroid in transgenic mice. The result is the availability of mouse models mimicking human thyroid diseases such as destructive hypothyroidism, hyperactive thyroid adenomas and thyroid cancers exhibiting varying levels of differentiation.Journal ArticleResearch Support, Non-U.S. Gov'tReviewinfo:eu-repo/semantics/publishe

Study of the promoter region of the human thyroglobulin gene

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Genomic organization of the human thyroglobulin gene: the complete intron-exon structure.

OBJECTIVE: In order to complete the knowledge of the genomic organization of the human thyroglobulin gene, the present work was designed to establish the intron-exon organization from exon 24 to exon 35 and to construct a more complete physical map of the gene. DESIGN: Screening of two genomic libraries, and subsequent restriction mapping, hybridization and sequencing were used to characterize the recombinant phages. METHODS: Two human genomic DNA libraries were screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. The Long PCR method was used to amplify the genomic DNA region containing exon 24. Intron-exon junction sequences were determined by using the Taq polymerase-based chain termination method. RESULTS: We isolated and characterized five lambda phage clones that include nucleotides 4933 to 6262 of the thyroglobulin mRNA, encompassing exons 25-35 of the gene. The remaining exon 24 (nucleotides 4817-4932) was sequenced from the amplified fragment. In total, 8010 intronic bases were analyzed. CONCLUSIONS: The present study shows that the five phages isolated and the amplified fragment include 59.4 kb genomic DNA, covering 1446 nucleotides of exonic sequence distributed over 12 exons, from exon 24 to exon 35. Using previous studies and our current data, 220 kb of the human thyroglobulin gene was analyzed, a physical map was constructed, and all exon-intron junctions were sequenced and correlated with the different domains of the protein. In summary, the thyroglobulin gene contains 48 exons ranging in size from 63 nucleotides to 1101 nucleotides.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe