Up to date with human thyroglobulin.

The coding region of the human thyroglobulin (TG) mRNA has been resequenced, and comparison with the TG sequence originally published in 1987 showed many variations. All of the variations were validated in 20--40 other alleles, and this resulted in the revision of 41 nucleotide positions. This review presents the revised wild-type human TG sequence, including all known exon/exon boundaries and additional data on the TG mRNA population, concerning alternative splicing and variability of the polyadenylation cleavage site. The amino acid sequence derived shows one additional, 12 changed, and 10 polymorphic residues. Protein characteristics, such as acceptor and donor tyrosine residues, N-glycosylation sites, cysteine-rich repeats, the proposed receptor domain, and antigenic epitopes, are included, and their relationship to the revised sequence is discussed. Furthermore, all reported TG mutations causing dyshormonogenesis in humans and animals are designated in the nucleotide and amino acid sequences. This up-to-date profile of the human TG molecule presents the features of importance for its complex role in thyroid hormonogenesis, and is the basis for future studies on the structure--function relationship.

Heterogeneity in polyadenylation cleavage sites in mammalian mRNA sequences: implications for SAGE analysis.

The analysis of a human thyroid serial analysis of gene expression (SAGE) library shows the presence of an abundant SAGE tag corresponding to the mRNA of thyroglobulin (TG). Additional, less abundant tags are present that can not be linked to any other known gene, but show considerable homology to the wild-type TG tag. To determine whether these tags represent TG mRNA molecules with alternative cleavage, 3'-RACE clones were sequenced. The results show that the three putative TG SAGE tags can be attributed to TG transcripts and reflect the use of alternative polyadenylation cleavage sites downstream of a single polyadenylation signal in vivo. By screening more than 300 000 sequences corresponding to human, mouse and rat transcripts for this phenomenon we show that a considerable percentage of mRNA transcripts (44% human, 22% mouse and 22% rat) show cleavage site heterogeneity. When analyzing SAGE-generated expression data, this phenomenon should be considered, since, according to our calculations, 2.8% of human transcripts show two or more different SAGE tags corresponding to a single gene because of alternative cleavage site selection. Both experimental and in silico data show that the selection of the specific cleavage site for poly(A) addition using a given polyadenylation signal is more variable than was previously thought.

A premature stopcodon in thyroglobulin messenger RNA results in familial goiter and moderate hypothyroidism.

Impaired thyroglobulin (Tg) synthesis is one of the putative causes for dyshormonogenesis of the thyroid gland. This type of hypothyroidism is characterized by intact iodide trapping, normal organification of iodide, and usually low serum Tg levels in relation to high TSH, and when untreated the patients develop goiter. In thyroid tissue from a 13-yr-old patient suspected of a thyroglobulin synthesis defect, the Tg mRNA was studied. The complete coding region of 8307 bp was directly sequenced and revealed a homozygous point mutation: a C886T transition in exon 7. Upon translation this mutation would result in a stopcodon at amino acid position 277, replacing the arginine residue. A Tg cDNA construct containing the mutation was expressed in rabbit reticulocyte lysate resulting in a truncated protein of 30 kDa. Expression in the presence of microsomal membranes resulted in a gel shift of this Tg molecule, indicating glycosylation ability. Two other siblings had a clinical presentation like the index patient, while their parents were unaffected. Additional restriction fragment length polymorphism analysis of the pedigree verified that the homozygous nonsense mutation cosegregated with the clinical phenotype. Clinically, hypothyroidism was not severe in the affected siblings because the truncated Tg glycoprotein was still capable of thyroid hormonogenesis.

The screening for mutations in the thyroglobulin cDNA from six patients with congenital hypothyroidism.

The six patients described in this study were clinically diagnosed with congenital hypothyroidism. Based on clinical and pathophysiological parameters, the cause of the thyroid dyshormonogenesis was suspected to be a defect in the synthesis of thyroglobulin, the matrix protein for thyroid hormone synthesis in the thyroid gland. After RNA isolation from six goitrous tissues and control thyroid tissues, RT-PCR was used to amplify 20 overlapping thyroglobulin (TG) cDNA fragments. Two alternative splice transcripts were identified: a transcript with a deletion of nucleotides 177-274 and a transcript with a deletion of nucleotides 3430-3736 that result in frame shifts and the introduction of premature stop codons. Two alternatively spliced transcripts not changing the reading frame were also identified: a transcript containing a deletion of nucleotides 4529-4699 and a transcript with a deletion of nucleotides 7301-7561. All these transcripts were expressed in thyroid tissue of both patients and controls. Nucleotide sequence analysis and comparison to the revised TG sequence (1997) revealed one revision and eight polymorphisms that did not result in amino acid changes and four polymorphisms that did change amino acid codons. In three patients a homozygous mutation was present at nucleotide position 229, causing a glycine to serine amino acid substitution. The clinical description 'thyroglobulin synthesis defect' in this population cannot be explained by major mutations in the coding region of the TG gene. Furthermore, the presence and level of expression of the alternatively spliced transcripts do not co-segregate with thyroid dyshormonogenesis, since in normal thyroid tissue the same alternatively spliced transcripts are present.

The revised 8307 base pair coding sequence of human thyroglobulin transiently expressed in eukaryotic cells.

We developed a transient transfection system for human thyroglobulin (TG) cDNA in both human thyroid cells and in COS-1 cells. Four overlapping TG cDNA fragments were amplified by reverse transcription-PCR from RNA of normal thyroid tissue. The most 5' fragment includes the natural translation initiation site and the sequence encoding the signal peptide (SP). After subcloning, the nucleotide sequence was determined and compared with the published human sequence, resulting in the detection of 30 nucleotide variations. For validation purposes, all variations were screened in 6-12 normal human alleles. Twenty-one were present in all screened alleles and have to be revised in the published nucleotide sequence. Since one variation concerns a triplet insertion, the coding sequence of the mature human thyroglobulin is 8307 nucleotides encoding 2750 amino acids. The TG cDNA constructs were transiently transfected in HTori 3 and COS-1 cells and protein expression was detected using a polyclonal anti-human-TG on fixed cells and after SDS-PAGE. In both cell-lines all four TG protein fragments were expressed. The mannose structures detected on the proteins by lectins and localization after expression in the cells suggest that only the N-terminal TG fragment (containing the SP) is directed to the endoplasmatic reticulum but is unable to reach the Golgi complex. The described expression system in human thyrocytes will be a helpful tool in studying the structure-function relationship of human TG in thyroid hormonogenesis.

[Complications of an intravenous administration system (Port-A-Cath) in children with an oncologic disease]. / Complicaties van een intraveneus toedieningssysteem (Port-A-Cath) bij kinderen met een oncologische aandoening.

OBJECTIVE: To describe complications of the use of an intravenous administration system (Port-A-Cath; PAC) in children. DESIGN: Retrospective record analysis. SETTING: Emma Children's Hospital, the Children Academic Medical Centre, Amsterdam. METHOD: From January 1989 to January 1992, 66 children aged 1 to 20 years were treated via a PAC system for malignant disease. 70 PACs were implanted with a cumulative period of use of 27,981 days. RESULTS: In one-third of the patients (36%) one or more complications arose. Most common were infection (16 times) and obstruction (8). Tip dislocation, secondarily infected haematoma, leakage, thrombosis and infiltrate occurred occasionally. Almost 50% of the complications could be treated effectively. In the other cases the PAC system had to be removed. CONCLUSION: The advantage of a PAC for oncological patients is the improvement of the quality of life, as it provides a simple and painless access to the venous system. Complications occur regularly, however. Reduction of the number of complications should be the start of further perfection of the PAC system.

The human low affinity immunoglobulin G Fc receptor IIC gene is a result of an unequal crossover event.

We have isolated and characterized three genes coding for hFc gamma RIIA, IIB, and IIC. Each gene spans approximately 15-19 kilobases of DNA and consists of eight exons. Two exons encode the 5'-untranslated region and signal peptides, two exons code for homologous Ig-like extracellular domains, a single exon encodes the transmembrane spanning region, and three exons encode the cytoplasmic domains and 3'-untranslated regions. Analysis of gene structures support the concept that the hFc gamma RIIA and hFc gamma RIIB genes originated via gene duplication and divergence processes. The hFc gamma RIIC gene, however, showed a remarkable homology at its 5' end with the hFc gamma RIIB gene, whereas its 3' region was highly homologous with the hFc gamma RIIA gene, suggesting that the hFc gamma RIIC gene results from an unequal crossover event between the hFc gamma RIIA and IIB genes. This hypothesis was supported by nucleotide sequence analyses of the putative break-point region. The proposed site of recombination was located approximately 300 nucleotides downstream from the sixth (C1) exon. These data provide a unique model for the evolutionary generation of a receptor family with multiple biological functions.