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Biomedical subjects

H M Targovnik

Publications and source records attributed to H M Targovnik.

At least 19 recordsLinked to original sources
PubMed2001

Congenital goiter with hypothyroidism caused by a 5' splice site mutation in the thyroglobulin gene.

In this work we have extended our initial molecular studies of a consanguineous family with two affected goitrous siblings (H.S.N. and Ac.S.N.) with defective thyroglobulin (Tg) synthesis and secretion because of a homozygotic deletion of a fragment of 138 nucleotides (nt) in the central region of the Tg mRNA, identified previously in H.S.N. In order to identify the intron/exon boundaries and to analyze the regions responsible for pre-mRNA processing corresponding to a 138 nt deletion, we performed a screening of a human genomic library. The intron/exon junction sequences were determined from one positive clone by sequencing both strands of the DNA template. The results showed that the deletion mapped between positions 5549 and 5686 of the Tg mRNA and corresponded to exon 30. The positions of the exon limits differed by three nucleotides from the previously reported data obtained from direct sequencing of the deleted reverse transcriptase-polymerase chain reaction fragment from H.S.N. These variations are because the intron/exon junctions in this region were not available at the time when the deletion was first described. The deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shortened by 46 residues. The same 138 nt deletion was observed in reverse transcriptase-polymerase chain reaction studies performed in the thyroid tissues from Ac.S.N. Genomic DNA analysis showed that a G to T transversion was observed at position +1 in the donor site of intron 30. Both affected patients (H.S.N. and Ac.S.N.) are homozygous for the mutation whereas the normal sister (At.S.N.) had a normal allele pattern. The functional consequences of the deletion are related to structural changes in the protein molecule that either could modify the normal routing of the translation product through the membrane system of the cell or could impair the coupling reaction. Probably the mutant Tg polypeptide might be functionally active in the production of thyroid hormone, because in the presence of a normal iodine ingestion (approximately 150 microg/day), Ac.S.N. was able to maintain normal serum levels of total triiodothyronine (T3) associated with relatively low serum total thyroxine (T4) with normal somatic development without signs of brain damage.

Adult↗
PubMed2001

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.

Base Sequence↗
PubMed2001

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.

Alternative Splicing↗
PubMed2000

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

OBJECTIVE: The purpose of the present work is to establish the intron-exon organization from exon 12 to exon 23 of the human thyroglobulin gene and to construct a physical map of the 5' terminal half of the gene. DESIGN: Screening of a genomic library and subsequent restriction map, hybridization and sequencing methods have been employed to characterize the recombinant positive phages. METHODS: A human genomic DNA library was screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. Intron/exon junction sequences were determined by the Taq polymerase-based chain terminator method. Finally, the thyroglobulin gene was mapped using the Gene Bridge 4 radiation hybrid clone panel. RESULTS: We isolated and characterized four lambda phage clones that include nucleotides 3002 to 4816 of the thyroglobulin mRNA, encompassing exons 12 to 23 of the gene. The exon sizes range between 78 and 219 nucleotides. We found that the GT-AG splicing sequences rule was perfectly respected in all the introns. A total of 7302 intronic bases was analyzed. Hormogenic tyrosine 5 and 1291 are encoded by exons 2 and 18. Also, seven alternative spliced variants are associated with the 5' region. Thyroglobulin gene maps to 5,5 centiRays from the AFMA053XF1 marker, in chromosome 8. CONCLUSIONS: The present study shows that the first 4857 bases of thyroglobulin mRNA are divided into 23 exons and the four phages isolated include 32.6 kb genomic DNA, covering 1815 nucleotides of exonic sequence distributed in 12 exons, from exon 12 to 23.

5' Untranslated Regions↗
PubMed1999

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.

Bacteriophage lambda↗
PubMed1999

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.

Adolescent↗
PubMed1999

[Molecular genetics of the thalassemias in Argentina].

PURPOSE: Was to establish the molecular genetics of thalassemias in Argentina. PATIENTS AND METHODS: Genomic DNA was amplified by PCR and six point mutations in the beta-globin gene were investigated by Dot Blot hybridization using oligonucleotide probes. The most frequent alpha-thalassemia deletions were studied by Southern Blotting. Patients were distributed in 4 groups: a) 109 beta-thalassemic carriers; b) 15 thalassemia major patients; c) 2 thalassemia intermedia patients and d) 14 probable alpha-thalassemic carriers. RESULTS: The distribution of mutated alleles in the group a) was: IVS-1 nt 1: 13.76%, IVS-1 nt 6: 7.34%, IVS-1 nt 110: 23.85%, codon 39: 39.45%, IVS-2 nt 1: 3.68% e IVS-2 nt 745: 1.83%, 10.01% could not be determined with the probes used; in the group b) the allelic distribution was similar and the compound genetic genotype were predominant related to homocygous ones; in the group c): we confirmed the presence of one beta-thalassemia mutation and a alpha gene triplication (alpha alpha alpha) in the 2 patients studied. The alpha-thalassemia character was confirmed in 8 patients of the group d) (6 had -alpha 3,7/alpha alpha genotype and 2,-alpha 3,7/-alpha 3,7 genotype). CONCLUSIONS: This study indicates that the analysis of 6 mutations in the beta-globin gene and the alpha-globin gene deletions are an effective strategy to identify thalassemias in Argentina.

Alleles↗
PubMed1998

Evidence for the segregation of three different mutated alleles of the thyroglobulin gene in a Brazilian family with congenital goiter and hypothyroidism.

We have previously reported a Brazilian family with congenital goiter, hypothyroidism, and marked impairment of thyroglobulin (Tg) synthesis. Analysis of the Tg mRNA in the goiter of one of the siblings revealed a cytosine to thymine transition creating a stop codon at position 1510. This point mutation is removed from the majority of Tg mRNA transcripts by the preferential generation in the goiter of a 171 nt deleted Tg mRNA by alternative splicing. The nonsense mutation destroys a TaqI site at this position in the mutant Tg gene. Using polymerase chain reaction (PCR) amplification and TaqI digestion we found that two siblings affected with goiter and hypothyroidism, as well as the father and three siblings with normal thyroid function, are all heterozygous for the nonsense mutation. This implies that an additional mutation must be present in the affected individuals, generating a compound heterozygote genotype. A new polymorphism within the thyroglobulin gene represented by three alleles has been detected. This was documented by the TaqI restriction enzyme and phTgM3 probe hybridization that showed a three allelic polymorphism with fragment sizes of 16.5 kb (allele A), 14.5 kb (allele B) and 11.0 kb (allele C). Segregation analysis of these alleles in the family indicated that the two affected siblings were homozygous for the allele C. In contrast the unaffected father and three other siblings, who carried the nonsense mutation, were heterozygous for alleles B and C. Analysis of the Tg genotypes implies that two additional mutations of the Tg gene must segregate in this family to account for the observed phenotypes.

Adult↗
PubMed1997

A new point mutation (M313T) in the thyroid hormone receptor beta gene in a patient with resistance to thyroid hormone.

Sequence analysis of the TR beta gene from a patient with the syndrome of resistance to thyroid hormone revealed a novel missense mutation in exon 9, changing thymidine in position 1123 to cytosine. The corresponding amino acid alteration is a substitution of a methionine (ATG) for a threonine (ACG) at codon 313 being the patient heterozygous for the mutation. In contrast, his parents had only the wild-type sequence, suggesting a de novo mutational event.

Child↗
PubMed1997

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.

Adenine↗
PubMed1996

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.

Animals↗
PubMed1996

Molecular analysis of sex determination in sex-reversed and true hermaphroditism.

The SRY (sex region of Y) gene determines testis formation but not all cases of sex reversal in humans can be explained by alterations in this gene. We studied one 46,XY female, four 46,XX males, and nine true hermaphrodites (TH): three with an XY and six with an XX chromosomal constitution. The SRY gene was identified in the XX males and the TH with a Y chromosome but was not demonstrated in the XY female and the six XX TH. The Y-heterochromatin region was also identified in one 46,XX male, indicating a low grade mosaicism undetected by cytogenetics. The amplification of the amelogenin gene showed the presence of a 977-bp band that belongs to the short arm of chromosome X in all patients but the absence of a 780-bp band of the short arm of chromosome Y in three 46,XX males and in all the 46,XX TH. These studies demonstrate that the molecular study of sex-reversed patients and TH will help to understand the complex mechanisms of sex determination. The SRY gene is involved but other genes on the X chromosome and autosomes still remain to be studied.

Adolescent↗
PubMed1995

A 138-nucleotide deletion in the thyroglobulin ribonucleic acid messenger in a congenital goiter with defective thyroglobulin synthesis.

Two siblings (HSN and AcSN) with congenital goitrous hypothyroidism were investigated in terms of clinical, biochemical, and molecular biology. Diagnosis of defective thyroglobulin (Tg) was based on findings of low serum T4, low normal or normal serum T3, a negative percholate discharge test, and the virtual absence of the serum Tg response to challenge by bovine TSH. Only minute amounts of Tg-related antigens were detected by RIA in the goitrous tissue (HSN, 0.82 mg/g, compared to 70-90 mg/g in normal thyroid tissue), as confirmed by sodium dodecyl sulfate-agarose gel electrophoresis that indicated the virtual absence of Tg. The Tg messenger ribonucleic acids (mRNAs) from controls and HSN thyroid tissue were first reverse transcribed and then divided into several portions from positions 57-8448; the resulting complementary DNAs were, in turn, amplified by reverse polymerase chain reaction. The amplification of nucleotides 5165-6048 from control thyroid tissue Tg mRNA showed a fragment of 884 base pairs (bp). In contrast, the fragment present in the HSN was +/- 750 bp and lacked the normal fragment. The sequencing of the smaller fragment revealed that 138 bp were missing between positions 5590-5727 of the HSN Tg mRNA. This deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shorter by 46 residues. A cysteine residue is maintained by the junction between the proximal T from leucine 1831 and the distal GT from cysteine 1877. DNA genomic polymerase chain reaction amplification excludes a deletion in the Tg gene and indicates that the deleted 138-nucleotide sequences lie in the same exon. The functional consequences of the deletion are not entirely clear, but it is conceivable that the excision of this segment of the Tg molecule could affect the protein structure, resulting in its premature degradation, very low colloid storage, and diminished thyroid hormone production rate.

Adult↗
PubMed1994

Molecular genetics of hereditary thyroid diseases due to a defect in the thyroglobulin or thyroperoxidase synthesis.

1. Hereditary goiter and the various degrees of thyroid hypofunction are the result of structural changes in the thyroglobulin (Tg) or thyroperoxidase (TPO) proteins, the inability to couple iodotyrosines or defective iodination, impairing or substantially altering the synthesis of T4 and T3. 2. The first mutations in the Tg and TPO genes responsible for human cases of dyshormonogenesis have been described. The mutation in two siblings with hereditary goiter and marked impairment of Tg synthesis was 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. 3. Genomic DNA studies identified a duplication of a 4-base sequence in the eighth exon of the TPO gene. Interestingly, besides abolishing the enzymatic activity by disrupting the reading frame of the messenger RNA and introducing stop codons, the GGCC duplication also unmasks a cryptic acceptor splice site in exon 9. 4. In conclusion, the identification of different molecular defects provided evidence that hereditary goiter associated with abnormal Tg or TPO synthesis is caused by heterogeneous genetic alterations.

Amino Acid Sequence↗
PubMed1993

Human thyroid tissue do not express thyroalbumin.

Thyroid tissue total RNAs from multinodular goiter (G2) and from hereditary goiter with defective Tg synthesis (JNA) were hybridized with a 5'albumin cDNA probe (F-47), a 3' albumin cDNA probe (B-44) and a thyroglobulin cDNA probe (phTgM3). JNA refers to tissue obtained from a patient with virtual absence of Tg in thyroid tissue and the presence of increased concentration of an albumin-like labeled protein in the thyroid. No hybridization signal was detected in both G2 and JNA with albumin probes at Northern Blot studies. Those results were confirmed by dot-blot analysis of total RNA where no hybridization signal was detected in G2 and JNA. To confirm that thyroid tissues do not express thyroalbumin total RNA from JNA and normal control thyroid tissue (C) were amplified by PCR using albumin and Tg primers. An expected fragment of 592 bp was observed in a human liver sample with the albumin primers. However JNA and C samples showed absence of an amplification product of the same size. We concluded that thyroid cells do not contain the albumin transcript. Albumin is probably taken up from circulation and iodinated by the thyroid follicular cell with subsequent release of iodoalbumin into the circulation.

Albumins↗
PubMed1993

Defective organification of iodide causing hereditary goitrous hypothyroidism.

We present a survey of the current state of knowledge about the prevalence of the syndrome involved in defective organification of iodide, and the mechanism of iodination and coupling catalyzed by the thyroid peroxidase (TPO) enzyme. A brief summary of the recent developments in molecular cloning of TPO and regulation of TPO gene expression is also included. Methods for purification of the enzyme and details about the assessment of TPO activity in tissue are briefly explained. The classification of defective organification of iodide is primarily based on the site of the biochemical defect, being quantitative (TPO absent) or qualitative (TPO structure, localization or apoenzyme are defectives). The presence of TPO inhibitors is also briefly described. The rare possibility of an absent source of peroxide (H2O2) causing defective iodide organification is discussed. Analysis of the 118 reported cases shows that the biochemical classification covers a spectrum of abnormalities and it is likely that further molecular biology studies will increase this heterogeneity as well as refining it. Genetic studies have suggested linkage between the TPO gene polymorphisms and the iodide organification defect and can be of importance for carrier detection and prenatal diagnosis. Neonatal screening for hypothyroidism is likely to expand the number of cases available for DNA analysis and possibly the molecular diagnosis. The importance of the mutations that would affect the histidine (His) residues in the translated protein was recently documented by the finding of a deletion removing part of exon 9 and thus also deleting a proximal His residue. The resulting TPO enzyme was inactive for iodide organification and coupling reaction. It is hoped that in time we will be able to expand our knowledge of the molecular diagnosis of the inborn errors of iodide organification.

Amino Acid Sequence↗
PubMed1993

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.

Amino Acid Sequence↗