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Biomedical subjects

L J DeGroot

Publications and source records attributed to L J DeGroot.

At least 91 records · Page 5Linked to original sources
PubMed1994

N-ras mutation: an independent prognostic factor for aggressiveness of papillary thyroid carcinoma.

BACKGROUND: The clinical importance of point mutations of ras oncogene in differentiated thyroid cancers has not been fully clarified. The purpose of this study is to determine the prognostic importance of ras mutation in papillary thyroid carcinoma. METHODS: Tumors of 91 patients with papillary carcinoma were studied; mean follow-up was 14.1 years (range, 1 to 40 years). Patients were grouped as follows: class I, intrathyroidal disease, n = 21; class II, cervical node metastases, n = 22; class III, extrathyroidal disease, n = 19; and class IV, distant metastases, n = 29. DNA was analyzed with polymerase chain reaction, oligonucleotide hybridization, and DNA sequence analysis techniques. RESULTS: Thirteen (14.3%) of 91 tumors showed an N-ras point mutation: 4.8% (1 of 21) patients in class I; 4.5% (1 of 22) patients in class II; 15.8% (3 of 19) patients in class III; and 27.8% (8 of 29) patients in class IV. Each mutation changed codon 61 from glutamine to arginine. Patients with distant metastases (8 of 29) had a significantly higher incidence of ras mutations than others (5 of 62, p = 0.01). Patients in classes III and IV also had a higher incidence of mutations (11 of 48) than patients in classes I and II (2 of 43, p = 0.01). The incidence of ras mutations was significantly higher in patients who died of papillary cancer (5 of 15, 33.3%) than in patients who are still alive (8 of 76, 10.5%) (p = 0.02). Kaplan-Meier survival curves also showed a greater mortality from tumor (p < 0.05) and a higher recurrence rate (p < 0.01) in ras-positive tumors than in the ras-negative group. Finally, in studies by multivariate analyses, positive ras mutation and age were shown to be two independent prognostic factors for prediction of death from papillary cancer and recurrence of cancer. CONCLUSIONS: Mutation of N-ras gene at codon 61 is an independent prognostic factor for aggressiveness of papillary thyroid carcinomas.

Adolescent↗
PubMed1993

Characterization of a novel mutant human thyroid hormone receptor beta in a family with hereditary thyroid hormone resistance.

OBJECTIVE: We wished to determine the abnormality responsible for Generalized Resistance to Thyroid Hormone in a family with this syndrome. DESIGN: Molecular biological studies were performed on a mutant human thyroid hormone receptor beta (hTR beta) cloned from fibroblasts of the patient. PATIENTS: The patient is from a previously reported family with typical features of Generalized Resistance to Thyroid Hormone, demonstrating goitre, elevated thyroid hormone levels, slightly elevated TSH, and retarded bone age. MEASUREMENTS: A cDNA for hTR beta 1 was cloned using specific oligonucleotide primers from fibroblast DNA. A mutant hTR beta 1 expression vector was constructed, and an in-vitro expressed mutant receptor was tested for T3 binding. Receptor binding to DNA was studied in a DNA cellulose assay and gel mobility shift assay. RESULTS: Two mutations were found in the cloned hTR beta. One was silent but the second changed arginine 438 to histidine. The mutation was present in RNA and genomic DNA, as shown by allele-specific amplification. The mutated receptor had reduced T3 binding affinity but demonstrated normal binding in a DNA cellulose assay and in a gel mobility shift assay. The receptor did not have altered heat sensitivity. CONCLUSIONS: In the T sibship with Generalized Resistance to Thyroid Hormone, resistance to thyroid hormone is apparently produced by a substitution of a histidine for arginine at amino acid 438, which causes reduced binding of receptor to T3, although the receptor remains able to bind to DNA and, for this reason, functions as a dominant negative in affected subjects who are heterozygous with one normal and one mutated allele.

Base Sequence↗
PubMed1993

Human histocompatibility leukocyte antigen-DQA1*0501 allele associated with genetic susceptibility to Graves' disease in a Caucasian population.

Graves' disease (GD) is an autoimmune disease of the thyroid gland. Genes of, or closely associated to, the HLA complex are assumed to contribute to the genetic predisposition to GD. We have previously reported an increased frequency of HLA-DR3/DQ2 in Caucasian patients with GD, and recently the importance of Dw24 encoded by DRB3 gene has been suggested. To further investigate the associations of GD and these genes, 94 unrelated patients with GD and 75 control subjects were typed for HLA-DRB3, -DRB1, -DQA1, and -DQB1, using sequence-specific oligonucleotide probes to analyze polymerase chain reaction amplified DNA (PCR-SSO). Three findings emerged from these studies. 1) The frequency of subjects positive for DQA1*0501 (GD, 73.4% vs. control 42.7%, P = 0.0001, Pc < 0.001, RR = 3.71) was significantly increased among patients. The frequency of DR3 (GD, 34.0% vs. control 17.3%, P = 0.0146, RR = 2.46), which is in tight linkage disequilibrium with DQA1*0501, was also increased; however, it was not significant when the P value was corrected for the number of antigens tested. Neither DQB1 nor DRB3 alleles were significantly increased in frequency. 2) After exclusion of DR3-positive subjects, DQA1*0501 was still significantly increased (GD, 59.7% vs. control 30.6%, P = 0.0012, Pc < 0.01, RR = 3.35) among patients. 3) The distributions of Dw24 and Dw25,26 (Dw25 or Dw26) did not differ between patients and controls on either DR3 positive or negative groups. These findings suggest that DQA1*0501, or a closely associated unknown gene, confers susceptibility to GD, while Dw24 is not directly involved. The importance of DR3, however, remains to be elucidated, because of the fixed linkage with DQA1*0501.

Alleles↗
PubMed1993

High affinity and specificity of dimeric binding of thyroid hormone receptors to DNA and their ligand-dependent dissociation.

The thyroid hormone receptors are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related steroid/thyroid hormone receptor superfamily. We report here the high affinity and specificity of dimeric DNA binding of human thyroid hormone receptor-alpha 1 (hTR alpha 1) and hTR beta 1 and the effect of T3 on its DNA binding. Gel mobility shift assay showed that hTR alpha 1 and -beta 1 bind to the rat GH-thyroid hormone response element (TRE) and rat malic enzyme (rME)-TRE as a monomer, dimer, and oligomer in the absence of T3 and bind to an irrelevant DNA sequence from the adenovirus 5 promoter only as a monomer. In competition studies using unlabeled TRE, dimer binding was displaced by lower concentrations of TRE than was the monomer, indicating that the dimeric binding has higher affinity than the monomer binding. These results suggest that the formation of dimers of TR increases the specificity and affinity for the response element. Surprisingly, T3 disrupted dimer binding and increased the intensity of monomer binding to rME-TRE in a dose-dependent manner. This does not occur with the rat GH-TRE. We also demonstrate that this disruption of dimeric binding by T3 occurred on a TRE formed by a direct repeat and not on a palindromic TRE. Furthermore, a mutant hTR beta (Mf), which has no detectable ligand-binding activity because of a glycine to arginine substitution at amino acid 340 in the hormone-binding domain, does not show dissociation from rME-TRE after the addition of T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1993

Effects of irradiation on the thyroid gland.

External irradiation to the thyroid, even at low doses, induces subsequent development of thyroid nodules and carcinomas, with an estimated incidence of five per one million people per year if they have been exposed to 1 rad. Larger doses are associated with hypothyroidism. External irradiation also induces thyroid hyperplasia and is associated with an increased incidence of thyroid autoimmunity and the development of thyroiditis and Graves' disease. 131I, used in the treatment of Graves' disease and thyroid cancer for nearly 50 years, has not been associated with an increased incidence of thyroid cancer. Nevertheless, caution is advisable in use of radioiodide therapy in adolescents. Low levels of fallout from nuclear explosions probably have minimal, if any, effect on the thyroid; but recent high-level exposure in individuals near Chernobyl is reported to be associated with the development of thyroid malignancies in children.

Adolescent↗
PubMed1993

Rat liver nuclear receptors for thyroid hormone: binding characteristics after stabilization and storage.

Conditions for both stabilization and storage of rat liver nuclear receptors for thyroid hormone for up to 28 days were determined using agents that might protect the receptor from spontaneous degradation. Without any protective agent a statistically significant (P < 0.05) diminution of 3,5,3'-triiodothyronine (T3) binding occurred at 0 degree C within 14 days. Dithiothreitol can maintain the initial T3 maximum binding capacity (MBC) for up to 7 days at 0 degree C. The compounds tested, including spermine, sodium molybdate, thioglycerol, and nitrogen gas, had no stabilization effect on nuclear receptors. A statistically significant (P < 0.05) stabilization for up to 28 days at 0 degree C of the initial MBC of T3 receptors was observed in the presence of 0.2 mol.l-1 phosphate anion or 20% (w/w) glycerol. The protective effect of phosphate anion was due to the phosphate ion itself rather than to the high ionic strength.

Animals↗
PubMed1992

Cloning and characterization of the human thyroid hormone receptor beta 1 gene promoter.

The promoter region of the human (h) thyroid hormone receptor (TR) beta 1 gene was isolated from a human placenta genomic library. Primer extension and S1 nuclease mapping confirmed a single transcriptional start site. DNA sequence analysis of the 5' upstream region revealed the existence of a putative thyroid response element (TRE) which is highly homologous to TREs found in several thyroid hormone responsive genes. Binding of hTR protein to the promoter region of the hTR beta 1 gene was confirmed by gel mobility shift assay. A transient transfection study demonstrated that hTR activated the expression of a reporter gene containing the promoter sequence of the hTR beta 1 gene in a hormone dependent manner. The TRE in the hTR beta 1 gene promoter may be involved in the autoregulation of hTR beta 1 gene expression.

Base Sequence↗
PubMed1992

Admission screening by thyroid function tests in an acute general care teaching hospital.

To determine the incidence of unrecognized thyroid disease among admissions to a large acute care university teaching hospital, 364 samples taken on consecutive admissions were assayed for thyroid-stimulating hormone (TSH) and free thyroxine index (FTI). Patients with abnormal test results were further evaluated by determination of antimicrosomal and antithyroglobulin antibodies, and charts were reviewed for evidence of prior diagnosis of thyroid disease, especially severe illness, drug treatment that might affect thyroid function tests, and prior diagnosis of thyroid disease. Results of subsequent thyroid function tests performed during the patient's hospitalization were correlated with the admission serum assays, and data on subsequent testing during the following 6 months were also obtained. A total of 3.9% of patients had significantly depressed TSH, and 11.1% of values were significantly elevated. A total of 11.3% of patients had significantly low FTI values, and 1% had significantly elevated values. A total of 7.4% appeared to have the euthyroid sick syndrome, 5.8% appeared to have unrecognized or undertreated primary thyroid failure, 6% had apparent subclinical hypothyroidism, 2% were thyrotoxic, and 2.8% (all women) had suppressed TSH levels for inapparent reasons. Limiting testing to patients over 49 years of age, or to women, would have missed many individuals with abnormal test results. Considering widespread availability of tests, relative costs, and value of the information obtained, it is suggested that the FTI determination would provide an appropriate screening test for patients in a population such as this entering a large, acute care general hospital.

Diagnostic Tests, Routine↗
PubMed1992

Pemphigus vulgaris in siblings: HLA-DR4 and HLA-DQw3 and susceptibility to pemphigus.

BACKGROUND: The association of pemphigus vulgaris with the HLA serotypes, DR4 and DRw6, and with the DQ-beta chain alleles, DQw1 and DQw3, suggests that there is a genetic predisposition to this disease. However, familial cases of pemphigus vulgaris are exceedingly rare. OBJECTIVE: We studied two siblings with pemphigus vulgaris and three unaffected family members to determine whether an HLA allele was associated with the development of pemphigus vulgaris in this family. METHODS: We utilized restriction fragment length polymorphism methods using the HLA-DR beta 1, HLA-DQ alpha, and HLA-DW beta 1 genes as cDNA probes. RESULTS: We found that the affected siblings share the haplotype HLA-DR4 and the DQw.3.2 allele. CONCLUSION: Our findings of gene sharing among siblings with pemphigus vulgaris lend support to previous studies of unrelated Caucasian patients, which implicated DQw3 allele polymorphisms in conferring susceptibility to pemphigus.

Adult↗
PubMed1992

Expression and function of a human thyroid hormone receptor-derived DNA-binding domain protein.

DNA binding domain proteins (DBDP) were prepared using a pET construct containing an insert coding for amino acids 49-122 of human thyroid hormone receptor (hTR) alpha and 103-179 of hTR beta. These proteins were expressed in Escherichia coli strain BL21 (DE3)-plysS after induction by isopropyl-D-thiogalactopyranoside (IPTG). The hTR alpha and hTR beta DBDP contain respectively 79 and 82 amino acids, including an amino terminal 4 amino acid extension derived from pET-3a or the synthesized initiation codon. Using a gel shift assay, both DBDPs were found to bind to a DNA oligonucleotide containing a thyroid hormone response element (TRE). The DBDPs competed with full length hTR alpha 1 for binding to the oligonucleotide. Apo-DBDPs (Zn2+ released by low pH) failed to bind to the palindromic TRE. DNA binding is restored however if apo-DBDP is preincubated in 500 microM Zn2+. When the DBDPs were expressed in COS-7 cells using a pCB6+ expression vector, they did not induce expression of a TRE-CAT fusion gene. hTR DBDPs thus can bind to DNA, presumably as monomers, since they do not contain the leucine zipper-like motif for dimerization. In COS-7 cells, they fail to cause transactivation of a TRE-CAT fusion gene. It is inferred that this may be because the DBDPs are not translocated to the nucleus or lack a transactivation domain.

Amino Acid Sequence↗
PubMed1992

Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform.

We characterized four antipeptide polyclonal antibodies (abs) able to specifically recognize each thyroid hormone receptor (TR) isoform. The abs immunoprecipitated both the in vitro synthesized receptor and the receptor expressed in E. coli and their specificity was confirmed by competition studies and immunohistochemistry. Ab activity measured by enzyme-linked immunosorbent assay decreased after preabsorption of each ab with the immunizing peptide or the specific receptor protein expressed in E. coli. No specific activity was detectable in enzyme-linked immunosorbent assay, no nuclear staining was observed after affinity column immunoabsorption, and the specific bands obtained in Western blot analysis disappeared after preabsorption with the specific TR isoform expressed in E. coli. By immunohistochemical studies we detected coordinate expression of each receptor isoform in most tissues examined. However, in heart and muscle, the beta-isoform is expressed at a very low level compared to the alpha-isoform in spite of the significant TR beta mRNA levels previously demonstrated by Northern blot analysis. We also demonstrated a different pattern of distribution of alpha- and beta-isoforms in rat testis. In this tissue the TR alpha is significantly expressed in spermatogonia nuclei, but in spermatids the beta-isoform is predominant, and only the TR beta is detectable in mature spermatozoa.

Animals↗
PubMed1992

Recessive inheritance of thyroid hormone resistance caused by complete deletion of the protein-coding region of the thyroid hormone receptor-beta gene.

Generalized resistance to thyroid hormone is a syndrome of reduced responsiveness of target tissues to thyroid hormone. The determination of amino acid sequences of the human thyroid receptor-beta (hTR beta), deduced from cDNA sequencing, has enabled evaluation of the genetic basis for this syndrome. Distinct point mutations in the ligand-binding domain of hTR beta have been identified in affected members of unrelated families, producing single amino acid substitutions that result in products with decreased or no hormone-binding activity. Inheritance in these families was autosomal dominant. We now report the molecular basis of generalized resistance to thyroid hormone in a consanguineous family unique for its autosomal recessive mode of inheritance. Deletion of the entire coding region of both hTR beta alleles in homozygous affected members of the family was demonstrated by the failure to amplify the coding exons 3-8 by the polymerase chain reaction using primers specific for flanking intronic sequences and by the demonstration of the presence of only two noncoding exons in Southern blots hybridized with exon-specific probes. As expected, obligate heterozygotes were phenotypically normal, since, in contrast to alleles with point mutations, the deleted allele could not act in a dominant negative fashion. Survival and maintenance of a euthyroid state are presumably mediated through expression of the hTR alpha gene, present in affected subjects, and the maintenance of high thyroid hormone levels. Furthermore, the clinical manifestations were relatively more mild that those observed in a homozygous patient with a single amino acid deletion in the hTR beta gene.

Amino Acid Sequence↗
PubMed1992

Proliferative responses of peripheral blood mononuclear cells from patients with autoimmune thyroid diseases to synthetic peptide epitopes of human thyroid peroxidase.

In order to analyze T cell epitopes of human thyroid peroxidase (TPO), 60 peptides based on the sequence of TPO were synthesized and used as antigens in a peripheral blood mononuclear cell (PBMC)-proliferation assay. PBMCs were obtained from 19 patients with Graves' disease, 19 patients with Hashimoto's thyroiditis, and 24 normal subjects. Significant proliferation of PBMC to these peptides occurred only among the autoimmune thyroid disease (AITD) patients, whereas normal subjects did not respond to any of the peptides with a stimulation index over 2. Many peptides induced isolated positive responses and eight produced stimulation of PBMCs from multiple patients on comparison to control PBMC responses. To confirm the significance of reactivity to the peptides PBMCs from four patients were studied on two occasions, and the proliferative responses found to be reproducible. Four peptides, designated according to the amino acid sequence as p110-129, p211-230, p842-861, and p882-901, stimulated patients' PBMCs in a dose-dependent manner. The optimal concentration was 10 micrograms/ml. An anti-HLA-DR monoclonal antibody directed against a monomorphic determinant of the DR molecule was able to block the responses. A significant correlation was found between the PBMC responses to these peptides and responses to microsomal antigen (McAg)/TPO. These data suggest that four peptides corresponding to the amino acid sequences 110-129, 211-230, 842-861 and 882-901 are T cell epitopes of TPO.

Adult↗
PubMed1992

HLA class II genes in Graves' disease.

Inheritance of Graves' disease has been linked to the HA-DR3 gene product which may function in some specific way in antigen presentation. To determine whether the first extracellular domain of this protein, which is specifically involved in antigen presentation, has the same sequence in patients with Graves' disease and in normal individuals, we have amplified the second exon using the polymerase chain reaction, and then cloned and sequenced the DNA segment. In eight subjects with Graves' disease, sequences identical to prototypic reported sequence for DRB1*0301 were recovered, and in two individuals sequences varied by a few nucleotides, leading to 1-3 amino acid substitutions which did not occur in a pattern. Sequences identical to the prototypic sequence known as DRB3*0101, also previously known as DRw52, were also recovered. Thus the HLA-DRB1 and B3 genes present in patients with autoimmune disease appear to be the same as those present in the general population. These observations indicate that a unique allele is not present in patients with autoimmune disease, but rather that the normal DR3 allele itself, in a manner yet to be described, increases the probability of developing autoimmune thyroid disease.

Amino Acid Sequence↗
PubMed1992

Site-specific anti-c-erb A antibodies recognizing native thyroid hormone receptors: their use to detect the expression and localization of alpha and beta c-erb A proteins in rat liver.

The cell-specific expression and tissue distribution of c-erbA proteins alpha and beta is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the alpha or beta form of thyroid hormone receptors. The cDNAs coding for h c-erbA beta 1, alpha 1 and alpha 2 were transcribed and the mRNAs were translated in vitro in the presence of 35S-methionine, and then their reactivity with the antisera was evaluated. The antiserum anti-beta 62-81 immunoprecipitated only the beta 1 receptor. The antiserum anti-alpha 144-162 determined precipitation of both alpha 1 and alpha 2 proteins but not of the beta 1 receptor. Anti-alpha 2 431-451 produced a selective precipitation of alpha 2, and had no effect on alpha 1 or beta 1 receptor. In order to study the interaction of the antibodies with native T3 receptor we evaluated the binding of antibodies to rat liver T3 receptors by Sephacryl S300 chromatography: both antisera anti-beta 62-81 and anti-alpha 144-162 caused a partial shift of the labeled T3-receptor complex to a higher molecular form, while the antibody directed against c-erbA alpha 2 did not produce any significant shift. The anti-peptide antibodies were then immunopurified by affinity chromatography and used to immunolocalize the different forms of c-erb A proteins in adult and fetal rat liver, by a sensitive immunohistochemical technique. All 3 antibodies stained mainly the nuclei of the majority of adult liver cells. No staining was detectable when the original antiserum was deprived of anti-peptide antibodies by running through the affinity columns or when the antibodies were pre-absorbed with the homologous peptide. No significant staining was present in the liver from rat fetus.

Animals↗