Prothrombin Greenville, Arg517-->Gln, identified in an individual heterozygous for dysprothrombinemia.

A 64-year-old white male was referred for evaluation of prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) obtained before elective surgery with initial PT and PTT results of 14.9 and 38.4 seconds, respectively, which corrected to normal in 1:1 mixes with normal plasma. Functional prothrombin assay indicated a level of 51% with thromboplastin as an activator. The prothrombin antigen was 102%. This discordance in the functional and immunologic prothrombin levels was evidence for dysprothrombinemia. Western blotting showed that thrombin was formed at a normal rate in diluted plasma consistent with a mutation within the thrombin portion of prothrombin. DNA was isolated from leukocytes and the thrombin exons were amplified by polymerase chain reaction, cloned, and sequenced. For exon 13, eight clones were sequenced with four clones showing a point mutation in the codon for Arg517, which would result in substitution by Gln. Arg517 is part of the Arg-Gly-Asp(RGD) sequence in thrombin and contributes to an ion cluster with aspartic acid residues 552 and 554. Mutation at this residue most probably distorts the structure of the Na+ binding site in thrombin. This is the first report indicating the critical role of Arg517 in the normal physiological interaction of thrombin with fibrinogen. This dysprothrombin is designated Prothrombin Greenville.

A novel C-terminal domain in the thyroid hormone receptor selectively mediates thyroid hormone inhibition.

Resistance to thyroid hormone (RTH) action is due to mutations in the beta-isoform of the thyroid hormone receptor (TR-beta). RTH patients display inappropriate central secretion of thyrotropin-releasing hormone (TRH) from the hypothalamus and thyrotropin (TSH) from the anterior pituitary in association with abnormal peripheral tissue responses to thyroid hormone. Whether TR-beta mutations cause a selective form of RTH, which only leads to abnormal pituitary TSH secretion (PRTH), is unclear. In a patient with PRTH, a novel mutation of a conserved arginine residue adjacent to the ninth heptad of TR-beta selectively disrupts TR homodimer formation. The mutant TR displays normal or enhanced function on stimulatory thyroid hormone response elements found in peripheral tissues, but has defective function on inhibitory thyroid hormone response elements found in the TRH and TSH subunit genes and explains the PRTH phenotype. This is the first report of a mutation in a member of the nuclear receptor superfamily that selectively abolishes hormone-dependent inhibition and localizes a novel C-terminal domain necessary for this property.

Divergent dimerization properties of mutant beta 1 thyroid hormone receptors are associated with different dominant negative activities.

Syndromes of resistance to thyroid hormones are caused by mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. The S receptor (deletion of THR332) is a potent dominant negative protein cloned from a kindred with generalized resistance to thyroid hormones. The G-H receptor (ARG311HIS) has compromised dominant negative function and was found in both normal individuals and in a patient with severe pituitary resistance to thyroid hormones. We have investigated the mechanism responsible for the difference in receptor phenotypes by analyzing the binding of S and G-H receptors to thyroid hormone response elements with electrophoretic mobility shift analysis. Wild-type human c-erbA beta 1 (WT), S, and G-H receptors were synthesized in reticulocyte lysate, reacted with a thyroid hormone response element consisting of a direct repeat with 4 base pairs (DR+4; AGGTCA CAGG AGGTCA), and the products analyzed by gel shift. G-H receptor homodimerization was greatly impaired; G-H formed predominantly monomeric complex compared with monomeric and homodimeric WT complexes. The G-H receptor was able to form heterodimeric complexes with cellular thyroid hormone receptor auxiliary protein (TRAP) factors including the human retinoid X receptor-alpha. When TRAP was limiting, the levels of G-H heterodimeric complex were 2- to 3-fold reduced compared with WT receptor. In contrast to the WT and G-H receptors, the S receptor formed almost exclusively homodimeric complex with DR+4; the approximate ratio of S:WT:G-H homodimeric complexes at equivalent concentrations of receptors was 60:20:1. A measurable increase (1.2- to 2.6-fold) in heterodimeric complex formation was observed with the S receptor relative to WT when TRAP was at limiting concentration. As reported previously by others, thyroid hormone significantly reduced the WT homodimeric complex with DR+4. There was no effect on the S homodimeric complex. Finally, the WT, S, and G-H receptors formed different complexes with the element consisting of an inverted repeat with 5 base pairs (IR+5; AGGTCA ACAGT TGACCT) and the IR element (AGGTCA TGACCT), which were differently regulated by thyroid hormone. The S receptor bound as a homodimer with IR+5, whereas the WT receptor bound as a homodimer only with thyroid hormone. No homodimeric complex formed with IR+5 and the G-H receptor. Qualitatively similar results were observed with the IR element. We conclude that the ARG311HIS mutation severely perturbs the homodimerization and, to a much less degree, heterodimerization functions of the c-erbA beta 1 receptor. Furthermore, the THR332 deletion mutation augments homodimerization of the c-erbA beta 1 receptor. These results indicate that different mutations in the c-erbA beta 1 thyroid hormone receptor have divergently affected dimerization activities which seem to influence the level of dominant negative activity in man.

Competitive polymerase chain reaction quantitation of c-erbA beta 1, c-erbA alpha 1, and c-erbA alpha 2 messenger ribonucleic acid levels in normal, heterozygous, and homozygous fibroblasts of kindred S with thyroid hormone resistance.

Mutations in the T3-binding domain of the thyroid hormone receptor gene c-erbA beta result in dominant negative proteins and thyroid hormone resistance syndromes. Variable clinical manifestations of resistance to thyroid hormones have been reported, including short stature and neuropsychological abnormalities. The molecular bases for heterogeneity of phenotype among and within kindreds have not been fully elucidated. Recent investigations have considered differential expression of mutant and wild-type beta 1-receptor alleles and the regulation thereof as a mechanism to explain differential sensitivity to thyroid hormones. We used reverse transcription-competitive polymerase chain reaction (PCR) to measure c-erbA beta 1, c-erbA alpha 1, and c-erbA alpha 2 mRNAs in skin fibroblasts cultured from normal subjects, heterozygotes, and a severely affected homozygous mutant of kindred S. The homozygous mutant of kindred S had severe growth and mental retardation. After reverse transcription with primers specific for each of the c-erbA mRNAs, first strand cDNAs were amplified by PCR using subtype-specific amplimers. Primer design allowed simultaneous detection of wild-type and mutant messages in heterozygous fibroblasts and showed an approximately 1:1 ratio of these mRNAs in three patients. Inclusion of competitive standard cDNAs of known concentration in the PCR reactions allowed quantitation of the absolute levels of the beta 1-, alpha 1-, and alpha 2 mRNAs by comparison of products on ethidium bromide-stained agarose gels. These studies showed no effect of the presence of the mutant beta 1-allele, as fibroblast RNA from normal subjects, heterozygotes, and the homozygote gave values of 56-184, 2.8-12, and 23-40 attomol/5 micrograms total RNA for beta 1-, alpha 1-, and alpha 2 mRNAs, respectively. We conclude that these sensitive methods allow the detection of molecular species present at levels as low as 10 molecules/cell, and that this potent dominant negative receptor does not disrupt c-erbA expression at the level of mRNA. The neuropsychological sequelae of the kindred S mutation are not due to relative overexpression of the mutant allele.

Dominant and nondominant negative C-erbA beta 1 receptors associated with thyroid hormone resistance syndromes augment 12-O-tetradecanoyl-phorbol-13-acetate induction of the collagenase promoter and exhibit defective 3,5,3'-triiodothyronine-mediated repression.

C-erbA receptors and v-erbA have been shown to functionally interact with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-inducible gene expression. These proteins enhance trans-activation by c-jun, and the c-erbA receptors in the presence of thyroid hormone repress TPA and c-jun induction of transcription. Also, v-erbA can abrogate T3-mediated repression. We have examined how dominant negative (S and CL) and nondominant negative (G-H) receptors cloned from various patients with thyroid hormone resistance syndromes affect expression of the collagenase promoter induced with TPA. The CL receptor (ARG315HIS mutation) has a 2-fold reduction in T3-binding affinity compared with human c-erbA beta 1 wild-type (WT) receptor, whereas the G-H receptor (ARG311HIS) and S receptor (deletion, THR codon 332) have T3-binding affinities reduced by 100-fold and greater than 100-fold, respectively. These mutant receptors were cotransfected with a collagenase promoter (-1200 to +63 base pairs) chloramphenicol acetyltransferase reporter gene (Col-CAT) into COS-7 cells. Levels of CAT reporter gene expression after transient transfection were determined in the presence or absence of 3-10 nM T3 and the presence or absence of 100 nM TPA. Unoccupied CL receptor and G-H and S receptors stimulated TPA-induced Col-CAT expression 1.5- to 9-fold. The CL receptor with thyroid hormone totally repressed TPA induction of the collagenase receptor. In the presence of thyroid hormone, the enhancing effects by S and G-H receptors on TPA-induced Col-CAT expression were unaffected and minimally diminished, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

An arginine to histidine mutation in codon 311 of the C-erbA beta gene results in a mutant thyroid hormone receptor that does not mediate a dominant negative phenotype.

We have examined the c-erbA beta thyroid hormone receptor gene in a kindred, G.H., with a member, patient G.H., who had a severe form of selective pituitary resistance to thyroid hormones (PRTH). This patient manifested inappropriately normal thyrotropin-stimulating hormone, markedly elevated serum free thyroxine (T4) and total triiodothyronine (T3), and clinical hyperthyroidism. The complete c-erbA beta 1 coding sequence was examined by a combination of genomic and cDNA cloning for patient G.H. and her unaffected father. A single mutation, a guanine to adenine transition at nucleotide 1,232, was found in one allele of both these members, altering codon 311 from arginine to histidine. In addition, a half-sister of patient G.H. also harbored this mutant allele and, like the father, was clinically normal. The G.H. receptor, synthesized with reticulocyte lysate, had significantly defective T3-binding activity with a Ka of approximately 5 x 10(8) M-1. RNA phenotyping using leukocytes and fibroblasts demonstrated an equal level of expression of wild-type and mutant alleles in patient G.H. and her unaffected father. Finally, the G.H. receptor had no detectable dominant negative activity in a transfection assay. Thus, in contrast to the many other beta-receptor mutants responsible for the generalized form of thyroid hormone resistance, the G.H. receptor appeared unable to antagonize normal receptor function. These results suggest that the arginine at codon 311 in c-erbA beta is crucial for the structural integrity required for dominant negative function. The ARG-311-HIS mutation may contribute to PRTH in patient G.H. by inactivating a beta-receptor allele, but it cannot be the sole cause of the disease.

An arginine to histidine mutation in codon 315 of the c-erbA beta thyroid hormone receptor in a kindred with generalized resistance to thyroid hormones results in a receptor with significant 3,5,3'-triiodothyronine binding activity.

Generalized resistance to thyroid hormones results from diverse mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor, and different kindreds have variable phenotypes. However, the T3-binding affinities of these mutant receptors studied in vitro have all been severely reduced compared to wild type. We report here a new kindred, CL, with a mutation further upstream than previously reported, a guanine to adenine base substitution at nucleotide 1244 in codon 315 changing an arginine to histidine. This base substitution was the only one found in codons 90-456 of genomic sequence and was formally shown to be a mutation by screening 51 random individuals. The kindred CL receptor complementary DNA was recreated, and the mutant receptor synthesized with rabbit reticulocyte lysate had a T3-binding affinity of 2.4 +/- 0.9 x 10(10) M-1 compared to the wild-type human placental receptor affinity of 5.2 +/- 1.6 x 10(10) M-1. Affected members of this kindred appeared clinically to have a relatively mild degree of resistance with mean total thyroxine of only 192 +/- 24 nmol/L and inappropriately normal TSH levels. Kindred CL is an example of mild generalized resistance to thyroid hormones correlated with a mutation in the beta-receptor that resulted in only a modest deficiency in T3-binding activity.

Variable transcriptional activity and ligand binding of mutant beta 1 3,5,3'-triiodothyronine receptors from four families with generalized resistance to thyroid hormone.

Mutations in the gene encoding the human beta 1 T3 receptor (hTR beta 1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR beta 1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T3 affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T3 affinity required 5- and 15-fold higher T3 concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTR beta 1s were able to inhibit the function of transfected normal hTR beta 1 and endogenous retinoic acid receptor in activating a palindromic positive T3 response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T3 concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTR beta 1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTR beta 1 was assessed by immunocytochemistry. The hTR beta 1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells. These data suggest that different mutations in the ligand-binding domain of the human hTR beta 1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T3 receptor dimers.

A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.

A new point mutation in the 3,5,3'-triiodothyronine-binding domain of the c-erbA beta thyroid hormone receptor is tightly linked to generalized thyroid hormone resistance.

Two different mutations in the c-erbA beta thyroid hormone receptor have recently been reported as genetic abnormalities responsible for the syndrome of generalized thyroid hormone resistance (GTHR). We have now found in a third kindred, D, in which GTHR is inherited as a dominant disease, a new point mutation in the T3-binding domain of c-erbA beta. A guanine to cytosine base substitution at nucleotide position 1305, which altered codon-335 from glutamine (CAG) to histidine (CAC), was found in one allele of 10 affected members and was not found in 6 unaffected members. This C-1305 sequence was not present in 106 random alleles, indicating that it was a mutation in c-erbA beta, and it was tightly linked to GTHR in kindred D, with a maximum logarithm of the odds score of 4.19 at a recombination fraction of 0. The tight linkage result confirms that GTHR maps to the c-erbA beta locus in multiple kindreds. In view of the tight linkage between the C-1305 mutation and GTHR, and that this mutation is a nonconservative alteration in a crucial region of the T3-binding domain, it is probably the genetic defect in kindred D responsible for GTHR. The kindred D receptor appears to result in a different phenotype of tissue resistance compared to the previously reported kindred. A receptor with a mutation in the carboxy-terminus of c-erbA beta.

Thyroid hormone and DNA binding properties of a mutant c-erbA beta receptor associated with generalized thyroid hormone resistance.

We have previously reported a family, Kindred A, with autosomal dominant generalized thyroid hormone resistance in which affected members were found to have a mutation in the carboxy-terminal domain of the c-erbA beta thyroid hormone receptor. In the current study, the thyroid hormone and DNA-binding properties of this mutant receptor were determined using c-erbA beta protein synthesized in vitro. Both the wild-type human placental c-erbA beta and Kindred A receptors bound [125I]-triiodothyronine, although the Kindred A receptor had decreased affinity for the hormone. The affinity for triiodothyronine was 4.5 x 10(9) M-1 and 2.3 x 10(10) M-1 for the mutant and wild-type receptors, respectively. No abnormality of DNA-binding was detected with the Kindred A receptor using a sensitive avidin-biotin DNA-binding assay with DNA fragments containing thyroid hormone response elements. The Kindred A mutant receptor which displays abnormal triiodothyronine-binding but normal DNA-binding activities in vitro acts as a dominant negative inhibitor of thyroid hormone action in man.