0021-972X/91/7305-0990$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 5 Printed in U.S.A.
Homozygosity for a Dominant Negative Thyroid Hormone Receptor Gene Responsible for Generalized Resistance to Thyroid Hormone* SEIGO ONO, I. DAVID SCHWARTZ, 0. THOMAS MUELLER, ALLEN W. ROOT, STEPHEN J. USALA, AND BARRY B. BERCU Division of Pediatric Endocrinology, Department of Pediatrics, University of South Florida College of Medicine, Tampa, Florida 33612; All Children's Hospital, St. Petersburg, Florida 33731; and the Department of Medicine, East Carolina University School of Medicine, Greenville, North Carolina 27858
ABSTRACT. Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-er6A/3 thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA/3 in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the /3-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4,330.8 pmol/L; free T3,82,719 fmol/ L). He displayed a complex mixture of tissue-specific hyperthy-
T
HE SYNDROME of generalized resistance to thyroid hormones (GRTH) was first described in 1967 by Refetoff et al. in a kindred, G (1). Although sporadic patients with GRTH have been reported, in most subjects the disorder has been inherited as an autosomal dominant trait (2-4). Patients with GRTH are usually euthyroid or mildly hypothyroid, with or without thyromegaly. The molecular defect for GRTH has now been eluciated; tight linkage between GRTH and the c-erbAff thyroid hormone receptor gene has been established in multiple kindreds with different phenotypes (5-7). Furthermore, several different mutations in the thyroid hormone-binding domain of the /?-receptor in patients with GRTH have now been reported (6, 8-10). All of these mutations are dominant negative, in that the mutant receptors are thought to act by inhibiting wild-type thyroid hormone receptors (c-erbA/3 and c-erbAa) and thyroid hormone action (11). There is one crucial exception to this rule which demonstrates this mechanism; the original Refetoff patient with GRTH from kindred G has
roidism and hypothyroidism. He had delayed growth (height age, 1 3/i2 yr at chronological age 2 9/i2 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardia The homozygous patient of kindred S is markedly different from a recently reported patient with no c-er6A /3-receptor. This difference indicates that a dominant negative form of c-erbAfi in man can inhibit at least some thyroid hormone action mediated by the cerbk a-receptors. {J Clin Endocrinol Metab 73: 990-994,1991)
recently been reported to have a homozygous deletion of c-erbAfi receptor genes resulting in the absence of /?receptors (12). However, the heterozygotes from this kindred, with only one c-erbA(3 allele, are phenotypically normal. We now describe a bizarre patient with unprecedented resistance to thyroid hormones in bone and pituitary tissues. The patient was mentally retarded, presumably also the result of severe thyroid hormone resistance. However, paradoxically, this patient was clinically tachycardic, markedly underweight, and had exopthalmus, suggesting that some tissues were relatively hyperthyroid. This patient was recently found to be homozygous for an amino acid deletion in the T3-binding domain of the c-erbAfi receptor (10). The clinical data presented here elaborate the only known case in man of homozygosity for a dominant negative form of the c-er6A/3 thyroid hormone receptor.
Subjects and Methods The diagnosis of GRTH in other affected members of this family, kindred S, was based on the presence of elevated serum levels of total and free T 4 and T3, inappropriately normal basal and post-TRH serum TSH levels, and a clinically euthyroid state (2). T4, T3, free T3, reverse T3, TSH, antithyroglobulin and
Received January 14,1991. Address all correspondence and requests for reprints to: Barry B. Bercu, M.D., Department of Pediatrics, All Children's Hospital, 801 Sixth Street South, St. Petersburg, Florida 33731-8920. * Presented in part to the Society for Pediatric Research, Anaheim, CA, 1987 and 1990.
990
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 00:02 For personal use only. No other uses without permission. . All rights reserved.
RESISTANCE TO THYROID HORMONE IN A HOMOZYGOTE
991
antimicrosomal antibodies, and thyroid binding inhibitory immunoglobulin were measured by commercially available RIA kits; free T4 was measured at Nichols Institute (San Juan Capistrano, CA). Thyroid-stimulating immunoglobulin, sex hormone-binding globulin, ferritin, and T4-binding globulin were measured by Nichols Institute. PRL was measured by RIA, using specific polyclonal antisera (13). Restriction fragment length polymorphisms (RFLP) studies Skin fibroblast cultures were established from family members by punch biopsy. DNA was extracted from confluent fibroblast cultures using 5% sodium lauryl sulfate and 1 mg/ mL proteinase-K [extracted twice with phenol and once with chloroform-isoamyl alcohol (24:1)]. DNA was precipitated with isopropanol, rinsed in 70% ethanol, and resuspended in TE (10 mmol/L Tris and 1 mmol/L EDTA, pH 8.0). DNA was digested in 5-/ig aliquots using 20 U BamW or EcoRV restriction endonucleases (Boehringer Mannheim Biochemicals, Indianapolis, IN). DNA electrophoresis was performed in 1% agarose gels and transferred to Nytran membrane (Schleicher and Schuell, Keene, NH) with 10 X SSC (1 X SSC = 0.15 mol/L sodium chloride and 15 mmol/L sodium /3-citrate). The thyroid hormone receptor /3 DNA probe pheA12 (14) was labeled with [32P]dTTP using the random oligonucleotide priming reaction to a specific radioactivity of 2-4 X 108 dpm/ Mg. Blots were prehybridized in 6 X SSC, 10 x Denhardt's solution, and 0.75% sodium dodecyl sulfate (SDS) for 2-16 h and hybridized in 50% formamide, 6 x SSC, 0.75% SDS, dextran sulfate, and 10 Mg/mL salmon sperm DNA at 42 C. Blots were washed twice in 1 x SSC and 0.5% SDS at 37 C and twice in 0.1 X SSC and 0.5% SDS at 65 C. These studies were approved by the University of South Florida College of Medicine and All Children's Hospital Institutional Review Boards, and written informed consent was obtained from the parents and members of the kindred.
I 123
I Thyroid Scans in
Postnatal Period
Case Report The male patient was born at 35 weeks gestation to a 20-yrold gravida 1, para 0, abortus 0 mother. The parents were maternal first cousins with documented GRTH. The birth weight was 1480 g, and the length was 46 cm, consistent with intrauterine growth retardation. Postnatal illnesses included respiratory distress, hyperbilirubinemia, and hypocoagulability. At 3.5 weeks of age, tachycardia (160-200 beats/min), exophthalmus, stare, and thyromegaly were noted (Fig. 1A). The results of initial thyroid function studies are summarized in Table 1. Antithyroglobulin and antithyroid microsomal (peroxidase) antibodies were negative. Bone age was less than term at 4 weeks of age. Automated chemical screen, skull x-ray, and brain ultrasound and magnetic resonance imaging were normal. At 3.5 weeks of age, 123I thyroid scan demonstrated a moderately enlarged thyroid gland with homogeneous uptake of radiolabeled iodide (Fig. IB); the uptake was 87% (normal range, 620%) at 4 h and 50% (normal range, 8-35%) at 24 h. After TRH treatment, serum TSH increased from a basal level of 317 mU/L to a peak of 413 mU/L 30 min later. Serum T4 reached a peak of 818.6 nmol/L at 4 months of age. Clinical
Normal Size Gland FIG. 1. A, Photograph of propositus at 3.5 weeks of age. B, Thyroid scan at 3.5 weeks of age. problems included feeding difficulties, diarrhea, recurrent otitis media and upper respiratory infections, and developmental and growth retardation. The patient grew poorly; by age 2 9/i2 yr he was significantly small (height age, 1 3/i2 yr; weight age, 7/i2 yr) and developmentally delayed. His clinical status was suggestive of tissuespecific hyperthyroidism and hypothyroidism (e.g. hyperthyroid cardiac status and hypothyroid skeletal maturation), although serum concentrations of thyroid hormones and TSH
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 00:02 For personal use only. No other uses without permission. . All rights reserved.
ONO ET AL.
992 TABLE 1. Laboratory investigations of the homozygous child
T4, nmol/L (94.0-193.1) Free T4, pmol/L (16.7-48.9) T3, pmol/L (1613-3210) Free T3( fmol/L (3994-7373) Reverse T3, pmol/L (154-538) T3RU, % (25-35) TSH, mU/L (0.7-5.0) TBG, mg/L (14-26) SHBG, nmol/L (6.9-48.5) Ferritin, jug/L (7-142) TBII, index (
Vol. 73, No. 5 Printed in U.S.A.
Homozygosity for a Dominant Negative Thyroid Hormone Receptor Gene Responsible for Generalized Resistance to Thyroid Hormone* SEIGO ONO, I. DAVID SCHWARTZ, 0. THOMAS MUELLER, ALLEN W. ROOT, STEPHEN J. USALA, AND BARRY B. BERCU Division of Pediatric Endocrinology, Department of Pediatrics, University of South Florida College of Medicine, Tampa, Florida 33612; All Children's Hospital, St. Petersburg, Florida 33731; and the Department of Medicine, East Carolina University School of Medicine, Greenville, North Carolina 27858
ABSTRACT. Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-er6A/3 thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA/3 in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the /3-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4,330.8 pmol/L; free T3,82,719 fmol/ L). He displayed a complex mixture of tissue-specific hyperthy-
T
HE SYNDROME of generalized resistance to thyroid hormones (GRTH) was first described in 1967 by Refetoff et al. in a kindred, G (1). Although sporadic patients with GRTH have been reported, in most subjects the disorder has been inherited as an autosomal dominant trait (2-4). Patients with GRTH are usually euthyroid or mildly hypothyroid, with or without thyromegaly. The molecular defect for GRTH has now been eluciated; tight linkage between GRTH and the c-erbAff thyroid hormone receptor gene has been established in multiple kindreds with different phenotypes (5-7). Furthermore, several different mutations in the thyroid hormone-binding domain of the /?-receptor in patients with GRTH have now been reported (6, 8-10). All of these mutations are dominant negative, in that the mutant receptors are thought to act by inhibiting wild-type thyroid hormone receptors (c-erbA/3 and c-erbAa) and thyroid hormone action (11). There is one crucial exception to this rule which demonstrates this mechanism; the original Refetoff patient with GRTH from kindred G has
roidism and hypothyroidism. He had delayed growth (height age, 1 3/i2 yr at chronological age 2 9/i2 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardia The homozygous patient of kindred S is markedly different from a recently reported patient with no c-er6A /3-receptor. This difference indicates that a dominant negative form of c-erbAfi in man can inhibit at least some thyroid hormone action mediated by the cerbk a-receptors. {J Clin Endocrinol Metab 73: 990-994,1991)
recently been reported to have a homozygous deletion of c-erbAfi receptor genes resulting in the absence of /?receptors (12). However, the heterozygotes from this kindred, with only one c-erbA(3 allele, are phenotypically normal. We now describe a bizarre patient with unprecedented resistance to thyroid hormones in bone and pituitary tissues. The patient was mentally retarded, presumably also the result of severe thyroid hormone resistance. However, paradoxically, this patient was clinically tachycardic, markedly underweight, and had exopthalmus, suggesting that some tissues were relatively hyperthyroid. This patient was recently found to be homozygous for an amino acid deletion in the T3-binding domain of the c-erbAfi receptor (10). The clinical data presented here elaborate the only known case in man of homozygosity for a dominant negative form of the c-er6A/3 thyroid hormone receptor.
Subjects and Methods The diagnosis of GRTH in other affected members of this family, kindred S, was based on the presence of elevated serum levels of total and free T 4 and T3, inappropriately normal basal and post-TRH serum TSH levels, and a clinically euthyroid state (2). T4, T3, free T3, reverse T3, TSH, antithyroglobulin and
Received January 14,1991. Address all correspondence and requests for reprints to: Barry B. Bercu, M.D., Department of Pediatrics, All Children's Hospital, 801 Sixth Street South, St. Petersburg, Florida 33731-8920. * Presented in part to the Society for Pediatric Research, Anaheim, CA, 1987 and 1990.
990
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 00:02 For personal use only. No other uses without permission. . All rights reserved.
RESISTANCE TO THYROID HORMONE IN A HOMOZYGOTE
991
antimicrosomal antibodies, and thyroid binding inhibitory immunoglobulin were measured by commercially available RIA kits; free T4 was measured at Nichols Institute (San Juan Capistrano, CA). Thyroid-stimulating immunoglobulin, sex hormone-binding globulin, ferritin, and T4-binding globulin were measured by Nichols Institute. PRL was measured by RIA, using specific polyclonal antisera (13). Restriction fragment length polymorphisms (RFLP) studies Skin fibroblast cultures were established from family members by punch biopsy. DNA was extracted from confluent fibroblast cultures using 5% sodium lauryl sulfate and 1 mg/ mL proteinase-K [extracted twice with phenol and once with chloroform-isoamyl alcohol (24:1)]. DNA was precipitated with isopropanol, rinsed in 70% ethanol, and resuspended in TE (10 mmol/L Tris and 1 mmol/L EDTA, pH 8.0). DNA was digested in 5-/ig aliquots using 20 U BamW or EcoRV restriction endonucleases (Boehringer Mannheim Biochemicals, Indianapolis, IN). DNA electrophoresis was performed in 1% agarose gels and transferred to Nytran membrane (Schleicher and Schuell, Keene, NH) with 10 X SSC (1 X SSC = 0.15 mol/L sodium chloride and 15 mmol/L sodium /3-citrate). The thyroid hormone receptor /3 DNA probe pheA12 (14) was labeled with [32P]dTTP using the random oligonucleotide priming reaction to a specific radioactivity of 2-4 X 108 dpm/ Mg. Blots were prehybridized in 6 X SSC, 10 x Denhardt's solution, and 0.75% sodium dodecyl sulfate (SDS) for 2-16 h and hybridized in 50% formamide, 6 x SSC, 0.75% SDS, dextran sulfate, and 10 Mg/mL salmon sperm DNA at 42 C. Blots were washed twice in 1 x SSC and 0.5% SDS at 37 C and twice in 0.1 X SSC and 0.5% SDS at 65 C. These studies were approved by the University of South Florida College of Medicine and All Children's Hospital Institutional Review Boards, and written informed consent was obtained from the parents and members of the kindred.
I 123
I Thyroid Scans in
Postnatal Period
Case Report The male patient was born at 35 weeks gestation to a 20-yrold gravida 1, para 0, abortus 0 mother. The parents were maternal first cousins with documented GRTH. The birth weight was 1480 g, and the length was 46 cm, consistent with intrauterine growth retardation. Postnatal illnesses included respiratory distress, hyperbilirubinemia, and hypocoagulability. At 3.5 weeks of age, tachycardia (160-200 beats/min), exophthalmus, stare, and thyromegaly were noted (Fig. 1A). The results of initial thyroid function studies are summarized in Table 1. Antithyroglobulin and antithyroid microsomal (peroxidase) antibodies were negative. Bone age was less than term at 4 weeks of age. Automated chemical screen, skull x-ray, and brain ultrasound and magnetic resonance imaging were normal. At 3.5 weeks of age, 123I thyroid scan demonstrated a moderately enlarged thyroid gland with homogeneous uptake of radiolabeled iodide (Fig. IB); the uptake was 87% (normal range, 620%) at 4 h and 50% (normal range, 8-35%) at 24 h. After TRH treatment, serum TSH increased from a basal level of 317 mU/L to a peak of 413 mU/L 30 min later. Serum T4 reached a peak of 818.6 nmol/L at 4 months of age. Clinical
Normal Size Gland FIG. 1. A, Photograph of propositus at 3.5 weeks of age. B, Thyroid scan at 3.5 weeks of age. problems included feeding difficulties, diarrhea, recurrent otitis media and upper respiratory infections, and developmental and growth retardation. The patient grew poorly; by age 2 9/i2 yr he was significantly small (height age, 1 3/i2 yr; weight age, 7/i2 yr) and developmentally delayed. His clinical status was suggestive of tissuespecific hyperthyroidism and hypothyroidism (e.g. hyperthyroid cardiac status and hypothyroid skeletal maturation), although serum concentrations of thyroid hormones and TSH
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 00:02 For personal use only. No other uses without permission. . All rights reserved.
ONO ET AL.
992 TABLE 1. Laboratory investigations of the homozygous child
T4, nmol/L (94.0-193.1) Free T4, pmol/L (16.7-48.9) T3, pmol/L (1613-3210) Free T3( fmol/L (3994-7373) Reverse T3, pmol/L (154-538) T3RU, % (25-35) TSH, mU/L (0.7-5.0) TBG, mg/L (14-26) SHBG, nmol/L (6.9-48.5) Ferritin, jug/L (7-142) TBII, index (
