0021-972x/92/7501-0213$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 75, No. 1 Printed in U.S.A.
A Point Mutation in the 3,5,3’-Triiodothyronine-Binding Domain of Thyroid Hormone Receptor-@ Associated a Family with Generalized Resistance to Thyroid Hormone YUJIN TAR0
SHUTO, OKAZAKI
ICHIJI
WAKABAYASHI,
NAOKI
AMURO,
Departments of Medicine (Y.S., I. W., N.A., S.M.) and Biochemistry School, Sendagi l-l-5, Bunkyoku, Tokyo 113, Japan
SHIRO
MINAMI,
AND
(Y.S., N.A., T.O.), Nippon
Medical
with
ABSTRACT A tight linkage between generalized resistance to thyroid hormone (GRTH) and the thyroid hormone receptor-0 (TRf3) gene is indicated. We evaluated a family with GRTH for the TRP gene. We found that a new point mutation, consisting of a cytosine to adenine replacement at nucleotide position 1642, resulted in substitution in codon 448 in the Ta-binding domain of TRP. This base substitution was found in only one allele of affected members, but not in unaffected members of the
family. The in uitro translation products of this mutant TR/3 gene demonstrated significantly reduced Ta-binding affinity. Previously, others have reported a kindred with GRTH, in that the same codon was subjected to proline to histidine replacement due to a mutation consisting of a cytosine to adenine replacement at nucleotide position 1643. There appeared to be a significant phenotypic difference between our kindred and that described by others. (J Clin Endocrinol Metab 75: 213-217, 1992)
T
hospitalized. History of delayed speech development, hyperactivity, or learning disability was not elicited. He was within the average in general performance and intelligence at school. His height was 148 cm, which was between 1-2 SD above the mean, and he weighed 41 kg. He has been a member of a soccer team and practices daily. The mean resting pulse was 94 beats/min, and the blood pressure was 90/60 mm Hg. Tremor or proptosis was not found. Liver and spleen were not enlarged. The relaxation phase of deep tendon reflexes was not prolonged. Serum cholesterol was in the normal range. Pituitary tumor was not visualized by a computed tomographic scan of the cranium. Thyroid function tests are shown in Table 1. The proband (subject 9 in Table 1) demonstrated high levels of thyroid hormones, with inappropriate secretion of TSH. Circulating antibodies to T3, Tl, TSH-binding inhibitory immunoglobulins, antimicrosomal antibodies, or antithyroglobulin antibodies were not detected. The TSH response to TRH was inhibited by T3 in a dose-dependent manner, while the response was clearly observed even when a supraphysiological dose of T) was admin-
HE SYNDROME of generalized resistance to thyroid hormone (GRTH) is characterized by an impaired response of pituitary and peripheral tissues to an adequate supply of the hormone (1). The actions of thyroid hormone are mediated through binding to one or more intracellular receptors, which, in turn, bind to specific regulatory sites in the chromosomesto influence genomic expression. There are two thyroid hormone receptor (TR) genes, TRCXand TRP (24). A tight linkage between GRTH and the TRP gene has been reported (5-10). In a kindred reported here, a new point mutation at nucleotide position 1642 resulted in proline to threonine substitution in codon 448. Usala et al. (6) described a kindred with GRTH, in that the same codon was subjected to a proline to histidine replacement due to a mutation consisting of cytosine to adenine at nucleotide position 1643. There appeared to be a significant phenotypic difference between our kindred and that described by others (11). Materials Clinical
and Methods
studies
The proband (subject 9 of the family pedigree in Fig. 1) was an llyr-old boy, who presented with a soft, symmetrically enlarged thyroid gland. He was born at fullterm and gained weight normally in the neonatal period. He had no history of hepatitis or jaundice. His height and weight had never been below the mean, He suffered occasional tonsillitis, but had no history of recurring infections. He had never been Received May 31, 1991. Address requests for reprints to: Dr. Ichiji Wakabayashi, Department of Medicine, Nippon Medical School, Sendagi l-l-5, Bunkyoku, Tokyo 113, Japan,
cl2
FIG. 1. Pedigree proband.
cl3
of a kindred
n4 T
with
GRTH.
o5 El6
The
arrow
213
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07
indicates
the
214 TABLE
SHUT0 1. Thyroid
function
Subject no. 1 2 3 4” 5 6 7 8” 9” 10 Normal
tests
of a kindred
Age 73 49 46 42 37 38 34 13 11 7
2. TRH
Free Ta 6.5 5.8 6.9 12.9 6.0 6.6 4.6 14.4 15.4 1.2
0.1-5.0
4.3-10.0
in Fig. 1. TBG,
T,-binding
C
T3 (nmol/L)
(pmol/L)
test
19 15 18 59 14 15 15 46 42 19
2.5 2.5 2.9 3.7 2.3 2.6 1.9 4.2 3.9 2.8
136 126 131 239 118 130 99 239 234 163
lo-23
1.5-3.1
644167
cycle parameters
were
14; Table above.
0 min
15 min
2.9 1.6 0.3
23.0 9.2 2.0
TBG (nmol/L)
T4 (nmol/L)
309 283 263 287 279 198 328 281 466 163-336
-_ globulin.
TSH (mu/L)
;:
JCE & M. 1992 Vol75.Nol
Free T,
(Pmow)
0.7 1.4 1.8 0.7 2.6 0.9 1.3 2.0 2.4 1.1
range
AL.
GRTH
TSH (mu/L)
(yr)
Subject numbers are the same as those ’ Affected member.
TABLE
with
ET
TRH test results before and after the administration of T, to the proband. Serum TSH was measured before and after TRH (jO0 pg) was given i.v. a, No T, was administered, b, 75 pg T,/day were administered for 21 days; c, 150 wg T3/day were administered for 14 days.
Cloning
3). PCR
the same
as those
described
and sequencing
The PCR-amplified products agarose gel (FMC, Rockland, ME) or pBluescript IISK+ (Stratagene clones were sequenced by the deoxy-CTP and a Sequenase land, OH).
were fractionated on 2% NuSieve GTG and subcloned into a pBluescript IIKS+ Cloning Systems, La Jolla, CA). Positive dideoxynucleotide method, using [“‘I’] kit (U.S. Biochemical Corp., Cleve-
Dot blot hybridization istered (Table 2). Thyroid function tests for available family members were performed (Table 1). Inappropriate secretion of TSH was found in his father and his elder sister (subjects 4 and 8 in Fig. 1 and Table 1). They also had small diffuse goiters, but they had no stigmata of thyrotoxicosis. His elder sister was 13 yr old, and her height was 140 cm, which was between l-2 SD below the mean. She was born at fullterm, gained weight normally in the neonatal period, and had no history of jaundice or recurring infections, She had no history of hyperactivity and did well in both elementary school and junior high school. Her mean resting pulse was 96 beats/min. Liver and spleen were not enlarged. Her serum cholesterol level was in the normal range. The proband’s father was 42 yr old, and his height was 164 cm, which was close to the mean. The mean resting pulse was 74 beats/min. Liver and spleen were not enlarged. Serum cholesterol was in the normal range. He was born at fullterm. He had always been in good health. He had no history of hyperactivity and delayed speech development. He had no history of being shorter than his siblings during childhood. He was graduated from senior high school and did well in his work.
DNA preparation and amplification polymerase chain reaction (PCR)
of
the TRfl gene using the
Blood samples were obtained from individuals, and high mol wt genomic DNA was isolated from leukocyte cells by standard procedures. PCR amplification of the T3-binding domain of the TRP gene, exons G and H, was carried out using 1 pg genomic DNA as the template and 100 pmol each of an appropriate pair of primers [we refer to the exons according to the available literature (12)] (Table 3 and Fig. 2). Fifty cycles of PCR reaction were performed, according to the following program. The first denaturation was set at 94 C for 5 min; this was followed by 49 cycles of 1 min of annealing at 58 C, 1 min of extension at 72 C, and 1 min of denaturation at 94 C. Exons B, C, D, E, and F were also amplified with appropriate pairs of primers (exon B, primers 5 and 6; exon C, primers 7 and 8; exon D, primers 9 and 10; exon E, primers 11 and 12; exon F, primers 13 and
Genomic DNA from available family members was also PCR amplified by the procedures described above. The amplified DNA samples were transferred to a Nitro Plus 2000 membrane (Micron Separations, Inc., Westboro, MA) and hybridized with allele-specific oligonucleotide probes containing either the normal or the mutant sequence (Table 3). Hybridization was performed overnight at 37 C in 5 x SSPE, 5 x Denhardt’s solution, and 0.5% sodium dodecyl sulfate (SDS). The blots were washed for 10 min at room temperature in 2 x SSC-0.5% SDS, then for 10 min at 55 C in 5 x SSC-0.5% SDS.
In vitro expression
of
normal
and mutant
TRP
pBST2-120 containing fragment II (nucleotides 1441-1692) of the mutant allele in the EcoRV site was digested with EcoRI and BglII. peAlO (a gift from Dr. Ronald M. Evans, The Salk Institute, La Jolla, CA) containing the entire coding region of the normal TR/3 gene was also digested with EcoRI and BglII. The resulting 1.3-kilobasepair EcoRI/BglII fragment of peAlO and the pBST2-120 fragment lacking the 131.basepair EcoRI/BglII fragment were ligated to produce pBST2120A. The structures of peAlO and pBST2-120A were confirmed by dideoxynucleotide sequencing. They were linearized with HindIII, transcribed, and translated in rabbit reticulocyte lysate, with or without [35S] methionine.
T3 binding
studies
The protein product (10 wL) of the unlabeled translation reaction was incubated overnight with various amounts (0.03-0.6 nmol/L) of [l?]T3 (115 MBq/pg) at 4 C. The binding was carried out in a total volume of 100 PL containing 0.2 mol/L KCl, 20 mmol/L Tris-HCI (pH 7.85), 5 mmol/L dithiothreitol, 10% glycerol, and 1 mmol/L MgCI,. Nonspecific binding was determined by the addition of a 1000.fold excess of nonradioactive T3. Protein-bound TX was separated from free T3 by gel filtration through Sephadex G-25 columns (13-15).
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MUTATION TABLE
3. PCR
primers
and allele-specific
PCR
IN THYROID
oligonucleotide
(ASO)
HORMONE
RECEPTOR-P
215
probes
primers 1
Sense Antisense Antisense Sense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
5’-TGCTGACATGAACTGGTTCT-3’ 5’.ATGAGAATGAATCCAGTCAG-3’ 5’-CTGAAGACATCAGCAGGACG-3’ 5’.CTTGCCTGTGTTGAGAGAAT-3’ 5’-AACCCTCTAGAAAATGGCCTTACAG-3’ 5’-ATCTGCATCCCTTTACATTTCTTCT-3’ -5’.TCCTGCAGAGGGTACATCCCCAGTT-3’ 5’-GGCtiTtiGCTTGCAGCCTTCACAC-3’ -5’.TTTCATCTAGAGTTTCTTTAGAAGA-3’ 5’.TCTACTTACtiGCTTTTGCCATGCC-3’ 5’-TCTCGAGTGGTGCTEGATGACAGCA-3’ -5’.GAAGCTTTACCAGGAATTTCCGTTT-3’ 5’.TCCTCCTTAGCCTGCAGACATTGGA-3’ -5’-ACATCCTGAGCTCACAAAACATAGG-3’ --
2 3 4 5 6 7 8 9 10 11 12 13 14
AS0 probes Normal Mutant
5’-TCTTCCCCCCTTTGTTCT-3’ 5’-TCTTCCCC&TTTGTTCT-3’
PCR primer 1 is an intronic oligomer, and primers 2-4 are exonic oligomers. Primers 5-14 are oligomers underlined nucleotides of PCR primers are base substitutions to create restriction sites. AS0 probe, normal, AS0 probe, mutant, has the mutant sequence, with the C to A substitution indicated by the underline.
Primer
3
4
--ix1429
5---
Exon G
IVS 1171
2
1441ck-
~
CO1692
Exon H
IVS 1429
of the exon-intron junction. The has the normal sequence, whereas
1430
3TAG 1698
I
4
IOObp
FIG. 2. Schematic diagram showing the 3’-end of the TRP gene with locations of the primers used for genomic were used in PCR to amplify fragment I (nucleotides 1171-1429), and primers 4 and 2 were used in PCR to amplify 1692). IVS, Intervening sequences.
Results Sequencing
of the TRP gene
Primers 1 and 3 amplified exon G of the TR/3 gene, which produced fragment I (nucleotides 1171-1429), and primers 4 and 2 amplified exon H of the TRP gene, which produced fragment II (nucleotides 1441-1692). Fragment I did not contain a variant sequence. In fragment II, there was a single base mutation at nucleotide position 1642, with the codon 448 CCT (proline) being converted to ACT (threonine) (Fig. 3). This mutation was detected in two of six independent genomic clones. The two mutant clones were derived from two separate PCR amplifications. Furthermore, multiple genomit clones containing exons B, C, D, E, or F showed no changes from the corrected TR/3 cDNA sequence (12).
Allele-specific
hybridization
The amplified II fragments of available family members (subjects 4, 5, 8, 9, and 10) were transferred to a Nitro Plus 2000 membrane and hybridized to allele-specific oligonucleotide probes (Fig. 4). The mutant cloned DNA and normal cloned DNA were also examined. The normal probe hybridized with amplified DNA from subjects 4, 5, 8, 9, and 10, and with the normal cloned DNA. The mutant probe hybrid-
ized 4, 8, react jects
amplification. Primers fragment II (nucleotides
1 and 3 1441-
with amplified DNA from affected members (subjects and 9) and with the mutant cloned DNA, but it did not with amplified DNA from unaffected members (sub5 and 10) or with the normal cloned DNA.
T3 binding
studies
Mutant and normal receptors were produced from pBST2120A and peAlO1, respectively. When the 35S-labeled in vitro translation products were analyzed by SDS-polyacrylamide gel electrophoresis, they displayed the expected 52to 55-kilodalton products (data not shown). Unlabeled translation products were used to study [1251]T3 binding; studies were performed in duplicate and were repeated three times. Binding affinities for T3 were calculated by Scatchard analysis. The mutant receptor could bind T3, but it had significantly reduced T3 affinity compared to that of the normal receptor (Table 4). The control translation product did not bind significant quantities of T3. Discussion The TRP gene is the locus of GRTH in multiple (6-10). Both dominant and recessive inheritance have been found in patients with the syndrome
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kindreds patterns of GRTH
216
SHUT0 Norma
I
ET AL.
JCE & M .1992 Vol75.Nol
Mutant
GATC
GATC
3.
3’ ------F-7
1650--sT-----
T ic r,
A T T T
T
Pro-446
650
Thr-448 id
C
z
c” T
w--i--l
1636-T//--
636 5’
5’
FIG. 3. Partial sequence of fragment II (nucleotides 1441-1692) of the TR/3 gene. The 252-basepair fragment of genomic DNA from the proband was amplified using primers 2 and 4 and was subcloned and sequenced as described in Materials and Methods. A point mutation was detected that changed codon 448 for proline (CCT) to one for threonine (ACT).
N AS0
4
5
8
9
10
M
Probes Normal
I
Mutant FIG. 4. Allele-specific hybridization of amplified genomic DNA. Genomit DNA from family members was amplified by PCR, dot blotted onto nylon membranes, and hybridized with allele-specific oligonucleotide (ASO) probes containing either the normal or the mutant sequence. Dot N contains the normal cloned DNA containing the normal sequence; M, the mutant cloned DNA containing the mutant sequence; dots 4, 5,8,9, and 10 contain DNA amplified from the genomic DNA of subjects 4, 5, 8,9, and 10 of this kindred, respectively.
TABLE
4. Ta-binding
affinity of normal and mutant receptors
Receptor K. (Lhl) Mutant 4.88 + 0.23 x log” Normal 1.07 + 0.06 x lOi Mutant and normal proteins were produced by in vitro translation in a rabbit reticulocyte lysate system. T3 binding studies were performed by incubation with [iz51]T3 overnight at 4 C. Protein-bound and free [Y]T3 were separated by gel filtration. Binding affinities for T3 were calculated by Scatchard analysis. The results are the mean f SD for three studies. ‘P < 0.001 for the comparison between two groups (by Student’s t test).
(1). In a majority of patients, the syndrome segregatesas an autosomal dominant disorder; distinct point mutations in the T3-binding domain of TR/3 have been identified in three unrelated kindreds (6-8). In the kindred originally described by Refetoff et al. (16, 17), the syndrome was inherited as an autosomal recessive trait, and a major deletion of the TR/3 gene was identified (9). We planned to locate the defect in our kindred, predicting that a mutation would be found within the regions of the T3-binding domain of the TRP gene.
After amplification with PCR and sequencing the fragment of the TRP gene, we identified a point mutation (CCT to ACT in codon 448) in one allele of the proband. The result of allele-specific hybridization was consistent with the presence of heterozygous mutation in affected members, as expected for a dominant disorder. This nucleotide substitution in the T3-binding domain of TRP would introduce a proline to threonine change at codon 448. Previous investigators have demonstrated that the extreme N (ligandi)- and C (ligand2)-terminals of the T,-binding domain are required for high affinity TJ binding to thyroid hormone receptor (1820). We ascertained that the mutation within the gene encoding ligand2 significantly reduced T3-binding affinity. Subjects with the syndrome of GRTH present with diverse phenotypic abnormalities (5-11). Different kindreds had different patterns of affected tissues, which led others to hypothesize that different mutations in the T3-binding domain of TR@result in variable tissueresistanceto thyroid hormone (7). Our observation may suggest that the mutation in the same codon does not necessarily result in the similar phenotypic abnormalities. The mutation of our kindred was in the samecodon as that of kindred A reported by Usala et al. (6, 11, 21). Hyperactivity syndrome, learning disability, and short stature are stressed as the phenotype of kindred A. Even though they have not been uniformly observed in their affected patients, the study of kindred A with 10 affected members demonstrated that they all had at least three of the following tissues resistant to thyroid hormone: pituitary, bone, liver, brain, and heart (11). In contrast, none of the affected members of our kindred showed short stature, bradycardia, or elevated serum cholesterol, as judged by the same criteria used by others (11). The affected members did not have hyperactivity syndrome or learning disability based on repeated and careful interviews with the subjects,parents, and siblings. Therefore, we conclude that there exists a
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MUTATION
IN THYROID
significant difference in the phenotypic characteristics affected members in kindred A and those in ours.
of
References 1. Refetoff S. 1990 Resistance to thyroid hormone revisited. Thyroid Today. 13:1-11. 2. Sap J, Muiioz A, Damm K, et al. 1986 The c-erb-A protein is a high-affinity receptor for thyroid hormone. Nature (Lond). 324:63540. 3. Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM. 1986 The c-e&A gene encodes a thyroid hormone receptor. Nature (Lond). 324:641-6. 4. Lazar MA, Chin WW. 1990 Nuclear thyroid hormone receptors. J Clin Invest. 86:1777-82. 5. Usala SJ, Bale AE, Gesundheit N, et al. 1980 Tight linkage between the syndrome of generalized thyroid hormone resistance and the human c-erbA/3 gene. Mol Endocrinol. 2:1217-20. 6. Usala SJ, Tennyson GE, Bale AE, et al. 1990 A base mutation of the c-erbA@ thyroid hormone receptor in a kindred with generalized thyroid hormone resistance. J Clin Invest. 85:93-100. 7. Usala SJ, Menke JB, Watson TL, et al. 1991 A new point mutation in the 3,5,3’-triiodothyronine-binding domain of the c-erbAP thyroid hormone receptor is tightly linked to generalized thyroid hormone resistance. J Clin Endocrinol Metab. 72:32-8. 8. Sakurai A, Takeda K, Ain K, et al. 1989 Generalized resistance to thyroid hormone associated with a mutation in the ligand-binding domain of the human thyroid hormone receptor /3. Proc Nat1 Acad Sci USA. 86:8977-81. 9. Takeda K, Balzano S, Sakurai A, DeGroot LJ, Refetoff S. 1991 Screening of nineteen unrelated families with generalized resistance to thyroid hormone for known point mutations in the thyroid hormone receptor fi gene and the detection of a new mutation. J Clin Invest. 87:496-502. 10. Usala SJ, Menke JB, Watson TL, et al. 1991 A homozygous deletion in the c-erbAP thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characteriza-
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tion of the mutant receptor. Mol Endocrinol. 5:327-35. 11. Magner JA, Petrick P, Menezes-Ferreira MM, Stelling M, Weintraub BD. 1986 Familial generalized resistance to thyroid hormones: report of three kindreds and correlation of patterns of affected tissues with the binding of [‘251]triiodothyronine to fibroblast nuclei. J Endocrinol Invest. 9:459-70. 12. Sakurai A, Nakai A, DeGroot LJ. 1990 Structural analysis of human thyroid hormone receptor p gene. Mol Cell Endocrinol. 71:83-91. 13. O’Donnell AL, Koenig RJ. 1990 Mutational analysis identifies a new functional domain of the thyroid hormone receptor. Mol Endocrinol. 4:715-20. 14. Koenig RJ, Warne RL, Brent GA, Harney JW, Larsen PR, Moore DD. 1988 Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor. Proc Nat1 Acad Sci USA. 85:5031-5. 15. Samuels HH, Tsai JS, Casanova J, Stanley F. 1974 Thyroid hormone action. J Clin Invest. 54:853-65. 16. Refetoff S, DeWind LT, DeGroot LJ. 1967 Familial syndrome combinding deaf-mutism, stippled epiphyses, goiter, and abnormallv high PBI: uossible target organ refractoriness to thvroid hormone. J Clin Endocrinol Meyab. 2?:279-94. 17. Refetoff S, DeGroot LJ, Benard 8, DeWind LT. 1972 Studies of a sibship with apparent hereditary resistance to the intracellular action of thyroid hormone. Metabolism. 21:723-56. 18. Forman BM, Samuels HH. 1990 Interactions among a subfamily of nuclear hormone receptors: the regulatory zipper model. Mol Endocrinol. 4:1293-301. 19. Horowitz ZD, Yang C, Forman BM, Casanova J, Samuels HH. 1989 Characterization of the domain structure of chick c-erbA by deletion mutation: in vitro translation and cell transfection studies. Mol Endocrinol. 3:148-56. 20. Forman BM, Yang C, Au M, Casanova J, Ghysdael J, Samuels HH. 1989 A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors. Mol Endocrinol. 3:1610-26. 21. Usala SJ, Wondisford FE, Watson TL, Menke JB, Weintraub BD. 1990 Thyroid hormone and DNA binding properties of a mutant cerbA@ receptor associated with generalized thyroid hormone resistance. Biochem Biophys Res Commun. 171:575-80.
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