A Homozygous Deletion in the c-erbA/3 Thyroid Hormone Receptor Gene in a Patient with Generalized Thyroid Hormone Resistance: Isolation and Characterization of the Mutant Receptor

S. J. Usala, J. BMenke, T.L.Watson, F. E. Wondisford, B. D. Weintraub, J. Berard, W. E. C. Bradley, S. Ono, 0. T. Mueller, and B. B. Bercu Department of Medicine East Carolina University School of Medicine (S.J.U., J.B.M., T.L.W.) Greenville, North Carolina 27858-4354 Department of Medicine Case Western Reserve University School of Medicine (F.E.W.) Cleveland, Ohio 44106 National Institute of Diabetes and Digestive and Kidney Diseases (B.D.W.) Bethesda, Maryland 20892 Institut du Cancer de Montreal (J.B., W.E.C.B.) Montreal, Canada H2L 4M1 Department of Pediatrics University of South Florida College of Medicine (S.O., O.T.M., B.B.B.) Tampa, Florida 33612

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 /3-receptor to thyroid hormone response elements of the human TSH/? (-12 to 43 bp) and rat GH (-188 to - 1 6 0 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA/3. (Molecular Endocrinology 5: 327335, 1991)

Different point mutations have been identified in the T3-binding domain of the c-erbA/? 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 ^-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/?

INTRODUCTION

Generalized thyroid hormone resistance (GTHR) is a syndrome of elevated circulating levels of free thyroid hormones, resistance to thyroid hormone action, and inappropriately normal or elevated levels of TSH (1, 2). There is a spectrum of refractoriness to thyroid hormone action among different tissues within a given patient with GTHR, and different kindreds have variable patterns of tissue resistance (2-5).

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MOL ENDO-1991 328

The gene for GTHR has been mapped to the c-erbA/3 locus in multiple kindreds; tight linkage has been established between GTHR and the c-erbA/3 thyroid receptor in three different kindreds (5-7). Three point mutations have been identified in the T3-binding domain of cenbA/3: in kindred A, a C ^ A at nucleotide position 1643, codon 448 (4); in kindred D, a G—»C at nucleotide position 1305, codon 335 (5); and in kindred Mf, a G—» C at nucleotide position 1318, codon 340 (8). These mutant receptors belong to a functional class of proteins called dominant negative inhibitors, in which a mutant form is coexpressed with the wild-type protein and disrupts normal activity (9). In the case of GTHR, a single dominant negative mutant allele results in inhibition of the function of normal thyroid hormone receptors. The fact that in man these mutant c-erbA/3 receptor genes act in a dominant negative fashion, and not through lack of activity, has now been demonstrated in the first kindred ever described with GTHR (10, 11). Takeda et al. (12) found in an affected patient from this kindred a deletion of the T3- and DNA-binding domains in both c-enbA/3 alleles. Although the precise extent of this deletion is unknown, homozygotes from this kindred have no functional /3-receptors. Importantly, the heterozygotes from this kindred are phenotypically normal (10-12), indicating that one c-enbA/? allele is sufficient for normal thyroid hormone action in man, and that the dominant mutant alleles that have been linked to GTHR (4, 5, 8) must produce receptors that inhibit wild-type receptor function. A single patient, S1, in a new kindred, S, with GTHR had been found with dramatically elevated free thyroid hormone and TSH levels, much higher than those of other affected members of this kindred and of other kindreds with GTHR (13, 14). He was the product of the consanguineous union of two affected members and was postulated to harbor a homozygous /3-receptor defect. Restriction fragment length polymorphism (RFLPs) were used to establish linkage between cenbA/3 and GTHR in this kindred (Mueller, 0. T., and B. Bercu, unpublished data), and patient S1 was indeed homozygous for /3-alleles (haplotype AA). We report here on the identification of a novel genetic abnormality, a 3-basepair (bp) deletion at nucleotide positions 1295-1297,1 in the T3-binding domain of cenbA/3 in kindred S. The mutant allele in kindred S behaved as a dominant negative gene, and the heterozygous affected members had thyroid hormone and TSH levels and clinical findings characteristic of other kindreds with GTHR. Patient S1 was determined to be homozygous for the /3-receptor defect, and with this genetic abnormality demonstrated a unique phenotype of extraordinary thyroid hormone resistance in the brain, bone, and pituitary. We have also cloned a partial

1 The nucleotide position coordinate system of first published by Weinberger et al. (15) for the cDNA isolated from the human placenta is used throughout this work. Several nucleotide corrections to this sequence have recently been noted (5,16).

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c-e/t>A/3 cDNA from fibroblasts of patient S1, created a full-length cDNA and synthesized the receptor in vitro, and found that the kindred S receptor does not bind thyroid hormone. This patient displays the effect in man of a dominant negative mutant form of c-enbA/3 in the absence of coexpressed wild-type /3-receptor.

RESULTS Phenotypic Characteristics of Kindred S The mean values and SDS of serum levels of thyroid hormones and TSH for affected and unaffected members of kindred S are compared in Table 1. Affected members of kindred S clearly had elevated free T4 levels with inappropriately normal TSH levels, the sine qua non of GTHR. The total T4, free T4, and total T3 levels of affected members from kindred S, with the exception of patient S1, were very comparable to those reported for other patients with GTHR (2, 4, 5, 8). However, patient S1, who was homozygous for c-enbA/? alleles inherited from consanguineous affected parents, had a profoundly elevated baseline TSH level of 136 mU/L when total T4 and free T4 levels were 45 /ug/dl (normal range, 5.6-10 ^g/dl) and 25 ng/dl (normal range, 1.33.8 ng/dl), respectively. Patient S1 also displayed differences in thyroid hormone action in the periphery compared to other affected members of his kindred. With established clinical parameters to measure thyroid hormone action (2, 4), affected members of kindred S demonstrated only moderate resistance to thyroid hormones in brain and minimal resistance in the bone compartment. In contrast, patient S1 suffered from marked delays in linear growth and was severely mentally retarded (13,14). Also, whereas affected heterozygote patients had normal pulse rates (cardiac resistance), patient S1 was tachycardic, suggesting that his cardiac tissue was relatively hyperthyroid. A Deletion in the T3-Binding Domain of c-erbA/? in Kindred S Since c-enbA/3 and GTHR were linked in kindred S, the c-enbA/? gene was examined for a mutation. Furthermore, since patient S1 was homozygous for c-enbA/3 alleles his genomic sequence was first studied because theoretically there was no confounding wild-type sequence. Exons were systematically amplified, subcloned, and sequenced using the oligomers described in Materials and Methods. The following exons, which included the entire DNA-binding domain and much of the T3-binding domain, were sequenced: exon nucleotides 569-669 (codons 90-123), exon nucleotides 670817 (codons 124-173), exon nucleotides 818-1023 (codons 173-241), exon nucleotides 1171-1429 (codons 291-377), and exon nucleotides 1430-1698 (codons 377-stop). At least six independent clones of each exon were sequenced. Genomic clones of exon 11711429 contained a 3-bp deletion, CAC, at nucleotides

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329

Mutant Thyroid Hormone Receptor

Table 1. Kindred S: Thyroid Hormones and TSH Member

Patient S1 (Homozygote; Age, 3 5/12) Affected (Heterozygote; n = 6) Unaffected (n = 5)

T«

Free T4

T3

TSH

(ng/dl)

(ng/dl)

mU/L

45.4 17.0 ± 2 . 5 8.8 ± 1 . 1

22.3 5.3 ±0.6 2.3 ± 0.1

831 238 ± 33 159 ±38

136 1.9 ±0.6 1.7 ±0.4

5.6-10 3.9-10.5

1.3-3.8 1.3-3.8

105-269 70-204

0.7-5.0 0.7-5.0

Reference ranges Child Adult

1295,1296, and 1297, resulting in a partial deletion of sequence from codons 332 and 333 (Fig. 1). The net result of this sequence change was deduced to be a deletion of the amino acid residue threonine at codon 332 and a consequent shortening of the kindred S receptor length to 455 amino acids. The mother (patient S2; Figs. 1 and 2) was found to be a heterozygote, as anticipated for a patient with the typical clinical syndrome of GTHR, with both a wild-type allele and an allele with the CAC 1295-1297 deletion. Several other base differences were found in these exons compared to the original human placental cerbAfi cDNA sequence (15). However, these were determined to be correct wild-type sequences (5) and in agreement with the human c-erbAfi cDNA sequence recently reported by Sakurai et al. (16). Finally, a c-erbA0 cDNA from codon 175 to stop

334 S2 Heterozygote WT-Allele

S1 Homozygote Mutant-Allele

VAL Fig. 1. Disease and Wild-Type Alleles in Kindred S The genomic sequences of exon 1171-1429 from patient S1 who was homozygous for /J-alleles and patient S2 who was heterozygous for /3-alleles are shown. The S1 sequence has a deletion of nucleotides 1295, 1296, and 1297, CAC, with a deduced net loss of threonine codon 332. There were two populations of genomic clones for patient S2, the wild type and one containing the deleted-sequence; the wild-type sequence is shown here.

12 3

4 5 7

A = 3 bp — A-1296

Fig. 2. Adenine Sequence of Seven Independent Genomic Clones of Exon 1171-1429 from Patient S2 The 3-bp deletion, nucleotides 1295-1297 (CAC), was screened in seven genomic clones of exon 1171-1429 from patient S2, who was heterozygous for two /3-alleles. Note that clones 6 and 7 were mixed together before sequencing. Deletion of adenine 1295 was detected in lanes 1,2,4, and 5. The CAC deletion was evident by the 3-base shift in phase of the AAAAA sequences (nucleotides 1309-1313) between lanes 2 and 3 and between lanes 4 and 6/7. Clones 6 and 7 were a mixture of wild-type and mutant. Clones 1,2,4, 5, and 6 or 7 were mutant, and clones 3 and 6 or 7 were wild type.

codon 457 was cloned using total RNA from fibroblasts of patient S1. This clone was sequenced and corroborated the genomic sequencing; it contained the CAC 1295-1297 deletion and was used to create a complete kindred S mutant receptor for the in vitro studies. Deletion CAC 1295-1297 Is a Mutation and Linked to GTHR The deletion of an amino acid in the T3-binding domain of c-enbA/3 was hypothesized to result in a major structural and functional change and not represent a benign polymorphism. To formally prove that the CAC 12951297 deletion was not a polymorphism, allelic-specific

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MOL ENDO-1991 330

amplification, shown in Figs. 3 and 4, was used to screen selected members of kindred S and 52 random individuals. This deletion was not found in the random individuals and also has not been reported by several different laboratories where c-enbA«1 and c-enbA/? has been sequenced (17-21). The data in Fig. 4 show the presence of only one mutant allele in five of five affected members (middle circle and patients 1, 2, 3, and 7) and the presence of two mutant alleles in patient S1. (Affected patient 10 was also positive for the mutant allele; only the mutant allele was tested for in him, since RFLP analysis indicated he was a heterozygote.) Also three of three unaffected members (patients 4, 6, and 8) did not harbor the CAC 1295-1297 deletion. Patients 5 and 9 were also unaffected and did not have the CAC 1295-1297 deletion, but were considered random individuals, since they were genetically unrelated to affected members of the first generation. RFLPs were uninformative in linking GTHR to c-erbA/3 in affected patients 1 and 10 and unaffected patients 4 and 6, but with allelic-specific amplification it was demonstrated that there was segregation of c-e/t>A/3 with the disease in these patients. This result changed the original lod score of 1.76 at a recombination fraction of 0 to 2.43, significantly increasing the case for linkage between GTHR and c-enbA/3 in kindred S (Mueller, 0. T., and B. Bercu, unpublished data). T3- and DNA-Binding Properties of the Kindred S Receptor To investigate the effects of the CAC 1295-1297 deletion on receptor function we quantified its T3-binding and DNA-binding properties. Schueler et al. (22) have recently shown that a filter binding assay can be used to compare T3-binding affinities of c-erbA/3 translation products. They also demonstrate that there can be variation in the Ka between separate experiments. We concurrently used a created kindred S receptor cDNA,

TZI

i

I 1429

1171

1293-GA I CAC I GGGGCCAGCTGAAAAATGGGGGTCT- 1322

[«

WT

Fig. 3. Allelic-Specific Amplification Strategy for Screening the Kindred S Deletion Oligomers L (see Materials and Methods) and (*) or WT were used to amplify a 199-bp segment from genomic DNA. The CAC sequence at nucleotides 1295-1297 in exon 11711429 was deleted in affected members of kindred S. Oligomer WT was a 27-mer with sequence of nucleotides 1296-1322, and * was a 27-mer with sequence of nucleotides 1293-1322 with CAC deleted (boxed sequence). The L,WT oligomers amplified the wild-type allele, and the L,* oligomers amplified mutant allele.

•

O O

*wr #wr *wr

1 2 3 4 5

6

7

8 9 10

r~p r~p r~p r~p r~p r~jnr~pr~pi i p i i * * * * * * * * * * C ****** ** *

Fig. 4. Screening of Alleles for Kindred S Deletion Oligomers L and * or WT were used for allelic-specific amplification, described in Fig. 3; WT produced a 199-bp band from wild-type alleles, and * produced a 199-bp band from kindred S alleles. The top panel shows results for a homozygous patient (darkened square), the heterozygous mother (half-darkened circle), and an affected member (open circle) from an unrelated kindred D, with a G—»C mutation at nucleotide position 1305 (5). The bottom panels show results for several members of kindred S; 1, 2, 3, 7, and 10 are affected, and 4, 5, 6, 8, and 9 were unaffected by TSH, T4, T3, and free T4 measurements. One hundred and four alleles from 52 random individuals did not demonstrate the kindred S deletion (data not shown). Lane C is an amplification reaction with oligomers L, WT, and * combined without any DNA template. A 123-bp ladder is in the top panel.

the wild-type human placental c-erbAp cDNA, and a created kindred A receptor cDNA (23) to produce mRNAs and program rabbit reticulocyte lysates. The translation products from the kindred S receptor cDNA did not differ in sizes compared to the products from wild-type cDNA (Fig. 5). When equal amounts of kindred S and wild-type receptors, as determined by trichloroacetic acid-precipitable counts and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, were used to bind [125I]T3 the kindred S receptor bound no T3 above that of control samples (Fig. 6). This was verified in four separate experiments (Table 2) where the wild-type Ka was determined to be 7.05 x 1010 ± 2.12 x 1010 M"1 (±SD), well within the range reported by Schueler et al. (22). The kindred A receptor did bind T3 and showed an 8-fold reduction in Ka compared to the wild-type /3-receptor, similar to previously reported results (Table 2) (23). As predicted, the ability of the kindred S receptor to bind to thyroid hormone response elements was intact. The avidin-biotin DNA-binding assay was used to compare the binding of the in vitro kindred S and wild-type receptors to the human TSH/3 gene encompassing - 1 2 to 43 bp and the rat GH gene encompassing -188 and -160 bp. Figure 7 shows that in this assay no differ-

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331

Mutant Thyroid Hormone Receptor

w

0.50

Fig. 5. Synthesis of Kindred S and Wild-Type Human c-erbA/3 in Vitro The construction of the cDNA of the kindred S receptor, with deletion of nucleotides 1295-1297 in the human placental c-erbAfi cDNA (peA 101) (15), is described in Materials and Methods. Complementary DNAs of kindred S and wild-type cenbAjS were used to generate capped mRNAs in vitro, which were subsequently translated in rabbit reticulocytes in the presence of [3SS]methionine. Two-microliter samples of the in vitro translation reactions (S, kindred S; W, wild type) were fractionated by electrophoresis on an SDS-10% polyacrylamide gel, and the bands were visualized with autoradiography. Lane C shows the products of in vitro translation with no added mRNA. Protein standards of 84, 58, 48.5, and 36.5 kDa mol wt are indicated.

ences were detected between mutant and wild-type receptors in the amount of binding to these thyroid hormone response elements.

DISCUSSION Information about mutant c-enbA/? receptors responsible for GTHR in man is now also available from the studies of Sakurai et al. (8), Takeda et al. (12), and Usala et al. .(4, 5, 23) and has various implications concerning the structure and function of the c-enbA/3 receptor. The kindred S mutation (deletion of threonine codon 332) reported here is clustered with the mutations of kindred Mf (glycine to arginine, codon 340) and kindred D (glutamine to histidine, codon 335). The kindred S receptor synthesized in vitro had no measurable ability to bind T3, as was also the case for the kindred Mf receptor (8). This suggests that the segment

0.25 0.12 0.06 0.03 '»I-T3 concentration (nM)

Fig. 6. T3-Binding Properties of Kindred S and Wild-Type Human c-enbA/3 in Vitro Receptors The ability to bind [125I]T3 was compared among in vitro kindred S and wild-type receptors, and translation mixture programmed with no mRNA (control). The amount of specifically bound [125I]T3 with SDS at concentrations of ligand from 0.03-0.50 nM are shown for 2-/ul aliquots of in vitro translation products and were determined with the filter binding assay (see Materials and Methods). Nonspecifically bound [125I]T3 was determined in the presence of greater than 500-fold excess of unlabeled T3, and all points were made in duplicate. Dark bars are data for kindred S receptor [K(S)], open bars for the control mixture (C), and hatched bars for the wild-type receptor (WT).

comprising codons 332-340 has a significant role in ligand binding. Of note, this segment lies outside a recently identified domain (codons 281-300) which is not crucial for T3 binding but appears to be involved in binding a nuclear protein and frans-activation (24, 25). In contrast to the kindred S and Mf receptors, the kindred A receptor (proline to histidine, codon 448) has detectable aifinity for T3. A conclusion from the present study is that the T3binding affinity of the mutant receptors, at least as measured in vitro, does not correlate with the degree of resistance to thyroid hormones and does not fully explain the differences in phenotype among kindreds. For example, the degree of pituitary resistance, as quantitated by TSH secretion, is comparable in kindreds A (2, 4) and S (Table 1). The greater Ka of the kindred A receptor did not result in a lower requirement of thyroid hormones for TSH suppression. Variations in phenotype may involve differences in complex interactions between mutated domains of the receptor and other frans-activating proteins. Various models have been proposed to explain how thyroid hormone receptors regulate gene expression (26-29). It has been inferred that thyroid hormone receptors bind as dimers to thyroid hormone response elements (27) and that dominant negative forms of cerbA, such as c-erbAcc2 and v-e/t>A, may block wildtype receptor binding and function at promoter sites (30-34). This is compatible with the existing data on the human mutant receptors; the in vitro kindred S and A receptors bind normally to TSHjS and rGH thyroid hormone response elements. However, further DNA-

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MOL ENDO-1991 332

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Table 2. Comparison of T3-Binding Affinities of in Vitro Mutant Receptors from Kindreds with GTHR In Vitro Receptor

T3-Binding Affinity (M" 1 )

Mutation8 Exp1" 0

Wild Type A S Mf d

10

C-+A(1643);P448H ACAC(1295-1297; A-T332 G-+C(1318);G340R

2.3 X10 4.5 X10 9

Exp 2 10

8.36 X 10 1.1 X 1010 None Detected

Exp 3-5 6.5 ± 2.2 x 1010 None Detected

None Detected

In each experiment, in vitro translation products, wild-type and A and/or S, were synthesized and tested simultaneously for T3binding affinity. Kindred S and wild-type receptors were simultaneously used for Exp 3, 4, and 5, and the mean ± SD for Ka are shown. None Detected, No binding of [12SI]T3 was detected above that for control translation products. a 1643 and 1318 are nucleotide positions according to Ref. 15. A is deletion of nucleotides CAC and deduced deletion of threonine. Codons with amino acid changes are shown. b These data previously reported in Ref. 23. c Wild-type refers to human placental c-erbAfi receptor (15). d These data were reported by Sakurai et al. (8).

AD5

rGH

hTSHB

Fig. 7. Binding of the Wild-Type Human Placental and Kindred S c-erbA(l in Vitro Receptors to DNA Fragments Containing the TSH0 Segment - 1 2 to 43 bp and Rat GH Gene Segment -188 to -160 bp. The negative control was a DNA fragment containing the adenovirus-5 long terminal repeat. The avidinbiotin DNA binding assay was employed with 1 pmol of the respective biotinylated DNA fragment (10 nwi), and equivalent amounts of 35S-labeled wild-type and kindred S in vitro cerbAfi translation products were used, as quantitated by SDSpolyacrylamide gel electrophoresis.

binding studies must be performed with these mutant receptors using extracts with recently identified transactivating factors (24, 25, 35, 36) to determine whether the in vivo DNA-binding affinity may be affected. There are also data to support a model for receptor dimerization in solution; Forman et al. (28, 29) have shown that DNA-binding domain deletion mutants of c-erbAa can act as dominant negative regulators. They have proposed a "leucine zipper" model for thyroid hormone receptor dimerization through eight hydrophobic heptad repeats between codons 329-425 (/3-receptor codons). The kindred S deletion at codon 332 would disrupt the phase of the first heptad, indicating that if dimerization is crucial for the dominant negative effect, the first heptad could not be structurally significant for dimerization. However, interestingly, all of the mutations reported to date lie outside of the other seven heptad

sequences, meaning that they could subserve a role in leucine zipper dimer formation. Patient S1 confirms the notion of the crucial role of c-enbA/3 in mediating thyroid hormone action in man. With two alleles for a dominant negative-acting receptor, this patient has severe pathology in brain development, growth, and TSH secretion. Strait et al. (37) have recently quantitated a r , «2-, and /3-receptor mRNA levels in tissues of developing and adult rats, and their data suggested that the fa-receptor may play a primary role in mediating the effects of T3. They measured a 40-fold rise in the level of fa mRNA in rat brain during the transition from fetus to neonate, indicative of a role for the /3-receptor in neurological development. The importance of the /3-receptor in mediating normal thyroid hormone action in brain is supported by the severe mental retardation seen in patient S1. In addition, the a-receptor must be involved in brain development, given the existence of patients without /3-receptor (10-12). The precise molecular mechanisms for the profound elevation in TSH in patient S1 are presently unclear. This may simply be the result of the removal of any repression by normal pituitary a,-receptor because of abundant mutant forms of /3-receptor. However, the patients without /3-receptor showed no evidence in clinical trials of repression of TSH secretion by T3 (38) [or stimulation after administration of propylthiouracil (11)], and little thyroid hormone regulation of the TSH/3 gene may be mediated through the arreceptor. One can speculate that the kindred S receptor [fa and /32 mutant forms) may act as a positive frans-activating factor on the TSH/3 gene. There are data to support this hypothesis from studies of the human TSH/3 gene transfected into human embroynal kidney 293 cells which indicate that unoccupied thyroid hormone receptors stimulate transcription from this promoter (39). Further studies are necessary to clarify the specific roles of the a- and /3-receptors in the regulation of TSH/3 gene expression. Finally, an observation can be made about the relative role of the c-enbA/3 receptor in cardiac tissue. Some of the metabolic effects of thyroid hormones were pre-

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Mutant Thyroid Hormone Receptor

served in the patients with no /3-receptor (11). In addition, patient S1 was tachycardic; although pulse rate has limitations as a parameter in quantitating the thyroidal status of cardiac tissue, this tachycardia suggested a relatively lower level of inhibition of thyroid hormone action by the mutant /3-receptors. One can infer from these observations that some degree of mediation of thyroid hormone action in the heart occurs through the a-receptor or other pathways. Further study of mutant forms of the c-enbA/3 receptor responsible for GTHR will provide insights on the mechanisms of gene regulation by thyroid hormones.

MATERIALS AND METHODS Clinical Studies Patient S1 had been serially followed shortly after birth in the Pediatric Endocrinology Outpatient Center at All Children's Hospital and the University of South Florida College of Medicine, where physical examinations, with measurements of linear growth, neurological development, and TSH and thyroid hormone levels, were obtained. Leukocytes for DNA and skin biopsies for culture of fibroblasts were also obtained after informed consent was given by patient S1 as well as other members of kindred S. Genomic Sequencing All oligomers were made at the East Carolina University School of Medicine Biotechnology Center. The following sets of intronic oligomers with underlined restriction sites were used to amplify exons for subcloning and sequencing: 1) exon nucleotides 569-669 (codons 90-123): 5'-TATATTTTTTAGAATTCCTTCTTTTAT-3' 5'-TGGAGCTGGATCCGGGCTAAGCTCTGT-3'2) exon (nucleotides 670-817; codons 124-173): 5'-TGATTTCAAGAATTCGAAACTGTTGTC-3' 5'-CATTCCTTGGATCCGAAGCAAGTGAAG-3'3) exon (nucleotides 8181023; codons 173-241): 5-TCTTGGGAGGATCCGTGTGCCTTGTCT-3' 5'-TAGAATTCACTGGCATATAAGTGAAAC-3'4) exon (nucleotides 1171-1429; codons 291-377): 5'-CTGGCATTTTGAATTCGTTCTTTGCTG-3' 5'-AAAGCTCTTTGGATCCCCACTAACGAG-3' and 5) exon (nucleotides 14301698; codons 377-stop): 5'-CCTTCCATCTCTGCAGCAATGTCCATC-3' 5'-GGAATTATGAGAATGAATTCAGTCACTThe polymerase chain reaction mixtures were made according to specifications in the GeneAmp DNA amplification kit (Perkin-Elmer-Cetus, Norwalk, CT), and the cycle conditions for the genomic studies were: denaturation, 94 C for 1 min; annealing, 55 C for 1 min; extension, 72 C for 2 min; for 30 cycles. Amplified exons were fractionated on and cut out of NuSieve GTG gels (FMC, Rockland, ME) and subcloned into pGEM3Z (Promega, Madison, Wl), and individual clones were isolated and sequenced using the Sequenase version 2.0 DNA Sequencing Kit (U.S. Biochemical Corp., Cleveland, OH). Single lane tracking for the adenine sequence was performed using dideoxyadenosine triphosphate (ddATP) termination mixture alone. Allelic-Specific Amplification The method of allelic-specific amplification or amplification refractory mutation system (40) was used to screen for the CAC 1295-1297 deletion. Intronic oligomer L in Fig. 3 was 5'CTGGCATTTTGAATTCGTTCTTTGCTG-3' and was comple-

333

mentary to the wild-type sequence. The wild-type and (*) oligomer sequences used to differentiate a wild-type from a mutant allele are given in Fig. 3. DNA specimens from members of kindred S and random individuals [courtesy of A. E. Bale (Department of Human Genetics, Yale University), O. T. Mueller (Molecular Genetics Laboratory, University of South Florida), and D. Accili (NIDDK, NIH)] were amplified with either (L,*) or (L,WT) for 30 cycles under the following conditions: denaturation, 94 C for 2 min; annealing, 62 C for 1 min; and extension, 72 C for 1 min. Complementary DNA Isolation and Sequencing Total RNA was prepared from primary fibroblasts by lysis in guanidine isothiocyanate (41). Reverse transcription of these specimens was accomplished with avian myeloblastosis virus reverse transcriptase, according to a previously published method (42). The antisense primer used for first strand synthesis contained a created EcoRI site: 5'-GGAATTATGAGAATGAAT7CAGTCAGT-3' (nucleotides 1672-1698, untranslated portion). The cDNA from this reaction were extracted with phenol, phenol-chloroform, and chloroform and recovered by ethanol precipitation. They were subsequently used in a polymerase chain reaction with sense oligomer (Kpn\ Site created): 5'-GTTGGCATGG7ACCAGATTTGGTGCTG-3' (nucleotides 799-825, codons 167-174) under cycling conditions of: denaturation, 94 C for 1 min; annealing, 55 C for 2 min; extension, 72 C for 3 min; 30 cycles. The cDNA products from this reaction were restricted with Pst\ and EcoRI and fractionated on a NuSieve agarose gel, and a band of about 890 bp was cut from the gel. The Psfl-£coRI cDNA fragment was subcloned into pGEM3Z and sequenced. This cDNA from patient S1 was then used to construct a full-length mutant cDNA for in vitro transcription. Construction of Kindred S Receptor The partial kindred S cDNA just described was restricted with Sty\ (one site at nucleotide position 987) and fig/I I (nucleotide position 1563), and the fragment was recovered by fractionation in a NuSieve gel. The fragment was ligated into the corresponding site in peA 101 (wild-type /Vreceptor cDNA in pGEM3) (15), exchanged for the wild-type segment. The CAC 1295-1297 deletion was verified by sequencing the kindred S cDNA construct from nucleotide 826 (before the Sty\ insertion site) to the stop codon. The construction of the kindred A receptor cDNA has been previously reported (23). In Vitro Transcription and Translation Messenger RNAs of the kindred S, kindred A, and wild-type receptor cDNAs were prepared according to Promega specifications, using m7G(5')pppp(5')G from Pharmacia (Piscataway, NJ). The in vitro translation reactions were prepared using the DuPont-New England Nuclear reticulocyte lysate L[35S]methionine translation kit (Wilmington, DE). The amount of product was quantitated by trichloroacetic acid-precipitable counts and the intensity of bands on autoradiographs of 10% SDS-polyacrylamide gels. Ts-Binding Studies The nitrocellulose filter binding assay used in the present study has been described in detail by Inoue et al. (43), Schueler et al. (22), and Usala et al. (23). L-[ 125 I]T 3 (SA, 2200 Ci/mmol) was purchased from DuPont-New England Nuclear, diluted in buffer, and directly added to 500 fi\ ligand-binding incubations containing 2 n\ translation products. Final concentrations of [125I]T3 of 0.5-0.01 nM were used to generate Scatchard plots. Nonspecific binding was determined in the presence of a 1000fold excess (0.5 MM) of unlabeled T3.

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Vol 5 No. 3

MOL ENDO-1991 334

DNA Binding Studies The avidin-biotin DNA binding assay used to measure kindred S and wild-type receptor binding to fragments of the TSH/3 and rat GH genes and a fragment of the long terminal repeat of adenovirus-5 has been described for the kindred A receptor (23). Each DNA fragment contained 11 biotinylated residues and was quantitatively precipitated by a streptavidin-agarose matrix. The amount of receptor bound to precipitated fragments was quantitated using liquid scintillation counting.

9. 10.

11.

12.

Acknowledgments We thank Dr. Cary Weinberger of the Scripps Clinic (San Diego, CA) for peA 101. We also thank Mr. Michael Bennett and Dr. James Coleman, Department of Microbiology, East Carolina University School of Medicine, for synthesis of the oligomers.

13.

14. Received November 6, 1990. Revision received December 17,1990. Accepted December 21,1990. Address requests for reprints to: Stephen J. Usala, M.D., Ph.D., Department of Medicine, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354. This work was supported by Grant 1R29-DK-42807-01 (to S.J.U.).

15. 16.

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