0021-972X/91/7201 J032$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society

Vol. 72, No. 1 Printed in U.S.A.

A New Point Mutation in the 3,5,3'-TriiodothyronineBinding Domain of the c-erbA/3 Thyroid Hormone Receptor Is Tightly Linked to Generalized Thyroid Hormone Resistance STEPHEN J. USALA, JAY B. MENKE, TRACEY L. WATSON, JACQUES BERARD, W. EDWARD C. BRADLEY, ALLEN E. BALE, ROBERT W. LASH*, AND BRUCE D. WEINTRAUB Department of Medicine, East Carolina University School of Medicine (S.J.U., J.B.M., T.L.W.), Greenville, North Carolina 27858-4354; Institut du Cancer de Montreal (J.B., W.E.C.B.), Montreal, Canada H2L 4M1; the Department of Human Genetics, Yale University (A.E.B.), New Haven, Connecticutt 065108005; and the Molecular, Cellular, and Nutritional Endocrinology Branch, National Institutes of Health (R.W.L, B.D.WJ, Bethesda, Maryland20892

ABSTRACT. Two different mutations in the c-erbkfi 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-erbkfi. 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-er&A/9, 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-er&A/? 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-erbhp. (J Clin Endocrinol Metab 72: 32-38, 1991)

G

ENERALIZED thyroid hormone resistance (GTHR) refers to a syndrome where thyroid hormone action is inhibited in peripheral tissues and the pituitary (1). This resistance syndrome most commonly appears as a familial autosomal dominant disorder (2). There is a spectrum of refractoriness to thyroid hormone action among different tissues within a given patient with this syndrome, and different kindreds with GTHR have variable patterns of tissue resistance (2, 3). The variable phenotypes of kindreds with GTHR have suggested that there are heterogeneous genetic abnormalities responsible for this syndrome. There are 2 putative thyroid hormone receptor genes, c-erbkfi at 3p22->3p24.1 (4) and c-erbAa at 17qll.2-> 17q21 (5). The /?-gene encodes at least 2 receptors, c-

erbA/31, which was first isolated from human placenta (6), and c-erbA02, which appears to be expressed only in the pituitary (7). The c-erbAf31 and c-erbA(32 receptors share common DNA- and T3-binding domains, but differ at the N-terminus. The a-gene encodes multiple cDNAs (8-11); the c-er6Aal has in vitro properties of a T 3 receptor, but the c-erbAa2 protein does not bind T3. The gene for GTHR was first shown to map to the same region as the c-erbA(3 thyroid hormone receptor gene in 1 kindred, A (12). Tight linkage between c-erbA/3 and GTHR strongly suggested that the c-erbA(3 gene is important in man as a thyroid hormone receptor and identified a putative c-erbA(5 mutant phenotype with short stature and central nervous system, pituitary, liver, and basal metabolic abnormalities (12). Subsequently, a base substitution, cytosine to adenine at nucleotide position 1643, was found in codon-448 in the T3-binding domain of the 0-receptor which altered a proline residue to histidine (13). This single base substitution was found in only 1 of the 2 alleles of all 7 affected members of kindred

Received June 6, 1990. Address all correspondance and 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. * Present address: Division of Endocrinology, University of Maryland School of Medicine, Baltimore, Maryland 21201. 32

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A NEW POINT MUTATION IN c-erbAp

A, but was not present in 10 unaffected family members; it was shown to be a mutation by examining random alleles. Recently, the kindred A mutation has been inserted into a wild-type c-erbkfi cDNA, expressed in vitro, and found to bind T 3 with a 5-fold reduced affinity compared to the wild-type receptor (14). Interestingly, this mutated receptor bound normally to a DNA fragment of the human TSH/3 gene containing a thyroid hormone response element (15). Another c-erbAj3 sequence abnormality has been reported by Sakurai et al. (16) in the T3-binding domain of the receptor in a different family with GTHR. A guanine to cytosine replacement at nucleotide position 1318 resulted in a glycine to arginine subsitution in codon 340 in one of two alleles. The /^-receptor with this base alteration was expressed in vitro, and T3-binding activity was not detected. The kindred A mutation was not present in two other kindreds, B and D, where GTHR was also linked to the /3-receptor (13). Kindreds B and D had tissue patterns of thyroid hormone action distinct from those of kindred A (13). Short stature was not a clinical manifestation of GTHR in these two kindreds, and there was significant resistance to the chronotropic effects of thyroid hormone in these kindreds compared to kindred A. However, affected members of kindred D did have cognitive impairments similar to those of kindred A. It was hypothesized that different mutations in the c-erbAfi receptor were probably responsible for the variant phenotypes of thyroid hormone resistance in kindreds B and D. We have tested the hypothesis that different mutations in the T3-binding domain of c-erbAf3 result in variable tissue resistance to thyroid hormones by examining the genomic sequence of affected members of kindred D. We found a single base substitution in affected members of kindred D, again in the T3-binding domain of c-erbA(3, different from the other two base substitutions previously reported (13, 16). This base substitution was a mutation, since it was not found in over 100 random alleles. Finally, this mutation was tightly linked to GTHR in kindred D, confirming that the c-erbAfi receptor is indeed the abnormal genetic locus and suggesting that this mutation is the genetic abnormality causing thyroid hormone resistance in kindred D.

33

from the kindred provided blood for thyroid function studies and DNA from collection in the field. Height measurements, but not IQ, cholesterol, sleeping pulse, or pulse wave arrival time, were obtained from these families. All serum T4, free T4, T3, and TSH measurements were performed at the NIH Clinical Center and Hazelton Biotechnologies (Vienna, VA). Genetic studies Periperal blood was used to prepare high mol wt DNA (12). All oligomers were made at the East Carolina University Biotechnology Center. The two exons of c-er6A/3 containing codons 291-457 (stop codon) were amplified using the following two sets of 27mers: exon (nucleotides 1430-1698), 5'-1698-GGAATTATGAGAATGAATTCAGTC AGT-3' (exonic oligomer, in italics is base substitution to create EcoRl site), 5'CCTTCCATCTCTGCAGCAATGTCCATC-3' (intronic oligomer, in italics are base substitutions to make PstI site); exon (nucleotides 1171-1429), 5'-AAAGCTCTTTGGATCCCC ACTAACGAG-3' (intronic oligomer, in italics is base substitution to make BamHI site), 5'-CTGGCATTTTGAATTCGTTCTTTG CTG-3' (intronic oligomer, in italics are base substitutions to make JBCORI site). The polymerase chain reaction mixtures were made according to specifications in the GeneAmp DNA amplification kit (Perkin-Elmer-Cetus, Norwalk, CT), and the cycle conditions were: denaturation, 94 C for 1 min; annealing, 55 C for 1 min; and extension, 72 C for 2 min, for 30 cycles. DNA from patient 2 (Fig. 1) was first used to amplify the exons. The amplification products were restricted with EcoRl, Pstl (exon 1430-1698) or BamHI, EcoRl (exon 1171-1429), fractionated on NuSieve GTG gels (FMC, Rockland, ME), and subcloned into pGEM3Z (Promega Biotec, Madison, WI). Eight separate clones were picked and sequenced using the SEQUENASE version 2.0 DNA-sequencing kit (U.S. Biochemical, Cleveland, OH). Several clones were rapidly screened for the C-1305 base substitution by single lane sequence analysis (17). Rapid screening for the C-1305 mutation DNA samples from 16 kindred members with whom linkage could be tested were used to amplify exon 1171-1429. The reaction mixtures were phenol-chloroform extracted and KINDRED D

a

Materials and Methods Subjects IS

Clinical data on members of kindred D were obtained during serial hospitalizations at the Clinical Center of the NIH. The clinical parameters used in this study have been previously detailed (2, 13). All patients had entered into Clinical Center protocols and gave informed consent. Several nuclear families

16

"

FIG. 1. Pedigree of kindred D. Males are shown by squares, and females by circles. Darkened symbols are affected members of the kindred. N signifies that a member was tested for generalized thyroid hormone resistance and is unaffected. Symbols with diagonal denote deceased members, and blank symbols denote untested members.

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USALA ET AL.

34

ethanol precipitated, and the resuspended products were digested with Puull. The PuuII-restricted products were fractionated on 2% NuSieve GTG-1% agarose gels, and the 16 members were haplotyped for the C-1305 allele. The frequency of the C1305 sequence in random alleles was assessed in 53 individuals. DNA specimens of these 53 random individuals were obtained from the Department of Human Genetics, Yale University, the NIDDK (courtesy of D. Accili), and the University of South Florida Pediatrics Laboratories (Tampa, FL; courtesy of 0. T. Mueller). Linkage analysis of c-erbA(3 and GTHR in kindred D Pairwise linkage analysis was performed with the MLINK option of the LINKAGE package (Department of Human Genetics, Yale University). The logarithm of the odds (lod) scores were computed using a C-1305 allele frequency ranging from 1 in 100 to 1 in 10,000, since the actual frequency was determined to be less than 1 in 100.

TABLE 1. Kindred D: thyroid function tests Member no. [2] [3] 4° 5 [6]" [7] [8] 9C [10] [11]" [12] 13 14

15 [16] [17] Normal range

Results Phenotype of kindred D The pedigree of kindred D is shown in Fig. 1 and demonstrates the autosomal dominant transmission of GTHR in this kindred. The average thyroid hormone levels, and TSH and general laboratory and clinical parameters of tissue responsiveness to thyroid hormones in this kindred have been reported previously (13). Table 1 shows the TSH and thyroid hormone levels of kindred members 2-17 used to assign the affected and normal phenotypes. The affected assignments could be unambiguously made on the basis of elevated free T4 (range, 3260 pmol/L) with inappropriately normal or elevated TSH. Member 11, who was assigned to the affected group, had elevated free T4 as well as elevated free T3; surprisingly, her total T4 and total T 3 were within normal limits. Many of these patients had been followed serially over several years, and the assignments repeatedly verified. The overall pattern of thyroid hormone action in peripheral tissues in affected members of kindred D has been reported as minimal resistance in bone, resistance in basal metabolism, and severe resistance in heart and brain (13). The clinical and laboratory measurements that were used to establish this phenotype are shown in Table 2. The height of the kindred members was not related to GTHR. There was a reduction in intelligence, inappropriately normal or elevated serum cholesterol, and near bradycardia in affected members of the kindred. The level of pituitary resistance was assessed by TRH stimulation as well as basal TSH. The results of TRH stimulation in affected members are shown in Table 3 and demonstrate approximately a 10-fold rise in TSH in the setting of elevated basal free T4.

JCE & M • 1991 Vol 72 • No 1

TSH (mU/L)

T4 (nmol/L)

Free T4 (pmol/L)

T3 (nmol/L)

5.4 11.0 4.8

216 197 73 90

48 41 10 15

2.7 2.9 2.3 2.8

2.1 2.0 1.5 2.1 1.9

173 221 133 195 140

33 41 16 41 32

3.1 3.7 3.4 3.6 2.4

3.3

173

34

4.8

3.7 3.9

105 101

21

3.0

2.7

84

20 14

2.5 1.8

4

2.4

189

37

3.7

0.25

5.0

Age (yr) 52 44 48 42 19 20 16 25 29 1 1.5

8 6

3.5

0.43-3.8

60 73-145

15-27

0.9-3.0

Brackets indicate that member was assigned affected phenotypes for linkage study. ° Member was on an iodine-containing medication when these tests were made. He had a normal T 4 (102 nmol/L) 1 yr previously. 6 There were no TSH or thyroid hormone levels available on this member, now deceased. However, she must have had GTHR because members 10, 11, 16, and 17 clearly are affected (see Fig. 1). c Pregnant. d Free T 3 was 5.6 pmol/L (normal range, 3.2-5.0 pmol/L). Free T4 2 yr earlier was 34 pmol/L.

Point mutation in the T^-binding domain of c-erbA(3 Because 2 mutations had been previously found in the T3-binding domain of c-erbkfi in 2 different kindreds with GTHR, a mutation in exon (nucleotides 1171-1429) (16) and a mutation in exon (nucleotides 1430-1698) (13), we first sequenced these exons in both alleles of kindred D.1 Six of 6 independent genomic clones of amplified exon 1430-1698 did not contain a variant sequence. However, a single base substitution of guanine to cytosine at nucleotide position 1305 was found in exon 1171-1429 in one allele (Fig. 2). This base change was found in 5 of 12 independent genomic clones of amplified exon 1171-1429 (Fig. 3). This base substitution altered codon-335 from CAG (glutamine) to CAC (histidine). Five additional base differences in the coding sequence of codons 291-457 were found in both alleles of kindred D compared to the previously reported human c-erbA(31 cDNA sequence (Table 4), but these are wild-type sequences since they were found on sequencing multiple random human alleles and have been reported in the human c-er6Aal and rat c-erbA(3l cDNAs (19, 20). We next determined the incidence of the C-1305 base 1 The genomic structure of the DNA- and T3-binding domains of cerbkfi has been determined by Berard et al. (18). The 5' region has not been reported, and we, therefore, refer to the two exons by nucleotides rather than exon number.

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A NEW POINT MUTATION IN c-erbkp

35

TABLE 2. Kindred D: clinical and laboratory parameters of thyroid hormone action Member no. [2] 4 [6] [7] [8] 9

13 14 15

F M F F F F F F F M M M

[16]

F

[10] [11] [12]

Brain (IQ verbal/ performance/full scale)

Liver (cholesterol, mmol/L)°

144 160 167 151 144 152 158

81/83/82

157°

67/76/70

Bone (ht, cm)

Sex

Heart Sleeping pulse

QKd

7.55

64

190-207

4.44

68 75

197-210 173-178

5.63 6.18

64

212-239

4.18 4.13 4.73

116 72 64 86

78/76/76

75 135 130 88"

87/96/91 90/95/91

Affected members are bracketed. ° Normal range of cholesterol (1.93-5.16 mmol/L) varies by age and sex. 6 QKd is pulse wave arrival time; hypothyroid range, more than 250 ms; hyperthyroid range, less than 150 ms. c Fifty-fifth percentile for height for chronological age. d Fiftieth percentile for height for chronological age. TABLE 3. Kindred D: TRH stimulation in affected members

12 3 4 5 6

TSH (mU/L)

Time (min) -15 0 5 10

4.0 3.2 9.3 11.3

15

13.1

20 30 60 120

14.2 21.2 17.8

2.1

8

10

2.0

1.5

23.9 36.5

24.2

9.5

11

1.9 2.2 6.1 11.5

16

2.5 2.2

17.4 22.6 26.7 19.8

12.3 14.8 16.6 18.2 17.2 11.0

9.2

3.5

Member no.

QATC

CTAG

-1305

1305-

DISEASE

334

WILD-TYPE

G-1305 —

FlG. 3. Single lane sequencing of six independent genomic clones of exon (nucleotides 1171-1429). Genomic clones were obtained using the polymerase chain reaction, as described in Materials and Methods. Sequencing reactions were run with dideoxycytidine triphosphate only and show the presence (wild-type) or absence (mutation) of a guanine at position 1305. There is a 1:1 proportion of mutant to wild-type sequences after amplification of exon 1171-1429 from patient 2 (Fig. 1). TABLE 4. c-erbAfi wild-type sequence differences from the human cer6A/3 cDNA sequence reported by Weinberger et al. (6) Nucleotide

Codon/amino acid

1254-A 1295-C 1353-G 1636-T 1651-T

318-CAG/PRO 332-ACA/THR 351-CTG/LEU 446-TTC/PHE 451-TTG/LEU

FIG. 2. Sequences of disease and wild-type c-er6A/3 alleles of kindred D. The sequences shown are from patient 2 (see Fig. 1). One population of genomic clones (see Materials and Methods) contained a guanine to cytosine base substitution at nucleotide position 1305 which changed glutamine codon-335 (GLN/CAG) to histidine (HIS/CAC). Another population of genomic clones contained the wild-type sequence.

These sequence corrections are only for the coding regions of exon (nucleotides 1171-1429) and exon (nucleotides 1430-1698). Sakurai et al. (16) report corrections to the 3'-untranslated portion of exon 10.

substituion in all members of kindred D and in 106 random alleles. The strategy used to screen for this base substitution is shown in Fig. 4. The G—»C alteration at

nucleotide position 1305 eliminated a Pvull restriction site at position 1305, which could be easily visualized after amplification of exon 1171-1429 and neighboring

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36

USALA ET AL. FIG. 4. Strategy for screening kindred D and random alleles for C-1305 mutation. Oligomers L and R (see Materials and Methods) were used to amplify exon (nucleotides 1171-1429) of DNA samples. The C-1305 mutation eliminated a Pvull site as shown. Products of amplification were digested with Pvull and gel fractionated. A wild-type allele could be easily distinguished from the kindred D mutant allele as two bands of 165 and 135 basepairs vs. a single band of 299 basepairs.

JCE & M • 1991 Vol 72 • No 1

PVUII

PVUII 27

I

I

JJU

I 1171

intronic sequences and digestion with Pvull. (An additional Pvull site was present at the left intron/exon border, which made it impossible to detect this base substitution by conventional genomic Southern analysis.) There was perfect segregation of the C-1305 base substitution with all 10 affected members of kindred D (Fig. 5). Furthermore, none of the 6 unaffected members contained this change, and the 10 affected members had a normal allele. Finally, 106 random alleles (53 individuals) were screened for this base substitution and did not harbor it (data not shown). This means the C-1305 base substitution is a mutation in kindred D and not a polymorphism.

27

14

R

I 14M

CA|G]CTG-WT CAISJCTG-KINDRED D

I 1305 PVUII FRAGMENTS WT:

165 bp, 135 bp, (48 bp)

KINDRED D:

299 bp, (48 bp)

gene in kindred D mapped to the c-erbA/3 locus and, therefore, was probably caused by the C-1305 mutation. Given that the mutant allele frequency was no greater than 1 in 100, a maximum likelihood estimate for the

segregation of GTHR with the C-1305 mutation was obtained at a recombination fraction of zero with a lod score of 4.19 using the MLINK option of LINKAGE (21) (Table 5). This lod score means that there is an approximately 1 in 15,000 probability that the C-1305 mutation segregated with GTHR by chance alone. A lod score of 3.0 or greater is accepted as significant evidence for linkage (22). This analysis demonstrated tight linkage between GTHR and the C-1305 mutation in c-erbAfi in kindred D.

Linkage between GTHR and mutation C-1305 We originally reported linkage between c-erbA(3 and GTHR in kindred D with a lod score of 0.71 (13). In this kindred, restriction fragment length polymorphisms were not informative in most members. However, the above studies enabled linkage between GTHR and c-erbA(3 to be better quantitated. We examined whether the disease 4 5 2 1 3 I I I I

123bp—*

Discussion This study confirms the earlier observation by us and others that c-erbAfi is the gene locus of GTHR in multiple kindreds (12-14, 23). It demonstrates in another kindred tight linkage (lod score >3.0) between c-erbA(3 and thyroid hormone resistance. The C-1305 base substitution is the third mutation reported in familial generalized thyroid hormone resistance; all of these mutations lie within the T3-binding domain in exon 11711429 and exon 1430-1698. Although we have not proved that the C-1305 base mutation is the one responsible for the syndrome in kindred D, this is highly likely given that 1) this mutation is tightly linked to the disease; 2) the predicted glutamine to histidine residue change in codon 335 is a nonconservative change which alters the charge of the amino acid in the T3-binding domain; and 3) this mutation is in close proximity to the C-1318 mutation of another GTHR family reported by Sakurai et al. (16). Furthermore, we have sequenced another TABLE 5. Pairwise lod scores for linkage between GTHR and c-er&A/3

FlG. 5. Screening of kindred D for C-1305 mutation. Members 1-17 were analyzed as described in Fig. 4. Affected members (no. 2, 3, 6, 7, 8, 10, 11, 12, 16, and 17; Fig. 1) show a mutant allele (299-basepair band) and a wild-type allele (165 and 135-basepair bands). Unaffected members (no. 4, 5, 9, 13, 14, and 15; Fig. 1) show only the wild-type allele. Member 1 is a normal individual. Fifty-three random individuals did not demonstrate the mutant allele (data not shown).

Recombination fraction

Kindred D

0.0

0.1

0.2

0.3

0.4

4.19

3.45

2.64

1.72

0.74

Computed with data from Figs. 1 and 5, C-1305 gene frequency less than 0.01, and using the MLINK option of LINKAGE (21).

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A NEW POINT MUTATION IN c-erbAQ important functional domain, the entire DNA-binding domain of the c-er6A/3 gene in kindred D (sequences including codons 90-172), and multiple genomic clones containing the DNA-binding domain showed no changes from the published fii sequence (data not shown). The C-1305 mutation of kindred D was associated with a phenotype distinct from that of kindred A. Kindred D demonstrated resistance to thyroid hormone action in the heart and to some degree in the liver, but there was no clinical abnormality in bone tissue (stature). In contrast, the A-1643 mutation of kindred A, with a proline to histidine change in codon 448 in the carboxy-terminus, was associated with short stature. Also, cardiac resistance to thyroid hormones was not a constant finding in kindred A as it was in kindred D. The phenotype of the patients reported by Sakurai et al. appears to be similar to that of kindred D, and short stature was not present in their patients either. It is possible that the kindred A receptor represents a class of c-erbA mutations that specifically inhibits normal bone development, and that the mutations in exon 1171-1429 of kindred D and the other GTHR family are c-erbA mutants that can specifically inhibit cardiac responses to thyroid hormones. The mutations in both exons were associated with cognitive deficiencies and/or the attention deficit disorder with hyperactivity. Identification of other c-er&A/3 mutations in different GTHR kindreds will enable these correlations to be further examined. The mutation in kindred D was present in only one cerbA(3 allele as expected for a dominant disorder. The exact mechanism of how the mutant receptor functions as a dominant negative inhibitor of the wild-type receptor is unknown. The T3-binding domain of the /?i-receptor, defined by in vitro mutagenesis studies and by analogy to other c-erfcA-like receptors, is broad and approximately encompasses amino acid codons 230-456 (2426). The C-1318 mutation, with a glycine to arginine change at codon 340, resulted in an in vitro receptor with undetectable T 3 binding (16), and one would expect a glutamine to histidine change at codon 335 to alter the T 3 affinity of the mutant receptor. However, simply a reduction in the T 3 affinity of the mutant /^-receptor would probably not explain the distinct kindred D phenotype, since the kindred A mutation resulted in vitro in reduced T 3 affinity (14). Forman et al. (27) have reported on a domain in the thyroid hormone and retinoic acid receptors containing leucine zipper-like motifs which may mediate dimerization of these receptors. They provide in vivo data using genetically engineered c^-receptor mutants to support a model in which a region of eight heptad repeats in the T3-binding domain is crucial for a dominant negative function. The kindred D mutation is in the first heptad of their model, although it does not interupt a leucine in the 1, 5, or 8 position. The location

37

of the kindred D mutation suggests that the first heptad would have to have a function in T 3 binding and/or binding with other transactivation factors (28, 29) in addition to or instead of contributing to dimerization. However, the other heptad repeats identified by Forman et al. could result in heterodimers of the C-1305 mutant receptor and wild-type receptor, inhibiting normal receptor action. It is presently unclear how different mutant c-erbAfi receptors result in variable thyroid hormone action and different tissue phenotypes. Although all of the mutations identified to date lie within the T3-binding domain, there may be significant differences in T3-binding affinities between certain mutant receptors (14, 16). Structural and functional differences between mutant receptors, in homo- or hetero-dimer form, may result in variable interactions with trans-activation factors (28, 29) that could differentially modulate thyroid hormone action depending upon cell type. Identification of further GTHR mutations will help elucidate the mechanisms of action of the thyroid hormone receptors and the dominant negative interactions of the various mutant receptor forms. Studies are in progress to define the fundamental properties of this mutant receptor, including T3-binding, DNA-binding, and transcriptional activities. Comparison of such functional properties as well as the phenotypic features of the C-1305 mutant receptor with those of the two previously reported mutant receptors will provide further insights on thyroid hormone action in

Acknowledgments We thank Dr. Herbert H. Samuels, Departments of Medicine and Pharmacology, New York University School of Medicine, for reviewing the manuscript and insightful discussions. We also wish to thank Dr. Timothy H. McCalmont, Department of Pathology, Bowman Gray School of Medicine, for providing DNA from a member of kindred D. We thank Drs. Richard Kleinman (Charleston, WV) and Walter Fening (Middleton, OH), who referred the initial kindred members to the NIH. We thank Dr. Peter Hauser, NIH, for assistance in obtaining blood specimens. We are grateful to Dr. James P. Coleman, Department of Microbiology, East Carolina University, for synthesis of the oligomers used in this study.

References 1. Refetoff S. Syndromes of thyroid hormone resistance. Am J Physiol. 1982;243:E88-98. 2. Magner JA, Petrick P, Menezes-Ferreira M, Weintraub BD. Familial generalized resistance to thyroid hormones: report of three kindreds and correlation of patterns of affected tissues with the binding of [125I]triiodothyronine to fibroblast nuclei. J Endocrinol Invest. 1986;9:459-69. 3. Smallridge RC, Parker RA, Wiggs EA, Rajagopal KR, Fein HG. Thyroid hormone resistance in a large kindred: physiologic, biochemical, pharmacologic, and neuropsychologic studies. Am J Med. 1989;86:289-96. 4. Drabkin H, Kao FT, Hartz J, et al. Localization of human ERBA2 to the 3p22—»3p24.1 region of chromosome 3 and variable deletion

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5.

6. 7. 8. 9. 10. 11.

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