J. Endocrinol. Invest. 15. 573-579,1992
Generalized thyroid hormone resistance: Identification of an arginine to cystine mutation in codon 315 of the c-erb Abeta thyroid hormone receptor1, 2 KD. Burman*, Y.Y. Djuh*, D. Nicholson*, P. Rhooms*, L. Wartofsky*, H.G. Fein**, S.J. Usala***, E.-H. Hao***, W.E.C. Bradley****, J. Berard****, and R.C. Smallridge** *Endocrine-Metabolic Service, Kyle Metabolic Unit, Departments of Medicine and Clinical Investigation, Walter Reed Army Medical Center, Washington, DC, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, **Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC, ***Department of Medicine, East Carolina University School of Medicine, Greenville, NC, USA, and ****Institut du Cancer de Montreal, Montreal, Canada ABSTRACT. The present report studies a large kindred (WR) with generalized thyroid hormone resistance that has varying degrees of neuropsychological dysfunction, hyperactivity, poor attention span, decreased IQ and/or abnormalities in spatial perception. In this kindred, there has been found tight linkage of the syndrome with the c-erb A beta gene. The present study was performed to identify the presence of a possible gene mutation as a cause for this syndrome. DNA from peripheral leukocytes was isolated from 15 unaffected and 8 affected individuals from the kindred. Primers encompassing exons 9 (nucleotides 1171-1429) and 10 (nucleotides 1430-1698) were synthesized and used in PCR reactions to amplify these exons. Direct sequencing revealed a consistent substitution in each affected subject, but in none of the unaffected individuals, of a C to T change in one allele from nu-
cleotide 1243, resulting in an arg to cys change in codon 315. The mutant and wild-type human beta 1 receptors were prepared and their translated proteins were analyzed for T3 binding. The WR T3 receptor from affected patients had reduced T3 binding affinity, with values approximately 2.5 x 10 10 M-1 compared to about 5 x 1010 M-1 in normals. In summary, we have: i) identified a consistent and reproducible mutation of a C to T change in nucleotide 1243 in each of the affected but in none of the unaffected individuals of a large weil characterized kindred with generalized thyroid hormone resistance; and ii) noted that the WR allele causes an approximate 50% decrease in the T3 binding affinity. Further studies analyzing the mechanism by which a single point mutation in one allele results in the biochemical and clinical manifestations of generalized thyroid hormone resistance are warranted.
INTRODUCTION
ration, low basal metabolic rate, and decreased urinary hydroxyproline; sex hormone binding globulin (SHBG), ferritin, angiotensin converting enzyme (ACE) and cholesterol levels mayaiso be abnormal. Mild abnormalities in neuropsychologic function occur in some individuals. This syndrome is inherited mainly as an autosomal dominant trait, although rarely it may be autosomal recessive, Since its initial description in 1967 (1), approximately 150 cases have been described with marked clinical heterogeneity being observed between families. It is likely this syndrome is more common than the reports would indicate, perhaps accounting for varied levels of thyroid hormone resistance in many patients that may escape customary detection, In contrast, very severe cases may be incompatible with life and these individuals may die in utero. Search for the underlying defect has focused on
Thyroid hormone resistance is a familial syndrome characterized by elevations of serum total and free T4 and T3, inappropriately detectable serum TSH and peripheral manifestations of impaired thyroid hormone action (1-3). These parameters denoting impaired action may include delayed bone matu-
'The opinions or assertions contalned herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Oepartment of the Army or the Oepartment of Oefense. 2Portions of these data were presented at the Endocrine Society Annual Meeting in Washington, OC, June, 1991.
Key-words.· Generalized resistance, mutation, thyroid hormone receptor. Correspondence: Or. Kenneth Burman, Endocrinology Clinic, 70. Walter
Reed Army Medical Center, Washington, OC 20307. Received January 2, 1992; accepted June 11, 1992.
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the T3 receptor (4-6). T3 binding studies using nuclear receptor preparations from fibroblasts and Iymphocytes have defined, in some families, altered binding capacity and/or dissociation constant (Kd). The binding capacity is usually normal, but in other studies has been found to be increased or occasionally decreased; the Kd is either normal or increased. Most of these studies have analyzed peripheral white cells which have a low number of T3 receptors and may not represent a T3 responsive tissue. Fibroblasts are a more representative source of cells that may obviate the above criticisms of white cells, but even fibroblast studies have not demonstrated a consistent defect in T3 binding. An important advance was realized in 1986 when two c-erb A proto-oncogene proteins were identified as being putative T3 receptors (7-10). The c-erb A beta cONA sequence has about 1710 bases and the translated protein has an amino terminal domain that binds ONA, whereas the carboxy terminal contains a domain that is ligand or T3 binding (9). This thyroid hormone receptor is part of a larger superfamily which includes the estradiol, glucocorticoid and vitamin 0 receptors. The c-erb A beta gene has been found to have tight linkage with several families who have peripheral thyroid hormone resistance (12). In further studies of separate kindreds, Usala et al. (13) have noted a single base substitution, cytosine to guanine, in position 1643 (proline to histidine codon 448) and Sakurai et al. (14) found a single substitution of cytosine for guanine which resulted in a glycine to arginine change in codon 340. The family reported herein has different clinical characteristics from the earlier families under study; moreover, the present kindred is larger, containing 89 persons spanning four generations. In addition, we have extensively analyzed the laboratory and clinical characteristics of this family, including the performance of detailed neuropsychologic testing (12). Earlier re ports which examined the structure of the T3 receptor usually only sequenced the ONA from that specific involved area in very few patients, and either compared it to the reported sequence in normal, or performed some variation of allelic specific hybridization to identify this sequence substitution in other affected family members (13-16). The entire coding region of the thyroid hormone beta gene has been sequenced, however, in a few affected individuals (14). In the present study, we detected a specific base substitution in affected members of our kindred. We sequenced the entire ligand binding area (exons 9 and 10) of 14 affected members and 14 unaffected members of the family. This procedure eliminated the possibility that another defect that would not be detected by allelic hybridization techniques may be present in these ar-
eas (i.e. a compound mutation). In addition, we used asymmetric primer amplification with direct sequencing, thus, obviating the requirement for extensive and timely subcloning procedures. MATERIALS AND METHODS Clinical studies
The clinical and laboratory characteristics of our kindred have recently been reported; this kindred has been denoted "WR" (12). This family has 89 members spanning four generations. Blood was obtained from 17 unaffected and 9 affected family members, with ONA of sufficiently high quality to allow sequencing reactions in 15 unaffected and 8 affected individuals. Generalized thyroid hormone resistance had been identified in 14 of 45 family members tested; autosom al dominant inheritance occurs in the family. Although there is marked heterogeneity of the clinical expression of thyroid resistance in affected family members, generally individuals are considered to be euthyroid and have not received exogenous thyroid hormone treatment. Informed consent was obtained from each subject and this protocol had been approved by the Hospital's Clinicallnvestigation Committee. Peripheral blood was obtained by routine venipuncture. Because the majority of the kindred live in Utah or Florida, blood was either drawn by local practitioners and se nt to our laboratory in Washington, OC arriving within 48 hours of being drawn, or one of us (HF) obtained the blood and personally transported it to our laboratory. ONA was extracted by standard techniques (17); attempts at isolating RNA proved futile due to its lability. Molecular studies The exon-intron borders for the two ligand binding domains were mapped (WEB, JB) (18). Further definition of the entire genome is continuing, but at present, these two exons are referred to as "exon 9" (nucleotides 1171-1429) and "exon 10" (nucleotides 1430-1698) (19). ONAwas quantitated by spectrophotometric analysis. Primers Primers were prepared by a Pharmacia Gene Assembler and purified by an "Oligonucleotide Purification Cartridge" (OPC), AB61 #400771, Applied Biosystems Inc, Foster City, CA.
Exon 9 Primer 1; sense; 5'-GACTGGCATTTTGCATTIGTIC3' Primer 2; sense with universal; 5'-TGTAAAAC-
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111 - 3.0 111 of DNA template was mixed with 2-4 111 (004 pmol/Ill)dye primer (A, C, G, T) and with 2-4 111 dideoxynucleotide (A, C, G, T) and 1-2 111 Taq Polymerase (5 Units/Ill). The sequencing reactions were allowed to proceed as recommended in the ABI User Manual. Each of 14 affected and nonaffected patients had their DNA analyzed and in every patient the entire exon 8 and 9 were sequenced. Sequencing gels (0.6% acrylamide 25 x 42 cm, 5 mm non-fluorescent glass, 66lanes) were used to identify peaks that were computer analyzed and designated as a particular nucleotide. The defect was confirmed by standard radiolabelied deoxynucleotide techniques.
GACGGCCAGTGCATTTGTTCTTTGCTGACA-3' Prim er 3; antisense; 5'-AGACMGCAAAAGCTCffiG3' Primer 4; antisense reverse universal; 5'CAGGAAACAGCTA TGACCGCTCTTTGGATGCCCACT-3' Exon 10 Primer 5: sense; 5'-CTTCCCCCTTCCATCTCTGA-3' Primer 6 ; sense with universal; 5'-TGTAAAACGACGGCCAGTCTGAATCAATGTCCATC-3' Primer 7; antisense; 5'-AAGAGCTAGGCAATGGAATGA-3' Primer 8; antisense with reverse universal; 5'CAGGAAACAGCTATGACCAATGGAATGAAATGAC-3'
Construction of the WR receptor cONA and T3 binding studies The splicing overlap extension method was used to create the WR receptor cDNA (20). Briefly, a 5'oligomer overlying a single Sty I site at nucleotide 987 (5'-TGG CGA CCA ACG CCC AAG GCA GCC ACT-3') and a 3'-0Iigomer overlying a single Bglll site at nucleotide 1563 (5'-TAT CAT CCG CAG ATC TGT CAC CTT CAT-3') were used with complementary oligomer pairs containing the T-1243 base substitution to create an Sty-Bgl II fragment with the mutation. This Sty-Bgl II fragment was then exchanged with the corresponding segment in peA 101 (pGEM3 wild type human beta-receptor cDNA) (7). The Sty I-Bglll segment was sequenced in the final plasmid to exclude a spurious PCR artifact. The mutant and wild-type human beta 1 receptors were synthesized using cDNAs in pGEM3 and rabbit reticulocyte Iysate as previously described (19). The in vitra translation reactions were prepared with the Dupont-New England Nuclear reticulocyte Iysate L[35 S]- methionine translation kit (Wilmington, DE). The WR and wild-type receptors were analyzed for the apprapriate size on 10% SDS-polyacrylamide gels. The nitrocellulose filter binding assay was used as described elsewhere (21, 22). T3 binding affinities were computed from Scatchard plots that were generated from saturation curves using L-125-T3 (specific activity 2200 Ci/mmol; Dupont-New England Nuclear) over a final concentration range of 0.001004 nM. Scatchard points were determined from duplicate measurements and nonspecific binding was measured in duplicate in the presence of 0.5 11M of unlabeled T3.
Polymerase chain reaction (PCR) Asymmetrie PCR was accomplished by generally following the guidelines in User Bulletin 13 fram the Model 370 A DNA Sequencing System, Applied Biosystems, Inc., Foster City, CA The initial PCR reaction used 10 x (10 111) reaction buffer, 1.25 mM (8 111) dNTP mix, 0.5111 Taq Polymerase (5 Units/Ill), 20 pmoles of primer in both amplification steps, 5 pmoles of the second primer (only used in the initial amplification), and 1 Ilg DNA template. 100111 was the total volume. The amplification reaction then occurred at 96 degrees C for 30 sec, 55 C for 1 min, and 72 C for 1 min; the amplification occurred over 30 cycles. The second amplification reaction used: 1011110 x reaction buffer, 1.6 111 1.25 mM dNTP mix, 0.5 111 Taq Polymerase (5 Units/Ill), 50 pmoles of one primer in both amplification steps, 3 pmoles limiting primer and 10 pg DNA template. The amplification reaction occurred at 96 C for 30 sec, 50 C for 1 min, and 72 C for 2 min. Autoextension occurred for 5 sec at 72 C. The amplification reaction praceeded for"20 cycles; 5 111 of sampie was used for gel electrophoresis (1 % agarose, 3% Nusieve), to check the size and amount of the DNA obtained. Both single and double stranded DNA could be visualized; ethidium bromide stains the single stranded DNA about 10 fold less intensely than does double stranded DNA
Automated sequencing Automated fluorescence-based sequencing was performed using equipment (Model 370A) purchased fram Applied Biosystems, Foster City, CA Single stranded DNA was prepared as noted above using asymmetrie PCR reaetions. The universal or reverse universal sequences were incorporated into the primers so that direct sequencing could occur. 1.5
RESULTS Sequencing Using automated sequeneing techniques, a consistent substitution of C to T in nucleotide 1243 was identified in one allele from each of 8 affected individuals
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K.D. Burman, Y Y. Ojuh, D. Nicholson, et al.
mal individuals (102 alleles) each demonstrated a Hha I site, thus allowing the T-1243 base substitution to formally be designated a mutation.
(Fig. 1). This substitution caused an arg to cys change in codon 315. None of 15 unaffected family members had this base abnormality . This mutation was confirmed by standard dideoxynucleotide sequencing techniques in which a heterozygous allele at base 1243 was observed and an additional C, as weil as the wild type T, was observed (Fig. 2). No other consistent abnormalities or substitutions were found in exons 9 or 10 with either technique. We did confirm that corrections to Weinberger's original wild type cDNA were: base 1295 C, base 1254 A, base 1353 G, base 1636 T, base 1651 T.
T3 Binding
Repeated Scatchard analyses demonstrated that the WR allele decreased the affinity of T3 binding from approximately 5 x 10 10 M-1 to approximately 2.5 x 10 10 M -1 (Fig. 3). Three separate experiments showed a ratio of the WR Ka to the normal Ka wildtype of 0.45±0.08. The maximum T3 binding capacity of the translation products for the wild type and WR mutation were similar, being 0.019 and 0.014, respectively.
Hha I digestion
None of 102 alleles from randomly selected normal individuals contained the C to T mutation noted.
DISCUSSION
Polymorphism
We have identified a specific nucleotide substitution in each of 8 family members affected with generalized thyroid hormone resistance who were studied; this mutation was absent in 15 unaffected famil y members. This defect was the only consistent abnormality detected in the genes coding for the ligand binding domain (exons 9 and 10). In situ mutagenesis with subsequent translation and analysis of the products identified a modestly reduced (approxi-
Fifty-one random presumably normal individuals were screened for a possible mutation in codon 315 as part of an earlier study (23). The endogeneous Hha I site at nucleotide 1232 was obliterated with amplification by the primers chosen for use in this screening procedure (23) and the additional Hha I site at 1242 was expunged by the base substitution in the WR kindred. 51 randomly selected nor-
A
Fig. 1 - Automated fluorescence -ba sed sequencing (Applied BIOsystems . Model 370A Foster City, CA) of an unaffected (Panel A) and an affectecl (Panel B) patient from kindred WR wtth generalized thyroid hormone resistance A representative portion of exon 9 is 11lustrated with the arrow depicting the substitution in one allele of a C to T tn nueleotide 1243. (Panel B) Under actu al laboratory conditions, each nueleotide is associated with different color on the graph, making the identtflca!ion of abnormalities simpler than demonstrated in a black and white flg ure.
B
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Generalized hormone resistance
1.0 •
WT Ko.S.O)(, Q'·U-I
•
WR Ko.2.5xlO"U-·
0.8
"-
..
0.6
"CD
0.4
.~ ....~f----
0.2
T
C
--
0.0 0.000'
0.605
0 .610
0.020
B ( nM )
Fig . 3 - Seatehard plot of T3 bin ding data with the WR and human plaeental beta reeeptor. The WR kindred and wild-type beta reeeptor were synthesized with rabbit retieuloeyte Iysate and 2 J.11 aliquots of the translation mixtures were ineubated with 0.001-0.4 nM eoneentrations of 125-1 T3. Bound 125-1 T3 was separated from free with a filter-bin ding assay. Non-speeifie bin ding was measured in the presenee of 1000-fold exeess (0.5 J.1M) unlabeled T3. The representative plot demonstrates a WR T3-affinity of 2.5 x 10 ' 0 M ' and a wild-type T3-a ffinity of 5 x 10 10
Mt
G
A
T
c odds ratio was 3.67, a value very suggestive of a specific defect in the T3 receptor . Our present report extends these earlier findings and confirms them by actually identifying a specific mutation . We knew this defect would be different than the C to A change in nucleotide 1643 wh ich had been identified in Kindred A because of our allelic specific hybridization analysis (12). Kindred WR has been extensively studied (2 , 12). Physiologic studies demonstrated serum T4 values that ranged from about 12-20 1l9/dl, Free T4 from about 3 - 4 ng/dl, T3 200-300 ng/dl, Free T3 500700 pg/dl , and TSH 1-6 IlU/ml in affected individuals. SHBG , ACE, and ferritin serum levels were generally within the normal range , and did not demonstrate a pattern consistent with that noted in thyrotoxic patients . Detailed neuropsychologic testing showed mild impairments that were not thought to be specific for thyroid hormone resistance . It is possible that the modest T3 binding affinity defect in the WR kindred accounts for the relatively mild form of resistance observed. In another kindred, Sakurai et al. (14) identified a guanine to cytosine substitution in codon 340 (amino acid change glycine to arginine) in patients with GTRH (Kind red MF). In vitra translated protein apparently did not bind T3 . It is difficult to ascertain, but it seems that only an affected father and son had direct nucleotide analysis performed, with
Fig. 2 - An autoradiogram from a di-deoxynueleotide sequeneing reaetion whieh had been applied to a gel. Eaeh lane refleets an individual nucleotide reaetion as labeled at the bottom. The mutation is shown as the substitution of a C to T nucleotide at position 1243 in one allele; the normal Tat position 1243 in the unaffeeted allele is also depieted.
mately 50%) T3 affinity, as compared to the normal or wild type translation products (14, 16). Since this C to T defect was not present in 102 alleles from randomly selected normal individuals, we believe that this substitution constitutes an allelic mutation rather than a nonspecific polymorphism. Our studies provide sequence data on a large number of affected and nonaffected family members. Parenthetically, we did find several alterations in the previously reported cDNA code that probably represent corrections from the original sequence (7 , 13, 14, 19). We cannot determine with certainty if other defects are present in the genome coding for the DNA binding area or in other exons . Transfection and binding studies are supportive of a single nucleotide substitution being critical, but even these studies may not be truly representative of the in viva milieu . We have previously reported that there was tight linkage between the human c-erb A beta gene and the presence of generalized thyroid hormone resistance in this kindred (12) . The logarithm of the
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K.O. Burman, Y. Y. Ojuh, D. Nicholson, et al.
one being sequenced and the other having allelic specific amplification. Usala et al. (13) have reported a cytosine to adenine substitution in nucleotide 1643 (praline to histidine codon 448) in Kindred A in association with an approximate ten fold decrease in T3 affinity. Kindred 0, studied by Usala et al. (11) had a guanine to cytosine substitution in base 1305 (glutamine to histidine codon 335) in 10 affected members but not in 6 unaffected family members. Certain cognitive function abnormalities are found in Kindreds A and D. Allelic specific hybridization or endonuclease digestion screening techniques demonstrated the same genetic abnormality in family members, but only one affected and one nonaffected individual from Kindred 0 had exons 9 and 10 sequenced. Recently, another kind red (S) has been described in which a CAC deletion occurred at bases 12951297 (a threonine at codon 332 deleted) (19). Kindred CL (24) has been noted to have an arginine to histidine mutation in codon 315 (guanine to adenine at 1244), the same codon we have found to be mutated in the present study. To the best of out knowledge, these kindreds are unrelated. A larger deletion has been reported in a family with generalized thyroid hormone resistance (15). Takeda et al. (15) used PCR with one set of primers designed to identify two previously reported defects (glycine to arginine-340 and histidine for proline-448), and a second set of primers wh ich would identify normal alleles. Using these techniques, 18 of the 19 representative families studied showed amplification with only the normal probe indicating the absence of the substitutions reported earlier. In marked contrast, one family had affected members whose DNA could not be amplified either with normal or mutant primers. Southern blot studies suggested that these family members possessed a major deletion encompassing both DNA and hormone binding domains. In contrast to all other families under study, this single family with the broad genomic deletion had GTHR inherited as an autosom al recessive trait, and, therefore, even unaffected individuals must have this deletion in one allele. Direct sequencing of these areas is not yet reported. With regard to our present study, a single base mutation altered T3 binding and resultant action to a clinically significant degreeWe have not conclusively praven that this single substitution accounts for the entire clinical spectrum observed, but the correlation of decreased T3 binding with the presence of the mutation is of interest We do not yet understand how differences in the T3 receptor result in such varied clinical effects both between and among families. Our understanding of the mechanism by which various tissues exhibit differential T3
resistance is similarly sketchy, and we wonder about the relevance of the fact that most of the reported abnormalities in this syndrome occur in exon 9. Of potential importance, Krishna et al. (25) and Sakurai et al. (26) have recently suggested that mutant receptors may be capable of decreasing the transcriptional activity of normal or wild type receptors. Further investigations in this area will focus on the described abnormalities and will attempt to further understand the pracess by which these mutations are translated into biochemical and clinical disease. ACKNOWLEDGMENTS The authors would like to thank Vicky Guo, Laboratory 01 Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes 01 Health, Bethesda, MD lor advice and assistance in the perlormance 01 the automated sequencing. Dr. Fran Carr, Department 01 Clinicallnvestigation, Walter Reed Army Medical Center, Washington, DC provided consultation and encouragement. The authors would also like to thank Jay Menke at East Carolina University lor sequencing the mutant cDNAS.J'v' was supported by DHHS-PHS grant DK 42807.
REFERENCES 1. Refetoff S., Dewind LT, DeGroot L.J. Familial syndrome combining deaf mutism, stippled epiphyses, goiter and abnormally high PBI: possible target organ refractoriness to thyroid hormone. J. Clin. Endocrinol. Metab. 27: 279,1967. 2. Smallridge R.C., Parker RA, Wiggs EA, Rajagopal K.R., Fein H.G. Thyroid hormone resistance in a large kindred: physiologie, biochemieal, pharmacologic, and neuropsychologie studies. Am. J. Med. 86: 289,1989. 3. Refetoff S. Syndromes of thyroid hormone resistance. Am. J. Physiol. 243: E88, 1982. 4. Eil c., Fein H.G., Smith T.J., Furlanetto RW., Bourgeois M., Stelling MW., Weintraub BD. Nuclear binding of 125-1 triiodothyronine in dispersed cultured skin fibroblasts from patients with resistance to thyroid hormone. J. Clin. Endocrinol. Metab. 55: 502, 1982. 5. Menezes-Ferreira M.M., Eil C., Wortsman J, Weintraub BD. Decreased nuclear uptake of 1-125 triiodothyronine in fibroblasts from patients with resistance to thyroid hormone. J. Clin. Endocrinol. Metab. 59: 1081,1984. 6. Ichikawa K., Hughes IA, Horwitz AL., DeGroot L.J. Characterization of nuclear thyroid receptors of cultured skin fibroblasts from patients with resistance to thyroid hormone. Metabolism 36: 392, 1987.
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7. Weinberger C., Thompson C.C., Ong E.S., Lebo R., Gruol D.J., Evans R.M. The c-erb-A gene encodes a thyroid hormone receptor. Nature 324: 2641, 1986. 8. Sap J., Munoz A, Damm H., Goldberg Y., Ghysdael J., Lentz A, Beug H., Vennstrom B. The c-erb A protein is a high affinity receptor for thyroid hormone. Nature 324: 635, 1986. 9. Evans R.M. The steroid and thyroid hormone receptor super-family. Science 240: 889, 1988.
17. Bromley S.E., Klupt S.F. Genomic DNA isolation system. Focus 11:32,1989. 18. Berard J., Gareau P., Bradley W.E.C. The structure of the c-erb A beta gene [Abstract]. Am. J. Hum. Genet. 45: A175, 1989. 19. Usala S.J., Menke J.B., Watson T.L., Wondisford F.E., Weintraub B.o., Berard J., Bradley W.E.C., Ono S., Mueller O.T., Bercu B.B. A homozygous deletion in the c-erb A beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: Isolation and characterization of the mutant receptor. Mol. Endocrinol. 5: 327, 1991.
10. DeGroot L.J., Nakai A, Sakurai A, Macchia E. The molecular basis of thyroid hormone action. J. Endocrinol. Invest. 12: 843, 1989. 11. Usala S.J., Menke J.B., Watson TL, Berard J., Bradley W.E.C., Bale AE., Lash RW., Weintraub B.o. A new point mutation in the 3,5, 3'-Triiodothyroninebinding domain of the c-erb A beta thyroid hormone receptor is tightly linked to generalized thyroid hormone resistance. J. Clin. Endocrinol. Metab. 72: 32, 1991. 12. Fein H.G., Burman K.o., Djuh Y.-Y., Usala S.J., Bale AE., Weintraub B.o., Smallridge R.C. Tight linkage of the human c-erb A Beta gene with the syndrome of generalized thyroid hormone resistance is present in multiple kindreds. J. Endocrinol.lnvest. 14:219,1991. 13. Usala S.J., Tennyson G.E., Bale A.E., Lash RW., Gesundheit N., Wondisford E.E., Accili D., Hauser P., Weintraub B.o. A base mutation of the C-erb A Beta thyroid hormone receptor in a kindred with generalized thyroid hormone resistance: molecular heterogeneity in two other kindreds. J. Clin. Invest. 85: 93, 1990. 14. Sakurai A, Takeda H., Ain K., Ceccarelli P., Nakai A, Seino S., Bell G.I., Refetoff S., DeGroot L.J. Generalized resistance to thyroid hormone associated with a mutation in the ligand-binding domain of the human thyroid hormone receptor Beta. Proc. Natl. Acad. Sci. USA 86: 8977,1989.
20. Higuchi R. Simple and rapid preparations of sam pies for PCR. In: Erlich HA (Ed.), PCR Technology. Stockton, New York, NY, 1989, p.31. 21. Inoue A, Yamakawa J., Yakioka M., Morisawa S. Filter-binding assay procedure for thyroid hormone receptors. Anal. Biochem. 134: 176, 1983. 22. Schueler PA, Schwartz J.L., Strait KA, Mariash C.N., Oppenheimer J.H. Binding of 3, 5, 3'-triiodothyronine (T3) and its analogues to the in vitra translational products of c-erb A protooncogenes: Differences in the affinity of the alpha- and beta-forms for the acetic acid analog and failure of the human test is and kidney alpha-2 products to bind T3. Mol. Endocrinol. 4: 227, 1990. 23. Usala S.J., Menke J.B., Cugini C.o. Jr., Leidy JW. Jr., Zangeneh F., Chertow B.S., Driscoll HK A Mutation in codon 315 of c-erb A beta in a kindred with generalized thyroid hormone resistance and dyslexia. Clin. Res. 39: 293A, 1991. 24. Cugini C.D., Leidy JW., Chertow B.S., Berard J., Bradley W.E.C., Menke J.B., Hao E.-H., Usala S.J. An arginine to histidine mutation in codon 315 of the c-erb A Beta thyroid hormone receptor in a kind red with generalized resistance to thyroid hormones results in a receptor with significant 3, 5, 3'-triiodothyronine binding activity. J. Clin. Endocrinol. Metab. 74: 1164, 1992.
15. Takeda K., Balzano S., Sakurai A., DeGroot L.J., Refetoff S. Screening of nineteen unrelated families with generalized resistance to thyroid hormone for known point mutations in the thyroid hormone receptor beta gene and the detection of a new mutation. J. Clin. Invest. 87: 496, 1991.
25. Krishna V., Chatterjee K., Nagaya T., Madison L.o., Datta S., Rentoumis A, Jameson L.J. Thyroid hormone resistance syndrome: Inhibition of normal receptor function by mutant thyroid hormone receptors. J. Clin. Invest. 87: 1977, 1991.
16. Usala S.J., Wondisford F.E., Watson TL, Menke J.B., Weintraub B.o. Thyroid hormone and DNA binding properties of a mutant c-erb A beta receptor associated with generalized thyroid hormone resistance. BBRC 171: 575, 1990.
26. Sakurai A, Miyamoto T., Refetoff S., Degroot L.J. Dominant negative transcriptional regulation by a mutant thyroid hormone receptor-beta in a family with generalized resistance to thyroid hormone. Mol. Endocrinol. 4: 1988, 1990.
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Generalized thyroid hormone resistance: Identification of an arginine to cystine mutation in codon 315 of the c-erb Abeta thyroid hormone receptor1, 2 KD. Burman*, Y.Y. Djuh*, D. Nicholson*, P. Rhooms*, L. Wartofsky*, H.G. Fein**, S.J. Usala***, E.-H. Hao***, W.E.C. Bradley****, J. Berard****, and R.C. Smallridge** *Endocrine-Metabolic Service, Kyle Metabolic Unit, Departments of Medicine and Clinical Investigation, Walter Reed Army Medical Center, Washington, DC, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, **Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC, ***Department of Medicine, East Carolina University School of Medicine, Greenville, NC, USA, and ****Institut du Cancer de Montreal, Montreal, Canada ABSTRACT. The present report studies a large kindred (WR) with generalized thyroid hormone resistance that has varying degrees of neuropsychological dysfunction, hyperactivity, poor attention span, decreased IQ and/or abnormalities in spatial perception. In this kindred, there has been found tight linkage of the syndrome with the c-erb A beta gene. The present study was performed to identify the presence of a possible gene mutation as a cause for this syndrome. DNA from peripheral leukocytes was isolated from 15 unaffected and 8 affected individuals from the kindred. Primers encompassing exons 9 (nucleotides 1171-1429) and 10 (nucleotides 1430-1698) were synthesized and used in PCR reactions to amplify these exons. Direct sequencing revealed a consistent substitution in each affected subject, but in none of the unaffected individuals, of a C to T change in one allele from nu-
cleotide 1243, resulting in an arg to cys change in codon 315. The mutant and wild-type human beta 1 receptors were prepared and their translated proteins were analyzed for T3 binding. The WR T3 receptor from affected patients had reduced T3 binding affinity, with values approximately 2.5 x 10 10 M-1 compared to about 5 x 1010 M-1 in normals. In summary, we have: i) identified a consistent and reproducible mutation of a C to T change in nucleotide 1243 in each of the affected but in none of the unaffected individuals of a large weil characterized kindred with generalized thyroid hormone resistance; and ii) noted that the WR allele causes an approximate 50% decrease in the T3 binding affinity. Further studies analyzing the mechanism by which a single point mutation in one allele results in the biochemical and clinical manifestations of generalized thyroid hormone resistance are warranted.
INTRODUCTION
ration, low basal metabolic rate, and decreased urinary hydroxyproline; sex hormone binding globulin (SHBG), ferritin, angiotensin converting enzyme (ACE) and cholesterol levels mayaiso be abnormal. Mild abnormalities in neuropsychologic function occur in some individuals. This syndrome is inherited mainly as an autosomal dominant trait, although rarely it may be autosomal recessive, Since its initial description in 1967 (1), approximately 150 cases have been described with marked clinical heterogeneity being observed between families. It is likely this syndrome is more common than the reports would indicate, perhaps accounting for varied levels of thyroid hormone resistance in many patients that may escape customary detection, In contrast, very severe cases may be incompatible with life and these individuals may die in utero. Search for the underlying defect has focused on
Thyroid hormone resistance is a familial syndrome characterized by elevations of serum total and free T4 and T3, inappropriately detectable serum TSH and peripheral manifestations of impaired thyroid hormone action (1-3). These parameters denoting impaired action may include delayed bone matu-
'The opinions or assertions contalned herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Oepartment of the Army or the Oepartment of Oefense. 2Portions of these data were presented at the Endocrine Society Annual Meeting in Washington, OC, June, 1991.
Key-words.· Generalized resistance, mutation, thyroid hormone receptor. Correspondence: Or. Kenneth Burman, Endocrinology Clinic, 70. Walter
Reed Army Medical Center, Washington, OC 20307. Received January 2, 1992; accepted June 11, 1992.
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the T3 receptor (4-6). T3 binding studies using nuclear receptor preparations from fibroblasts and Iymphocytes have defined, in some families, altered binding capacity and/or dissociation constant (Kd). The binding capacity is usually normal, but in other studies has been found to be increased or occasionally decreased; the Kd is either normal or increased. Most of these studies have analyzed peripheral white cells which have a low number of T3 receptors and may not represent a T3 responsive tissue. Fibroblasts are a more representative source of cells that may obviate the above criticisms of white cells, but even fibroblast studies have not demonstrated a consistent defect in T3 binding. An important advance was realized in 1986 when two c-erb A proto-oncogene proteins were identified as being putative T3 receptors (7-10). The c-erb A beta cONA sequence has about 1710 bases and the translated protein has an amino terminal domain that binds ONA, whereas the carboxy terminal contains a domain that is ligand or T3 binding (9). This thyroid hormone receptor is part of a larger superfamily which includes the estradiol, glucocorticoid and vitamin 0 receptors. The c-erb A beta gene has been found to have tight linkage with several families who have peripheral thyroid hormone resistance (12). In further studies of separate kindreds, Usala et al. (13) have noted a single base substitution, cytosine to guanine, in position 1643 (proline to histidine codon 448) and Sakurai et al. (14) found a single substitution of cytosine for guanine which resulted in a glycine to arginine change in codon 340. The family reported herein has different clinical characteristics from the earlier families under study; moreover, the present kindred is larger, containing 89 persons spanning four generations. In addition, we have extensively analyzed the laboratory and clinical characteristics of this family, including the performance of detailed neuropsychologic testing (12). Earlier re ports which examined the structure of the T3 receptor usually only sequenced the ONA from that specific involved area in very few patients, and either compared it to the reported sequence in normal, or performed some variation of allelic specific hybridization to identify this sequence substitution in other affected family members (13-16). The entire coding region of the thyroid hormone beta gene has been sequenced, however, in a few affected individuals (14). In the present study, we detected a specific base substitution in affected members of our kindred. We sequenced the entire ligand binding area (exons 9 and 10) of 14 affected members and 14 unaffected members of the family. This procedure eliminated the possibility that another defect that would not be detected by allelic hybridization techniques may be present in these ar-
eas (i.e. a compound mutation). In addition, we used asymmetric primer amplification with direct sequencing, thus, obviating the requirement for extensive and timely subcloning procedures. MATERIALS AND METHODS Clinical studies
The clinical and laboratory characteristics of our kindred have recently been reported; this kindred has been denoted "WR" (12). This family has 89 members spanning four generations. Blood was obtained from 17 unaffected and 9 affected family members, with ONA of sufficiently high quality to allow sequencing reactions in 15 unaffected and 8 affected individuals. Generalized thyroid hormone resistance had been identified in 14 of 45 family members tested; autosom al dominant inheritance occurs in the family. Although there is marked heterogeneity of the clinical expression of thyroid resistance in affected family members, generally individuals are considered to be euthyroid and have not received exogenous thyroid hormone treatment. Informed consent was obtained from each subject and this protocol had been approved by the Hospital's Clinicallnvestigation Committee. Peripheral blood was obtained by routine venipuncture. Because the majority of the kindred live in Utah or Florida, blood was either drawn by local practitioners and se nt to our laboratory in Washington, OC arriving within 48 hours of being drawn, or one of us (HF) obtained the blood and personally transported it to our laboratory. ONA was extracted by standard techniques (17); attempts at isolating RNA proved futile due to its lability. Molecular studies The exon-intron borders for the two ligand binding domains were mapped (WEB, JB) (18). Further definition of the entire genome is continuing, but at present, these two exons are referred to as "exon 9" (nucleotides 1171-1429) and "exon 10" (nucleotides 1430-1698) (19). ONAwas quantitated by spectrophotometric analysis. Primers Primers were prepared by a Pharmacia Gene Assembler and purified by an "Oligonucleotide Purification Cartridge" (OPC), AB61 #400771, Applied Biosystems Inc, Foster City, CA.
Exon 9 Primer 1; sense; 5'-GACTGGCATTTTGCATTIGTIC3' Primer 2; sense with universal; 5'-TGTAAAAC-
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111 - 3.0 111 of DNA template was mixed with 2-4 111 (004 pmol/Ill)dye primer (A, C, G, T) and with 2-4 111 dideoxynucleotide (A, C, G, T) and 1-2 111 Taq Polymerase (5 Units/Ill). The sequencing reactions were allowed to proceed as recommended in the ABI User Manual. Each of 14 affected and nonaffected patients had their DNA analyzed and in every patient the entire exon 8 and 9 were sequenced. Sequencing gels (0.6% acrylamide 25 x 42 cm, 5 mm non-fluorescent glass, 66lanes) were used to identify peaks that were computer analyzed and designated as a particular nucleotide. The defect was confirmed by standard radiolabelied deoxynucleotide techniques.
GACGGCCAGTGCATTTGTTCTTTGCTGACA-3' Prim er 3; antisense; 5'-AGACMGCAAAAGCTCffiG3' Primer 4; antisense reverse universal; 5'CAGGAAACAGCTA TGACCGCTCTTTGGATGCCCACT-3' Exon 10 Primer 5: sense; 5'-CTTCCCCCTTCCATCTCTGA-3' Primer 6 ; sense with universal; 5'-TGTAAAACGACGGCCAGTCTGAATCAATGTCCATC-3' Primer 7; antisense; 5'-AAGAGCTAGGCAATGGAATGA-3' Primer 8; antisense with reverse universal; 5'CAGGAAACAGCTATGACCAATGGAATGAAATGAC-3'
Construction of the WR receptor cONA and T3 binding studies The splicing overlap extension method was used to create the WR receptor cDNA (20). Briefly, a 5'oligomer overlying a single Sty I site at nucleotide 987 (5'-TGG CGA CCA ACG CCC AAG GCA GCC ACT-3') and a 3'-0Iigomer overlying a single Bglll site at nucleotide 1563 (5'-TAT CAT CCG CAG ATC TGT CAC CTT CAT-3') were used with complementary oligomer pairs containing the T-1243 base substitution to create an Sty-Bgl II fragment with the mutation. This Sty-Bgl II fragment was then exchanged with the corresponding segment in peA 101 (pGEM3 wild type human beta-receptor cDNA) (7). The Sty I-Bglll segment was sequenced in the final plasmid to exclude a spurious PCR artifact. The mutant and wild-type human beta 1 receptors were synthesized using cDNAs in pGEM3 and rabbit reticulocyte Iysate as previously described (19). The in vitra translation reactions were prepared with the Dupont-New England Nuclear reticulocyte Iysate L[35 S]- methionine translation kit (Wilmington, DE). The WR and wild-type receptors were analyzed for the apprapriate size on 10% SDS-polyacrylamide gels. The nitrocellulose filter binding assay was used as described elsewhere (21, 22). T3 binding affinities were computed from Scatchard plots that were generated from saturation curves using L-125-T3 (specific activity 2200 Ci/mmol; Dupont-New England Nuclear) over a final concentration range of 0.001004 nM. Scatchard points were determined from duplicate measurements and nonspecific binding was measured in duplicate in the presence of 0.5 11M of unlabeled T3.
Polymerase chain reaction (PCR) Asymmetrie PCR was accomplished by generally following the guidelines in User Bulletin 13 fram the Model 370 A DNA Sequencing System, Applied Biosystems, Inc., Foster City, CA The initial PCR reaction used 10 x (10 111) reaction buffer, 1.25 mM (8 111) dNTP mix, 0.5111 Taq Polymerase (5 Units/Ill), 20 pmoles of primer in both amplification steps, 5 pmoles of the second primer (only used in the initial amplification), and 1 Ilg DNA template. 100111 was the total volume. The amplification reaction then occurred at 96 degrees C for 30 sec, 55 C for 1 min, and 72 C for 1 min; the amplification occurred over 30 cycles. The second amplification reaction used: 1011110 x reaction buffer, 1.6 111 1.25 mM dNTP mix, 0.5 111 Taq Polymerase (5 Units/Ill), 50 pmoles of one primer in both amplification steps, 3 pmoles limiting primer and 10 pg DNA template. The amplification reaction occurred at 96 C for 30 sec, 50 C for 1 min, and 72 C for 2 min. Autoextension occurred for 5 sec at 72 C. The amplification reaction praceeded for"20 cycles; 5 111 of sampie was used for gel electrophoresis (1 % agarose, 3% Nusieve), to check the size and amount of the DNA obtained. Both single and double stranded DNA could be visualized; ethidium bromide stains the single stranded DNA about 10 fold less intensely than does double stranded DNA
Automated sequencing Automated fluorescence-based sequencing was performed using equipment (Model 370A) purchased fram Applied Biosystems, Foster City, CA Single stranded DNA was prepared as noted above using asymmetrie PCR reaetions. The universal or reverse universal sequences were incorporated into the primers so that direct sequencing could occur. 1.5
RESULTS Sequencing Using automated sequeneing techniques, a consistent substitution of C to T in nucleotide 1243 was identified in one allele from each of 8 affected individuals
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K.D. Burman, Y Y. Ojuh, D. Nicholson, et al.
mal individuals (102 alleles) each demonstrated a Hha I site, thus allowing the T-1243 base substitution to formally be designated a mutation.
(Fig. 1). This substitution caused an arg to cys change in codon 315. None of 15 unaffected family members had this base abnormality . This mutation was confirmed by standard dideoxynucleotide sequencing techniques in which a heterozygous allele at base 1243 was observed and an additional C, as weil as the wild type T, was observed (Fig. 2). No other consistent abnormalities or substitutions were found in exons 9 or 10 with either technique. We did confirm that corrections to Weinberger's original wild type cDNA were: base 1295 C, base 1254 A, base 1353 G, base 1636 T, base 1651 T.
T3 Binding
Repeated Scatchard analyses demonstrated that the WR allele decreased the affinity of T3 binding from approximately 5 x 10 10 M-1 to approximately 2.5 x 10 10 M -1 (Fig. 3). Three separate experiments showed a ratio of the WR Ka to the normal Ka wildtype of 0.45±0.08. The maximum T3 binding capacity of the translation products for the wild type and WR mutation were similar, being 0.019 and 0.014, respectively.
Hha I digestion
None of 102 alleles from randomly selected normal individuals contained the C to T mutation noted.
DISCUSSION
Polymorphism
We have identified a specific nucleotide substitution in each of 8 family members affected with generalized thyroid hormone resistance who were studied; this mutation was absent in 15 unaffected famil y members. This defect was the only consistent abnormality detected in the genes coding for the ligand binding domain (exons 9 and 10). In situ mutagenesis with subsequent translation and analysis of the products identified a modestly reduced (approxi-
Fifty-one random presumably normal individuals were screened for a possible mutation in codon 315 as part of an earlier study (23). The endogeneous Hha I site at nucleotide 1232 was obliterated with amplification by the primers chosen for use in this screening procedure (23) and the additional Hha I site at 1242 was expunged by the base substitution in the WR kindred. 51 randomly selected nor-
A
Fig. 1 - Automated fluorescence -ba sed sequencing (Applied BIOsystems . Model 370A Foster City, CA) of an unaffected (Panel A) and an affectecl (Panel B) patient from kindred WR wtth generalized thyroid hormone resistance A representative portion of exon 9 is 11lustrated with the arrow depicting the substitution in one allele of a C to T tn nueleotide 1243. (Panel B) Under actu al laboratory conditions, each nueleotide is associated with different color on the graph, making the identtflca!ion of abnormalities simpler than demonstrated in a black and white flg ure.
B
576
Generalized hormone resistance
1.0 •
WT Ko.S.O)(, Q'·U-I
•
WR Ko.2.5xlO"U-·
0.8
"-
..
0.6
"CD
0.4
.~ ....~f----
0.2
T
C
--
0.0 0.000'
0.605
0 .610
0.020
B ( nM )
Fig . 3 - Seatehard plot of T3 bin ding data with the WR and human plaeental beta reeeptor. The WR kindred and wild-type beta reeeptor were synthesized with rabbit retieuloeyte Iysate and 2 J.11 aliquots of the translation mixtures were ineubated with 0.001-0.4 nM eoneentrations of 125-1 T3. Bound 125-1 T3 was separated from free with a filter-bin ding assay. Non-speeifie bin ding was measured in the presenee of 1000-fold exeess (0.5 J.1M) unlabeled T3. The representative plot demonstrates a WR T3-affinity of 2.5 x 10 ' 0 M ' and a wild-type T3-a ffinity of 5 x 10 10
Mt
G
A
T
c odds ratio was 3.67, a value very suggestive of a specific defect in the T3 receptor . Our present report extends these earlier findings and confirms them by actually identifying a specific mutation . We knew this defect would be different than the C to A change in nucleotide 1643 wh ich had been identified in Kindred A because of our allelic specific hybridization analysis (12). Kindred WR has been extensively studied (2 , 12). Physiologic studies demonstrated serum T4 values that ranged from about 12-20 1l9/dl, Free T4 from about 3 - 4 ng/dl, T3 200-300 ng/dl, Free T3 500700 pg/dl , and TSH 1-6 IlU/ml in affected individuals. SHBG , ACE, and ferritin serum levels were generally within the normal range , and did not demonstrate a pattern consistent with that noted in thyrotoxic patients . Detailed neuropsychologic testing showed mild impairments that were not thought to be specific for thyroid hormone resistance . It is possible that the modest T3 binding affinity defect in the WR kindred accounts for the relatively mild form of resistance observed. In another kindred, Sakurai et al. (14) identified a guanine to cytosine substitution in codon 340 (amino acid change glycine to arginine) in patients with GTRH (Kind red MF). In vitra translated protein apparently did not bind T3 . It is difficult to ascertain, but it seems that only an affected father and son had direct nucleotide analysis performed, with
Fig. 2 - An autoradiogram from a di-deoxynueleotide sequeneing reaetion whieh had been applied to a gel. Eaeh lane refleets an individual nucleotide reaetion as labeled at the bottom. The mutation is shown as the substitution of a C to T nucleotide at position 1243 in one allele; the normal Tat position 1243 in the unaffeeted allele is also depieted.
mately 50%) T3 affinity, as compared to the normal or wild type translation products (14, 16). Since this C to T defect was not present in 102 alleles from randomly selected normal individuals, we believe that this substitution constitutes an allelic mutation rather than a nonspecific polymorphism. Our studies provide sequence data on a large number of affected and nonaffected family members. Parenthetically, we did find several alterations in the previously reported cDNA code that probably represent corrections from the original sequence (7 , 13, 14, 19). We cannot determine with certainty if other defects are present in the genome coding for the DNA binding area or in other exons . Transfection and binding studies are supportive of a single nucleotide substitution being critical, but even these studies may not be truly representative of the in viva milieu . We have previously reported that there was tight linkage between the human c-erb A beta gene and the presence of generalized thyroid hormone resistance in this kindred (12) . The logarithm of the
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K.O. Burman, Y. Y. Ojuh, D. Nicholson, et al.
one being sequenced and the other having allelic specific amplification. Usala et al. (13) have reported a cytosine to adenine substitution in nucleotide 1643 (praline to histidine codon 448) in Kindred A in association with an approximate ten fold decrease in T3 affinity. Kindred 0, studied by Usala et al. (11) had a guanine to cytosine substitution in base 1305 (glutamine to histidine codon 335) in 10 affected members but not in 6 unaffected family members. Certain cognitive function abnormalities are found in Kindreds A and D. Allelic specific hybridization or endonuclease digestion screening techniques demonstrated the same genetic abnormality in family members, but only one affected and one nonaffected individual from Kindred 0 had exons 9 and 10 sequenced. Recently, another kind red (S) has been described in which a CAC deletion occurred at bases 12951297 (a threonine at codon 332 deleted) (19). Kindred CL (24) has been noted to have an arginine to histidine mutation in codon 315 (guanine to adenine at 1244), the same codon we have found to be mutated in the present study. To the best of out knowledge, these kindreds are unrelated. A larger deletion has been reported in a family with generalized thyroid hormone resistance (15). Takeda et al. (15) used PCR with one set of primers designed to identify two previously reported defects (glycine to arginine-340 and histidine for proline-448), and a second set of primers wh ich would identify normal alleles. Using these techniques, 18 of the 19 representative families studied showed amplification with only the normal probe indicating the absence of the substitutions reported earlier. In marked contrast, one family had affected members whose DNA could not be amplified either with normal or mutant primers. Southern blot studies suggested that these family members possessed a major deletion encompassing both DNA and hormone binding domains. In contrast to all other families under study, this single family with the broad genomic deletion had GTHR inherited as an autosom al recessive trait, and, therefore, even unaffected individuals must have this deletion in one allele. Direct sequencing of these areas is not yet reported. With regard to our present study, a single base mutation altered T3 binding and resultant action to a clinically significant degreeWe have not conclusively praven that this single substitution accounts for the entire clinical spectrum observed, but the correlation of decreased T3 binding with the presence of the mutation is of interest We do not yet understand how differences in the T3 receptor result in such varied clinical effects both between and among families. Our understanding of the mechanism by which various tissues exhibit differential T3
resistance is similarly sketchy, and we wonder about the relevance of the fact that most of the reported abnormalities in this syndrome occur in exon 9. Of potential importance, Krishna et al. (25) and Sakurai et al. (26) have recently suggested that mutant receptors may be capable of decreasing the transcriptional activity of normal or wild type receptors. Further investigations in this area will focus on the described abnormalities and will attempt to further understand the pracess by which these mutations are translated into biochemical and clinical disease. ACKNOWLEDGMENTS The authors would like to thank Vicky Guo, Laboratory 01 Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes 01 Health, Bethesda, MD lor advice and assistance in the perlormance 01 the automated sequencing. Dr. Fran Carr, Department 01 Clinicallnvestigation, Walter Reed Army Medical Center, Washington, DC provided consultation and encouragement. The authors would also like to thank Jay Menke at East Carolina University lor sequencing the mutant cDNAS.J'v' was supported by DHHS-PHS grant DK 42807.
REFERENCES 1. Refetoff S., Dewind LT, DeGroot L.J. Familial syndrome combining deaf mutism, stippled epiphyses, goiter and abnormally high PBI: possible target organ refractoriness to thyroid hormone. J. Clin. Endocrinol. Metab. 27: 279,1967. 2. Smallridge R.C., Parker RA, Wiggs EA, Rajagopal K.R., Fein H.G. Thyroid hormone resistance in a large kindred: physiologie, biochemieal, pharmacologic, and neuropsychologie studies. Am. J. Med. 86: 289,1989. 3. Refetoff S. Syndromes of thyroid hormone resistance. Am. J. Physiol. 243: E88, 1982. 4. Eil c., Fein H.G., Smith T.J., Furlanetto RW., Bourgeois M., Stelling MW., Weintraub BD. Nuclear binding of 125-1 triiodothyronine in dispersed cultured skin fibroblasts from patients with resistance to thyroid hormone. J. Clin. Endocrinol. Metab. 55: 502, 1982. 5. Menezes-Ferreira M.M., Eil C., Wortsman J, Weintraub BD. Decreased nuclear uptake of 1-125 triiodothyronine in fibroblasts from patients with resistance to thyroid hormone. J. Clin. Endocrinol. Metab. 59: 1081,1984. 6. Ichikawa K., Hughes IA, Horwitz AL., DeGroot L.J. Characterization of nuclear thyroid receptors of cultured skin fibroblasts from patients with resistance to thyroid hormone. Metabolism 36: 392, 1987.
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7. Weinberger C., Thompson C.C., Ong E.S., Lebo R., Gruol D.J., Evans R.M. The c-erb-A gene encodes a thyroid hormone receptor. Nature 324: 2641, 1986. 8. Sap J., Munoz A, Damm H., Goldberg Y., Ghysdael J., Lentz A, Beug H., Vennstrom B. The c-erb A protein is a high affinity receptor for thyroid hormone. Nature 324: 635, 1986. 9. Evans R.M. The steroid and thyroid hormone receptor super-family. Science 240: 889, 1988.
17. Bromley S.E., Klupt S.F. Genomic DNA isolation system. Focus 11:32,1989. 18. Berard J., Gareau P., Bradley W.E.C. The structure of the c-erb A beta gene [Abstract]. Am. J. Hum. Genet. 45: A175, 1989. 19. Usala S.J., Menke J.B., Watson T.L., Wondisford F.E., Weintraub B.o., Berard J., Bradley W.E.C., Ono S., Mueller O.T., Bercu B.B. A homozygous deletion in the c-erb A beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: Isolation and characterization of the mutant receptor. Mol. Endocrinol. 5: 327, 1991.
10. DeGroot L.J., Nakai A, Sakurai A, Macchia E. The molecular basis of thyroid hormone action. J. Endocrinol. Invest. 12: 843, 1989. 11. Usala S.J., Menke J.B., Watson TL, Berard J., Bradley W.E.C., Bale AE., Lash RW., Weintraub B.o. A new point mutation in the 3,5, 3'-Triiodothyroninebinding domain of the c-erb A beta thyroid hormone receptor is tightly linked to generalized thyroid hormone resistance. J. Clin. Endocrinol. Metab. 72: 32, 1991. 12. Fein H.G., Burman K.o., Djuh Y.-Y., Usala S.J., Bale AE., Weintraub B.o., Smallridge R.C. Tight linkage of the human c-erb A Beta gene with the syndrome of generalized thyroid hormone resistance is present in multiple kindreds. J. Endocrinol.lnvest. 14:219,1991. 13. Usala S.J., Tennyson G.E., Bale A.E., Lash RW., Gesundheit N., Wondisford E.E., Accili D., Hauser P., Weintraub B.o. A base mutation of the C-erb A Beta thyroid hormone receptor in a kindred with generalized thyroid hormone resistance: molecular heterogeneity in two other kindreds. J. Clin. Invest. 85: 93, 1990. 14. Sakurai A, Takeda H., Ain K., Ceccarelli P., Nakai A, Seino S., Bell G.I., Refetoff S., DeGroot L.J. Generalized resistance to thyroid hormone associated with a mutation in the ligand-binding domain of the human thyroid hormone receptor Beta. Proc. Natl. Acad. Sci. USA 86: 8977,1989.
20. Higuchi R. Simple and rapid preparations of sam pies for PCR. In: Erlich HA (Ed.), PCR Technology. Stockton, New York, NY, 1989, p.31. 21. Inoue A, Yamakawa J., Yakioka M., Morisawa S. Filter-binding assay procedure for thyroid hormone receptors. Anal. Biochem. 134: 176, 1983. 22. Schueler PA, Schwartz J.L., Strait KA, Mariash C.N., Oppenheimer J.H. Binding of 3, 5, 3'-triiodothyronine (T3) and its analogues to the in vitra translational products of c-erb A protooncogenes: Differences in the affinity of the alpha- and beta-forms for the acetic acid analog and failure of the human test is and kidney alpha-2 products to bind T3. Mol. Endocrinol. 4: 227, 1990. 23. Usala S.J., Menke J.B., Cugini C.o. Jr., Leidy JW. Jr., Zangeneh F., Chertow B.S., Driscoll HK A Mutation in codon 315 of c-erb A beta in a kindred with generalized thyroid hormone resistance and dyslexia. Clin. Res. 39: 293A, 1991. 24. Cugini C.D., Leidy JW., Chertow B.S., Berard J., Bradley W.E.C., Menke J.B., Hao E.-H., Usala S.J. An arginine to histidine mutation in codon 315 of the c-erb A Beta thyroid hormone receptor in a kind red with generalized resistance to thyroid hormones results in a receptor with significant 3, 5, 3'-triiodothyronine binding activity. J. Clin. Endocrinol. Metab. 74: 1164, 1992.
15. Takeda K., Balzano S., Sakurai A., DeGroot L.J., Refetoff S. Screening of nineteen unrelated families with generalized resistance to thyroid hormone for known point mutations in the thyroid hormone receptor beta gene and the detection of a new mutation. J. Clin. Invest. 87: 496, 1991.
25. Krishna V., Chatterjee K., Nagaya T., Madison L.o., Datta S., Rentoumis A, Jameson L.J. Thyroid hormone resistance syndrome: Inhibition of normal receptor function by mutant thyroid hormone receptors. J. Clin. Invest. 87: 1977, 1991.
16. Usala S.J., Wondisford F.E., Watson TL, Menke J.B., Weintraub B.o. Thyroid hormone and DNA binding properties of a mutant c-erb A beta receptor associated with generalized thyroid hormone resistance. BBRC 171: 575, 1990.
26. Sakurai A, Miyamoto T., Refetoff S., Degroot L.J. Dominant negative transcriptional regulation by a mutant thyroid hormone receptor-beta in a family with generalized resistance to thyroid hormone. Mol. Endocrinol. 4: 1988, 1990.
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