0021-972x/92/7401-0049$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 74, No. 1
Printed in U.S.A.
Recessive Inheritance of Thyroid Hormone Resistance Caused by Complete Deletion of the Protein-Coding Region of the Thyroid Hormone Receptor-@ Gene* KYOKO TAKEDA, AKIHIRO AND SAMUEL REFETOFF Thyroid Study Unit, Departments Kennedy, Jr., Mental Retardation
SAKURAI,
LESLIE
J. DEGROOT,
of Medicine (K.T., A.S., L.J.D., S.R.) and Pediatrics (S.R.) and the J. P. Research Center (S.R.), University of Chicago, Chicago, Illinois 60637
ABSTRACT. Generalized resistance to thyroid hormone is a syndrome of reduced responsiveness of target tissues to thyroid hormone. The determination of amino acid sequences of the human thyroid receptor-6 (hTR/3), deduced from cDNA sequencing, has enabled evaluation of the genetic basis for this syndrome. Distinct point mutations in the ligand-binding domain of hTRB have been identified in affected members of unrelated families, producing single amino acid substitutions that result in products with decreased or no hormone-binding activity. Inheritance in these families was autosomal dominant. We now report the molecular basis of generalized resistance to thyroid hormone in a consanguineous family unique for its autosomal recessive mode of inheritance. Deletion of the entire coding region of both hTRB alleles in homozygous affected members of
the family was demonstrated by the failure to amplify the coding exons 3-8 by the polymerase chain reaction using primers specific for flanking intronic sequences and by the demonstration of the presence of only two noncoding exons in Southern blots hybridized with exon-specific probes. As expected, obligate heterozygotes were phenotypically normal, since, in contrast to alleles with point mutations, the deleted allele could not act in a dominant negative fashion. Survival and maintenance of a euthyroid state are presumably mediated through expression of the hTRa gene, present in affected subjects, and the maintenance of high thyroid hormone levels. Furthermore, the clinical manifestations were relatively more mild that those observed in a homozygous patient with a single amino acid deletion in the hTR6 gene. (J Clin Endocrirwl Metab 74: 49-55,1992)
G
hormone receptor (TR) (9). However, the opportunity to test this hypothesis directly did not present itself until 20 yr later, after the isolation of cDNAs that encode proteins with hormone-binding properties similar to those found previously in cell nuclei extracts (10, 11). The four isoforms of the TR so far identified ((Ye,(Ye,pl, and &) are generated by alternative splicing of two genes (TRcY and TRP) located on human chromosomes 17 and 3, respectively (11-16). With the exception of the a2 isoform, the other three bind thyroid hormone and function as thyroid hormone receptors (11, 14, 17, 18). The availability of these new probes soon led to the identification of point mutations in 1 of the 2 alleles of the human (h) TR/3 gene of members from two unrelated families affected by GRTH (19, 20). These nucleotide substitutions produced single amino acid changes in the hormone-binding domain of the molecule, which in 1 case reduced (21) and in the other abolished (19) the binding of TB. To search for nucleotide substitutions in the hTR/? gene identical to those identified in these 2 families, we recently screened by the allele-specific amplification technique, DNAs from 24 subjects with GRTH belonging to 19 unrelated families (22). One of the unexpected findings was our failure to amplify 2 of
ENERALIZED resistance to thyroid hormone (GRTH) is a syndrome of reduced tissue sensitivity to thyroid hormone (l-3). The syndrome is characterized by 1) elevated serum thyroid hormone levels without abnormalities in thyroid hormone transport, 2) the absence of clinical and laboratory manifestations of thyroid hormone excess, 3) nonsuppressed TSH, and 4) reduced tissue responses to the administration of supraphysiological doses of thyroid hormone. The heritable nature of the syndrome has been demonstrated in 181 subjects with GRTH belonging to 47 families (4). Although in the majority of reported families inheritance appears to be autosomal dominant, recessive transmission was suggested in about 10% of the families on the basis of consanguinity (5-8). Soon after the description of the syndrome, it was postulated that the defect resides at the cellular level and that it is most likely caused by an abnormal thyroid Received April 1, 1991. Address all correspondence Refetoff, M.D., Thyroid Study 5841 South Marvland Avenue, *This work was supported and DK-00055.
and requests for reprints to: Samuel Unit, Box 138, University of Chicago, Chicago. Illinois 60637. in pak’by USPHS Grants DK-15070 49
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50
TAKEDA
the coding exons (exons 7 and 8) in all affected subjects of 1 of the 19 families with GRTH, the only one with recessive inheritance. Since this was observed using oligonucleotide primers complementary to the mutant and wild-type sequences, our preliminary conclusion was that affected members of this family might have a major deletion in both alleles of their hTRp gene. In the current study we present detailed analysis of this unique defect. The deletion spans at least 40 kilobases (kb) of the hTRp gene, encompassing all eight coding exons, with preservation of only the first two noncoding exons, 0 and 00. In contrast, analysis of their hTRa! gene revealed no major deletions. Clinical and laboratory data obtained from homozygous and hererozygous members of this family with complete hTR/3 deletion are compared to those from subjects of another family who express a mutant hTRP. Materials
and Methods
Providers of biological reagents, including modifying and restriction enzymes, were Sigma (St. Louis, MO), Bethesda Research Laboratories (Gaithersburg, MD), and New England BioLabs (Beverly, MA). [cy-“‘P]dCTP was obtained from Amersham Corp. (Arlington Heights, IL). Synthetic oligonucleotides were synthesized by fi-cyanoethyl chemistry in a DNA synthesizer (model 308B, Applied Biosysterns, Foster City, CA). Genomic DNA from three affected and eight unaffected members in the family and from two unrelated normal subjects was prepared from peripheral blood leukocytes, as previously described (23), and from cultured skin fibroblasts, using a nucleic acid extractor (model 340A, Applied Biosysterns).
ET AL.
JCE & M. 1992 Vol74.Nol
gene are diagrammed in Fig. 2. PCR products subjected to 1% agarose gel electrophoresis were also transferred onto nitrocellulose paper after alkaline denaturation. Hybridization was carried out under the same conditions, except for the omission of dextran sulfate. The probe used was hTR& cDNA (pheA12), isolated by Weinberger et al. (11).
Results Patient identification to thyroid hormone
and evidence for tissue resistance
The family pedigree (Fig. 1) identifies affected and normal subjects studied and their relationship. It includes two more generations since the initial report in 1967 (5). Note that the three affected and four normal siblings were born to consanguineous parents; the maternal grandfather and paternal great grandmother were brother and sister. Affected subjects had high concentrations of TB, Tq, and free Tq in serum, with nonsuppressed TSH levels. Extensive clinical and metabolic studies of this family, the first described with GRTH, have been previously reported (5, 9). Briefly, in addition to the above-stated hormonal abnormalities, affected subjects were deaf-mute and exhibited stippled epiphyses during childhood (5), one of several manifestations of hypothyroidism. Other tissues appeared to be euthyroid, as was their basal metabolic rate, with some manifestations of hormonal excess, such as tachycardia (9). Evidence for normal penetration of thyroid hormone into tissues and conversion of Tq to TS by cultured skin fibroblasts but attenuated cell responses to the hormone has been presented (26-28).
DNA amplification by PCR The coding exons 3-8 of the hTR@ were each amplified using oligonucleotide primers complementary to their flanking intronic sequences, listed in Table 1. One hundred picomoles of the primer pairs were annealed, in separate reactions, to 1.5 pg genomic DNA and submitted to 30 polymerase chain reaction (PCR) cycles (24). Each cycle consisted of denaturation for 30 s at 94 C, annealing for 1.5 min at 55 C, and extension for 1.5 min at 72 C. The resulting fragments were resolved in 1% agarose gels and stained with ethidium bromide. Southern blotting analysis Genomic DNA samples (10 rg each) were digested with EcoRI, electrophoresed on 1% agarose gel, and, after alkaline denaturation, transferred onto nitrocellulose paper (Schleicher and Schueller, Keene, NH). Hybridization conditions were identical to those previously reported (25). The probes used were specific for each of five hTRp exons (26) and a hTRap cDNA probe (pke711) (13). The exon-specific hTRP probes were released by enzymatic digestion of recombinant bacteriophage clones (XEhTR&1, with BamHI; XEhTRP-2, undigested; hChTR/3-1, with EcoRI; hChTR&3, with EcoRI; XChTRP-4, with EcoRI and HindIII). Their size and location on the hTR/3
Amplification
of coding exons by PCR
The structure of the hTR/3 gene is shown in Fig. 2. It contains 2 noncoding exons (0 and 00) and 8 coding exons, numbered from l-8. Six pairs of synthetic oligonucleotide 20-mers complementary to known intronic sequences flanking exons 3-8 (Table 1) were used for amplification of genomic DNA by PCR. When DNAs from unaffected subjects and the parents of the affected subjects (no. 4 and 12 in Fig. 1) were used as template, each of 6 pairs of primers generated DNA fragments of the predicted size, which, after transfer to nitrocellulose paper, hybridized with hTR& cDNA (phe A12) probe (lane N in Fig. 3). When genomic DNA from each of the 3 affected siblings was used as template in the same reaction, no amplification occurred (lanes 1, 2, and 3 in Fig. 3). It should be noted that these DNA samples could direct amplification using primers specific for the human Tr-binding globulin gene (30) (data not shown). Furthermore, the same 2 pairs of primers used to amplify exons 7 and 8 were also used successfully to amplify genomic DNA from 21 unrelated patients with GRTH, 13 unaf-
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THYROID TABLE
1. Sequence and location of oligonucleotide
HORMONE
RECEPTOR-/9 GENE DELETION
primers used for amplification
of exons 3-8 of the hTR@ gene
Primer Exon” 3 (0
4 (D)
5 0%
6 (F)
7 G)
8u-u
51
Size of amplified DNA fragment (bp)
Sequence
Orientation
5’-gcaagccaattttgtctctg-3’
Sense
5’-tgtggacccagggcaattac-3’
Antisense
5’-ctgttgtcttgggtctttgt-3’
Sense
5’-gcaagtgaagatctacttac-3’
Antisense
5’-tgtgcatcagtggtcccact-3’
Sense
5’-caccagtatcccaaggtgat-3’
Antisense
5’-caggatatcagttcagaaga-3’
Sense
5’-ccagtattcctggaaactga-3’
Antisense
Y-tgctgacatgaactggttct-3’
Sense
5’-cactaacgagtctaggcgtac-3’
Antisense
5’-ggcctggaattggacaaag-3’
Sense
5’-Flanking intron
Exon
3’-Flanking intron
Total
99
101
20
220
31
148
20
199
90
206
90
386
102
147
67
316
26
259
25
310
73 5’-ATGAGAATGAATCCAGTCAG-3’ Antisense Lower casing and capital letters show intronic and noncoding exonic sequences, respectively. ’ Letters in parentheses correspond to exon identification published by Sakurai et ol. (29).
242
21 (noncoding)
336
FIG. 1. Pedigree of the family. ID numbers are on the wper right of symbols. Numbers within the symbols indicate the number of siblings of the same gender. The significance of high frequency of miscarriage (9 of a total of 17 pregnancies) in the 3 normal female siblings (no. 10, 5, and 7), who are products of the consanguineous union, is unclear.
Iii 13 x.::../
fected relatives, and 5 normal controls (22). We were unable to amplify exons 00, 0, 1, and 2 due to incomplete intron sequence information or the presence of repetitive, and thus nonspecific, flanking sequences. Localization of exons by hybridization of Southern blots of genomic DNA with exon-specific probes While EcoRI digestion of genomic DNA produces fragments corresponding to each of the 10 exons of the hTRp gene (29), hybridization of Southern transfers failed to
distinguish exons 2 and 6 because of similarity in size and was inconsistent in demonstrating exon 00 (22). Thus, genomic DNA digested to completion with EcoRI and transferred to nitrocellulose paper was hybridized with probes specific for exons 00, 0, 2, 3, and 6, shown in Fig. 2. As shown in Fig. 4, the affected member of the family exhibited bands corresponding to exon 00 (-25 kb) and exon 0 (5.1 kb). The size of the latter was slightly altered compared to that generated from normal DNA digest. The bands generated with probes specific to exon 2 (5.7 kb), exon 3 (2.1 kb), and exon 6 (5.5 kb) using
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52
TAKEDA
EXONS
06
5”ALA-... PROBES -- AEh
3 W-1
AEh
TR!$-2
-ACh
TRB-I
mACh
45 TRB-3
676 -
hCh
TR@-4
FIG. 2. Structure of the hTR(3 gene and diagrammatic representation of the localization and size of exon-specific probes used for hybridization. Bores representing exons are joined by lines representing introns. Black areas within the boxes are coding sequences, and exons containing such sequences are numbered 1-8. Note the lo-fold scale difference in the size in the diagrammatic representation of exons and introns. The intron size at breakpoints is not known. Bacteriophage clones containing the gene inserts shown below were endonuclease digested, and the fragments isolated on agarose gel electrophoresis (depicted as thick bars) were used for labeling and hybridization. Note that the entire insert containing exon 0 was used owing to our failure to isolate a smaller DNA fragment containing the exon. Also, the fragment of DNA containing exon 3 could not be separated from an intronic sequence of a similar size.
DNA from the normal control subject were not present in the DNA from the affected members of the family (Fig. 4), but were present in the 2 parents (data not shown). Exon 1 was not present in the Southern blot hybridized with the complete hTR& cDNA (22). In contrast, hybridization of the same Southern blots with a hTRu! cDNA probe generated the same 4 bands in DNA from the normal subject and an affected member of the family (Fig. 5), indicating the absence of gross deletions in the TRa gene. Discussion In this work we have demonstrated that GRTH was caused in one family by the deletion of virtually all coding
FIG. 3. Specific amplification of individual exons of the hTRp gene using primers complementary to flanking intronic sequences (see Table 1). Lanes labeled N contain DNA from a normal subject, and those labeled 1, 2, and 3 contain DNA from the affected members of the family, 1, 2, and 3, identified in Fig. 1. A, Ethidium bromide stain of agarose gels. B, Radioautograph of the same gels after the transfer of DNA onto nitrocellulose paper and hybridization with “LP-labeled hTR(3 cDNA. The theoretical (calculated) fragment size is given below. The actual size was as predicted and can be estimated with the help of the molecular size markers (HaeIII digested 4X 174).
ET AL.
JCE & M. 1992 Vol74.Nol
sequences in both alleles of the hTR/3 gene. Indeed, with the possible exception of exon 1, the absence of each coding exon was demonstrated by either amplification using oligonucleotide primers specific for sequences flanking each exon and/or hybridization of Southern blots with hTR/3 exon-specific probes. The latter technique was used to demonstrate the presence of the two 5’-flanking noncoding exons. The proximal site of the deletion is most likely located in the intron between exons 0 and 1 because of the slightly altered size of exon 0 generated by EcoRI digestion of DNA from affected members of the family compared to that from normal controls. The distal point of the deletion, however, remains unknown. Southern fragments of known size covering the area of deletion provides a minimal deletion size of 40 kb. This is undoubtedly an underestimate, considering the recent report indicating that the TRP gene spans more than 250 kb (31). Compared to single base substitutions, major gene deletions are infrequently the cause of inherited diseases in man. With Duchenne muscular dystrophy being an exception to this rule (32), deletions of 40 basepairs or more account for less than 10% of mutations causing such conditions as Lesch-Nyhan syndrome (33, 34) and hemophilia A and B (35, 36). Of 4 unrelated families with GRTH examined by Usala et al. (20, 37-39) for mutations of the hTRp gene and 18 additional families studied by us (19, 22), the family reported herein is the only one to exhibit a major deletion, resulting in an overall frequency of 5%. Furthermore, while in most instances clinical manifestations are present in the het-
A~N123N123N123N123N123N123 ------------------------1353L 603~ gF
B
Exon3 Exon4 Exon5 Exon6 ----s-B--------------__
Exon7
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Exon8
THYROID
HORMONE
M ---
ExonOO
ExonO
Exon2
Exon 3
RECEPTOR-8
NM
Exon 6
FIG. 4. Southern blot analysis of EcoRI-digested DNA from a normal individual (N) and an affected member of the family, no. 3 (M). After transfer of the DNA onto nitrocellulose paper, the pairs of strips were hybridized with each of the four exon-specific “‘P-labeled probes (see Fig. 2). Note that the high background of hybridization with exon Oand exon 3-specific probes is due to their greater content of intronic sequences (see Fig. 2). The affected subject had only sequences recognized by the probes specific for the two 5’-flanking noncoding exons (00 and 0).
N
M
--
kb 97. LI . 80r
i
. 54/-
17. FIG. 5. Southern blot analysis of EcoRI digested DNA, as described in Fig. 4. In this instance the blots were hybridized with “P-labeled hTRa* cDNA. The pattern of both samples is identical.
erozygous state, in this family only homozygous subjects with hTR@ deletion had GRTH. Interference of a mutant gene at the protein level, known as dominant negative mutation (40), has been postulated as being responsible for the autosomal dominant inheritance of GRTH (18, 19). This hypothesis is supported by the demonstration that coexpression of a mutant hTR&, which does not bind TO (hTR&-Mf),
GENE
DELETION
53
with either the wild-type hTR& or hTRal interfered with the truns-activating function of the latter two (41). Thus, it could be predicted that complete deletion of a single hTR/3 allele, as in the case of the obligate heterozygous parents of affected children of this family, is unlikely to produce hormone resistance. This is now proven to be the case. More surprising was the finding that complete absence of the hTRP gene was not only compatible with survival, but that some effects of thyroid hormone could be demonstrated, albeit at a higher hormone concentration (9). Indeed, responses to thyroid hormone in terms of increases in urinary creatine and hydroxyproline and the development of tremor and cardiac gallop were observed. Therefore, it is logical to conclude that the hTRa, expressed in virtually all tissues (42, 43), is functional. Since, contrary to sporadic thyroid hormone-deficient cretins, the three homozygous affected subjects achieved normal growth and mental development, it is inconceivable that hTRa! action is mediated through a thyroid hormone-independent mechanism. Rather, the resting tachycardia, shortened systolic time interval, and decreased deep tendon relaxation time observed in these patients (9) most likely represent the effect of high thyroid hormone levels on nerve tissues mediated through the hTRa. We also considered the possibility that in the absence of hTR/3, the hTRa gene is overexpressed. This hypothesis could be tested by measurement of hTRaI mRNA or protein. Unfortunately, both the cy- and &receptors are expressed in fibroblasts at a low level, since repeated attempts failed to generate a positive Northern blot using normal fibroblast mRNA. Similarly, antibodies directed to both receptors failed to yield positive results. Additional mechanisms, other than a simple increase in TRa expression or saturation by the high hormone levels, can be invoked in the preservation of thyroid hormone responsiveness mediated by the hTRcv serving as a single truns-activating factor. For example, protein-protein interaction among various nuclear factors, similar to that reported in modulation of the transcriptional activity of the major low density lipoprotein apoprotein-B-100, may be involved (44). Indeed, recent evidence has been advanced for the presence of a nuclear binding enhancement factor or Ta receptor auxiliary protein (45, 46). This as yet poorly characterized protein has a relatively higher potency in facilitating binding of rat TRa than rat and human TRP to thyroid hormone response elements of the rat GH and pituitary glycoprotein a-subunit genes, which are regulated by TRs (46). From the foregoing it may be speculated that homozygous subjects harboring a mutation that leads to the expression of only a malfunctioning hTR@ would be more severely affected than the patients described herein, who
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TAKEDA ET AL.
54
TABLE 2. Comparison of thyroid function tests in heterozygous and homozygous individuals complete deletion of the hTRfi
Subject ID (ref. no.) Heterozygous Complete deletion (this family) Single amino acid deletion (kindred S) Homozygous Complete deletion (this family)
Mother* (9,22) Fathe@ (9,22) Mother (47) Maternal grandfather
(47)
mGh (9,22, 23) vGh (9, 22, 23) maG” (9,22,23) Proband’ (47,48)
TT, (nmol/L) 116 128 244 196 257 307 344 653
Single amino acid deletion (kindred S) (653) 64-154 Normal range ’ Above (+) or below (-) the upper limit of normal. b Average of three to six separate determinations over the period of lo-15 yr. ’ Age 2 9/12 yr and value from 3 weeksof age in parentheses. d Upper limit of normal in infants is 3.55 nmol/L, and in prepubertal children is 3.22 nmol/L.
do not express this receptor at all. Such an observation would be indicative of a functional hTRa, since the effect could be only mediated through the antagonism of the mutant hTR/3 with hTRa. The recent report that a homozygous subject of kindred S expressed a hTR@ devoid of Ts-binding activity due to a single amino acid deletion (threonin-337) from the hormone-binding domain (38) provided us with the opportunity of testing the above hypothesis. The homozygous propositus manifested severe delays in linear growth and central nervous system development. Since results on peripheral tissue responses to thyroid hormone are not available, we compared the potency of thyroid hormone to negatively regulate TSH. Data are presented in Table 2. Obligate heterozygotes from our family with complete hTRp deletion showed no abnormalities, while those from kindred S, with a single amino acid deletion, had clearly elevated thyroid hormone levels, which failed to suppress their TSH. More importantly, the only homozygous subject from family S had 2- to &fold higher elevation of thyroid hormone levels than the average for affected homozygots of our family. Despite such high hormone levels, TSH in the homozygous patient from kindred S remained exceptionally high, about 20-fold above the levels found in the homozygous patient described in this communication. Furthermore, our patients showed no growth or mental retardation (5, 9). The deaf-mutism and mild somatic abnormalities found in our patients have not been described in other subjects with GRTH and may, thus, be a consequence of the deletion of genetic material other than the hTR@ gene. Acknowledgments We wish to thank Dr. Cary Weinberger for provision of the pheAl2 clone, Graeme I. Bell for invaluable advice, Mr. Paul
JCE & M. 1992 Vol74.Nol
exhibiting
either a single amino acid deletion or
TT3 (nmol/L)
FT, (%)”
1.81
-21 -18 +96 +53
3.1 1.8 1.0 2.2
+88 +130 +125 +1017 (+575) -56to0
5.5 5.8 1.6 389
4.58 3.52 4.53 12.75 4.75 20.72 (15.94) 1.38-2.84d
TSH bU/L)
(102) 0.5-4.0
Gardner for the synthesis of oligonucleotide primers, and Mrs. Yolanda W. Richmond for preparation of the manuscript.
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THYROID
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33. 34.
35.
36. 37.
38.
39.
40. 41.
42. 43. 44. 45. 46.
47. 48.
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