0021-972X/92/7404-0712$03.00/0 Journal of Clinical Endocrinology Copyright 0 1992 by The Endocrine

and Metabolism Society

Vol. 74, No. 4 Printed in U.S.A.

Rapid Localization of Mutations in the Thyroid Hormone Receptor-@ Gene by Denaturing Gradient Gel Electrophoresis in 18 Families with Thyroid Hormone Resistance* KYOKO

TAKEDA,

ROY E. WEISS,

AND SAMUEL

REFETOFF

Departments of Medicine (K.T., R.E. W., S.R.) and Pediatrics (S.R.) and the J. P. Kennedy, Retardation Research Center (S.R.), University of Chicago, Chicago, Illinois 60637

ABSTRACT. Generalized resistance to thyroid hormone (GRTH) is an inherited syndrome of reduced tissue responsiveness to thyroid hormone. Point mutations in the human thyroid hormone receptor-@ (hTR@ gene of these patients, causing single amino acid substitutions, appear be different in unrelated individuals affected by the same syndrome. To localize mutations in the hTR@ gene, GC-clamped DNA fragments from affected individuals belonging to 21 families with GRTH were generated by the polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis (DGGE). Putative mutations in the hTRp gene of 18 unrelated individuals with GRTH were identified, and their nature has been confirmed in 9 by sequencing. All were in the hormone-

binding domain of the receptor, and 13 of 18 mutations were in its center (exon 7). In 3 families we were unable to identify mutations in hTRP, suggesting the existence of mutations at other loci, possibly the hTRol gene or other proteins involved in the thyroid hormone-dependent transactivation system. Sequencing of DNA fragments negative for the presence of putative mutations by DEEG confirmed the absence of sequence differences. DGGE of amplified DNA fragments can rapidly and reliably localize the sites of mutations in the hTR@ gene of patients with GRTH. The procedure enabled mapping the regions in the hTRB harboring mutations associated with GRTH. (J Clin Endocrinol Metab 74: 712-719,1992)

G

resistance to thyroid hormone ENERALIZED (GRTH) is an inherited syndrome of reduced target tissue sensitivity to thyroid hormone (1,2). Although heterogeneous in their presentation, affected subjects lack the typical clinical and laboratory manifestations of thyroid hormone excess despite elevated serum levels of both T4 and Ts. Furthermore, reduced responses to the administration of supraphysiological doses of thyroid hormone have been demonstrated in. uiuo (3, 4) and in vitro

Jr. Mental

acids located in the hormone-binding domain of the hTRP, resulting in the expression of receptor proteins with either reduced (11-13) or no (7, 10) Ts-binding activity. The failure to identify in 19 unrelated families with GRTH 2 of the previously described nucleotide substitutions suggested that different mutations may be responsible for GRTH in unrelated families (14). Characterization of mutations by systematic sequencing of the entire hTRP gene would be laborious, because sequencing of both alleles is required owing to the autosomal dominant mode of GRTH inheritance. This task could be greatly simplified by a screening method capable of localizing sites of the mutation to a lOO- to 500basepair (bp) region of the gene. Of the several techniques available to detect point mutations (l&20), we chose the method of denaturing gradient gel electrophoresis (DGGE) (20) performed on gene fragments amplified by the polymerase chain reaction (PCR) (21). As described by Myers et al. (20), the method is based on the principle that partial denaturation of double stranded DNA (strand separation or melting) reduces its compactness and, thus, retards its mobility on polyacrylamide gel electrophoresis. Since the stability of DNA is determined by its base composition, in DNA fragments

(5).

It has long been suspected that GRTH is caused by abnormalities in the thyroid hormone receptor (TR) (6). However, this hypothesis was only recently confirmed by the identification of mutations in the human (h) TRP gene of affected members from unrelated families with GRTH (7-12). These mutations altered single amino Received July 10, 1991. Address all correspondence and requests for reprints to: Dr. Samuel Refetoff, University of Chicago, MC 3090, 5841 South Maryland Avenue, Chicago, Illinois 60637-1470. * Presented in part at the 73rd Annual Meeting of The Endocrine Society, June 19-22, 1991, in Washington, D.C. This work was supported in part by USPHS Grants DK-15070 and RR-00055. K.T. and R.E.W. contributed equally to this work and are both considered first authors. 712

LOCALIZATION

OF MUTATIONS

smaller than 400 bp, melting occurs at one end or the other. When submitted to electrophoresis in a gel containing a linear gradient of denaturants, migration is sharply reduced when a double stranded DNA fragment reaches the concentration of denaturant that causespartial melting. This allows separation of 2 DNA fragments differing by a single base because they melt at different concentrations of denaturant. To ensure identification of sequence differences at both ends of a DNA fragment, a 40-nucleotide synthetic oligomer composed only of guanidines and cytosines (GC clamp) (21, 22) is covalently attached to one end and then at the other end of the amplified DNA fragment (see Fig. lb). No radioactive probes are required, since DNA fragments can be visualized by staining with ethidium bromide. Using this technique we were able to localize putative mutations in the hTRP gene of individuals with GRTH from 18 of 21 unrelated families. All were located in the hormone-binding domain and were confined to 2 areas, the middle and carboxyl-terminal regions.

Materials

and Methods

Subjects

Twenty-three patients with GRTH belongingto 21 unrelated families and 5 unaffected subjects were studied. All affected (a) $?z!+l0.000bq EXON DNA

00

-

3

45

676

~~.GJ+&cH”’ ,,,,,

i’.,

F 2’ ;’ ,’ ,, :

:

3

mRNA

713

individuals were clinically euthyroid despite elevated serum free T, and T, concentrations. All also had reduced pituitary sensitivity to thyroid hormone, as documented by the inability of supraphysiological doses of TS and/or T4 to normally suppress serum TSH. The hormonal data of affected individuals

and the presumedmode of GRTH inheritance, basedon pedigree analysis, DNA extraction

are provided

in Table

1.

and amplification

High mol wt DNA was extracted from peripheral blood leukocytes, as previously described (23), or from cultured skin fibroblasts using a nucleic acid extractor (model 340A, Applied Biosystems, Foster City, CA). Five coding exons (no. 4-8), which include the entire TB-binding domain and most of the DNA-binding domain of hTR@ (Fig. la), were separately amplified using synthetic oligonucleotide primers complementary to their flanking nonprotein-coding sequences (Table 2). In the first amplification reaction, 100 pmol 20-mer or 23mer primer pairs, without GC clamp sequence adaptor, were annealed to 1.5 pg genomic DNA and submitted to 30-40 PCR cycles (Perkin-Elmer/Cetus thermal cycler, Norwalk, CT) without mineral oil (24). Each cycle consisted of denaturation for 30 s at 94 C and, depending on the primer pair used, annealing for l-2 min at 55-64 C and extension for 0.5-2 min at 72 C. Two microliters of product of the first PCR reaction were submitted to a second amplification under similar conditions, except one of the primers had a 40-nucleotide GC clamp sequence (Fig. lb). The oligonucleotide primers were designed to enable the generation of DNA fragments with the GC clamp at both the 5’-end (5’-GC) and the 3’-end (3’-GC; Table 2). To confirm the generation of DNA fragments and their appropriate size, 5-10 PL from each PCR reaction were resolved in 1% agarose gel and stained with ethidium bromide. DGGE

Protein

(W CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCG

---?. IVS v ;,“;I GC-clamped

-double

stranded

DNA

fragment

1. a, Structure of the hTRp gene, its mRNA, and protein. The hTR/3 gene has eight protein-coding exons (black boxes numbered 1-8) and two exons, designated 00 and 0 (open boxes), that encode only the 5’-untranslated region of the mRNA. hTRp has 461 amino acids. b, Exon amplification with attachment of a GC clamp adaptor. Two primers, A and B, each containing 20-23 nucleotides complementary to intron sequences flanking the exon, were used for amplification by PCR. Primer A has a IO-mer adaptor sequence (GC clamp). The resulting double stranded DNA fragment is tightly bound at one end (GC-clamped) owing to its relative abundance of Gs and Cs. FIG.

IN GRTH

The DGGE apparatus, describedby Traystman et al. (22), was purchased from Green Mountain Lab Supply, Inc. (Waltham, MA). Polyacrylamide gel slabs containing a gradient of denaturants were made by mixing different proportions of two solutions: a 0% denaturant mix [6.5% acrylamide in 40 mM Tris-20 mM sodium acetate and 1 mM EDTA (TAE) buffer, pH 7.41 and a 100% denaturant mix (6.5% acrylamide with 40% formamide and 7 M urea in TAE buffer). Ammonium persulfate and N,N,N’,N’-tetramethylethylenediamine were added to each chamber of the gradient maker just before gradient gel casting. Eight to 10 PL of the final PCR product were mixed with 2 PL 80% glycerol, 10 mM Tris, 25 mM EDTA, and 1% bromphenol blue. Electrophoreses were run at 60 C and 70 V. Gels were stained with ethidium bromide and photographed under UV transillumination. A trial gradient gel with a broad denaturant range (O-80%) was first used to analyze the PCR products. The range of denaturant was then narrowed, and the electrophoresis time was varied (14-22 h) to obtain the best separation between

normal and mutant fragments. Fragments containing known mutations,

when available,

were used as controls

[exon 7 (fam-

TAKEDA,

714 TABLE 1. Hormone concentration,

JCE& M -1992

WEISS, AND REFETOFF

presumed mode of inheritance,

and localization

Vol74.No4

of nucleotide differences in the hTR@ gene

I

Presumed mode of inheritance” D

II

D

232

5.34

+80

8.0

IV

D

192 205

4.30 3.24

+39 +55

15.5d 5.0

V

D

256

4.38

+89

2.7

7 (V, 3’)

VI

D

270

3.99

+261

7.0

8 (5’)

VII

D

284

5.13

+ss

7.6

7 (51, 3’)

VIII

D

275

4.93

+40

1.3

7 (5’, 3’)

IX

D

192

4.02

+62

X

A

224

4.96

+67

3.3

8 (5’)

XI

D

288

3.86

+57

3.0

8 (5’)

XII

D

245

4.30

+105

5.9

7 (5’, 3’) 8 (5’)

XIII

NK

286

5.02

+149

4.4

7 (3’)

XIV

NK

230

4.22

+40

18.5

None

D

256

4.02

+83

3.1

7 (3’)

NK

237

5.13

+100

2.0

7 (5’, 3’)

D

228 217

3.44 5.23

+59 +70

1.3 3.3

None None

XVIII

NS

360

3.15

+171

4.2

7 (3’)

XIX

D

225

4.44

+75

3.0

6 (5’) 7 (5’, 3’)

xx

D

108

2.75

-11

XXI

NS

205

4.39

+85

2.0

7 (5’, 3’)

D

193

3.36

+82

3.5

8 (5’)

64-154

1.38-2.84

Family no.

xv

Subject no.

1

XVI XVII

XXII Normal range

1 3

2

T3 bM)

FTII @Jb

TSH bU/L)

334 227

5.82 3.69

+139 +107

3.2 1.4

Putative mutation site (exon)’ 7 (5’. 3’) 7 (5’; 3lj 7 (5’, 3’), 8 (5’)

260d

134d

None None

8 (5’)

7 (3’)

0.5-4.0 -43 to 0 a D, Dominant; A, adopted; NK, not known (possible new mutation); NS, family not studied. * +, Above the upper limit of normal; -, below the upper limit of normal. ’ Sequence differences detected in 5’-GC-clamped DNA fragments (5’) and in 3’-GC-clamped DNA fragment (3’) appear in parentheses. d Partial ablation of the thyroid gland.

ily I) and exon 8 (family II)]. The final conditions of DGGE were: exon 4,35-55% denaturant gradient ran for 15 h; exon 5, 35-55% for 16 h; exon 6,30-50% for 17 h; exon 7,35-65% for 20 h; and exon 8,35-60% for 18.5h. DNA sequencing

DNA fragments found to contain sequencedifferences on DGGE were reamplified, cloned into Ml3 bacteriophage, and

sequencedby the dideoxynucleotide chain termination method (25), usingthe Sequenaseversion 2.0 DNA sequencingkit (U.S. Biochemical Corp., Cleveland, OH) and [a-32P]ATP (Amersham Corp., Arlington Heights, IL). At least six separatetemplates of each DNA fragment were sequenced,and the authenticity of each mutation was verified by sequencingproducts of two separate PCR amplifications, by allele specific amplification (7), or by digestionwith specificrestriction endonucleases.

LOCALIZATION TABLE

2. Sequences

of oligonucleotide

primers

and the fragment

OF MUTATIONS size generated

IN GRTH

715

by PCR Fragment

Exon

4

Primer

sequence”

Orientation

(A) 5’-ctgttgtcttgggtctttgt-3’

Sense

(B) 5’-gcaagtgaagatctacttac-3’

Antisense

(C) 5’-GC

Sense

Primer pair

amplification Exon size

Total size

AB

5

(D)

+ primer

(A)-3’

5’-tgtgcatcagtggtcccact-3’

199 148

BC

239

DE

386

Sense

(E) 5’-caccagtatcccaaggtgat-3’

Antisense

(F) 5’-GC (G) 5’-GC

Sense Antisense

206

6

(H)

+ primer + primer

(D)-3’ (E)-3’

5’-caggatatcagttcagaaga-3’

EF DG

426 426

HI

316

Sense

(I) 5’-ccagtattcctggaaactga-3’

Antisense

(J) 5’-GC (K) 5’-GC

Sense Antisense

147

7

+ primer + primer

(H)-3’ (I)-3’

(L) 5’-cagtatgttgttcctgactggca-3’

Sense

(M)

Antisense

5’-cactaacgagtctaggcgtac-3’

IJ HK

356 356

LM

350 259

8

(N) 5’-GC (0) 5’-GC

+ primer + primer

(L)-3’ (M)-3’

(P) 5’-GC

+ tctgaatcaatgtccatcttc-3’

Sense Antisense

MN LO

390 390

PQ*

337

Sense

(Q) 5’-ATGAGAATGAATCCAGTCAG-3’

Antisense

(R) 5’-caataaaggcctggaattgg-3’ (S) 5’-GC + primer (Q)

Sense Antisense

269

GC + includes the sequence shown in Fig. 1. ’ Lowercase and capital letters show intron and noncoding exon sequences, respectively. * A 5’-GC-clamped DNA fragment containing exon 8 could be generated by a single PCR

Results An example of DGGE analysis presenting sequence differences in exon 8 of the hTR@ gene is shown in Fig. 2b. Of the eight unrelated subjects with GRTH analyzed on this gel, three (VI-l, XI-l, and X-l) had multibanded patterns, indicating the presence of a mutation in one of the two alleles. All three showed two bands (a close doublet in the case of subject X-l) that melted first, i.e. at lower concentrations of denaturants. They represent the two heteroduplexes, thus confirming the heterozygous state of the subjects. Also, all showed a common band representing the normal homoduplex (arrow in Fig. 2b). The mutant homoduplex of subject VI-l melted after the normal homoduplex, predicting an A or T replacement by G or C (A-pattern). In contrast, the mutant homoduplex of subject X-l melted before the normal

RQ

343 383

RS

reaction

using

the genomic

DNAs

as template.

homoduplex, predicting G or C replacement by A or T (B-pattern). Finally, the position of the mutant homoduplex of subject XI-l coincided with that of the normal homoduplex, predicting a G*C or A-T substitution (Cpattern). The accuracy of these predictions was confirmed in nine of the unrelated subjects with GRTH whose DNA fragments were sequenced (see below). As previously noted (20), the ability to identify mutations by DGGE depended on the location of the GC clamp. As shown in Fig. 3, the mutation in exon 7 found in subject I-l could be detected when the GC clamp was on either end of the PCR product. In contrast, the mutation in exon 8 found in subject II-1 could be seen only when the GC clamp was placed at the 5’-end of the DNA fragment. These findings provided encouraging confirmation regarding the ability of this method to

TAKEDA,

716

WEISS, AND REFETOFF

(a>

IN --

JCE & M * 1992 Vol74*No4

IN --

1 Mutant allele

PCR G&clamp

i

E.-----

Normal Mutant

=----

1

Homo duplexes

Normal /Mutant 3 Heterduplexes

60%

FIG. 2. DGGE. a, Pictorial representation of the PCR products from a DNA template, one allele of which contains a point mutation. Since cDNA fragments pair even if they differ by a few basepairs, amplified DNA from a heterozygous individual can form four possible double stranded DNA molecules: two homoduplexes (normal and mutant) and the two types of heteroduplexes. b, Positive picture of a DGGE of DNA fragments containing exon 8 of the hTRp gene. Note the three patterns of band separation according to the position of the mutant homoduplex relative to that of the normal homoduplex (common band indicated by the arrow on the right). A-pattern (lane VI-l), when the mutant homoduplex is more resistant to the denaturant; B-pattern (lane X-l), when melting of the mutant homoduplex precedes that of the normal homoduplex; C-pattern (lane XI-l), when the mutant and normal homoduplexes comigrate. Bands arrested earlier in the migration path represent heteroduplexes, since they are completely mismatched at the III mutation site.

predict the location of nucleotide substitutions, at the middle of exon 7 in subject I-l and the 3’-end of exon 8 in subject II-l. The results of DGGE analyses of exons 4-8, which include the T,-binding and most of the DNA-binding domains, are summarized in Table 3. No differences in the sequences of exons 4 and 5 were evident in any of the individuals tested. Sequence variations were observed in DNA fragments containing exons 6-8. Subjects from 13 families may have mutations in exon 7, and those from 7 families may have mutations in exon 8. Mutations at 2 sites, exons 7 and 6 and exons 7 and 8, were found in 1 and 2 families, respectively. No abnormalities in DGGE patterns were observed in affected subjects from 3 families (IV, XIV, and XVII). Results were verified for their reproducibility by repeated DGGEs on products from different PCRs. Similarly, no abnormalities were found in any of the amplified DNA fragments from the 5 unaffected controls. Mutations identified by DNA sequencing are shown in Fig. 4. The ability of DGGE to localize the mismatched nucleotides on DNA fragments by the detection of abnormalities relative to the position of the GC clamp was confirmed in each of the nine subjects by sequencing.

5’-GC 3’-GC

EXON

7

S-GC

3'-GC

EXON 8

3. Composite negative pictures of DGGEs showing the influence of GC clamp placement relative to the location of the mutation. The mutation in exon 7, located close to the center of the DNA fragment (148 nucleotides from the 3’-end of a 350-bp fragment), was detected with the GC clamp at either end (5’-GC and 3’-GC). That in exon 8, near the 3’-end of the fragment (50 nucleotides from the 3’-end of a 343-bp fragment), was detected only with the GC clamp placed at the 5’-end (5’-GC). N, Corresponding fragments amplified using DNA from a normal subject. FIG.

Indeed, subjects XV-l and XVIII-l who had mutations at the 5’-end of exon 7 exhibited abnormalities on DGGE only when the GC clamp was placed at the 3’-end of the corresponding DNA fragment. Subjects I-l, I-2, VII-l, and VIII-l, with mutations in the middle of exon 7, showed abnormal DGGE patterns with the GC clamp placed on either end of their DNA fragments. Finally, subjects II-l, X-l, XI-l, and XII-l, with mutations confirmed at the 5’-end of exon 8, revealed DGGE abnormalities only with the GC clamp located at the 3’-end of the corresponding DNA fragments (Table 1 and Fig. 3). The relative positions of the normal and mutant homoduplexes also accurately predicted the type of nucleotide substitution. Pattern A, exhibited by subject XV-l, was associated with a T + C substitution. Pattern B was found in subjects II-l, VII-l, VIII-l, X-l, and XVIII-l with G + A, C + T. or C + A substitutions (see Fig. 3 for II-1 and Fig. 2 for X-l). Of the subjects with pattern C on ,DGGE, three were analyzed by sequencing. Subjects I-l and I-2 had G + C substitutions (Fig. 3), and subject XI-1 had a C insertion (Fig. 2). Twelve to 20 templates prepared from each of 4 DNA fragments from 4 subjects with GRTH that failed to

LOCALIZATION 3. Localization

TABLE

Subject no.

I

1 2 1 1 2 1 1 1 1

II IV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII

Exon 5

5’-GC clamp

5’-GC clamp

-

-

XXI XXII

3’-GC clamp -

717

5’-GC clamp -

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1

ND’

ND

2

ND

ND

3’-GC clamp -

-

-

ND ND

+ ND ND

DNA

Exon 7

-

-

1

xx

Exon 6

-

-

1 1 1 1 1 1 1 1 3 1 1 1

XVIII XIX

IN GRTH

of nucleotide differences in the hTR@ gene by DGGE of GC-clamped, PCR-amplified Exon 4

Family no.

OF MUTATIONS

-

5’-GC clamp + + + +

3’-GC clamp + + + +

+ +

+ -

-

+ + +

ND ND

Exon 8

+ -

+ + + + + + + + -

5’-GC clamp +

3’-GC clamp -

+ -

-

+

-

+ + + -

-

-

Mutation site (exon) 7n.b 7a.b 7a.b

8”

None None 7&b 8” y,b

7”,b 8” 8” 8”

-

y,b

-

7b None 7b

8”

7a.b

None None 7b

6” y,b +

lb ND ND

7a.b

8”

’ Mutation detected in 5’-GC-clamped DNA fragment. b Mutation detected in 3’-GC clamped DNA fragment. ’ ND, not done.

FAMILY ~2 CODON II) NORMAL>

WR CL 320 320 Ai” Ai”

S 337 T;’

Discussion

D 340 Gi”

MUTANT 3

FAMILY =I) CODON 3 NORMAL 3 MUTANT 3

310 Met t Thr

317 Ala t Thr

vi 332 Gly t Arg

i vi 345345 Gly Gly tt Arg Asp

Xii 417 Phe t Phe*

XII 438 Arg t His

Xl 454 Leu t Phe

453453 PrOPrO tt SW His

wa+me shift

4. Mutations in the hTR@ localized by DGGE in subjects with GRTH. The DNA-binding and Ta-binding domains and the regions of the hTRp protein encoded by each exon (numbers in open boxes) are indicated. The number of GRTH families with mutations localized to each exon are circled below the corresponding exon. The amino acid replacements deduced by gene sequencing are indicated below the diagram. Those in four families studied by Usala et al. (9-12) are shown above the diagram. *, Second silent mutation in family XII. FIG.

show abnormalities on DGGE were sequenced. These were exon 7 of subjects IV-2 and XI-l and exon 8 of subjects I-l and XVII-3. None had nucleotide sequence abnormalities compared to the wild type. Of the 2 nucleotide substitutions detected in subject XII-l, 1 produced a silent mutation (Fig. 4).

A previous search for mutations in the hTR/3 gene identical to those found in 2 patients with GRTH indicated that mutations are probably unique to affected members of each unrelated family (14). In the current study we used DGGE to localize mutations in the coding sequences of the hTRp gene of 21 families. Five of the 8 coding exons (no. 4-8), representing 72% of the amino acid sequence of the hTR& were analyzed. They cover the entire Ts-binding domain (exons 6-8) and 74% of the DNA-binding domain (exon 4). Unfortunately, we were unable to amplify exon 3, which encodes the remaining 26% of the DNA-binding domain of the receptor, and we had difficulty in generating unique DNA fragments with the GC clamp located at the 3’-end of exon 4. However, failure to demonstrate DGGE abnormalities in exon 4 with the GC clamp placed at the 5’end indicates the absence of putative mutations in the functionally important second zinc finger (26). Since at the time of the study, mutations in the hTRP gene probably responsible for GRTH were known in 2 families (I and II) (7, 8), we could use DNA from these subjects to test the ability of GC-clamped DNA fragments generated by PCR to reveal known mutations

718

TAKEDA,

WEISS,

when submitted to DGGE. The technique proved to properly localize their nucleotide substitutions to exons 7 and 8, respectively. Nucleotide differences in the hTR/l gene of subjects with GRTH were identified in 18 of the 21 families tested. All were located in the 3 exons (6, 7, and 8) that encode the Ta-binding domain of the receptor. Although all of the nucleotide substitutions characterized to date appear to result in the synthesis of abnormal proteins (7, lo), we cannot exclude the possibility that some of those not yet sequenced may be silent or reside in intron sequences of the amplified DNA fragments. Several characteristics related to mutations of the hTRp gene in GRTH have emerged from the current study. Nine of the mutations so far identified by sequencing are located in exon 7, at about the center of the Tsbinding domain, and involve 1 of 35 contiguous amino acids (codons 310-345) that are conserved in both (Yand p thyroid hormone receptors (27) and across species, from human to frog (27-31). The DGGE results suggest that eight additional families may have mutations in this region. Mutations located in exon 8 appear to involve amino acids at the carboxyl-terminus of the hTR@ This conclusion is based on their detection by DGGE only when the GC clamp was placed at the 5’-end of the DNA fragment and was confirmed in four families by sequencing. Finally, two different mutations have been identified in each of codons 320, 345, and 453. Thus, mutations of the hTRp in patients with GRTH are clustered in the middle and carboxyl-terminal regions of the Ts-binding domain and appear to involve specific codons. It is likely that alterations of these regions of the receptor produce more important alterations of hormone binding, thus resulting in GRTH. It is also possible that the localization of mutations exclusively in the hormone-binding domain is the consequence of selection bias produced by the fact that the syndrome is only detected in euthyroid individuals with elevated thyroid hormone levels. Based on pedigree analysis, inheritance was autosomal recessive in 1 family, dominant in 15, and unknown in 6 of the 22 families with GRTH we have so far studied for mutations in the hTR/3 gene (Refs. 14 and 32 and this report). Affected individuals from the family showing recessive inheritance had deletion of the hTRP gene in both alleles, while their obligatory heterozygous parents were clinically and biochemicalIy normal (32). In 9 of the 21 families with the more common, dominantly inherited, form of GRTH, we have demonstrated a mutant and a normal hTR/3 allele by gene sequencing (Ref. 7 and this report). The presence of heteroduplexes on DGGE confirmed the dominant inheritance in 9 more families, including 5 of the 6 families with insufficient pedigree information. In 3 families (IV, XIV, and XVII), there were no DGGE detectable sequence differences in exons 4-8 of the hTR@ gene located on chromosome 3

AND

REFETOFF

JCE & M. 1992 Vol74.No4

(33), suggesting that the thyroid hormone resistance in these families may be due to a mutation in the hTRa gene on chromosome 17 (34). The hTRa1 product of this gene has been shown to be functional (35, 36) and appears to be responsible for the mediation of thyroid hormone action in subjects that completely lack the hTR/3 gene (32). However, the possibility remains that GRTH in these 3 families is related to a deficiency of the TR auxiliary protein that stabilizes the binding of TR to thyroid response elements located in thyroid hormone-responsive genes (37, 38). Note

added

in proof

While this communication was in press, Parrilla et al. (39) reported point mutations in subjects with GRTH from seven additional families. Their case 1, from family F-W is subject VII-l in the current communciation. This subject with GRTH, first reported by Kaplowitz et al. (40) and subsequently studied by us (5, 14), moved to another state following his adoption. Also in the interim, ‘we confirmed by sequencing the presence of point mutations in exons of subjects from three additional families (XIII-l, XIX-l, and XX-l) as predicted by DGGE; a total of 12 positive predictions. In addition, we found no mutations in three more exons that showed no abnormalities on DGGE. However, no mutations were found in exon 7 of two subjects (II-l and XII-l) in whom a mismatch was predicted by DGGE. In these 2 out of 9 apparent false-positive DGGE analyses, we cannot rule out the possibility of mismatches in intronic regions which were included in the DNA fragment used for DGGE but were not sequenced. Finally, sequencing revealed a mutation in exon 8 of subject IV1 who failed to show abnormality n DGGE; 1 false negative out of 13. Acknowledgments

We thank the following individuals for the referral of patients or provision of material in the form of whole blood or skin fibroblasts: Dr. Nives Dumbovic (families I and X), Dr. Charles Eil (family II), Dr. Gregory B. Pehling (family V), Drs. PhilIip DeNayer and J. V. Vandalem (family VI), Dr. Paul B. Kaplowitz (family VII), Dr. Alan Chait (family VIII), Dr. Randolph Seed (family IX), Dr. Jacob0 Wortsman (family XI), Dr. I. A. Hughes (family XII), Dr. Barry H. Rich (family X111), Drs. Desmond Schatz and William E. Winter (family XV), Dr. Deborah V. Edidin (family XVI), Dr. Claudio Marcocchi (family XVII), Drs. Catherine M. Edwards and P. W. Stacpoole (family XVIII), Dr. Corbin P. Roudebush (family XIX), Drs. Victoria C. Mussey and Terry J. Smith (family XX), Drs. J. Weill and J. Martial (family XXI), and Dr. J. Wing (family XXII). Dr Graeme Bell is specially acknowledged for his advice and encouragement and, together with Drs. Kenneth Polonsky and David Ehrmann, for the review of the manuscript. The

LOCALIZATION assistance of Mrs. Yolanda W. Richmond the manuscript is also acknowledged.

in the preparation

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Rapid localization of mutations in the thyroid hormone receptor-beta gene by denaturing gradient gel electrophoresis in 18 families with thyroid hormone resistance.

Generalized resistance to thyroid hormone (GRTH) is an inherited syndrome of reduced tissue responsiveness to thyroid hormone. Point mutations in the ...
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