Vol. July

178, No. 31, 1991

SINGLE THYROID

2, 1991

BASE

BIOCHEMICAL

MUTATION

HORMONE

HORMONE

IN THE

RECEPTOR

RESISTANCE

+Princess Received

June

10,

HORMONE

RESEARCH

BINDING

p GENE

IN

BY

OF THE

SINGLE

THYROID STRANDED

P. E. Hickman+,

D. P. Cameron+

Institute Medical Research, Alexandra

DOMAIN

ANALYSIS

B. T. Teh+, N. K. Hayward*, and

COMMUNICATIONS Pages 606-612

GENERALISED

POLYMORPHISM

G. J. Ward+,

*Queensland

BIOPHYSICAL

DEMONSTRATED

CONFORMATION

C. V. Boothroyd*,

AND

Herston,

Hospital, Woolloongabba

4006 Australia 4102, Australia

1991

Thyroid hormone resistance is a syndrome of considerable clinical heterogeneity. Three mutations in the c-e& A p gene encoding the human B thyroid hormone receptor have been described in different kindreds. We report here, in a family affected with peripheral thyroid hormone resistance, a unique point mutation in the ligand binding domain of the c-em A B gene resulting in histidine replacement of an arginine residue at position 438. The region in which the mutation occurred was identified by single stranded conformation polymorphism analysis and confirmed by subcloning and sequencing of the mutant alleles from each of the affected members. Binding of tri-iodothyronine to isolated nuclei from family members was normal suggesting the mechanism of thyroid hormone resistance in this family is not mediated by abnormal binding of ligand and receptor. 0 1991 Academic Press, Inc.

Generalised thyroid hormone resistance ,

a syndrome of considerable clinical

heterogeneity, is characterised by varying degrees of peripheral resistance to the action of thyroid hormones with elevated levels of thyroxine

and tri-iodothyronine

the presence of normal or marginally elevated thyrotropin

after exclusion of other

causes of euthyroid hyperthyroxinemia(1).

in

Following recognition that the c-erb-A ,9

gene encodes the B thyroid hormone receptor(2), distinct mutations in three different kindreds affected with GTHR were described(3,4,5).

All mutations are in the ligand-

binding domain, two in exon G and the other in exon H, (as named in the reported sequence of the human thyroid hormone receptor B gene (6)) close to the carboxyl Abbreviations: GTHR, generalised thyroid hormone resistance; T4, thyroxine; T3, tri-iodothyronine; TSH, thyrotropin; SSCPA single stranded conformation polymorphism analysis. 0006-291X/91 Copyight All rights

$1.50 0 1991 bx Academic Press. of reproduction in any form

Inc. reserved.

606

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

terminus of the fi thyroid hormone receptor.

in a newly described family with GTHR

we sought to determine whether the disease was caused by one of the three previously characterised mutations of c-e&A p. We show in this article that GTHR in our family is caused by a novel mutation of this gene.

MATERIALS AND METHODS Clinical data The proband, a male aged 26, was diagnosed as thyrotoxic, treated with propylthiouracil and ultimately subtotal thyroidectomy (normal thyroid tissue was repotted on pathological examination) before referral to our unit. Clinical examination showed moderate residual goitre but was otherwise unremarkable. His siblings and father similarly were developmentally and physically normal. Free thyroxine was assayed using Amerlex-MAB free thyroxine RIA (Amersham Int. product number IM5054)and tri-iodothyronine, using Amerlex-M free T3 RIA (Amersham Int. product number IM3101). TSH was assayed using reagents supplied by Bioclone Australia (TSH-IRMA kit, product no. 20-260-125) Measurement of albumin, prealbumin and thyroxine binding globulin were performed according to the described method(7). Oliaonucleotide Primer Synthesis Oligonucleotide primers for polymerase chain reaction (PCR) were designed using the known sequence of the thyroid hormone receptor p gene(6) with an artificial restriction site at each 5’ end to facilitate subcloning (primers A and D with f3amHl site and primers B and C with EcoRl site) as follows: primers A and B covering exon G (codon 303-375) 5’-TCGCGGATCCTGCCATGTGAAGACCAGAT-3’ and 5’TCCGGAATTCTGAAGACATCAGCAGGACG-3’ respectively and primers C and D covering exon H (codon 392-462) 5’-CAGTGAATTCTTGCCTGTGTTGAGAGAGAATA-3’ and 5’-CGTAGGATCCATGAGAATGAATCCAGTCAG-3’ respectively. PCR amplification of aenomic DNA DNA was extracted from transformed lymphoblastoid cell lines according to the salting out method(8). The PCR mixture (total volume 25 J) contained 50 pmol each primer (A & B or C & D), approximately 1 ,,g DNA, 0.2 mM each dCTP, dllP, dATP and dGTP and 0.5 ~1 a [?$]dATP (37 TBq/mmol in 20 mM dithiothreitol), 2.5 U Taq polymerase (Promega) in 50 mM KCI, 10 mM TrisHCl (pH 9.0), 1.5 mM MgCI,, 0.01% gelatin, 0.1% Triton X-100. This mixture was overlaid with 25 ~1 mineral oil. Amplification was over 25 cycles of 1 min at 94OC, 2 mins at 55OC and 2 min at 72°C using a Perkin-Elmer Cetus Thermocycler. Sinale stranded conformation oolvmorohism analvsis SSCPA was performed with some modifications according to the method described(g). Two microlitres of PCR product were removed from below the oil, added to 0.5 ~1of 10% SDS and 1 M EDTA and 2 PI of 95% formamide with 20 mM EDTA, 0.05%. bromophenol blue, 0.05% xylene cyanol. After heating to 90°C for 3 min, 2.5 pl were loaded onto a 5% acrylamide gel, (BioRad Sequi-Gen sequencing cell 0.4 mm thick, 50 cm X 21 cm) in 90 mM Tris borate pH 8.3, 4 mM EDTA. This was run over 4-6 hours at 30W. The temperature of the buffer tank did not exceed l?C and this was achieved by running the apparatus in a 4OC cold room. The gel was transferred to blotting paper, dried and placed on Kodak XAR5 X-ray film without intensifying screens for l-3 days. Subclonina and sesuencinq PCR amplification was performed as described above but with 0.2 mM dNTPs final Phenol and chloroform extraction and ethanol precipitation were concentration. performed prior to digestion overnight with EcoRl and BamHl and subcloning of the 607

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COMMUNICATIONS

Sequencing was performed using the digested PCR product into M13mpl8. Amersham Multiwell DNA sequencing system (l7 polymerase). Nuclear bindina studies T3 binding to isolated lymphoblastoid cell nuclei was studied in the four affected family members and six unrelated controls. Extraction of nuclear thyroid hormone receptors was performed as previously reported (10). Binding assays were performed with increasing concentrations of [‘?]T3 (0.1-l .O nmol/l) in the presence or absence of excess unlabelled T3 (1.0 pmol/l) and the binding of T3 was measured using hydroxyapatite as described(l1). T3 binding results were analysed according to the method of Scatchard(l2) using a computer programme(13).

RESULTS The endocrinological table

1.

Despite

the elevation

shown

support

parameters

albumin, prealbumin Antibodies

and biochemical

of free thyroid

the clinical impression

and thyroxine

patients

of euthyroidism.

binding globulin were

family tree are shown

antithyroid

(112) has marked

mutation lay between

previously

therapy(l4).

the

Binding

to

elevation of

the sequences

substitution

have two extra bands

in the control samples. homologous

of guanine

to primers

to adenine

These predicted

NORMAL

freeT3

TSH

SHBG’

(12-28 PmW

(2.5-7.5 pmoUl)

(~4 mUA)

(nmoVl)

55

11.1

1.6

46 41 51

12.7 13 10.7

1.7 13.0 0.5

37 22 25 38

at position

1613

Ferritin

ACE’

(25-400

(35-130

Pgn)

UN

86 96 81 87

63 87 52

CASE

*SHBG = sex hormone binding globulin, female 30-W nmoVl *ACE = angiotensin converting enzyme.

normal ranges male lo-50

608

the

C and D in exon H. A

Table 1. Biochemical data of affected family members (see figure 1) with generalized thyroid hormone resistance freeT4

in patients

Results of SSCPA and

in figure 1. Affected family members

the two bands demonstrated

I2 II1 II2 II3

in

normal (data not shown).

rate and this has been described

with GTHR treated with inappropriate

base

in these

are shown

to levels of free T4 and free T3 despite clinical evidence of euthyroidism

and normal basal metabolic

single

hormone

to T3 and T4 were not detected. The proband

TSH compared

between

data of affected family members

nmol/l,

(corrected

Vol.

178, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fiaure 1. Singlestrandedconformationpolymorphismanalysisof a family with GTHR. The presence of two extra bands (arrowed) predicted that there was a mutation bstween primersC and D. The unaffected mother (II) was not tested by SSCPA. 0 =affected 0 =normal Q =unrelated control

nomenclature as in (6)) was found in 3 out of 6 clones sequenced (figure 2) from case 112.This was confirmed in all the remaining affected family members (2/3 clones for 113,112for III and 3/4 for 12). The remaining clones sequenced from affected family members were wild-type. This is in keeping with previous reports (3,4,5) and implies that the mutation occurs in only one allele and is dominant. The mutation results in replacement of arginine 438 (codon CGC) by a histidine residue (codon CAC) in the ligand-binding domain of c-erbA B. A further ten unrelated controls were subjected to SSCPA after PCR amplification of exon H and each showed only two bands indicating that the observed mutation is not likely to be a genetic polymorphism. Misincorporation by Tag polymerase was found in approximately 1:4000 bases which 609

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mutant

2, 1991

wild

BIOCHEMICAL

type

AND

BIOPHYSICAL

RESEARCH

MAXIMUM BINDING CAPACITY

BINDING AFFINITY (Ko)

zo-

COMMUNICATIONS

1

A 1

15--

o

-I

0

0

10-m

ol

0x5-37

i 0 0

02

03

ACGTACGT

100

OJ

NORMAL

I RESISTANT

10 NORMAL

RESISTANT

Figure 2. Sequence analysis of genomic DNA from patient 112with GTHR. The autoradiographof the normalnucleotidesequence of part of exon H of the c-eb A p gene is shown on the right. Adenine substitution for guanine at position 1613 is shown on the left causing histidine(mutant) replacementof an arginine (wild-type). Figure 3. Binding affinity and maximumbinding capacity of affected family members and unrelated controls. Mean values are marked as bars. Comparisonof values showed no difference between affected family membersand controls for either variable (Mann Whitney Wilcoxon test p>O.5).

was random except at position 1262 where adenine was replaced by guanine in one clone from the proband and at the same position replacement by cytosine in one clone from member I1 was found. The binding affinity of controls (mean 6.83 X 10’ I/mol +/- 4.5 X IO’) and affected family members (mean 7.23 X 10’ I/mol +/- 2.939 X IO’) and the maximum binding capacity of controls (mean 33 fmol/mg protein +/- 1.75) and affected family members (mean 27 fmol/mg protein +/- 0.76) were calculated.

Comparison of results (see

figure 3) revealed no difference between the affected family members with GTHR and controls (Mann Whitney Wilcoxon test p>O.5).

DISCUSSION

We have described a unique mutation in a kindred affected with generalised thyroid hormone resistance.

The mutation results in replacement of an arginine residue by

a histidine residue which is unlikely to produce a major conformational change in the secondary structure of the protein (as predicted by Garnier computer analysis of protein structure).

However changes in interactions

dependent on ionic charge

and/or hydrophobicity (perhaps between ligand and receptor or between receptorligand complexes in dimerization) may result from this amino acid substitution. Studies of mutant B thyroid hormone receptors indicate loss of hormone binding 610

A -h

Vol.

178,

No.

capacity

2, 1991

BIOCHEMICAL

and/or dimerization

the carboxyterminal show

binding

previously

BIOPHYSICAL

with retinoic acid receptor

end of the B thyroid hormone

affinity

lymphoblastoid

AND

and

maximum

order

that dimerization

of thyroid

hormone

normal.

The apparent

resistance

of hormone-receptor

clinical compensation

of magnitude

on

as reported

described

that many more mutations

are responsible

is difficult to confirm on clinical grounds

binding

studies

to confirm the diagnosis

and results

variable

(10,17).

mutation

involves synthesis

harbouring

the previously

reported

mutations.

(confirmed

interactions

two bands

strands).

on DNA sequence,

is postulated

The T,

of clinical

of

ten clones from each family

according

Denatured

four bands

DNA

to its sequence strand)

(wild type and mutant

so and

each with

The nature of the bands formed on SSCPA is dependent and buffer concentration

of unidentified

mutations

(20).

of pituitary thyroid hormone

of c-et-b p-2 which is selectively expressed

The ability to generate large amounts

SSCPA suitable for use in children

Use of SSCPA may

within regions of interest, for example it

that the more unusual syndrome

is due to an abnormality

Descriptions

(wild type and its complementary

produce

temperature

facilitate localisation

kindreds.

of time-consuming

during SSCPA is limited.

control samples

complementary

that

of exon H did. Current understanding

DNA of varying conformation

family members

by

of primers to amplify the exons

by sequencing

moves as single stranded produce

in

In our case SSCPA of PCR product

member tested) whilst SSCPA of PCR product and interstrand

receptor

abound and are of limited conclusiveness(l8,19).

for a predicted

of intrastrand

p thyroid

appears

(3,4,5) are all different and it is likely

Screening

no mutations

binding

for GTHR in different

disease

exon G predicted

may be the basis

of the disease, it is not surprising

mutations

have been accordingly

with

is in keeping with the model proposed

this and the three previously

manoeuvres

complexes

for the abnormal

Takeda et al(16). Given the clinical heterogeneity

cells(21).

T3 receptors

in this family since ligand-receptor

this family by elevation of thyroid hormones

affected

of

(11). These studies, if indicative of in vivo binding of T3, are compatible

the interpretation

of

Our binding studies

capacity

the same

COMMUNICATIONS

may occur with mutations

receptor(l5).

binding

cells of approximately

RESEARCH

of genomic

and neonates

following

resistance

in anterior pituitary

material by PCR makes collection

of tissue from

buccal smear.

Acknowledoments:

The assistance

binding investigations,

of Dr.Graham

Marilyn Walters

cell lines and Dr. Alan Ma who assisted

McLellan who performed

who assisted

the protein-

in the culture of lymphoblastoid

in analysis of binding assay results is gratefully

acknowledged. 611

Vol.

178,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Refetoff S (1962) Am. J. Physiol. 243: E66-E96 Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM (1966) Nature 324641-6 Sakurai A, Takeda K, Ain K, Ceccavelli P, Nakai A, Seino S, Bell GI, Refetoff S, and DeGroot W. (1989) Proc.Nat.Acad.Sci.USA. 66:6977-6961 Usala SJ, Bale AE, Gesundheit N, Weinberger C, Lash RW, Wondisford FE, McBride OW, and Weintraub BD. (1996) J.C/in./nvest. 6593-100 Usala SJ, Menke JB, Watson TJ, Berard J, Bradley WE, Bale AE, Lash RW, Weintraub BD. (1991) J.C/in EndocrinMetab. 72:32-a Sakurai A, Nakai A, and DeGroot W. (1990) Mo/.Ce//.Endocrino/. 71:63-91 Stockigt JR, Dyer SA Mohr VS, White EL, Barlow JW. (1966) J.Clin.Endocrin.Metab.

a. 9. 10. 11. 12. 13. 14.

J. EndocrinoLlnvest.

15. 16. 17. ia.

62:230-3.

Miller SA, Dykes DD, Polesky HF (1966) Nucleic Acids Research 16:1215 Orita M, Suzuki Y, Sekiya T, Hayashi K. (1969) Genomics 5:674-g lchikawa K, Hughes IA, Horwitz AL, DeGroot J. (1967) Metabolism 36:392-g Barlow JW, Denayer P. (1966) Acta Endocrinol. 117:327-32 Scatchard G. (1949) Ann. N Y Acad. Sci. 51: 660-6 MacPherson GA (1965) J. Pharm. Methods 14:213-a Magner JA, Petrick P, Menezes-Ferreira MM, Stelling M, Weintraub BD. (1966) 9:459-702

Glass Ck, Lipkin Sm, Devary OV, Rosenfeld MG (1969) Cell 59:697-766 Takeda K, Balzano S, Sakurai A, DeGroot W, Refetoff S. (1991) J. C/in. Invest. 87:496-502 Ceccarelli P, Refetoff S, Murata Y. (1966) J.Clin.Endocrin.Metab. 65:242-6 Smallridge RC, Parker RA, Wiggs EA, Rajagopal KR, Rein HG. (1969) Am. J. Med. 66:269-96

19.

Sarne

DH,

Refetoff

J.C/in. Endocrin. Metab.

20.

Orita

M,

lwahana

Proc.Natl.Acad.Sci.

21.

S,

Rosenfield

RL,

Farriaux

JP.

(1988)

66:740-6

H,

Kanazawa

H, Hayashi

K, Sekiya

T.

(1989)

66:2766-70

Hodin RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen PR, MooreDD, Chin WW. (1969) Science 244:76-a

612