Biochimica et Biophysica Acta, 1139 (1992) 307-310

307

© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00

BBADIS 61176

Sequencing of the variant thyroxine-binding globulin (TBG)-San Diego reveals two nucleotide substitutions Richard Bertenshaw a, David Sarne c, Joyce Tornari a, Michael Weinberg a and Samuel Refetoff a,b Department of Medicine Division of Biological Sciences, The University of Chicago, Chicago, IL (USA), b Department of Pediatrics, DiL'ision of Biological Sciences, The University of Chicago, Chicago, IL (USA) and c Department of Medicine, University of lllinois at Chicago, Chicago, IL (USA)

(Received 24 December 1991)

Key words: Thyroxine-bindingglobulin; TBG-San Diego; Sequencing; Variant; Mutation; Deficiency; Polymorphism;(Thyroid) Thyroxine-binding globulin (TBG) is a liver glycoprotein that transports thyroid hormone in serum. In 1989, a variant TBG was reported with reduced binding affinity for thyroxine (T4) and triiodothyronine (T 3) which results in low serum T4 and T 3 levels. This variant, TBG-San Diego (TBG-SD), also displays reduced heat stability but has a normal isoelectric focusing pattern. We now report the sequence of the entire coding region of TBG-San Diego. It reveals two nucleotide substitutions: one located in exon 1 which results in the replacement of the normal Ser-23 (TCA) with threonine (ACA) and the other, located in exon 3, changes the normal codon 283 of TFG (leucine) with that of TTI', (phenylalanine). Allele specific amplification was used to search for both nucleotide substitutions in four affected members of the family. Results confirmed the co-segregation of these nucleotide substitutions with the TBG-SD phenotype. The substitution in codon 283 has been previously described and exists as a polymorphism in some ethnic groups or in combination with other TBG variants with different physical characteristics. Thus, it appears that the replacement of Ser-23 with threonine is responsible for the observed alterations in physical properties of TBG-San Diego.

Introduction Thyroxine-binding globulin ( T B G ) is a 54 kDa glycoprotein of hepatic origin [1], which is the major thyroid hormone transport protein in blood [2]. The T B G gene has been shown to be carried on the X-chromosome [3] and its sequence and structure have been determined [4,5]. T B G abnormalities are inherited as X-linked traits and have been reported to present with excess, reduced or absent T B G in serum [6]. Males carry only a single copy of the T B G gene (hemizygous), while females may be either homozygous or heterozygous. The presence of a variant T B G does not alter the metabolic status but could alter the concentration of total thyroxine (TT 4) and total triiodothyronine (TT 3) in serum [7]. In 1989, we reported a variant thyroxine binding globulin (TBG) with reduced binding affinity for thyroxine (T 4) and triiodothyronine (T 3) in a male of English, Scottish, Irish and Cherokee extraction.

Correspondence to: S. Refetoff, Thyroid Study Unit (Box 138), The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637-1470, USA.

This variant, TBG-San Diego (TBG-SD), has a normal isoelectric focusing (IEF) pattern and minimally reduced heat stability [8]. As a result of decreased ligand affinity, serum T B G concentration derived from binding capacity assays run at non-saturating conditions underestimate the T B G concentration. We now report the amino-acid sequence of TBG-San Diego deduced by nucleotide sequencing of this variant T B G gene. Nucleotide substitutions were found in two codons, resulting in the replacement of the normal Ser-23 with threonine and the normal Leu-283 with phenylalanine. The substitution at amino acid 283 has previously been described and, when occurring alone, produces no detectable alterations of the T B G molecule. Although this substitution at codon 23 is the source of the observed phenotypic alterations of TBGSan Diego, the replacement at codon 283 may contribute to the variant phenotype, since mutations in three other unique T B G gene variants have been found in association with the presence of Phe-283 [9-11]. Materials and Methods Sequencing o f the T B G - S a n Diego gene. D N A was isolated from peripheral white blood cells of an af-

3O8 TABLE I

Sequences and h)cations of oligont,'h'otidc primet:~ The bold letters are the substituted nucleotides of TB(;-San [)icg(L (s), sense; (a). antisensc Primer

Sequence

Location

A(s) B (a) C (s) D (a) E(s) F(a) G (s) t1 (a) J (s) K(a) L(s)

5'-CCCTGATGAGCACATCATCA-3' 5-'CAGTGGAGCAGATCACTGTG-3' 5'-CTTGTTGGTACTGATACCGG-3' 5'-CTTGGCATATTCTAGTGATC-3' 5'-AAGCTTGATATGGTGATTGC-3' 5'-TCAGAACTGCATCTCACCA-3' 5'-CGCCAGAGAATGATTGACTA-3' 5'-CCATTGCAATACACACGTGC-3' 5'-TGCCACTCTCTACAAGATGT-3' 5'-TGCCACTCTCTACAAGATGA-3' 5'-GTCTTGAATTAGACCCACAA-3' 5'-CCCACAGATGGGTTGACTTG-3'

M (s)

5'-CCCACAGATGGGTTGACTTT-3'

144 nucleotides 5' from exon I 108 nucleotides 3' from exon I 57 nucleotides 5' from exon 2 116 nucleotides 3' from exon 2 48 nucleotides 5' from exon 3 68 nucleotides 3' from exon 3 171 nucleotides 5' from exon 4 within exon 4, 45 nucleotides downstream from TAG witihin exon 1, 108 nucleotides downstream from AT(} within exon 1, 108 nucleotides downstream from ATG within exon 1,573 nucleotides downstream from AT(I 7 nucleotides 5' from exon 3 and extending 13 nucleotides into cxon 3 7 nucleotides 5' from exon 3 and extending 13 nucleotides into cxon 3

I

(s)

fected male from the San Diego family as previously described [12]. Each of the four coding exons of the TBG gene were amplified separately by the polymerase chain reaction (PCR) using specific oligonucleotides for flanking non-coding regions (introns or a non-coding exon) (Table I). Amplified D N A segments were isolated on DEAE-nitrocellulose paper, and ligated into M13. Sequencing was carried out with a modified T7 DNA polymerase by the dideoxynucleotide chain termination method (Sequenase Version 2.0, United States Biochemical, Cleveland, OH). Endonucleases were purchased from New England Biolabs, Beverly, MA.

Screening of family members by allele specific amplification. Whole blood samples were obtained from four members of the family. On the basis of T B G typing by criteria previously established [13,14], three were hemizygous for TBG-San Diego and one was heterozygous, TBG-San D i e g o / T B G - C (common-type TBG). DNA from an unrelated male with TBG-C was used as a control. Their D N A was screened for the substitutions in codon 23 (exon 1) and for that in codon 283 (exon 3) by the method of allele specific amplification (ASA) [15]. The specific 'mutant' and 'wild-type' oligonucleotides used contained the substituted or normal nucleotide at the 3' terminal position of one of each pair of primers (Table I). The PCR product was resolved by electrophoresis on 1% agarose gels and photographed under UV light after staining with ethidium bromide. Results

Fig. 1 shows the strategy of TBG-San Diego gene sequencing. The sequences of the coding exons and their adjacent introns were identical to those of TBG-C with two exceptions. In exon 1, a T-to-A transversion

was detected in the codon for amino acid 23 which resulted in the substitution of the normal serine (TCA) with threonine (ACA), (Figs. 1 and 2). In exon 3 a G-to-T transversion in the codon for amino acid 283 replaced the normal leucine (TTG) with phenylalanine (TTT) (Fig. 1). Both nucleotide substitutions were confirmed by sequencing the products of two separate PCR amplifications of each of the two exons. Fig. 3 depicts the results of TBG typing by both direct TBG analysis and by ASA. Members of the family hemizygous for TBG-San Diego or heterozygous (TBG-San D i e g o / T B G - C ) and an unrelated male with TBG-C were identified by direct analysis of serum. To determine whether one or both nucleotide substitutions were associated with the presence of the TBG-San Diego allele, oligonucleotides were synthesized and positioned so as to direct allele specific amplification. The strategy is depicted in Fig. 3. As expected from their close proximity, the two substituted nucleotides cosegregated. As shown in Fig. 3, DNA from hemizy-

x

ATG

A 'a

I

I~ B .~

500 bp

I

C o~--

TAG

I~ ;D

E:~

(23)

(283)

Ser TCA

Leu TTG

ACA Thr

TTT Phe

)

~ . F ~ H

)

Fig. 1. Schematic representation of the TBG gene and the strategy of sequencing. Exons are depicted by boxes (coding sequences in black) and locations of translation initiation (ATG) and termination (TAG) codons are indicated. Arrows indicate regions and directions sequenced. The two mutations and their resulting amino acid substitutions are shown. Letters A - H represent positions of oligonucleotides used for exon amplification from genomic DNA.

309 (21) L y s M e t AAG & / ~

Set

Set

~

~::C

from the non-affected control male expressing only TBG-C was amplified with only the 'wild' oligonucleotide primer pair (I and L); and both primer pairs amplified DNA obtained from the heterozygous member of the family expressing both the 'wild' and 'mutant' TBGs.

T i e (25) ATT

G TBG-C

A

T e

Discussion

Although two mutations were detected in affected individuals, the newly described mutation causing the replacement of serine-23 by threonine in the TBG molecule is the likely cause of the altered properties in TBG-San Diego for two reasons. Firstly, the substitution has been shown by ASA to co-segregate with those persons who are affected. Secondly, and perhaps more importantly, the substitution of Leu-283 by phenylalanine when present alone, has been shown not to alter the physical properties of TBG. TBG-San Diego most closely resembles TBG-A, found in 40% of Australian Aborigines, both in its physical properties and gene substitutions [8,11,13]. Both of these variants have reduced affinity for thyroid hormone but relatively well preserved levels of immunoreactive TBG. Both TBG-A and TBG-SD contain the Phe-283 substitution associated with an additional unique substitution in exon 2 for TBG-A and

TBG-SD

A~

Thr (23)

Fig. 2. Section of a sequencing gel displaying the exon 1 substitution in TBG-San Diego (SD). Thymidine in codon 23 of TBG-C is replaced with adenine in TBG-SD. The resulting amino-acid substitution is depicted. The substitution in codon 283 has been previously demonstrated [1.1].

gous affected persons, whose serum contained exclusively TBG-San Diego, was amplified only with the 'mutant' oligonucleotide primer pair (J and M); DNA

, n

m

r--~

n

m

r--I

n

m

I-'--1

n

~

m

n

m

r----t

I TBG-SD J TBG-C

A,.

E .....

5

n m

n m

n m

n m

'

~

3

'

n m L TBG-SD M TBG-C

B

5' WS

__

~ON 3

~IVS i

309 - -

Fig. 3. Allele specific amplification (ASA) to search for Thr-23 and Phe-283 in members of the family expressing TBG-SD. Four affected family members were studied. An unrelated male with TBG-C served as a control. Their phenotype determined by TBG analysis in serum and family relation is depicted in the pedigree. A S A was carried out by PCR using pairs of oligonucleotide primers having either both normal (n) sequences or one mutant (m) sequence (depicted by the schematic representations on the right, not drawn to scale). (A), ASA of Thr-23 and (B), ASA of Phe-283. Note the co-segregation of the two nucleotide substitutions. Marker: phiX174 Hae III digest. For specific primer sequences see Table I.

310 TABLE ll Comparison of physical properties o]" TBG variants with a Phe-283 substitution

N1, normal; D, decreased. Variants with Phe-283

Additional substitution

AffiNity for T4

IEF pattern

Heat stability

Rcf.

San Diego Aborigine CD-5 Quebec Polymorphism

Exon 1 Ser-23 --, Thr Exon 2 Ala-191 ~ Thr Exon 2 Leu-227 ~ Pro Exon 4 His-331 --, Tyr None

D D complete deficiency D NI

NI NI

D D

Shift N1

D NI

This report 11 II) 9 ltl

exon 1 for T B G - S D . It is likely t h a t t h e a r e a s of t h e T B G m o l e c u l e e n c o d e d by b o t h exon 1 a n d 2 play m a j o r roles in t h e f o r m a t i o n of t h e h o r m o n e b i n d i n g site. Study o f t h e s e a n d o t h e r v a r i a n t s h o p e f u l l y will h e l p to e l u c i d a t e f u r t h e r the s t r u c t u r e o f the h o r m o n e b i n d i n g site, which has b e e n h a m p e r e d so far by t h e inability to crystallize T B G for t h r e e d i m e n s i o n a l studies. T h e Phe-283 s u b s t i t u t i o n has now b e e n d e m o n s t r a t e d in f o u r o t h e r v a r i a n t T B G g e n e s a s s o c i a t e d with u n i q u e s e c o n d m u t a t i o n s ( T a b l e II). T h e s e m u t a tions w e r e d e t e c t e d in subjects o f widely d i f f e r e n t e t h n i c b a c k g r o u n d s and, unless t h e previously r e p o r t e d relatively low allele f r e q u e n c y [10] p r o v e s to b e e r r o neous, its a s s o c i a t i o n with o t h e r n u c l e o t i d e substitutions is unlikely t h e result o f a f o u n d e r effect. A l t h o u g h , w h e n f o u n d alone, Phe-283 d o e s n o t a l t e r the p r o p e r t i e s o f t h e m o l e c u l e , it m a y c o n t r i b u t e to the c h a n g e s o b s e r v e d in a s s o c i a t i o n with o t h e r single a m i n o - a c i d substitutions. To study t h e relative contrib u t i o n s o f Phe-283 a n d its a s s o c i a t e d u n i q u e m u t a t i o n s to t h e a l t e r a t i o n s o b s e r v e d in t h e p a r t i a l deficiency variants, it will b e n e c e s s a r y to c o n s t r u c t r e c o m b i n a n t T B G clones having the two s u b s t i t u t i o n s a l o n e a n d in c o m b i n a t i o n . T h e i r e x p r e s s i o n w o u l d allow d i r e c t analysis of t h e p r o p e r t i e s of the g e n e p r o d u c t .

Acknowledgements W e t h a n k t h e ' S a n D i e g o ' family for t h e i r c o o p e r a tion. W e a r e i n d e b t e d to Dr. L o u i s L i n a r e l l i for h e l p ing o b t a i n i n g b l o o d s a m p l e s a n d to Dr. J e r a l d N e l s o n for b r i n g i n g t h e family to o u r a t t e n t i o n . W e a p p r e c i a t e the h e l p f u l c o m m e n t s o f Dr. O n n o E. J a n s s e n a n d t h e assistance a n d p r e p a r a t i o n of t h e m a n u s c r i p t by Mrs.

Y o l a n d a W. R i c h m o n d . This w o r k was s u p p o r t e d in p a r t by U,S. Public H e a l t h Service grants DK-15070, D K 40181. Dr. B e r t e n s h a w was s u p p o r t e d by the End o c r i n o l o g y T r a i n i n g g r a n t DK-0701 1.

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