A Novel Mutation Causing Complete Thyroxine-Binding Globulin ...

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T4-binding globulin (TBG) is the major thyroid hormone transport protein in human serum. Inherited TBG abnormalities do not usually alter the metabolic status ...

0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society

Vol. 85, No. 10 Printed in U.S.A.

A Novel Mutation Causing Complete Thyroxine-Binding Globulin Deficiency (TBG-CD-Negev) among the Bedouins in Southern Israel YOSHITAKA MIURA, ELI HERSHKOVITZ, AKEMI INAGAKI, RUTI PARVARI, YUTAKA OISO, AND MOSHE PHILLIP First Department of Internal Medicine, Nagoya University School of Medicine (Y.M., A.I., Y.O.), Nagoya, Japan; Department of Pediatrics (E.H., M.P.) and Laboratory of Molecular Genetics (R.P.), Soroka Medical Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84101, Israel ABSTRACT T4-binding globulin (TBG) is the major thyroid hormone transport protein in human serum. Inherited TBG abnormalities do not usually alter the metabolic status and are transmitted in X-linked inheritance. A high prevalence of complete TBG deficiency (TBG-CD) has been reported among the Bedouin population in the Negev (southern Israel). In this study we report a novel single mutation causing complete TBG deficiency due to a deletion of the last base of codon 38 (exon

T

4-BINDING

GLOBULIN (TBG) is the principal transport protein for thyroid hormones in serum. TBG is an approximately 54-kDa acidic glycoprotein synthesized by the liver. Its single chain polypeptide core is composed of 395 amino acids (1). The human TBG gene is located on the long arm of chromosome X (Xq22.2) (2). The human TBG gene consists of five exons spanning 5.5 kbp. Its exon-intron organization is similar to that of other members of the serine protease inhibitor family (3). The first exon (exon 0) is a short noncoding sequence (4). Hereditary TBG abnormalities are manifested as complete TBG deficiency (TBG-CD), partial deficiency (TBG-PD), or TBG excess. By definition, complete deficiency is the absence of detectable TBG in serum of affected hemizygous males based on the current ability to detect TBG levels as low as 0.03% of the average normal levels in adults (16 mg/L) (5, 6). The prevalence of TBG deficiency varies from 1:3,000 to 1:15,000 (7, 8). TBG-CD is characterized by low total T4 and T3 levels, but normal free T4, free T3, and TSH levels. Although it is a harmless condition, it may cause undue concern among patients (parents) and physicians, resulting in unnecessary evaluation and therapy for presumed hypothyroidism (9). A high prevalence of TBG deficiency has been recently reported among the Bedouin newborns in the Negev area (Southern Israel) (10). In this study we demonstrate a new type of TBG-CD (TBG-CD-Negev). This new variant was Received December 16, 1999. Revision received June 27, 2000. Accepted June 30, 2000. Address all correspondence and requests for reprints to: Eli Hershkovitz, M.D., Pediatric Department, Soroka Medical Center, P.O.B. 151, Beer Sheva 84101, Israel. E-mail: [email protected]

1), which led to a frame shift resulting in a premature stop at codon 51 and a presumed truncated peptide of 50 residues. This new variant of TBG (TBG-CD-Negev) was found among all of the patients studied. We conclude that a single mutation may account for TBG deficiency among the Bedouins in the Negev. This report is the first to describe a mutation in a population with an unusually high prevalence of TBG-CD. (J Clin Endocrinol Metab 85: 3687–3689, 2000)

found in all the Bedouin subjects with TBG deficiency studied. Subjects and Methods Subjects Eight subjects (seven males and one female) from five different nuclear families of two Bedouin clans who had TBG-CD were studied after informed consent had been obtained from their parents. The pedigree of one family is presented in Fig. 1. These patients were detected between 1992 and 1996 by the national neonatal hypothyroidism screening program based on total T4 measurement on the second or third postpartum day. The diagnosis of TBG-CD was based upon the following findings: clinical euthyroidism, low serum levels of total T4 associated with normal serum TSH and free T4 levels, and the absence of immunoreactive TBG in the serum, using an assay capable of detecting at least 0.03% of the average normal concentrations (10). All of the infants were otherwise healthy.

Tests of thyroid function Serum total T4 was measured by RIA. TSH and free T4 were measured by enzyme-linked immunosorbent assay and competitive enzymelinked immunosorbent assay, respectively. Native TBG was measured by ultrasensitive RIA in the serum of three patients (the other patients were their relatives) at Prof. Samuel Refetoff’s laboratory as previously described (10).

Isolation and amplification of DNA Genomic DNA was obtained from the eight infants with inherited TBG-CD by extraction from peripheral blood monocytes. All exons (exons 0 – 4) and adjacent exon/intron junctions of the TBG gene were amplified by DNA thermal cycler (Perkin-Elmer Corp./Cetus, Norwalk, CT), using the oligonucleotide primers and PCR conditions as previously described (11).

DNA sequencing PCR products were isolated and subjected to autosequencer (PerkinElmer Corp./Cetus, Norwalk, CT). We sequenced both directions (sense

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MIURA ET AL.

and antisense) of the five exons and the exon-intron boundaries to confirm the data.

PmlI digestion Exon 1’s PCR products of the TBG gene were isolated and digested with PmlI. These samples were analyzed on 1.5% agarose gel electrophoresis.

Results

All subjects (seven males and one female) have normal TSH and free T4 concentrations, indicating a clinically euthyroid state (Table 1). TBG-CD was documented by the absence of immunoreactive TBG using an assay capable of detecting at least 0.03% of the average normal concentrations. Sequencing all of the exons and the adjacent exon/intron junctions of the TBG gene in the affected hemizygous males showed a deletion of the last base of the codon for amino acid 38 (exon 1), which led to a frame shift resulting in a prema-

FIG. 1. Pedigree of a Bedouin family with TBG-CD-Negev. Roman letters indicate generations I–III. f, Affected individuals.

TABLE 1. Thyroid function tests of the eight Bedouin infants with TBG-CD-Negev Test

Mean

Range

Normal values

Total T4 (nmol/L) Free T4 (pmol/L) TSH (mIU/L)

20.64 19.35 1.13

15.48–32.25 16.12–22.53 0.8–2.2

92.88–185.76 14.19–24.51 0.4– 4

FIG. 2. The structure of the normal TBG gene (TBG-N) is compared to that of the TBG-CD-Negev gene. A deletion of T in codon 38 causes a frame shift downstream, and a stop codon appears at codon 51, presumably resulting in a truncated peptide of only 50 residues.

ture stop at codon 51 (Fig. 2). Interestingly, the female subject has been found to be homozygous for this mutation. The mutation was confirmed by restriction endonucleotide digestion of amplified DNA. As the nucleotide deletion creates a new PmlI site, PCR products of exon 1 of the TBG gene from the affected subjects as well as those from a normal control were digested with PmlI. A 794-bp fragment from the affected subjects was digested to 528 and 266 bp, whereas the same 795-bp fragment from the normal control was resistant to this enzyme (Fig. 3). Discussion

Extensive molecular studies of the TBG gene in recent years have found that there are no hot spots for mutations in familial TBG deficiency, and there is no correlation between the degree of TBG deficiency and the location of the mutation (12). However, the mutation type usually determines the degree of TBG deficiency. Until now, six variants of TBG-CD have been characterized. Four (TBG-CD-Japan, TBG-CDYonago, TBG-CD-6, and TBG-CD-Buffalo) present premature stop codons. TBG-CD-5 is the only known variant causing complete deficiency due to a missense mutation. TBGCD-Kankakee has a mutation in the acceptor splice junction of intron 2, causing a frame shift and a premature stop at codon 195 (6, 11, 13–16). TBG-PD variants are caused by missense mutations (12). In this study we present the TBG gene analysis of Bedouin subjects with TBG-CD-Negev. All of the subjects have the same deletion of a single nucleotide in codon 38 in exon 1. We did not examine the biological expression of this mutant product. However, by analogy to TBG-CD-Japan, it can be assumed that the complete deficiency results from a secretion defect due to truncation of the TBG molecule, as lack of secretion by COS-1 cells of the TBG-CD-Japan mutant protein into the medium was demonstrated in an expression study (17). Although genetic variants of TBG-PD have been reported in increased prevalence in certain populations (e.g. TBGPD-A in Australian Aborigines and TBG-PD-S in African American) (18, 19), and a single mutation (TBG-CD-Japan) has been found in 30 Japanese males with TBG-CD (20), our report is the first to describe a mutation in a population with an unusually high prevalence of TBG-CD. This prevalence of 1:900 (or 1:450 in males) is significantly higher than those reported in other populations (10). We can assume that TBG-

COMPLETE TBG DEFICIENCY IN BEDOUINS (TBG-CD-NEGEV)

FIG. 3. Electrophoresis of a restriction digestion by PmlI of exon 1 PCR products of the TBG gene on 1.5% agarose gel (with ethidium bromide staining). A new restriction site for PmlI arises in exon 1 of TBG-CD-Negev gene. The predicted 794-bp fragment (due to one base deletion) from affected subjects was digested to 528- and 266-bp short fragments, whereas the intact 795-bp fragment from normal control (TBG-N) was resistant to this enzyme.

CD-Negev is probably the only mutation in this population, because it was demonstrated in subjects from both clans in whom TBG deficiency had been demonstrated. An early founder effect should be highly considered as the cause of this mutation. As the Bedouin population of the Negev has a similar genetic background as the Arab population of the Arab Peninsula (21, 22), our findings may apply to a much larger populations in the Middle East. References 1. Refetoff S. 1989 Inherited thyroxine-binding globulin abnormalities in man. Endocr Rev. 10:275–293. 2. Mori Y, Miura Y, Oiso Y, Hisao S, Takazumi K. 1995 Precise localization of the human thyroxine-binding globulin gene to chromosome Xq22.2 by fluorescence in situ hybridization. Hum Genet. 96:481– 482. 3. Huber R, Carrell RW. 1989 Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry. 28:8951– 66.

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4. Hayashi Y, Mori Y, Janssen OE, et al. 1993 Human thyroxine-binding globulin gene: complete sequence and transcriptional regulation. Mol Endocrinol. 7:1049 –1060. 5. Refetoff S, Murata Y, Vassart G, Chandramouli V, Marshall JS. 1984 Radioimmunoassays specific for the tertiary and primary structures of thyroxinebinding globulin (TBG): measurement of denatured TBG in serum. J Clin Endocrinol Metab. 59:269 –77. 6. Mori Y, Takeda K, Charbonneau M, Refetoff S. 1990 Replacement of Leu227 by Pro in thyroxine-binding globulin (TBG) is associated with complete TBG deficiency in three of eight families with this inherited defect. J Clin Endocrinol Metab. 70:804 – 809. 7. Mandel S, Hanna C, Boston B, Sesser D, LaFranchi S. 1993 Thyroxine-binding globulin deficiency detected by newborn screening. J Pediatr. 122:227–230. 8. Fisher DA. 1990 The thyroid. In: Kaplan SA, ed. Clinical paediatric endocrinology, 2nd Ed. Philadelphia: Saunders; 118. 9. Arisaka O, Hosaka A, Shimura N, Yabuta K. 1993 Thyroxine-binding globulin deficiency misdiagnosed as hypothyroidism. J Pediatr. 123:333–334. 10. Hershkovitz E, Leiberman E, Refetoff S, Pilpell, Phillip M. 1995 High prevalence of thyroxine-binding globulin deficiency among Bedouin infants in southern Israel. Isr J Med Sci. 31:500 –502. 11. Yamamori I, Mori Y, Hisao S, et al. 1991 Nucleotide deletion resulting in frameshift as a possible cause of complete thyroxine-binding globulin deficiency in six Japanese families. J Clin Endocrinol Metab. 73:262–267. 12. Refetoff S, Murata Y, Mori Y, et al. 1996 Thyroxine-binding globulin: organization of the gene and variants. Horm Res. 45:128 –38. 13. Ueta Y, Mitani Y, Yoshida A, et al. 1997 A novel mutation causingcomplete deficiency of thryoxine-binding globulin. Clin Endocrinol (Oxf). 47:1–5. 14. Li P, Janssen OE, Takeda K, Bertenshaw RH, Refetoff S. 1991 Complete thyroxine-binding globulin (TBG) deficiency caused by a single nucleotide deletion in the TBG gene. Metabolism. 40:1231–1234. 15. Carvalho GA. Weiss RE, Vladutiu AO, Refetoff S. 1998 Completedeficiency of thyroxine-binding globulin (TBG-CD Buffalo) caused by a new nonsense mutation in the thyroxine-binding globulin gene. Thyroid. 8:161–165. 16. Carvalho GA, Weiss RE, Refetoff S. 1998 Complete thyroxine-binding globulin (TBG) deficiency produced by a mutation in acceptor splice site causing frameshift and early termination of translation (TBG-Kankakee). J Clin Endocrinol Metab. 83:3604 –3608. 17. Miura Y, Kambe F, Yamamori, et al. 1994 A truncated thyroxine-binding globulin due to a frameshift mutation is retained within the rough endoplasmic reticulum: a possible mechanism of complete thyroxine-binding globulin deficiency in Japanese. J Clin Endocrinol Metab. 78:283–287. 18. Takeda K, Mori Y, Sobieszczyk S, et al. 1989 Sequence of the variant thyroxine-binding globulin of Australian Aborigines: only one of two amino acid replacements is responsible for its altered properties. J Clin Invest. 83:1344 –1348. 19. Waltz MR, Pullman TN, Takeda K. 1990 Molecular basis for the properties of the thyroxine-binding globulin-slow variant in American Blacks. J Endocrinol Invest. 13:343–349. 20. Inagaki A, Miura Y, Mori Y, Saito H, Seo H, Oiso Y. 1996 Gene screening of thryoxine-binding globulin (TBG) deficiency in the Japanese: only two mutations account for TBG deficeincy in the Japanese. J Clin Endocrinol Metab. 81:580 –585. 21. Parvari R, Hershkovitz E, Kanis A, et al. 1998 Homozygosity and linkagedisequilibrium mapping of the syndrome of congenital hypoparathyroidism, growth and mental retardation, and dysmorphism to a 1-cM interval on chromosome 1q42– 43. Am J Hum Genet. 63:163–169. 22. Diaz GA, Gelb BD, Ali F, et al. 1999 Sanjad-Sakati and autosomal recessive Kenny-Caffey syndromes are allelic: evidence for an ancestral founder mutation and locus refinement. Am J Med Genet. 85:48 –52.

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