T4-BINDING globulin (TBG) is a 54-kDa glycoprotein

Transcription

1 X/98/$03.00/0 Vol. 83, No. 10 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1998 by The Endocrine Society Complete Thyroxine-Binding Globulin (TBG) Deficiency Produced by a Mutation in Acceptor Splice Site Causing Frameshift and Early Termination of Translation (TBG-Kankakee)* GISAH A. CARVALHO, ROY E. WEISS, AND SAMUEL REFETOFF Departments of Medicine (G.A.C., R.E.W., S.R.), Pediatrics (S.R.), and the J. P. Kennedy, Jr. Mental Retardation Research Center (S.R.), The University of Chicago, Chicago, Illinois ABSTRACT Fourteen T 4 -binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T 4 because of fatigue associated with a low concentration of serum total T 4. Fifteen family members were studied showing low total T 4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids. (J Clin Endocrinol Metab 83: , 1998) T4-BINDING globulin (TBG) is a 54-kDa glycoprotein synthesized in the liver (1). TBG defects are inherited as X chromosome-linked traits, compatible with the presence of a single copy of the TBG gene on the X chromosome (2, 3). TBG deficiencies are divided into three types depending on the level of TBG in serum: complete deficiency (TBG-CD), partial deficiency (TBG-PD), and excess (TBG-E) (1, 2). These defects are fully manifested in hemizygous males but only partially in heterozygous females, because dosage compensation for X chromosome-linked genes between males and females is achieved by random inactivation of one of the X chromosomes in females (4). Although in most instances random inactivation produces TBG levels in heterozygous females that are intermediary between those in affected and nonaffected males (2), variable phenotypes have been observed in some instances of selective inactivation of the X chromosome carrying one of the two alleles (5). Fourteen distinct mutant TBGs have been identified at the gene level. Six mutations produce TBG-CD: TBG-CD5 (6), TBG-CD6 (7), TBG-CDJ (Japan) (8), TBG-CDY (Yonago) (9), TBG-CDB (Buffalo) (10), and TBG-CDBe (Bedouin) (11). Received May 18, Accepted June 22, Address all correspondence and requests for reprints to: Samuel Refetoff, The University of Chicago, MC3090, 5841 South Maryland Avenue Chicago, Illinois refetoff@medicine.bsd.uchicago.edu. * This work was supported in part by the National Institutes of Health Grants DK and DK and the Seymour J. Abrams Thyroid Research Center. Presented in part at the 70th Annual Meeting of the American Thyroid Association, October 15 19, 1997, Colorado Springs, Colorado. This paper is dedicated to the memory of Dr. Niall O Meara who as a fellow in the Section of Endocrinology contributed patients to this study and whose enthusiasm for endocrinology was an inspiration to all of us. TBG-CD5 has a single amino acid substitution causing aberrant posttranslational processing. The other five TBG-CDs have truncated molecules caused by early termination of translation caused by a single nucleotide substitution (TBG- CDB) or by a frameshift caused by a nucleotide deletion (TBG-CD6, TBG-CDJ, TBG-CDY, and TBG-CDBe) (Fig. 1). Until now, no mutations have been identified in noncoding regions of the TBG gene that cause TBG abnormalities. We describe here the first such mutation in a North American family of German ancestry with the TBG-CD phenotype. Sequence analysis of genomic DNA (gdna) revealed an A to G transition in the acceptor splice junction of intron II (ag3gg). Genotyping, using a mutation specific restriction site, confirmed the presence of this nucleotide substitution in all affected family members. Sequencing of TBG complementary DNA (cdna) transcribed from fibroblast messenger RNA (mrna) confirmed a shift in the ag acceptor splice site resulting in the insertion of a G inexon 2 that produces a frameshift with a premature stop codon. This premature stop in the transcript predicts a truncated TBG lacking the C-terminal 201 amino acids. Patients Materials and Methods The proband (II-3), a 55-yr-old woman, was treated for 20 yr with L-T 4 for what was presumed to be hypothyroidism based on low total T 4 (TT 4 ) concentration and fatigue. The diagnosis of TBG deficiency was confirmed when 15 family members were studied, some showing low TT 4 inherited as an X chromosome-linked trait. Skin biopsies were obtained from subjects II-3 and II-2 (Fig. 2A) for fibroblast cultures. All subjects gave informed consent for genetic testing in accordance with the University of Chicago Institutional Review Board. 3604

2 SPLICE SITE MUTATION OF TBG-CD KANKAKEE 3605 FIG. 1. Mutations causing TBG-CD (left) and TBG-PD (right) and their location on the gene. Both mutant nucleotide and resulting amino acid change are shown in bold letters. Y, Yonago; Be, Bedouin; B, Buffalo; J, Japan; SD, San Diego; G, Gary; M, Montreal; S, Slow; A, Aborigine; Cgo, Chicago; and Q, Quebec. For detailed description see Ref. 2. FIG. 2. Pedigree and genotype of family with TBG-CD Kankakee. A, Pedigree aligned with restriction enzyme analysis of gdna samples from respective individuals. B, Genotype analysis. Replacement of an A to a G at acceptor splice site in intron II creates a new recognition site for enzyme HaeIII that digests amplified fragment reducing its size from 467 to 411 bp. Affected females are heterozygous; affected and normal males show only mutant (411 bp) or normal (467 bp) allele, respectively. Mol wt, DNA size marker ( x, digested with HaeIII). Tests of thyroid function Serum TT 4, total T 3 (TT 3 ), total reverse T 3 (TrT 3 ), TSH, and TBG concentrations were measured by RIAs. The serum free T 4 index (FT 4 I) was calculated as the product of the serum TT 4 concentration and the T 4 -resin uptake value. P values were calculated using the unpaired Student s t test. Preparation of RNA, cdna, and gdna Total RNA was extracted from cultured skin fibroblasts and peripheral blood leukocytes from one normal individual, one heterozygous female, and one affected hemizygous male. RNA was extracted with phenol/guanidine isothiocyanate (Trizol, Gibco BRL, Gaithersburg, MD). The integrity of the RNA was assessed by formaldehyde-agarose gel electrophoresis. cdna was synthesized by RT of very small amounts of TBG mrna present in fibroblasts (illegitimate transcription) (12). Avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI) and oligo-dt primer were used to prepare the first strand of cdna. gdna was isolated from peripheral blood leukocytes (13). Sequencing of TBG gene gdna fragments were amplified by PCR using specific oligonucleotide primers (7). Amplified sequences included the entire coding re-

3 3606 CARVALHO ET AL. JCE&M 1998 Vol 83 No 10 gion, splice junctions, and the promoter region of the TBG gene. Amplified DNA segments were subcloned into M13 bacteriophage (New England Biolabs, Inc., Beverly, MA) or pgem T plasmids (Promega Corp.) and sequenced by the dideoxynucleotide chain termination method (Sequenase version 2.0, United States Biochemical Corp., Cleveland, OH). Two consecutive PCRs were needed to amplify a TBG cdna fragment across exon 2, Oligonucleotide primers for the first PCR were: 5 -GCATCTGATCTGTTCACTGAATTTC-3 sense, nucleotide (nt) and 5 -GTTTCAGACCATTGTCCTCT-3 antisense, nt A second, nested PCR, used the following oligonucleotides: 5 -CTAAT- TCAAGACCTCAAGC-3 sense, nt and 5 -GAAAACTTTG- GAACAAAC-3 antisense, nt The conditions of the PCR reactions were as follows: initial denaturation at 94 C for 3 min, followed by 35 cycles consisting of 94 C for 1 min, 55 C for 1 min, 72 C for 1 min, and a final extension at 72 C for 15 min. Automated fluorescence-based cycle sequencing (ABI, Perkin Elmer, Foster City, CA) was performed directly with the PCR products. The oligonucleotide primers used were the same as those used in the second PCR. Genotyping The replacement of the normal nucleotide A withagintheacceptor splice junction of intron II (ag3gg) creates a new restriction site for the enzyme HaeIII. gdna from peripheral blood leukocytes was amplified by PCR using the primers employed to amplify exon 2 from gdna for sequencing (7). Amplified gdna fragments were separated by electrophoresis in 3% agarose gel before and after digestion with HaeIII and photographed under ultraviolet light after staining with ethidium bromide. The allele containing the wild-type sequence resists the endonuclease and retains its original size of 467 bp, whereas the mutant allele is digested into two fragments, 411 and 56 bp (Fig. 2B). Results Thyroid function tests The clinical diagnosis of TBG deficiency was confirmed in 10 of 15 family members who showed cosegregation of the phenotype of low total serum iodothyronine levels and low TBG concentration inherited in a X chromosome-linked pattern (Figs. 2A and 3). The 4 affected males (II-1, II-2, II-4, and II-9) presented with low TT 4 levels of nmol/l (mean sd) compared with those of unaffected subjects of nmol/l, P Serum TT 3 was also significantly lower in the affected males ( nmol/l, P ) as compared with the unaffected individuals ( ). Serum TBG level of all affected males was undetectable, 0.5 mg/l (normal range mg/l, P ). The 6 affected females (I-1, I-2, I-5, II-3, II-6, and II-7) had on the average low serum TT 4 ( nmol/l, P 0.001) and TT 3 ( nmol/l, P ) levels. However, in only 3 of them were values below the lower limit of normal. In contrast, all affected females had serum TBG concentrations below the limit of normal ( mg/l). The 5 unaffected members of the family showed normal levels of total iodothyronines and TBG in serum (Fig. 3). All 15 individuals had normal serum FT 4 I and TSH, except for one affected female with suppressed TSH caused by treatment with L-T 4. Serum FT 4 I was significantly lower in the affected males, (normal range , P 0.01), as compared with unaffected individuals ( ) caused by underestimation of the resin T 4 uptake value in samples from the affected males. Genetic analysis gdna from an affected male with TBG-CD (II-2) was sequenced. The sequences of the exons and the promoter region did not show any abnormality. However, sequences of the flanking introns revealed an A to G transition in nu- FIG. 3. Tests of thyroid function in individual members of family. One affected female with suppressed TSH was on L-T 4 therapy.

4 SPLICE SITE MUTATION OF TBG-CD KANKAKEE 3607 cleotide 1671 of the acceptor splice site in intron II (Fig. 4). The mutation was present in all affected family members as confirmed by genotyping. Affected and normal males showed only the mutant allele or normal allele, respectively, and heterozygous females had both normal and mutant allele (Fig. 2B). To evaluate the effect of this mutation on the splicing pattern, it was necessary to determine the sequence of TBG mrna. TBG mrna is transcribed in liver and therefore is not readily accessible for sampling. We attempted illegitimate amplification of TBG mrna from peripheral blood leukocytes, as previously accomplished for the TSH receptor and other mrna (14), and failed on several attempts, though the extracted RNA was of good quality. However, we did succeed in synthesizing TBG cdna using RNA extracted from cultured skin fibroblasts. The sequence of this TBG cdna transcribed from fibroblasts of a normal subject was identical to that transcribed from liver mrna of an unrelated person with normal TBG, confirming the same pattern of splicing in both tissues. To determine the effect of the mutation on the patient s TBG transcripts, RNA from fibroblasts was processed in the same manner. Direct sequencing of the PCR cdna product from an affected male (II-2) revealed a shift of one nucleotide upstream of the authentic consensus ag acceptor splice site. Sequencing of cdna from a heterozygous female (II-3) demonstrated both the mutant and the normal allele. This mutation resulted in the insertion of the G in exon 2 causing a frameshift with a premature stop at codon 195 (Fig. 5). As a result of this mutation, the new acceptor splice site better matches the ideal consensus sequence compared to the wildtype sequence (Fig. 6). Discussion Mutations are randomly distributed throughout the TBG gene. There are no hot spot areas, and there is no correlation between the degree of TBG deficiency and location of the mutation (2). Until now, six TBG-CDs have been described, FIG. 4. Section of a sequencing gel of TBG gene from gdna displaying A to G transition of nucleotide 1671 in intron II at acceptor splice site junction. Lower case letters are in intron II; upper case letters are in exon 2, and substituted nucleotide is in bold and detached. FIG. 5. Direct sequencing of TBG gene from cdna showing codons 186 through 189. Normal sequence (common type, TBG-C) is compared with sequence of affected male (hemizygous). Latter sequence shows insertion of G (in bold) in exon 2 causing a frameshift downstream to amino acid 187 and ultimately a stop codon 195. Altered codons 188 and 189 are also indicated in bold. FIG. 6. mutation a3g in nucleotide 1671 (*) creates a new ag acceptor splice site (four nucleotides shown in bold) in intron II. Compared with wild-type sequence (also in bold), new acceptor recognition sequence is a better match to consensus sequence. This shifts the ag splice site resulting in insertion of a G(short arrow) in exon 2 and causing a frameshift that produces an early stop at codon 195. all located in the coding region of the gene. Five present early stop codons (nucleotide deletion or nonsense mutation) and one has a missense mutation (6 11). Herein we describe the first splice junction mutation causing TBG-CD (TBG-CD Kankakee). A novel A3 G transition in the acceptor splice site of intron II resulted in an one nucleotide shift in the acceptor splice site, predicting a truncated protein containing only the first 194 of 395 amino acids in the wild-type molecule. Similar to the other five TBG-CDs described, this mutation has an early stop codon. Instead of deletion of a nucleotide in a coding exon, the frameshift is caused by a single nucleotide insertion in exon 2. It has been estimated that up to 15% of known single base pair substitutions causing human genetic disease disrupt the normal splicing of mrnas (15). For genes expressed in a tissue-specific manner, analysis of the consequences of such defects on mrna processing has often been hindered by the inaccessibility of the mrna transcripts. Ectopic transcript analysis (illegitimate transcription) is an invaluable tool for the characterization of defects of mrna splicing, although splicing patterns exhibited by normal and ectopic transcripts might not be identical (16). We found that processed TBG

5 3608 CARVALHO ET AL. JCE&M 1998 Vol 83 No 10 mrna transcripts from both expressing (liver) and nonexpressing (fibroblast) cells were indistinguishable at the site of the mutation. Although illegitimate transcription should, theoretically, amplify minute amounts of any mrna present in all tissues, the amount of mrna in nonexpressing tissues depends not only on gene-specific but also tissue-specific influences (17). This explains why illegitimate amplification of TBG mrna from peripheral blood leukocytes failed, whereas that from skin fibroblasts was successful. The splice junction mutation causing TBG-CD Kankakee creates a new acceptor splice site one nucleotide upstream of the authentic acceptor splice site adding an intronic G to the downstream exon. This is in agreement with the majority of acceptor splice site mutations reported (15, 18). Exonic and intronic recognition sequences have an established role for splice-site selection, also important are the consensus sequences in the immediate vicinity of the exon-intron borders (19 21). A consensus value (CV) can be calculated by comparing the actual nucleotide sequence with the mutation (CVM) to the ideal consensus sequence (CVN) (22). A perfect match would have a CV score of 1. The calculated CV for the four nucleotide ( 1 to 3) wild-type acceptor splice recognition sequence using the primate nucleotide weight table of Shapiro and Senapathy (22) is 0.66 compared with the CV of 0.83 for the new sequence resulting from the mutation. We have, therefore, described a novel mechanism for the molecular basis of complete TBG deficiency identified by sequencing the intron exon junction of the TBG gene. Although this mechanism is not uncommon in other genetic defects, it has not been previously described in TBG defects of which 15 have been so far identified. Acknowledgments We thank Dr. Neal H. Scherberg and the Technical Staff of the Endocrinology Laboratory for performing the tests of thyroid function, and all family members for their consent to participate in this study. References 1. Bartalena L Recent achievements in studies on thyroxine-binding globulin (TBG). Endocr Rev. 11: Refetoff S, Murata Y, Mori Y, Janssen O, Takeda K, Hayashi Y Thyroxine-binding globulin: organization of the gene and variants. Horm Res. 45: Trent JM, Flink IL, Morkin E, Van Tuinen P, Ledbetter DH Localization of the human thyroxine-binding globulin gene to the long arm of the chromosome (Xq21 22). Am J Hum Genet. 41: Lyon MF The X inactivation centre and X chromosome imprinting. Eur J Hum Genet. 2: Okamoto H, Mori Y, Tani Y, et al Molecular analysis of females manifesting thyroxine-binding globulin (TBG) deficiency: selective X chromosome inactivation responsible for the difference between phenotype and genotype in TBG deficient females. J Clin Endocrinol Metab. 81: Mori Y, Takeda K, Charbonneau M, Refetoff S Replacement of Leu 227 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: Li P, Janssen OE, Takeda K, Bertenshaw RH, Refetoff S Complete thyroxine-binding globulin (TBG) deficiency caused by a single nucleotide deletion in the TBG gene. Metabolism. 40: Yamamori I, Mori Y, Seo H, et al Nucleotide deletion resulting in frameshift as a possible cause of complete thyroxine-binding globulin deficiency in six Japanese families. J Clin Endocrinol Metab. 73: Ueta Y, Mitani Y, Yoshida A, et al A novel mutation causing complete deficiency of thyroxine-binding globulin. Clin Endocrinol (Oxf). 47: Carvalho GA, Weiss RE, Vladutiu AO, Refetoff S Complete deficiency of thyroxine-binding globulin (TBG-CDBuffalo) caused by a new nonsense mutation in the thyroxine-binding globulin gene. Thyroid. 8: Inagaki A, Miura Y, Mizuno Y, et al Sequence analysis of TBG Deficiency in Bedouins. Folia Endocrinol Japn. 3[Suppl 2]: 507 (Abstract). 12. Cooper DN, Berg LP, Kakkar VV, Reiss J Ectopic (Illegitimate) transcription: new possibilities for the analysis and diagnosis of genetic disease. Ann Med. 26: Bell GI, Karam JH, Rutter WJ Polymorphic DNA region adjacent to the 5 end of the human insulin gene. Proc Natl Acad Sci USA. 78: Sunthornthepvarakul T, Gottschalk M, Hayashi Y, Refetoff S Resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med. 332: Cooper DN, Krawczak M Human gene mutations. Oxford, UK: Bios Scientific Publishers, Ltd.; Roberts RG, Bentley DR, Bobrow M Infidelity in the structure of ectopic transcripts: a novel exon in lymphocyte dystrophin transcripts. Human Mutation. 2: Kaplan J-C, Kahn A, Chelly J Illegitimate transcription: its use in the study of inherited disease. Human Mutation. 1: Maquat LE Defects in RNA splicing and the consequences of shortened translational reading frames. Am J Hum Genet. 59: Cooper TA, Mattox W The regulation of splice-site selection and its role in human disease. Am J Hum Genet. 61: Maniats T Mechanisms of alternate pre-mrna splicing. Science. 251: Krawczak M, Reiss J, Cooper DN The mutational spectrum of single base-pair substitutions in mrna splice junctions of human genes: causes and consequences. Human Genet. 90: Shapiro MB, Senapathy P RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15: