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Complete Thyroxine-Binding Globulin (TBG) Deficiency in Two Families without Mutations in Coding or Promoter Regions of the TBG Genes: In Vitro Demonstration of Exon Skipping

Departments of Medicine (S.Reu., P.E.M., S.Ref.) and Pediatrics (S.Ref.), the J. P. Kennedy Jr. Mental Retardation Research Center (S.Ref.), and the Committee on Genetics (A.D., S.Ref.), The University of Chicago, Chicago, Illinois 60637; University of Mississippi Medical Center (G.W.M.), Jackson, Mississippi 39216; and University Clinic for Internal Medicine III (H.V.), A-1090 Vienna, Austria

Address all correspondence and requests for reprints to: Samuel Refetoff, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: . refetoff{at}medicine.bsd.uchicago.edu

Abstract

Inherited thyroxine-binding globulin (TBG) deficiency is caused by mutations in the TBG gene located on the X-chromosome. We now describe two families (K and H) with X-linked complete TBG deficiency without mutations in the coding or promoter regions of the TBG gene. The propositi of both families presented with euthyroid hypothyroxinemia and were found to have undetectable TBG in serum. Affected females had approximately half the normal serum TBG concentration except for one woman who also had undetectable TBG (family H). All four of her children (two boys and two girls) were affected.

Affected members of family K had no mutations in any of the five exons or in the minimal promoter region of the TBG gene. However, a G to A substitution, five base pairs downstream from exon 3, was associated to the phenotype of TBG deficiency (TBG-Jackson) and was not present in 100 normal alleles. In contrast to individuals without this mutation, no TBG mRNA could be detected in fibroblasts of the propositus, expressing solely TBG-Jackson. In vitro transcription of genomic DNA containing the mutant intron in an exon trapping system showed that this mutation, reducing the consensus value on the 5' donor splice site, affects the normal splicing process. The transcript of TBG-Jackson lacks exon 3 and is unstable. The deduced amino acid sequence has a frameshift and an early stop codon at position 325.

Affected subject of family H had no mutations in the TBG gene including all exons, all introns, the minimal promoter, and the 3' untranslated sequence. However, an intragenic A/G polymorphism (125 bp upstream from exon 2) was identified. It allowed us to confirm a cosegregation of the phenotype to the TBG gene and to show that the single female with complete TBG deficiency was homozygous for the polymorphic TBG allele. The cause of TBG deficiency in this family remains unknown.

THYROXINE-BINDING GLOBULIN (TBG) is a 54 kDa acidic glycoprotein synthesized by the liver (1). It is composed of a single polypeptide chain of 395 amino acids and is encoded by a single gene copy located on the long arm of human X-chromosome (Xq22.2) (2, 3, 4). With a single exception (5), inheritance of TBG abnormalities, including TBG deficiency and TBG excess, follows the X-linked pattern (6).

Complete TBG deficiency (TBG-CD) is defined as undetectable TBG concentration in hemizygotes (XY males or XO females) who express only the mutant allele (6). To date, 10 distinct mutations have been reported to cause TBG-CD (7, 8, 9, 10, 11, 12, 13, 14). These mutations are randomly distributed throughout the TBG gene. Six of these mutations (7, 8, 9, 10, 11, 13) occur in the coding region, leading to premature stops and truncated TBG molecules or a failure to secrete variant TBG molecules due to an aberrant posttranslational processing. Only one mutation, TBG-Kankakee (12), occurs in a noncoding region.

We describe herein two families with X-linked TBG-CD without mutations in the coding or promoter regions of the TBG gene. In one family, TBG deficiency may be associated with an unstable TBG mRNA caused by a mutation at 5' donor splice site of intron IV. However, the cause of TBG deficiency in the other family remains unknown.

Patients and Methods

Patients

The propositus of family K was a 3-yr-old boy who was found to have euthyroid hypothyroxinemia at birth but who developed normally without treatment. The diagnosis of TBG-CD was subsequently confirmed. Family members including his parents and his twin brother were studied. Results revealed that his mother had partial TBG deficiency, consistent with X-linked mode of inheritance. Punch skin biopsies were obtained from the propositus, his mother, and two normal individuals for fibroblast culture.

The propositus of family H was a 30-yr-old male with low serum total T₄ (TT₄) and total T₃ (TT₃) concentrations and a normal TSH level. No TBG was detected in serum. Blood samples from family members including his parents and three siblings were obtained for studies of thyroid function and genotyping.

Family K was Caucasian and lives in the United States, and family H was of Lebanese origin. The study protocols were approved by the institutional review board, and informed consent was obtained from all individuals who participated in the study.

Test of thyroid function

Serum TT₄, TT₃, total reverse T₃ (TrT₃), TSH, and TBG concentrations were measured by RIAs. TBG was measured by binding capacity and by RIA, as previously described (15, 16). The serum free T₄ index (FT₄I) was calculated as the product of the serum TT₄ concentration and the T₄-resin uptake ratio. In some serum samples, free T₄ was also measured by equilibrium dialysis (Nichols Institute Diagnostics, San Juan Capistrano, CA). Titers of thyroid peroxidase (TPO) and thyroglobulin (TG) autoantibodies were measured by agglutination.

Preparation of RNA and amplification of cDNA

RNA was obtained from cultured skin fibroblasts of the propositus of family K (II-1), his mother (I-2), and two normal individuals. Total RNA was extracted with phenol/guanidine isothiocyanate (TRIZOL, Life Technologies, Inc., Gaithersburg, MD). cDNA was synthesized with reverse transcriptase (Promega Corp., Madison, WI) using an oligo(dT) primer. Two consecutive PCRs were required to amplify a TBG cDNA fragment across exon 3. The forward primer used in the first round of PCR was 5'-CTGAGATTGTGGAGAACCTTG-3' (located in the middle of exon 1), and the reverse was 5'-CCATGGATCCAATGGCCTTTTTCCCAACTA-3' [located in the 3' untranslated (3'UT) region of exon 4]. PCR conditions were: denaturation at 94 C for 1 min, annealing at 58 C for 1.15 min, and extension at 72 C for 1.30 min; a total of 40 cycles. The forward primer used in the second round of PCR was 5'- ACTATCACCTAGTGGATAT-3' (located in exon 2), and the reverse was 5'-TTTTCAGGCTGATCCGAAAG-3' (located in exon 4). PCR conditions were: denaturation at 94 C for 1 min, annealing at 52 C for 1 min, and extension at 72 C for 1 min; a total of 38 cycles. The cDNA fragment size was 440 bp.

Sequencing of the TBG gene

Genomic DNA (gDNA) was isolated from peripheral blood leukocytes (17). Fragments of gDNA were amplified by PCR using specific oligonucleotide primers (Fig. 1). Amplified sequences included all five exons with splice junctions and the promoter region of the TBG gene. Primers used to amplify exons 2, 3, and 4 were as previously described (11). Primer sequences used to amplify exon 1, exon 0, and the promoter region are shown in Table 1. In family H, we also amplified all the introns and the 3'UT region. For primer sequences location and sizes of fragments amplified, see Table 1 and Fig. 1. PCR conditions were: denaturation at 94 C for 1 min, annealing at temperatures shown in Table 1 for 1 min, and extension at 72 C for 1 min; a total of 38 cycles. All PCRs were performed in a volume of 100 µl with 8 mmol/liter of each primer and buffer containing 2.5 mmol/liter MgCl₂, 10 nmol/liter deoxynucleotide triphosphates, and 0.2 U Taq DNA polymerase. PCR products were sequenced directly using an automated fluorescence-based cycle sequencer (ABI, Perkin-Elmer Corp., Foster City, CA). Primers used for sequencing were the same as those used to perform PCRs. Additionally, for exon 1, intron I, intron II, and intron III, internal primers were needed for sequencing, as listed in Table 1.

View larger version (11K): [in this window] [in a new window]   Figure 1. Sequencing strategy. The TBG gene is drawn to scale, with exons depicted as boxes (coding areas in black) and introns as connecting lines. Double-headed arrows identify amplified regions using primers listed in Table 1 (A-G) and primers previously described (PD) (11 ). The cross-hatched rectangle denotes the DNA region amplified to generate the fragment used in the exon trapping experiment.

In family K, the replacement of the normal G for an A at the donor splice junction of intron IV creates an additional restriction site for the enzyme Tsp 509I. Peripheral blood leukocytes gDNA from family members and 50 random normal females were amplified by PCR using the forward primer 5'-ATAAGCTTGATATGGTGATTG-3' and the reverse primer 5'-AAAATAAGAAAGTTGAATCgATTG-3' (mismatched nucleotide shown in lowercase). The mismatched nucleotide was created to eliminate another restriction site for enzyme Tsp 509I that is present in both normal and mutant sequences. This resulted in a bigger size and a better separation of PCR products after digestion. PCR conditions were: denaturation at 94 C for 1 min, annealing at 54 C for 1 min, and extension at 72 C for 30 sec; a total of 38 cycles. The size of the PCR product was 275 bp. Amplified gDNA fragments were separated by 2% agarose gel electrophoresis before and after endonuclease digestion with Tsp 509I. The allele containing the normal sequence yielded two fragments of 244 and 31 bp after digestion, whereas the mutant allele yielded three fragments of 202, 42, and 31 bp.

In family H, an A to G substitution in intron II is polymorphic because the TBG sequence reported by Akbari et al. (18) has a G at the same position. The presence of a G creates an additional restriction site for the enzyme HincII. After extraction from family members’ peripheral leukocytes, gDNA fragments were amplified by PCR using the forward primer 5'-TGGGTATGATTCCTGGCTCT-3' and the reverse primer 5'-TTATCAACTTACATTGGAAAGTTT-3'. PCR conditions were: denaturation at 94 C for 1 min, annealing at 60 C for 1 min, and extension at 72 C for 30 sec; a total of 38 cycles. The size of the PCR product was 232 bp. The amplified fragments were separated by 2% agarose gel electrophoresis before and after endonuclease digestion with HincII. After digestion, the allele containing an A yielded two fragments of 205 and 27 bp, whereas that containing a G yielded three fragments of 143, 62, and 27 bp.

Southern blot

Despite two consecutive PCRs performed using cDNA from fibroblasts, we were unable to visualize the fragment amplified from the propositus’ sample, although it was present in those of two normal individuals. To detect small amounts of PCR product that might exist, a Southern blot was performed by standard technique and was probed using a ³²P-labeled TBG specific primer (5'-GCATCTGAACTGCGCACTGAATTTTC-3').

In vitro analysis of splicing

The exon trapping system (Life Technologies, Inc.) was used to investigate the effect of the mutation in the donor splice site of intron IV. gDNAs from the propositus and from a normal individual containing exons 3 and 4 and the intervening intron IV sequence of the TBG gene were amplified using the primers 5'-ctgtgagtttggagccatgtattgac-3' and 5'-CAAGCTCACATCAATCACACCAGGCT-3' at an annealing temperature of 58 C for 35 cycles. The 1078 bp PCR products were gel-purified (QIAGEN Inc., Valencia, CA) and subcloned into the pGEM-T Easy vector (Promega Corp., Madison WI). After confirmation that the sequences were correct, the inserts were cloned into the NotI site of the pSPL3 vector (New England Biolabs, Inc. Beverly, MA). Plasmids were amplified in Escherichia coli JM109, extracted, purified (QIAGEN Inc.), and used for transfection into COS 7 cells.

Cells were grown in 10-cm dishes in DMEM (Life Technologies, Inc.) supplemented with 10% bovine calf serum (Life Technologies, Inc.) and 50 mg/liter gentamycin in a 10% CO₂ atmosphere at 37 C. When cells reached approximately 50% confluence, 10-cm dishes were transfected with 10 µg of plasmid DNA using SuperFect reagent (QIAGEN Inc.). Plasmids consisted of normal TBG, TBG-Jackson, and a control pSPL3. Twenty-four hours later, cells were harvested, total RNA was extracted with TRIZOL (Life Technologies, Inc.), and cDNA was synthesized. After two rounds of PCR with intervening BstXI digestion, according to the manufacturer’s instructions, the products were gel-purified, cloned into the pAMP10 vector, and then sequenced (ABI, Perkin-Elmer Corp.).

Results

Family K

The pedigree of family K and results of thyroid function tests are shown in Fig. 2. The serum TT₄ and TT₃ concentrations of the propositus were low, whereas his TSH level was normal, with FT₄I above the upper limit of normal; serum TBG was undetectable. His mother (I-2) also had low serum TT₄ and TT₃ concentrations, although her TBG capacity was half normal, a finding typical for heterozygotes in families with TBG-CD (6). Results of thyroid function tests in serum samples from his father (I-1) and his twin brother (II-2) were within the normal range.

View larger version (38K): [in this window] [in a new window]   Figure 2. Pedigree, phenotype, and genotype of family K with TBG-CD. A, The phenotype determined from the concentrations of TT3, TT4, and TBG. Abnormal values are in bold numbers. Free T3 was also measured in the propositus and was found to be mildly elevated at 5.1 pg/ml (normal range, 2.3–4.2 pg/ml). B, Pedigree of the family aligned with results of thyroid function tests and the gel electrophoresis data. C, Confirmation of cosegregation of the mutant allele (G to A at the 5' splice junction of intron IV) with the TBG-CD phenotype by genotyping with Tsp 509I endonuclease digestion. The undigested DNA fragment is 275 bp. In the presence of the mutation, the digested fragment is reduced from 244 to 202 bp.

View larger version (63K): [in this window] [in a new window]   Figure 3. Sequencing gel showing the mutation (G to A) at the 5' splice junction of intron IV (left), compared to a normal sequence (right). Uppercase letters indicate nucleotides of exon 3, and lowercase letters indicate nucleotides in intron IV.

View larger version (70K): [in this window] [in a new window]   Figure 4. Southern blot analysis of TBG cDNA in the propositus (left lane) and two normal individuals (center and right lanes). This TBG band is absent in the propositus.

View larger version (39K): [in this window] [in a new window]   Figure 5. Pattern of splicing in normal TBG and TBG-Jackson using exon trapping system. A, Electrophoresis of the processed normal TBG, TBG-Jackson, and control pSPL3 after completion of the exon trapping protocol. The abnormal product of splicing in TBG-Jackson has two bands: the 325 bp band found in normal TBG and a 177 bp band representing the loss of exon 3 characteristic of TBG-Jackson. B, Diagram showing the processing of the TBG gene fragments cloned into pSPL3 vector. Chromatograms obtained from the processed normal TBG (top) show the retention of exon 3 sequence flanked by the vector sequences (V). In contrast, the chromatogram of the lower size product (bottom), derived from the processing of the TBG-Jackson, shows only the connected vector sequences without exon 3. Note that excision of exon 4 is expected due to the absence of the donor splice site. C, Alignment of the normal TBG amino acid sequence with that of TBG-Jackson in which exon 3 is skipped. The resulting frameshift at codon 280 and the nonsense amino acid sequence with a stop at codon 325 are shown in red. It is unlikely that such an aberrant protein is synthesized owing to the instability of the corresponding mRNA. Negative numbers indicate the signal peptide, and positive numbers indicate the mature protein.

The pedigree and results of thyroid function tests are shown in Fig. 6. The propositus (II-1) and his brother (II-2) had a phenotype of TBG-CD with very low serum TT₄, TT₃, and TrT₃ concentrations, a normal TSH and an undetectable TBG concentration. Free T₄ measured by dialysis in subject II-2 was well within the range of normal. His two sisters (II-3 and II-4) had half normal TBG levels, suggesting a heterozygous state. Interestingly, his mother (I-1) displayed a TBG-CD phenotype, suggesting a homozygous state of TBG-CD. At the time her blood sample was obtained, she was taking L-T₄, explaining the suppressed serum TSH. TBG was measured also by RIA at Nichols Institute Diagnostics and by another, highly sensitive RIA (16). Values in the affected males were undetectable or less than 0.3 and 0.1 mg/liter, respectively.

View larger version (39K): [in this window] [in a new window]   Figure 6. Pedigree, phenotype, and genotype of family H with TBG-CD. A, The phenotype determined from the concentrations of TT3, TT4, and TBG. Abnormal values are in bold numbers. The TBG concentration of the propositus was remeasured in our laboratory with a more sensitive assay and was found to be less than 0.01 mg/liter (normal range, 11–21 mg/liter). B, Pedigree of the family, aligned with results of thyroid function tests and the gel electrophoresis data. C, Confirmation of cosegregation of the polymorphic allele (A to G in intron II) with the TBG-CD phenotype by genotyping with HincII endonuclease digestion. The undigested DNA fragment is 232 bp. In the presence of the polymorphic G, the digested fragment is reduced from 205 to 143 bp.

Discussion

We describe here two families with TBG-CD without mutations in the coding or promoter regions of their TBG gene. In family K, we found a substitution of a normal nucleotide G with an A at the 5' donor splice site of intron IV, 5 bp downstream from exon 3 (TBG-Jackson). Genotyping proved segregation of this substitution with the TBG-CD phenotype in an X-chromosome-linked pattern. This substitution is a mutation, because it was not present in 100 random alleles.

To evaluate the effects of this mutation on the TBG molecule, it was necessary to determine the TBG mRNA sequence. We synthesized TBG cDNA using RNA extracted from cultured skin fibroblasts, as successfully done previously (12). However, despite multiple attempts, we were unable to obtain TBG cDNA fragment from the propositus’ RNA, although it was present in two normal individuals. Only normal TBG cDNA could be synthesized from the mother’s fibroblasts, despite the fact that she is heterozygous by genotyping. The failure to obtain TBG cDNA from the fibroblasts of the propositus was unlikely due to technical problems, because together with our previous experience using different PCR primers (12), we were able to obtain TBG cDNA fragments from 6 of 7 individuals, except the propositus. Southern blot analysis confirms the lack of TBG mRNA in the propositus.

Because the mutation is located at the 5' donor splice site, this may cause an alteration of a normal mRNA splicing process. An intronic mutation at the 3' acceptor splice site was previously reported to produce TBG-CD (TBG-Kankakee) by causing a frameshift and early termination of translation. 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 border (19, 20, 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 score of 1. The calculated CVN for the eight nucleotide (-2 to +6) of the donor splice recognition sequence using the primate nucleotide weight table of Shapiro and Senapathy (22) is 0.87, compared with the CVM of 0.72 for the corresponding sequence in TBG-Jackson. A search revealed a sequence in intron IV, 251 bp downstream from exon 3 TGGTGAGA (a CV score of 0.83) as a putative alternative splice site, reaching a premature stop at codon 331.

In contrast to this prediction, direct analysis of the splice site using the exon trapping system showed that the mutation in TBG-Jackson results in the complete skipping of exon 3. The translation of such an aberrant mRNA would produce a frameshift at codon 280 and a premature stop (TGA) at codon 325 of the mature molecule. However, our failure to detect TBG mRNA in fibroblasts of the individual expressing solely the mutant allele suggests that TBG-Jackson mRNA is unstable. This is supported by the fact that the only form of TBG-mRNA in fibroblasts of the heterozygous mother had a normal sequence. The generation of some normally spliced TBG mRNA product in COS 7 cells transfected with the TBG-Jackson vector is most likely due to the properties of this artificial construct and system. Unfortunately, the experiment could not be performed in liver cells.

In family H, an attempt to find a mutation led us to sequence all the intronic and 3' untranslated regions, which failed to show a defect. No abnormalities were found in the 5776 bp of the TBG gene that were sequenced (Fig. 1). Genotyping using the variant nucleotide found in intron II cosegregated this allele to the TBG-CD phenotype. This indicates that the putative abnormality producing TBG-CD should be on the X-chromosome, very close or in the TBG gene, such as an upstream region in the promoter area. Unfortunately, we were not able to obtain skin biopsies to study TBG gene transcription in this family. Previous functional studies done by a series of 5' deletion of TBG gene revealed that the fragment -218 to +4 (D5'-218CAT) from the transcriptional start site has the highest promoter activity (3). This sequence was normal in family H. It is possible that the mutation may be located further upstream; however, the same study using a full length 2.65 kbp fragment from transcriptional start site showed little variation in the promoter activity compared with D5'-218CAT. Whether or not a mutation in this region or in an upstream sequence, inhibiting the promoter activity, is a cause of TBG deficiency in this family, remains unknown.

Acknowledgments

We thank Dr. Neal H. Scherberg and the technical staff of the Endocrinology Laboratory at the University of Chicago for performing some of the tests of thyroid function. We also thank Dr. Jerald C. Nelson from Nichols Institute Diagnostics at Quest Quagnostic for measuring some of the TBG and free T₄ concentration in serum from members of Family H. Special thanks are due to members of the two families for their consent to participate in this study and to Dr. Joachim Pohlenz for encouragement and help with the in vitro analysis of splicing.

Footnotes

This work was supported in part by NIH Grants RR 00055, DK 15070, and DK 07011 and in part by Tivoli Wien Katz.

Present address for P.E.M.: Via Posillipo 102/1, 80123 Napoli, Italy.

Abbreviations: CV, Consensus value; FT₄I, free T₄ index; gDNA, genomic DNA; TBG, thyroxine-binding globulin; TBG-CD, complete TBG deficiency; TG, thyroglobulin; TPO, thyroid peroxidase; TrT₃, total reverse T₃; TT₃, total T₃; TT₄, total T₄.

Received February 22, 2001.

Accepted November 15, 2001.

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