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Congenital Secondary Hypothyroidism Caused by Exon Skipping due to a Homozygous Donor Splice Site Mutation in the TSHß-Subunit Gene

Children’s Hospital of the Johannes Gutenberg University of Mainz (J.P., R.M., D.P.) and Städtisches Krankenhaus Hagen (U.A., G.K.), D-55101 Mainz, Germany; and Departments of Medicine and Pediatrics, J. P. Kennedy, Jr., Mental Retardation Research Center (S.R.), and Committee on Genetics (S.R., A.D.), University of Chicago, Chicago, Illinois 60637-1470

Address all correspondence and requests for reprints to: Samuel Refetoff, M.D., Thyroid Study Unit, Departments of Medicine and Pediatrics, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: refetoff{at}medicine.bsd.uchicago.edu

	Abstract

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Isolated TSH deficiency as a cause for congenital hypothyroidism is relatively uncommon. Even more rare is the identification of mutations in the TSHß gene, only four of which have been identified. We here report a 4-month-old girl with isolated TSH deficiency born to consanguineous parents. Sequencing of the TSHß-subunit gene revealed a homozygous G to A transition at position +5 of the donor splice site of intron 2. TSHß gene transcript could not be obtained from fibroblasts or white blood cells by illegitimate amplification. Thus, to investigate further the mechanism leading to TSH deficiency in this patient, we used an in vitro exon-trapping system. The mutation at position +5 of the donor splicing site produced a skip of exon 2. The putative product of translation from a downstream start site is expected to yield a severely truncated peptide of 25 amino acids. Surprisingly, a missense substitution affecting the 14th amino acid of the signal peptide (SigP A14T) was found in one allele of the mother and brother. SigP 14T is polymorphic with a frequency of 1.8% and has no functional consequence.

	Introduction

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TSH DEFICIENCY AS a cause of congenital hypothyroidism occurs in 1:20,000 to 1:50,000 newborns (1). Because newborn screening programs in most countries are based on the determination of TSH levels in blood, early diagnosis of this condition is missed in the majority of the cases. Affected individuals are brought to clinical attention later, usually in the first year of life because of symptoms consistent with hypothyroidism. The severity of hypothyroidism due to TSH deficiency varies. Even though secondary hypothyroidism is considered to be less severe, individuals with frank mental retardation have been described (2). Therefore, it is important to identify this condition early in life, allowing prompt hormonal substitution.

The gene encoding the TSHß-subunit in man was cloned in 1988 (3). It is located on chromosome 1 and consists of three exons: the first one is untranslated and the two other exons encode for a 118-amino acid mature TSHß protein after release of a 20-amino acid signal peptide. To date, four different mutations have been identified, all located in the coding region of the gene. Affected individuals are homozygous, expressing G29R (4); C105 fr sh, 114X (2, 5, 6, 7); E12X (8); and Q49X (9, 10). ¹

We now report a new genetic defect that leads to TSH deficiency caused by exon skipping. The proposita, who presented with isolated TSH deficiency, had a homozygous substitution of a G for an A in nucleotide +5 of the donor splice site of intron 2. When studied in vitro using an exon-trapping system, the mutation produced an mRNA lacking exon 2 of the TSHß-subunit gene. The putative product of translation is a severely truncated peptide of 25 amino acids with no biological activity.

	Subjects and Methods

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Patient

The proposita is the second child of a consanguineous marriage (parents are second degree cousins; their respective grandfathers are brothers). She was born at term after an unremarkable pregnancy. Shortly after she was discharged from the hospital, diminished alertness became apparent, and reduced food intake resulted in failure to thrive. Neurological development was delayed, which led to her referral to Children’s Hospital at 4 months of age for evaluation of psychomotor retardation.

She presented typical stigmata of hypothyroidism, with macroglossia, a depressed nasal bridge, a wide open anterior fontanel, and a puffy face. Her skin was cold and dry, and she had a small umbilical hernia. She had muscular hypotonia, and her psychomotor development was that of a 6-wk-old girl. Physical examination was otherwise unremarkable. Her thyroid gland was of normal size, a finding confirmed by ultrasound. X-Ray of the knee showed retarded ossification, compatible with hypothyroidism.

Thyroid function tests showed a decreased serum free T₄ level of 1.4 pmol/liter (normal, 10–22) and free T₃ of 1.27 pmol/liter (normal, 3.1–6.4) in combination with a low TSH concentration of 0.19 mU/liter (normal, 0.3–5 mU/liter), compatible with secondary hypothyroidism. Treatment with 50 µg L-T₄ was initiated, resulting in an increase in serum free T₄ and free T₃ concentrations to 11.5 and 6.6 pmol/liter, respectively, whereas the serum TSH level remained low at 0.07 mU/liter. Under continuing treatment with L-T₄ her psychomotor development improved, but remained retarded for age.

Her three-year old brother as well as her parents were healthy and were clinically euthyroid (Fig. 1).

View larger version (50K): [in this window] [in a new window]   Figure 1. Pedigree of the family showing the phenotype and haplotype, and results of thyroid function tests and DNA analysis. All data are aligned with each individual symbol on the pedigree. Abnormal values are in bold. Note that the affected child (II-2) was receiving L-T4, and was slightly overtreated at the time of blood sampling. The 456-bp fragment amplified from the DNA segment flanking exon 2 of the TSHß gene produces fragments of 255, 186, and 14 bp when digested with MslI. The mutation at position +5 of the donor splice site of intron 2 destroys recognition of one MslI restriction site, yielding only two fragments of 441 and 14 bp. Partial digestion confirms the heterozygous state of both parents and brother. Furthermore, the second nucleotide substitution in the 14th amino acid of the signal peptide (SigP A14T), an A replacing a G, enabled us to haplotype the family, as shown below each symbol.

Informed consent to participate in the clinical and genetic studies was given by both parents in their behalf and that of their minor children. These studies were approved by the respective institutional review boards. The initial free T₄ and free T₃ concentrations and TSH levels were measured using electrochemiluminescent immunoassays (Roche, Mannheim, Germany). Later, serum TSH, total T₄, total T₃, free T₄, and free T₃ concentrations (see Fig. 1) were measured by chemiluminescent immunoassays (Chiron Corp., Fernwald, Germany). Ultrasound of the thyroid gland was performed with an Acuson 128 linear 5.0-megahertz scanner (Acuson, Erlangen, Germany).

Genetic analysis

Skin fibroblasts from the proposita were obtained and cultured according to standard protocols. Genomic DNA from all available family members was isolated from peripheral blood leukocytes using the QIAamp Blood Kit (QIAGEN, Hilden, Germany). The TSHß-subunit gene was then amplified with primers flanking exons 1, 2, and 3, which contain all sequences incorporated into the mRNA, as previously described (6). PCR products were purified and sequenced using an automated sequencing system (A 377, PE Applied Biosystems, Weiterstadt, Germany).

Analysis of the splicing mechanism: construction of vector, transfection, RNA analysis, and sequencing

To study the effect of the mutation in the donor splice site of intron 2, an exon-trapping system (Life Technologies, Inc., Frederick, MA) was used. Genomic DNA from the proposita and from a normal individual containing intron 2, flanked by exons 2 and 3, was amplified using the primers 5'-gatcatatgcattgggatgg-3' and 5'-gctttatttcaggcaagcac-3' at an annealing temperature of 55 C for 35 cycles in a 2400 thermal cycler (Perkin-Elmer Corp., Foster City, CA). The 1.1-kb PCR products were electrophoresed on a 1.8% agarose gel, stained with ethidium bromide, visualized under UV light, gel-purified (gel extraction kit, QIAGEN), and then subcloned into the pGEM-T-easy vector (Promega Corp., Madison WI). After the correct sequence was confirmed by sequencing, the insert was cloned into the SacI and NotI sites of the pSPL3 vector (Life Technologies, Inc.). The plasmid DNA was amplified in Escherichia coli JM 109, extracted, purified (QIAGEN), and used for transfection.

COS-7 cells were grown in 10-cm dishes in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% bovine calf serum (Life Technologies, Inc.) and 50 mg/liter gentamicin in a 10% CO₂ atmosphere at 37 C. When cells reached approximately 50% confluence, they were transfected with 10 µg plasmid DNA (normal, mutant, or control pSPL3, containing an exon of known size)/10-cm dish with the SuperFect reagent (QIAGEN). Twenty-four hours later cells were harvested, and total RNA was extracted with TRIzol (Life Technologies, Inc.). Synthesis of cDNA was catalyzed by the Superscript II ribonuclease H^- reverse transcriptase primary PCR, and digestion with BstXI and secondary PCR were performed according to the manufacturer’s instructions supplemented with the exon-trapping system (Life Technologies, Inc.). The products of the secondary PCR were electrophoresed on a 1.8% agarose gel, stained with ethidium bromide, and visualized under UV light. If correct processing of the transfected sequences occurred, the expected size of the PCR product was 594 bp (see Fig. 4). Eventually the PCR products were gel purified (QIAGEN), cloned into the pAMP10 vector provided by the manufacturer, and then sequenced (PE Applied Biosystems).

View larger version (19K): [in this window] [in a new window]   Figure 4. Diagrammatic representation of TSHß gene organization and the transcription products of normal and mutant genes. A mutation at position +5 in the donor splice site of intron 2 results in the skipping of exon 2. Normally spliced product (shown on top) results in a transcript that encodes for the 118-amino acids TSHß-subunit. The mutant transcript (shown on the bottom) lacks exon 2 as well as the normal ATG start codon. The first alternative ATG located in exon 3 would produce a short nonsense peptide of 25 amino acids.

	Results

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View larger version (30K): [in this window] [in a new window]   Figure 2. Automated fluorescence-based sequencing chromatogram of the TSHß-subunit gene amplified from genomic DNA of a normal individual, a heterozygous member of the family, and the patient. The proposita is homozygous for a substitution of the WT guanidine (g) with an adenine (a) at position +5 of intron 2 (in red). The intronic sequence is indicated in small letters, whereas the exonic sequence is in capital letters. Furthermore, the consensus sequence of a donor splicing site is shown (u, uracil; r, purine).

View larger version (34K): [in this window] [in a new window]   Figure 3. Chromatograms of sequences at the splice junctions of transcripts from normal and mutant genomic DNAs. The PCR-amplified fragments were cloned into the exon-trapping pSPL3 vector, which was expressed in COS-7 cells. cDNA was synthesized from transcribed mRNA, amplified with primers complementary to flanking vector sequence and electrophoresed on a 1.8% agarose gel (inset in bottom right). Correct processing of the transfected sequences in the wild type produces a PCR product of 594 bp. In contrast, exon 2 was skipped in the mutant TSHß gene,producing a 431-bp fragment. The splice junctions in the wild-type and mutant products are shown on the chromatograms. The sizes of exons and flanking vector sequences are indicated. Note that the mutant cDNA lacks 163 bp that correspond to exon 2 of the TSHß gene.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Four different mutations causing isolated TSHß deficiency have been identified to date (4, 6, 8, 9). Two of these (C105 fr sh, 114X and Q49X) occurred in more than one unrelated family (2, 5, 6, 7, 9, 10). All are located in the coding region of the TSHß-subunit gene. We now describe a homozygous mutation in the donor splice site of intron 2 of the TSHß gene that causes isolated TSH deficiency. The mutation is novel and is the first described TSHß gene defect located in the noncoding, intronic sequence, affecting the splice donor site. It is known from other conditions that mutations in this site that alter the consensus sequence GURAGU can result in exon skipping, especially at position +5 (11, 12, 13, 14, 15). It was, therefore, tempting to speculate that exon skipping is the mechanism that causes TSH deficiency in this case. Because illegitimate amplification of TSHß mRNA from either skin fibroblasts or white blood cells was not successful, and pituitary tissue from affected individuals is not readily accessible, we used the exon-trapping system to determine whether the mutation in intron 2 produced an abnormal transcript by a defect in exon splicing. Indeed, using this technique, we were able to demonstrate that the mutation in the donor splice site caused exon skipping, resulting in TSH deficiency. Exon 2 of the mutant TSHß transcript is skipped, and the normal translational starting point, which is located in exon 2, is therefore missing in the mutant TSH molecule. It is possible that the mutant TSHß is translated from the first ATG in exon 3, which is in a different reading frame. In such an event, the mutant molecule would have a nonsense sequence of 25 amino acids and no biological activity.

Surprisingly, a second nucleotide substitution was found in one allele of the phenotypically normal mother and brother. The resulting missense mutation produces replacement of the normal alanine with a threonine at the 14th amino acid of the signal peptide. This change was not expected to cause a significant functional alteration because both mother and brother, who are compound heterozygote for this and the intronic mutation, had no thyroid test abnormalities. A random DNA survey revealed that the SigP 14T is polymorphic, with an allele frequency of 1.8%. Furthermore, the demonstration that an individual homozygous for SigP 14T had normal tests of thyroid function confirmed the lack of physiological consequence of this polymorphic variant.

Although secondary hypothyroidism is rare, unrelated individuals from different countries have been found to harbor identical TSHß gene mutations (2, 5, 6, 7). Accordingly, the prevalence of heterozygosity is likely to be more common than currently suspected. The systematic measurement of both TSH and T₄, as is the routine practice in some neonatal screening programs, should be recommended.

	Acknowledgments

 
We thank the family for their participation in the study, and Dr. Roy E. Weiss for review of the manuscript.

	Footnotes

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

¹ Numbering begins with the first amino acid of the mature protein, thus excluding the signal peptide (SigP).

Received June 22, 2001.

Accepted October 1, 2001.

	References

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