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A [R2223H]) Resulting in Fetal Goitrous Hypothyroidism
Service d’Endocrinologie, Centre Hospitalier Universitaire Rangueil (P.C., D.M.), 31403 Toulouse, France; Laboratorio de Biología Molecular, Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires (C.M.M., V.J.G., C.M.R., H.M.T.), 1120 Buenos Aires, Argentina; and Clinique Sarrus-Teinturiers (B.C.), 31076 Toulouse, France
Address all correspondence and requests for reprints to: Dr. Héctor M. Targovnik, Laboratorio de Biología Molecular, Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Av. Córdoba 2351, 4to piso-sala 5, 1120 Buenos Aires, Argentina. E-mail: htargovn{at}huemul.ffyb.uba.ar.
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Introduction |
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Tg is a large glycoprotein synthesized by the thyroid gland. It functions as a matrix where thyroid hormones (T4 and T3) are produced from the coupling of iodotyrosyl residues, catalyzed by TPO (27). Recently, the complete structure of the human Tg gene has been determined (28, 29, 30, 31). It is coded by a single copy gene, 270 kb long, that maps on chromosome 8q24 and contains an 8.5-kb coding sequence divided into 48 exons. The preprotein monomer is composed of a 19-amino acid signal peptide, followed by a 2749-residue polypeptide. Eighty percent of the monomeric primary structure is characterized by the presence of three types of repetitive units. The remaining 20%, which constitutes the carboxyl-terminal domain of the molecule, is not repetitive and shows a striking homology with acetylcholinesterase (ACHE) (32).
To date, only five mutations of the Tg gene have been identified in humans (11, 12, 13, 14, 15, 16, 17), resulting in structural defects and endoplasmic reticulum retention of Tg proteins, and have been linked to subsequent thyroid hormone-impaired and primary congenital hypothyroidism. In patients with Tg synthesis defects, most neonates present with congenital goiter, hypothyroidism may be moderate to severe, and the serum Tg concentration is low, especially in relation to the degree of TSH stimulation.
In the present study we have identified a compound heterozygous mutation in the Tg gene in a family with two affected siblings with congenital goitrous hypothyroidism. The paternal mutation consists of a cytosine deletion at nucleotide position 1143 in exon 9 (1143delC), resulting in a frameshift that generates a stop codon at position 382 in the same exon. The maternal mutation is a guanine to adenine substitution at position 6725 in exon 38, creating the R2223H missense mutation in the ACHE homology domain of Tg.
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Materials and Methods |
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In a 22-yr-old white female, a fetal goiter was diagnosed coincidentally by ultrasound at the sixth month of gestation during her first pregnancy (Fig. 1
). There was no historical evidence of iodine deficiency in the family, and the parents had no history of past thyroid diseases. On clinical examination, there were no symptoms compatible with hypo- or hyperthyroidism. Their thyroid volume was normal on clinical examination and by ultrasound evaluation (mother: right lobe, 45/15/12 mm; left lobe, 35/15/12 mm; father: right lobe, 44/15/12 mm; left lobe, 42/16/13 mm). A second ultrasound examination at 27 wk gestation showed further enlargement of the goiter (34/21 mm), but fetal vitality and amniotic fluid volume appeared normal. Percutaneous umbilical vein blood sampling under ultrasound guidance was performed at 27 wk gestation, showing important fetal hypothyroidism, with high serum TSH (336 mU/liter) and undetectable free T4 (fT4; <2.5 pmol/liter) levels (Table 1). An intraamniotic injection of 200 µg levothyroxine (Roche, Neuilly sur Seine, France) was performed at the beginning and end of the seventh month of gestation. Four weeks after the first levothyroxine injection, ultrasound evaluation revealed a fetal goiter size of 30/18 mm. The woman had an uncomplicated, spontaneous vaginal delivery at 41 wk gestation and delivered a female infant weighing 3.290 g (patient T.D., index patient), with an Apgar score of 10 at 1 and 5 min. Clinical examination of the neonate did not reveal hypothermia, bradycardia, jaundice, enlarged posterior fontanel, or umbilical hernia. The neonate had no visible goiter and did not present symptoms of airway obstruction. Ultrasonographic studies evidenced an enlarged thyroid gland; each thyroid lobe measured 32/15/14 mm. Cord blood analysis indicated elevated TSH (284 mU/liter) and low fT4 (5.5 pmol/liter) levels (Table 1). The serum Tg concentration was low (<0.8 ng/ml), suggesting that dyshormonogenetic goiter could be related to defective Tg synthesis. Treatment with levothyroxine (35 µg/d) was started on the first day of life, and the neonate was discharged on the fourth day. Serum TSH concentration was normalized on d 15 (0.31 mU/liter).
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). The fetus was treated by the injection of 500 µg levothyroxine into the amniotic fluid at 32 and 36 wk gestation. The dose of levothyroxine injected during the second pregnancy was based on the results observed in the first gestation, i.e. high TSH concentration present in the umbilical cord of the first neonate. The woman presented active labor at 37 wk and delivered a male infant weighing 2.380 g (patient P.D.). The Apgar score was 10 at 1 and 5 min. At birth no palpable goiter was present, and there were no clinical signs of hypothyroidism. In the cord blood the serum TSH concentration was 39 mU/liter, and the serum Tg level was low (0.7 ng/ml; Table 1). The infant was started on 25 µg/d levothyroxine on the day of delivery and was discharged on the fifth day of life. The serum TSH concentration was normalized on d 15 (0.2 mU/liter). Follow-up showed normal clinical growth and development of the two children on levothyroxine treatment after 4 and 3 yr, respectively. Written informed consent to participate in the clinical and genetic studies was given by both parents.
Thyroid function tests
fT4 (normal range, 8–19 pmol/liter) and fT3 (normal range, 3.0–9.2 pmol/liter) concentrations were determined using the two-step chromatographic separation, followed by RIA (Technogenetics Laboratory, Cassina de Pecchi, Italy). Serum TSH levels (normal range, 0.2–4.0 mU/liter) were measured by the luminescence immunoassay (LUMItest TSH, Behring Diagnostic, Rueil Malmaison, France). The serum Tg concentration (normal levels, <30 ng/ml) was determined using an immunoradiometric assay (DYNOtest Tg, Behring Diagnostic). Anti-TPO antibodies (normal level, <100 U/ml) were determined using the Dynotest anti-TPO kit (Behring Diagnostic). Anti-Tg antibodies (normal level, <100 U/ml) were measured by the Elisa-Tg kit (Pasteur-Bio-Rad, Marne la Coquette, France). Anti-TSH receptor antibodies (normal level, <10 U/liter) were measured by the Dynotest TRAK-human kit (Behring Diagnostic).
Genomic DNA isolation
At birth, the blood for DNA studies was obtained from cord blood for each neonate (T.D. and P.D.). A second blood sample was collected at 3 and 2 yr of age, respectively. Peripheral blood samples were collected from their parents and from 77 unrelated individuals. Genomic DNA was isolated from white blood cells by the sodium dodecyl sulfate-proteinase K method.
DNA amplification
The complete coding sequences of the human Tg gene, including regions of the Tg promoter and, splicing signals and the flanking intronic regions of each intron, were amplified from patient T.D. by PCR. The PCR amplifications were performed in 100 µl, using a standard PCR buffer (Invitrogen-Life Technologies, Carlsbad, CA), containing 100–300 ng genomic DNA, 2.5 mM MgCl2, 200 µM of each deoxy (d)-NTP (dATP, dCTP, dTTP, and dGTP), 4% dimethylsulfoxide, 2 U Taq polymerase (Invitrogen-Life Technologies), and 50 pmol of each forward and reverse primer. Forward and reverse intronic primers were specially designed for each one of the 48 Tg exons (28, 29, 30, 31).
The exons 9 and 10 were amplified in three (9A, 9B, and 9C) and two (10A and 10B) separated PCR fragments, respectively. A complete list of primers used for amplification of Tg by PCR will be provided by the authors upon request. M13 sequences (18 nucleotides long) have been incorporated at the 5' end of the primers. Samples were denatured at 95 C for 3 min, followed by 40 cycles of amplification. Each cycle consisted of denaturation at 95 C for 30 sec, primer annealing at 58 C for 30 sec, and primer extension at 72 C for 1 min. After the last cycle, the samples were incubated for an additional 10 min at 72 C to ensure that the final extension step was complete.
The amplified products were analyzed in 2% agarose gels. Purification of PCR fragments for sequence and cloning reactions was performed with Concert rapid gel extraction system (Invitrogen-Life Technologies).
DNA sequencing
DNA sequences from each amplified fragment were performed with the Taq polymerase-based chain terminator method (fmol, Promega, Madison, WI) and using either the M13 tags linked to the PCR fragments or the Tg-specific forward and reverse primers used in the DNA amplification. The results were analyzed using the PC gene (Intelligenetics, Geneva, Switzerland), DNASTAR (DNASTAR, Inc., University of California, San Francisco, CA), and Nucleotide BLAST (http://www.ncbi.nlm.nih.gov/BLAST) software programs.
Cloning of wild-type and mutated 9A PCR fragments
The 9A PCR fragment (606 bp) contains the first 474 nucleotides of exon 9. The sequences of primers used are as follows: forward intronic primer, 5'-gttctggcttcttactacct-3'; and reverse exonic primer, 5'-CTTCTTGTCTCCCTCCAT-3', both with M13 linkers attached. The 9A amplified fragment from patient T.D. and his father were T-A cloned into pGEM-T Easy vector (Promega). JM109 competent cells were used for transformations. After that, the recombinant clones were identified by color screening on indicator plates containing isopropylthio-ß-galactoside and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside, and plasmid DNA was isolated with the Concert Rapid Plasmid Miniprep System (Invitrogen-Life Technologies). DNA sequencing was performed as described above for wild-type and mutant allele clones, with the 9A forward intronic primer.
HaeII restriction analysis
The mutation detected by nucleotide sequencing at position 6725 in exon 38 destroyed a HaeII recognition site. The presence of the mutation was therefore independently confirmed by restriction analysis with HaeII (Promega) in the two affected siblings and their parents and in 77 unrelated individuals. A 414-bp fragment containing exon 38 was generated by PCR under identical PCR conditions as those described above, using the same forward (5'-cagaatgccagtggagagagc-3') and reverse (5'-ctgctactgagtcccatttgga-3') intronic exon 38 primers with M13 linkers attached. The PCR product (1 µg) was digested with 12 U HaeII overnight at 37 C. After digestion, the DNA fragments were separated on a 2% agarose gel and visualized with ethidium bromide. Digestion of the wild-type allele resulted in two fragments of 256 and 158 bp.
Protein analysis
Amino acid sequence homology between the ACHE-like domain of the mouse, rat, bovine, and human Tg and several ACHE were compared using the Protein BLAST and Search for Conserved Domains (http://www.ncbi.nlm.nih.gov/BLAST) software programs. The deduced C-terminal 525 amino acids in the ACHE-like domain of human Tg were submitted for computer analysis of the protein secondary structure prediction to the nnPredict-UCSF internet site (www.cmpharm.ucsf.edu/nomi/nnpredict.html).
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Results |
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The results of thyroid function tests of all family members are shown in Table 1. The two affected siblings (T.D. and P.D.) had goiter, high serum TSH concentrations, low fT4 levels, and low serum Tg concentrations. These findings are suggestive of the diagnosis of defective Tg synthesis. The parents were nonconsanguineous, and their thyroid parameters were in the normal range, with no circulating autoantibodies (anti-TPO, anti-Tg, and anti-TSH receptor).
Sequence analysis of Tg gene
All 48 exons of the Tg gene from index patient T.D. were analyzed as well as 198 bp of the Tg promoter and all of the flanking regions of each intron. A total of 14,345 bases were analyzed. The GT-AG splicing consensus sequences are rigorously respected in all introns. We identified one base deletion, 1143delC, in exon 9 (Fig. 2) and a guanine to adenine transition at position 6725 (CGC 
CAC) in exon 38 that replaces the normal arginine in codon 2223 with a histidine (R2223H) in the ACHE homology domain of Tg (data not shown). The former 1143delC altered an open reading frame, resulting in a frameshift at amino acid 362 with a premature stop at 382 (58 nucleotides downstream from deletion). However, the 362 glycine did not change. Analysis by direct sequencing of PCR products of exons 9, fragment 9A (Fig. 2A), and 38 from each member of the family showed that both siblings affected with goiter and hypothyroidism, T.D. and P.D., have inherited one copy of the 1143delC mutation from their father and one copy of the 6725GA mutation from their mother. The 1143delC heterozygous state of the patient T.D. and his father was confirmed by cloning and sequencing of both wild-type and mutant alleles (Fig. 2B). This finding established the compound heterozygous inheritance of the defect and the recessive manifestation of the phenotype.
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The guanine to adenine transition at position 6725 eliminates a restriction site for HaeII. This allowed independent confirmation of the mutation by restriction analysis. The mother and her two affected sibling (T.D. and P.D.) all have a wild-type allele containing guanine at position 6725 that is digested with HaeII, producing two fragments of 256 and 158 bp and a mutant allele (414 bp) resistant to digestion containing adenine at position 6725 (data not shown). In contrast, the father had only the wild-type sequence at this position.
We ruled out the possibility that it could be a common sequence polymorphism found in the general population because it was not detected in 154 chromosomes from the normal population.
Protein analysis
Comparison of the ACHE homology domain of human Tg with sequences found in the GenBank database, using the Blast network service, reveals that wild-type arginine residue at position 2223 is strictly conserved in all Tg and ACHE species analyzed (Fig. 3A). We submitted the amino acid sequence of the ACHE-like domain of the Tg to the nnPredict-UCSF internet site for protein secondary structure prediction to explore putative effects of the R2223H missense mutation on the structure of the human Tg. In the normal protein, residues 2220 and 2221 constitute a helix, whereas in the mutant protein the presence of a histidine at position 2223 extends the helix to the residue 2222 (Fig. 3B).
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Discussion |
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In the two siblings of the studied family with congenital goitrous hypothyroidism, the precise nature of the defect is a new compound heterozygote of the Tg gene, a 1143delC in exon 9 in combination with a 6725GA transition (R2223H) in exon 38. The 1143delC mutation produces a predicted premature stop codon that would result in a grossly truncated protein of 381 amino acids with limited ability to generate thyroid hormone (Fig. 4A). Five hormonogenic acceptor tyrosines have been identified and localized at positions 5, 1291, 2554, 2568, and 2747, and several other tyrosines localized at positions 130, 847, and 1448 have been proposed as donor sites that transfer a iodophenoxyl group to an acceptor iodotyrosine (27, 31). The most important hormonogenic acceptor site is at tyrosine 5, after coupling with the donor tyrosine at position 130 (36). The truncated form of Tg described here harbors both the acceptor tyrosine 5 and the donor tyrosine 130 residues. Therefore, it is assumed that the 1143delC mutation eliminates the carboxyl-terminal hormonogenic domain, resulting in the loss of thyroid hormone formation (Fig. 4A). An interesting but unsolved question due to the absence of mRNA analysis, because the thyroid tissue from both patients was unavailable, would have been to estimate the Tg transcript concentration in goiters. In general, mRNA-containing nonsense mutations are found in low concentration. This reduction is due to an accelerated degradation rate of the mutated mRNA, because the untranslated part of the messenger is not protected by ribosomes. To complement this scenario, it is not possible to exclude that an alternative splicing mechanism, by exon skipping or activation of cryptic splice sites, might restore the normal reading frame disrupted by the mutation and eliminate the stop codon, which would truncate the protein. As is reported for Afrikander cattle (37) and humans (12), a nonsense mutation at amino acid position 687 (exon 9) and position 1511 (exon 22), respectively, results in a preferential production of a smaller transcript lacking the mutated exon by alternative splicing. In these cases minor transcripts exist in low concentration in normal thyroid tissue. However, an alternative transcript involving exon 9 was not detected in normal and goitrous human thyroids (31). The other mutation found in these affected siblings generates the R2223H missense mutation in the ACHE homology domain of Tg (Fig. 4B). The putative function of the ACHE homologous domain in Tg is not yet clear. The congenital hypothyroidism observed in cog/cog mice (38) and rdw rats (39) was also caused by missense mutation in the ACHE-like domain of Tg. Both mutated Tg exhibit a severe defect in the exit from the endoplasmic reticulum (ER), causing a thyroidal ER storage disease. After translation of Tg proteins, intensive posttranslational processes take place in the ER and Golgi apparatus. In cog/cog mice full-length Tg is synthesized, but is defective in its folding, so most newly synthesized proteins never arrive at the Golgi complex (38). Accumulation of mutant Tg in the ER induces the synthesis of chaperones that prevent export of improperly folded proteins (38, 40). More importantly, the wild-type arginine residue at position 2223 is strictly conserved in all species for which suitable Tg and ACHE sequences have been reported. Computer analysis of the protein’s secondary structure showed that the R2223H mutation caused an extended stretch of the helix structure. Based on such observations we hypothesize that the arginine residue in this position plays a critical structural role in the Tg protein. Consequently, the R2223H mutation, as was observed in cog/cog mice and rdw rats, may cause structural instability, leading to deficient Tg export. Finally, in this family (Fig. 5), genotyping revealed that the euthyroid father carried the heterozygous 1143delC mutation, and the mother with normal thyroid function carried the heterozygous 6725GA mutation, indicating autosomal recessive inheritance. However, genetic studies have currently a limited importance in the clinical care of the patients with congenital hypothyroidism. Analysis at the molecular level may be useful for identification of affected newborns or gene carriers in families with mutation identified.
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In conclusion, we have reported two siblings with congenital goitrous hypothyroidism caused by compound heterozygous mutations of the Tg gene. A heterozygous 1143 cytosine deletion in codon 362 resulted in a conserved glycine followed by 19 altered amino acids and a premature stop codon due to the frameshift. The other allele carries a change of the 2223 highly conserved arginine to histidine in the ACHE-like domain of Tg. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of this neonatal hypothyroidism.
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Footnotes |
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P.C. and C.M.M. contributed equally to this study.
C.M.M. is a Research Fellow of the Universidad de Buenos Aires.
C.M.R. is a Research Fellow of the Argentine National Research Council.
H.M.T. is an Established Investigator with the Argentine National Research Council.
Abbreviations: ACHE, Acetylcholinesterase; d-, deoxy-; ER, endoplasmic reticulum; fT4, free T4; Tg, thyroglobulin; TPO, thyroperoxidase.
Received November 7, 2002.
Accepted May 6, 2003.
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, 6725G>A mutation; 
, 1143delC mutation. The arrow indicates the index patient.