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A Premature Stopcodon in Thyroglobulin Messenger RNA Results in Familial Goiter and Moderate Hypothyroidism Simone A. R. van de Graaf, Carrie Ris-Stalpers, Geertruda J. M. Veenboer, Marianne Cammenga, Cécilia Santos, Héctor M. Targovnik, Jan J. M. de Vijlder and Geraldo Medeiros-Neto J. Clin. Endocrinol. Metab. 1999 84: 2537-2542, doi: 10.1210/jc.84.7.2537 To subscribe to Journal of Clinical Endocrinology & Metabolism or any of the other journals published by The Endocrine Society please go to: http://jcem.endojournals.org//subscriptions/ Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online 0021-972X/99/$03.00/0 Vol. 84, No. 7 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright © 1999 by The Endocrine Society A Premature Stopcodon in Thyroglobulin Messenger RNA Results in Familial Goiter and Moderate Hypothyroidism SIMONE A. R. VAN DE GRAAF, CARRIE RIS-STALPERS, ´ GEERTRUDA J. M. VEENBOER, MARIANNE CAMMENGA, CECILIA SANTOS, HE´ CTOR M. TARGOVNIK, JAN J. M. DE VIJLDER, AND GERALDO MEDEIROS-NETO Academic Medical Center (S.A.R.v.d.G., C.R.-S., G.J.M.V., J.J.M.d.V.), University of Amsterdam, Emma Children’s Hospital AMC, Laboratory of Pediatric Endocrinology, Amsterdam, The ´ ´ ´ Netherlands; Laboratorio de Tireoide (LIM-25) (C.S., G.M.-N.), Hospital das Clınicas, Universidade de ˆ ˆ ´ ´ ´ Sao Paulo, Sao Paulo 01065–970, Brazil; Division Genetica (H.M.T.), Hospital de Clınicas “Jose de ´ ´ ´ San Martin”, Facultad de Medicina and Catedra de Genetica y Biologıa Molecular, Facultad de ´ Farmacia y Bioquımica, Universidad de Buenos Aires, 1120, Buenos Aires, Argentina ABSTRACT expressed in rabbit reticulocyte lysate resulting in a truncated protein Impaired thyroglobulin (Tg) synthesis is one of the putative causes of 30 kDa. Expression in the presence of microsomal membranes for dyshormonogenesis of the thyroid gland. This type of hypothy- resulted in a gel shift of this Tg molecule, indicating glycosylation roidism is characterized by intact iodide trapping, normal organifi- ability. Two other siblings had a clinical presentation like the index cation of iodide, and usually low serum Tg levels in relation to high patient, while their parents were unaffected. Additional restriction TSH, and when untreated the patients develop goiter. In thyroid fragment length polymorphism analysis of the pedigree verified that tissue from a 13-yr-old patient suspected of a thyroglobulin synthesis the homozygous nonsense mutation cosegregated with the clinical defect, the Tg mRNA was studied. The complete coding region of 8307 phenotype. Clinically, hypothyroidism was not severe in the affected bp was directly sequenced and revealed a homozygous point mutation: siblings because the truncated Tg glycoprotein was still capable of a C886T transition in exon 7. Upon translation this mutation would thyroid hormonogenesis. (J Clin Endocrinol Metab 84: 2537–2542, result in a stopcodon at amino acid position 277, replacing the argi- 1999) nine residue. A Tg cDNA construct containing the mutation was P RIMARY congenital hypothyroidism (CH) is caused by disorders of thyroid gland development (80%) or dy- shormonogenesis (20%). In thyroid dyshormonogenesis a polymorphisms have been identified in the thyroglobulin gene locus, of which 8 result in amino acid residue variation (8 –10). Furthermore, at least 12 alternative splice products mutation is expected in one of the genes encoding a key have been identified in normal Tg mRNAs (10 –16). Beside protein involved in the biosynthesis of thyroid hormones these wildtype variations, some mutations in the Tg gene (1, 2). have been identified in animal models and in man, resulting One of these proteins is thyroglobulin (Tg), the predom- in aberrant Tg protein expression and linked to subsequent inant glycoprotein (660 kDa) of the thyroid gland, which impaired thyroid hormone synthesis. functions as matrix protein in thyroid hormonogenesis. Cat- In Afrikander cattle a homozygous nonsense mutation, alyzed by thyroid peroxidase (TPO), tyrosine residues in the Arg697OPA (exon 9), results in the expression of a truncated Tg molecule are iodinated, and subsequently some specific Tg protein of 75 kDa. In this case also, an alternatively spliced ones are coupled to form mainly T4 and some T3 (2, 3). The mRNA lacking exon 9 sequence is observed, encoding a Tg human Tg gene, located on chromosome 8 (8q24.2– 8q24.3), protein of 250 kDa (17, 18). In Dutch goats, a homozygous is over 300 kb and contains over 37 exons (4 – 6). We recently nonsense mutation, Tyr296AMB, results in a truncated Tg revised the Tg messenger RNA (mRNA) sequence that was protein product (40 kDa in vivo) and causes hypothyroidism originally reported in 1987 (7). This revealed 8307 nucleo- with goiter (19, 20). Furthermore, in a mouse model, con- tides of coding sequence (instead of 8304), of which 66 triplets genital goiter (cog/cog) is linked to the Tg locus (21), and the (instead of 67) encode a tyrosine residue (8). Until now, 19 mutation has recently been identified as Leu2366Pro (22). So far in only three patients with congenital hypothyroid- ism and goiter has a mutation in the Tg gene been elucidated. Received December 30, 1998. Revised March 25, 1999. Accepted April 8, 1999. A homozygous mutation at the acceptor splice site of intron Address all correspondence and requests for reprints: Simone A.R. 3 results in the in-frame deletion of exon 4 sequences (nt 275 van de Graaf, Academic Medical Center G2–123, Laboratory of Pediatric - 478) from the mRNA, which results in an aberrant Tg Endocrinology, PO Box 22700, 1100 DE, Amsterdam, The Netherlands, protein lacking hormonogenic site Tyr130 (15). A homozy- Fax: **31.20.6916396, e-mail: S.A.vandeGraaf@amc.uva.nl. This work was supported in part by the Ludgardine Bouwman Foun- gous in-frame mRNA deletion is described of 138 bp (nt dation (The Netherlands) and by Grant 96/00998 from the Sao Paulo ˆ 5552–5789)(23). The preferential accumulation of a Tg mRNA State (Brazil) Research Foundation (FAPESP). alternative splice product with an in-frame deletion of 171 bp 2537 2538 VAN DE GRAAF JCE & M • 1999 Vol 84 • No 7 (nt 4529 – 4699, exon 22) has also been reported, linked to a cells and follicular lumen devoid of colloid. Immunostaining for Tg homozygous nonsense mutation at position 1510 (13). indicated the presence of Tg-related antigens only inside the cells. In the present study, we have identified a homozygous nonsense mutation in the thyroglobulin mRNA of a mod- RNA isolation and complementary DNA (cDNA) erately hypothyroid patient with goiter. preparation, genomic DNA isolation Total RNA was isolated from goitrous (patient IV-6) and control thyroid tissue using TRIzol®Reagent (Life Technologies BV). cDNA was Materials and Methods synthesized using random hexamers and reverse transcriptase accord- Patients ing to standard procedures. Genomic DNA of patient IV-6 was isolated from one of the TRIzol Figure 1 shows the pedigree of a Brazilian family with goiter in three fractions, and genomic DNA of the indicated family members was affected siblings (Table 1). isolated from white blood cells by the SDS-proteinase K method (24). Patient IV-2: female, first examined at the age of 17. She developed normally and had menarche at 15 yr of age. Her height was 149 cm, and DNA amplification her weight 43.5 kg. The thyroid gland was diffusely enlarged (65 g; normal: 7.4 2.2 g), and ultrasonography indicated a nodule of 15 mm PCR amplification (25) was performed using 100 ng cDNA as tem- in the left lobe with micro-calcifications. She has two unaffected children plate in a total reaction volume of 25 L. (V-1, V-2) from a consanguineous marriage. For nucleotide sequencing, fragments of 500 bp (with 20 –70 bp over- Patient IV-6 (index patient): male, first examined at the age of 13. At lap) were amplified with 2.5 units of AmpliTaq DNA polymerase (Per- presentation, he showed clinical signs of hypothyroidism and stunted kin Elmer) using the protocol: 2 min 94 C; 35 cycles of 15 sec 94 C, 1 min growth (bone age, 7 yr). His mental function was normal. The thyroid 60 C, 1 min 72 C; 10 min 72 C. The human Tg specific oligonucleotides gland was diffusely enlarged (60 g; normal: 7.4 2.2 g), and ultra- (synthesized on Expedite Nucleic Acid Synthesis System, Millipore sonography indicated a nodule of 13 mm in the right lobe. Corp.) coupled to M13 tags are already described (10). Reactions were Patient IV-10: male, first examined at the age of 2. He developed electrophoresed on 0.8 agarose gel and purified using the Quiaquick slowly and showed retarded growth (bone age, 3 months). His mental DNA gel extraction kit (Quiagen). function appeared to be normal. Ultrasonography of the thyroid gland For determination of alternative splice products, a cDNA fragment indicated a diffuse heterogeneous goiter of 13.5 g (normal: 4.1 1.1 g). ranging from exon 4 to exon 9 (nt 400-1350) was amplified with 2.5 units Patients IV-2 and IV-6 were subjected to subtotal thyroidectomy to of AmpliTaq DNA polymerase (Perkin Elmer) using the protocol: 2 min correct compressive symptoms and have received total thyroxine sup- 94 C; 35 cycles of 15 sec 94 C, 1 min 57 C, 1.5 min 72 C; 10 min 72 C. M13 plementation since that time. linked oligonucleotides 2F (nt 400) and 3R (nt 1350) were used (10). Both anti-TPO and anti-Tg antibody tests were repeatedly negative For subcloning purposes, the same conditions and oligonucleotides in all three patients. No data were available on iodine intake and urinary were used. secretion. For restriction fragment length polymorphism (RFLP) analysis, Microscopic examination of the goitrous tissue revealed the classic genomic DNA was amplified using the protocol: 5 min 95 C; 35 cycles macro-follicular pattern, with dilated follicles lined with high columnar of 1 min 95 C, 1 min 57 C, 1.5 min 72 C; 10 min 72 C, 2.5 units of AmpliTaq FIG. 1. Family pedigree of index pa- tient. PREMATURE STOP IN Tg mRNA IN A GOITROUS PATIENT 2539 TABLE 1. Clinical and laboratory data from the affected siblings No Year of birth TT4 nmol/L TT3 nmol/L TSh mU/L Serum Tg g/L: basal/48 ha % RAI uptake: at 2 h/at 24 hb IV-2 1970 82.3 2.6 13 1.5/1.6 55/54 IV-6 1976 38.6 1.5 112 3.5/4.0 54/71 IV-10 1985 25.7 1.2 96 2.8/5.6 8/35 Normal range 70 –160 1.1–3.1 4.5 12 6.5/34 16 a Serum Tg levels: basal and 48 h after 10 IU of bovine TSH im (mean and SD from ref. 27). b Administration of perchlorate in radioactive iodide uptake studies (RAI) had no effect in any of the siblings. DNA polymerase (Perkin Elmer), and the following oligonucleotides: Tg (26) coupled to protein A-sepharose CL-4B (Pharmacia Biotech) and (794) 5 TGGACCTTCCTTCCACCTTCACTG 3 and (1002) 5 CCTTC- were analyzed identically. CGTCTGGCACTGCA 3 . Restriction fragment length polymorphism analysis Nucleotide sequence analysis The mutation detected by nucleotide sequencing at position 886 cre- DNA amplification resulted in 20 fragments of approximately 500 bp ated an AlwN I recognition site. This enzyme was used according to the covering the entire thyroglobulin cDNA of patient IV-6. manufacturers protocol to screen for the presence of the mutation in the Both the sense and antisense strand were sequenced using either the amplified genomic DNA Tg fragment of the indicated family members M13 tags linked to the PCR fragments with the Big Dye Primer Cycle (III-1, III-2, IV-6, IV-10, IV-2, V-1, V-2) and of a wildtype control. The Sequencing Kit or the Tg-specific oligonucleotides with the Big samples were run on 2.5% agarose gel and stained with ethidium Dyedeoxyterminator Cycle Sequencing Kit, depending on the GC con- bromide. tent of the fragment (both kits from PE Applied Biosystems/Perkin Elmer). After electrophoresis on a sequencing gel, the samples were analyzed on the ABI Prism 377 DNA sequencer and aligned to the Tg Results cDNA sequence (8, 10) using AutoAssembler software (PE Applied The index patient (IV-6), suspected of having a defect in Biosystems/Perkin Elmer). thyroglobulin synthesis as a cause for hypothyroidism, was Determination of alternative splice products subjected to subtotal thyroidectomy, and thyroid tissue was available to screen for Tg mutations. RT-PCR was performed The coding region of nt 400-1350 (exon 4 –9) was amplified on mRNA on the total RNA isolated from this tissue, resulting in 20 of patient IV-6 and of wildtype, and the reactions were run on a 1.2% agarose gel stained with ethidium bromide. overlapping fragments of 500 bp each, covering the total coding region of 8307 bp. Direct sequencing revealed a cy- Subcloning of mutated Tg fragment tosine-to-thymidine mutation at nt position 886 (Fig. 2A). Its position in the gene near the end of exon 7 is schematically The TGI construct (8) containing wildtype Tg nucleotides 6 to 2110 in the pcDNA3 plasmid (TG-WT) was restricted with Bsu36 I, thereby given in Fig. 2B, and the supposed amino acid sequence after deleting the wildtype sequence from nt 686-1137, and purified from a translation is also shown. Instead of encoding for an arginine 0.8% agarose gel. The amplified product of 950 bp, containing the mu- residue on position 277, the triplet harboring the mutation tation at nt position 886 was also restricted with Bsu36 I. The mutant encodes a stopcodon. fragment of 451 bp was purified from a 1.2% agarose gel and ligated into the digested TG-WT resulting in TG-M. By automatic sequencing using Because the C886T transition induces a AlwN I restriction Tg specific primers, the nt sequence was validated. site, carriership for the mutation could be established using The protocols used for digesting with Bsu36 I (Biolabs) and for li- RFLP analysis. Therefore a fragment ranging from exon 7 to gation with T4 ligase (Boehringer Mannheim; rapid DNA ligation kit) 8 (including intron 7 of 200 bp) was amplified on genomic were according to the manufacturer. For gel purification, the Quiaquick DNA, isolated from blood of different family members: III-1, DNA gel extraction kit (Quiagen) was used. Standard heat shock trans- formation was performed with DH5 competent cells (Gibco BRL). III-2, IV-6 (index patient), IV-10, IV-2, V-1, and V-2 (Fig. 1). The wildtype amplified fragment of 400 bp was not digest- Expression of Tg in rabbit reticulocyte lysate and analysis ible by AlwN I. The AlwN I digestion of the mutated frag- ment resulted in two fragments of about 300 and 100 bp (Fig. For in vitro transcription and translation a TnT® T7 Coupled Reticu- locyte Lysate System was used (Promega Corp.), providing rabbit re- 2C). All affected siblings (IV-6, -10, -2) showed two fragments ticulocyte lysate, a reaction buffer, T7 RNA polymerase, and an amino after digestion (300 and 100 bp) and are homozygous for the acid mixture lacking cysteine. Reactions of 25 L were done, adding mutation. Both parents (III-1, -2) and the youngest offspring Rnasin® Ribonuclease Inhibitor (Promega Corp.), a mixture of 35S-me- (V-1, -2) showed three fragments after digestion (400, 300, thionine and 35S-cysteine (ICN Pharmaceuticals, Inc.; Tran35S-label). To obtain glycosylation, Canine Pancreatic Microsomal Membranes and 100 bp) and are heterozygous (carriers). (CPMM) (Promega Corp.) were added. In each reaction 300 ng plasmid To determine whether the nonsense mutation caused al- DNA was used as template (TG-WT or TG-M or positive control). Pos- ternative splicing of exon 7, a cDNA fragment ranging from itive control 1 was used to check for expression of a protein of 61 kDa exon 4 to 9 was amplified from mRNA of patient IV-6 and mol wt. Control sample 2 was used to check for glycosylation. Incubation a wildtype control (data not shown). No difference in either was done at 30 C for 90 min. Aliquots of 5 L (minus CPMM) or 10 L (plus CPMM) of reticulocyte splicing or abundance of the amplified product was detected. lysate reactions, together with a molecular weight marker (Biolabs; The mutation was expected to result in a truncated Tg Rainbow general), were subjected to SDS-PAGE according to Laemmli’s molecule upon translation, still harboring putative N-glyco- method (17.5%), and the gel was dried afterwards. The radioactive signal sylation sites. To examine translation and putative glycosyl- of expressed proteins was detected using a Phosphorimager and Image Quant software (Molecular Dynamics, Inc.). ation in relation to the mutation, a cell-free in vitro transcrip- Protein products from 40 L reticulocyte lysate reactions, were im- tion/translation system (rabbit reticulocyte lysate T7 RNA munoprecipitated using a rabbit polyclonal antibody specific for human polymerase) was used in which glycosylation conditions 2540 VAN DE GRAAF JCE & M • 1999 Vol 84 • No 7 FIG. 2. Screening for mutation in Tg mRNA. A, Part of wildtype and mutated (patient IV-6) thyroglobulin mRNA sequence. The arrow points to the homozygous C886T transition. B, Schematic drawing of thyroglobulin mRNA. Top: coding region from nt 1– 8307 (italics) appears as an open box, 5 and 3 untranslated regions are indicated as solid lines. Middle: part of the Tg gene showing exon 7 and exon 8 (introns as dashed lines). Bottom: coding nt sequences and corresponding aa sequences are shown (AlwN I recognition site is underlined). C, Agarose electrophoresis and ethidium bromide visualization of AlwN I RFLP analysis. Shown are the mol wt marker lane (bp), wildtype control plus or minus AlwN I, respectively, and the PCR amplified genomic DNA fragment (exon 7 - 8) of several family members after digestion. The open arrow indicates undigested wildtype fragment, and the filled arrows indicate mutant fragments resulting from AlwN I digestion. PREMATURE STOP IN Tg mRNA IN A GOITROUS PATIENT 2541 Mendelian pattern of inheritance of an autosomal recessive mutation. The affected family members all showed goiter with moderate hypothyroidism. Serum Tg levels were rel- atively low despite the high serum TSH levels and did not increase after exogenous bovine TSH stimulation (27). The absence of an iodide organification defect was based on the results of the radioactive iodide uptake studies (RAI): a high and rapid uptake and no iodide washout effect by admin- istered perchlorate ions (Table 1). These characteristics in- dicated a defect in thyroglobulin synthesis. After sequencing the total Tg cDNA of patient IV-6, a homozygous nonsense mutation was determined, which re- sulted in an AlwN I recognition site (Fig. 2). RFLP analysis demonstrated that both parents were heterozygous for this mutation and that all three affected siblings carried the same mutated Tg alleles. The mutation is a cytosine-to-thymidine transition at nt position 886 in exon 7, creating a stopcodon at amino acid Arg277. The change occurs in a CpG dinucle- otide and can be caused by deamination of a methylated cytosine to thymine (28). Furthermore, the CGA arginine codon is considered a “hot spot” for mutations (29). RNA transcripts containing a premature stopcodon may FIG. 3. In vitro expression of wildtype and mutant Tg fragment. In- be relatively unstable because the untranslated part of the cubations of rabbit reticulocyte lysates with wildtype (TG-WT) or messenger is not protected by ribosomes (13, 19). However, mutant (TG-M) or control (provided by the kit) templates or no tem- we have no indication that the mRNA of patient IV-6 was plate (none) were performed with 35S-labeled amino acids. After SDS- PAGE, proteins were visualized using the Phosphorimager. Open very unstable because, after total RNA isolation and RT-PCR arrows indicate wildtype Tg proteins, and filled arrows indicate mu- amplification of 500 - 950 bp fragments, the results of normal tant Tg proteins. Panels A and C correspond with the arrows on the and patient’s thyroid tissues were similar with respect to the left. In the reactions shown in panels B and D, microsomal mem- quantity of the generated products. The amount of tissue, branes were added to provide glycosylation (corresponding with ar- rows on the right). Panels C and D show the electrophoresis of im- however, was not sufficient to perform a Northern blot. munoprecipitated samples. The mol wt marker bands are indicated No differences were detected in expression of the de- (kDa). scribed alternative transcripts (10) compared with normals (data not shown). After RT-PCR amplification of a fragment from nt 400-1350 (exon 4 to 9) no differences in product could be established by addition of microsomal membranes length and abundance were detected between patient and (CPMM). For comparison two expression constructs were wildtype samples. This excluded an alternative splice event used containing the first 2110 bp of coding Tg sequence: of the Tg RNA, as is described for Afrikander cattle (17) and wildtype (TG-WT) and mutant (TG-M). 35S-labeled me- human (13), where a nonsense mutation at amino acid res- thionine and cysteine were incorporated, enabling visu- idue 687 and residue 1510, respectively, results in a relatively alization of the expressed protein after SDS-PAGE using increased expression of a smaller messenger RNA species the Phosphorimager. The apparent mol wts of the proteins lacking the mutated exon. The specific skipping of exons expressed from TG-WT and TG-M were respectively containing a nonsense mutation has also been described else- 76,000 and 30,000 (Fig. 3A). After addition of CPMM, both where (30). TG-WT and TG-M proteins were glycosylated, as observed Upon translation the mutated Tg transcript generated a Tg by a shift in apparent molecular weight, although the protein, validated by immunoprecipitation with a specific expression of the TG-WT was low (Fig. 3B). The controls antihuman-Tg antibody, with an apparent mol wt of 30,000 1 and 2 provided by the manufacturer showed that both (Fig. 3). This was in good accordance with the predicted mol translation (Fig. 3A, lane control 1) and glycosylation (Fig. wt of 30,778 (276 amino acid residues). Three putative N- 3B, lane control 2) occurred. To validate that the expressed linked glycosylation sites are still present in this truncated proteins were human thyroglobulin fragments, the reac- protein, of which two have been shown to be glycosylated in tion samples were immunoprecipitated with an anti- the mature protein (Asn57 and Asn179) (31). The use of human-Tg polyclonal. Specific recognition of the Tg wild- microsomal membranes in the in vitro expression assay in- type and mutant proteins with and without glycosylation dicated that the aberrant Tg protein could indeed be is shown in Fig. 3C and 3D respectively. glycosylated. It has been reported that the phenotypic expression of Discussion defective Tg protein varies considerably when different fam- In this paper we present the results of studies conducted ilies or affected siblings within the same family are compared in a family in which goiter and hypothyroidism occur. Three (32). Although the laboratory tests were performed at a siblings out of ten, from a consanguineous marriage, showed younger age in patient IV-10 than in patients IV-2 and IV-6, thyroid function abnormalities (Fig. 1), in accordance with a it seems that the consequences of the defective Tg synthesis 2542 VAN DE GRAAF JCE & M • 1999 Vol 84 • No 7 in patient IV-2 are less severe. Apparently the goiter is able roglobulin variant: the guanine-to-adenine transition resulting in substitution of arginine 2510 by glutamine. Thyroid. 7:587–591. to compensate for the hypothyroid status with a somewhat 10. van de Graaf SAR, Cammenga C, Ponne NJ, et al. 1999 The screening for elevated TSH. Her brothers show diminished total T4 levels mutations in the thyroglobulin cDNA from six patients with congenital hy- while their total T3 levels are in the normal range (Table 1). pothyroidism. Biochimie. 81:425– 432. 11. Mason ME, Dunn D, Wortsman J, et al. 1995 Thyroids from siblings with Thyroid hormone synthesis involves a two-step modifi- Pendred’s syndrome contain thyroglobulin messenger ribonucleic acid vari- cation of tyrosine residues. Iodination and subsequent cou- ants. J Clin Endocrinol Metab. 80:497–503. pling take place at the apical membrane of the cell, and both 12. Bertaux F, Noel M, Lasmoles F, Fragu P. 1995 Identification of the exon structure and four alternative transcripts of the thyroglobulin-encoding gene. reactions are catalyzed by thyroid peroxidase (2, 3). The Gene. 156:297–301. specific iodinated tyrosine residues that are involved in the 13. Targovnik HM, Medeiros-Neto G, Varela V, Cochaux P, Wajchenberg BL, Vassart G. 1993 A nonsense mutation causes human hereditary congenital coupling reaction can either accept (hormonogenic sites) or goiter with preferential production of a 171-nucleotide-deleted thyroglobulin donate iodinated phenyl groups. The most important accep- ribonucleic acid messenger. J Clin Endocrinol Metab. 77:210 –215. tor site in all vertebrate species examined is at Tyr5, while 14. Targovnik HM, Cochaux P, Corach D, Vassart G. 1992 Identification of a minor Tg mRNA transcript in RNA from normal and goitrous thyroids. Mol priority for hormonogenesis at the other acceptor sites Cel Endocrinol. 84:R23–R26. Tyr1291, Tyr2554, and Tyr2747 varies among species (2). For 15. Ieiri T, Cochaux P, Targovnik HM, et al. 1991 A 3 splice site mutation in the human Tg, three potential donor sites have been identified thyroglobulin gene responsible for congenital goiter with hypothyroidism. J Clin Invest. 88:1901–1905. so far (Tyr130, Tyr847, Tyr1488) (33). The truncated form of 16. Bertaux F, Noel M, Malthiery Y, Fragu P. 1991 Demonstration of a hetero- ` Tg described here harbors both the acceptor Tyr5 and the genous transcription pattern of thyroglobulin mRNA in human thyroid tis- sues. Biochem Biophys Res Commun. 178:586 –592. donor Tyr130 residues. This feature, as well as its size and 17. Ricketts MH, Simons MJ, Parma J, Mercken L, Dong Q, Vassart G. 1987 A ability to become glycosylated, makes it comparable to the nonsense mutation causes hereditary goitre in the Afrikander cattle and un- truncated Tg product in the goitrous Dutch goats. In these masks alternative splicing of thyroglobulin transcripts. Proc Natl Acad Sci USA. 84:3181–3184. animals the glycosylated Tg fragment (mol wt of 40,000) was 18. Tassi VPN, Di Lauro R, van Jaarsveld P, Alvino CG. 1984 Two abnormal able to synthesize T4 in vivo, and the amounts produced were thyroglobulin-like polypeptides are produced from Afrikander cattle congen- comparable to normal when 1 mg iodide/day was admin- ital goiter mRNA. J Biol Chem. 259:10507–10510. 19. Veenboer GJM, de Vijlder JJM. 1993 Molecular basis of the thyroglobulin istered, although goiter remained (34). It has also been re- synthesis defect in Dutch goats. Endocrinology. 132:377–381. ported that, in man, oral administration of excess iodine can 20. Sterk A, van Dijk JE, Veenboer GJM, Moorman AFM, de Vijlder JJM. 1989 Normal sized thyroglobulin mRNA in Dutch goats with a thyroglobulin syn- partially correct the hypothyroid condition in patients with thesis defect is translated into a 35,000 molecular weight N-terminal fragment. defective Tg synthesis (35). Therefore iodide administration Endocrinology. 124:477– 483. may explain in part the variability in the clinical presentation 21. Taylor BA, Rowe L. 1987 The congenital goiter mutation is linked to the thyroglobulin gene in the mouse. Proc Natl Acad Sci USA. 84:1986 –1990. of the affected individuals within this family. 22. Kim PS, Hossain SA, Park YN, Lee I, Yoo SE, Arvan P. 1998 A single amino In conclusion, molecular analysis of a family with hered- acid change in the acetylcholesterase-like domain of thyroglobulin causes itary hypothyroidism and goiter reveals a novel autosomal congenital goiter with hypothyroidism in the cog/cog mouse - a model of human endoplasmatic storage diseases. Proc Natl Acad Sci USA. 95:9909 –9913. recessive mutation in the thyroglobulin gene. The mutation 23. Targovnik HM, Vono J, Billerbeck AEC, et al. 1995 A 138-nucleotide-deletion is a C-to-T transition at nt position 886 in exon 7, creating a in the thyroglobulin ribonucleic acid messenger in a congenital goiter with defective thyroglobulin synthesis. J Clin Endocrinol Metab. 80:3356 –3360. stopcodon at amino acid Arg277. The expressed truncated Tg 24. Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular cloning-a laboratory protein has a mol wt of 30,778, can be glycosylated, and is still manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. able to produce thyroid hormone. 25. Saiki RK, Gelfland DH, Stoffel S, et al. 1988 Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 239:487– 491. Acknowledgments 26. Den Hartog MT, de Boer M, Veenboer GJM, de Vijlder JJM. 1990 Generation and characterization of monoclonal antibodies directed against noniodinated We thank J. Vono, M.D., for the clinical work on this family. and iodinated thyroglobulin, among which are antibodies against hormono- genic sites. Endocrinology. 127:3160 –3165. References 27. Medeiros-Neto G, Marcondes JA, Cavaliere H, Wajchenberg BL, Knobel M. 1985 Serum thyroglobulin (Tg) stimulation with bovine TSH: an useful test for 1. de Vijlder JJM, Vulsma T. 1996 Hereditary metabolic disorders causing hy- diagnosis of congenital goitrous hypothyroidism due to defective Tg synthesis. pothyroidism. In: Braverman LE, Utiger RD, eds. Werner’s & Ingbar’s The Acta Endocrinol (Copenh). 110:61– 65. Thyroid. Philadelphia: J.B. Lippincott Company; 749 –755. 28. Coulondre C, Miller JH, Farabaugh PJ, Gilbert W. 1978 Molecular basis of 2. Dunn JT. 1996 Thyroglobulin: chemistry and biosynthesis. In: Braverman LE, base substitution hotspots in Escherichia coli. Nature. 274:775–780. Utiger RD, eds. Werner’s & Ingbar’s The Thyroid. Philadelphia: J.B. Lippincott 29. Antonarakis SE, Kazazian HH. 1988 The molecular basis of hemophilia A in Company; 85–95. man. Trends Genet. 4:233–237. 3. de Vijlder JJM, den Hartog MT. 1997 Anionic iodotyrosine residues are 30. Dietz HC, Valle D, Francomano CA, Kendzior RJ, Pyeritz RE, Cutting GR. required for iodothyronine synthesis. Eur J Endocrinol. 138:227–231. 1993 The skipping of constitutive exons in vivo induced by nonsense mutations. 4. Baas F, Bikker H, Geurts van Kessel A, et al. 1985 The human thyroglobulin Science. 259:680 – 683. gene: a polymorphic marker localized distal to c-myc on chromosome 8 band 31. Yang S-X, Pollock HG, Rawitch AB. 1996 Glycosylation in human thyroglob- q24. Hum Genet. 69:138 –145. ulin: location of the N-linked oligosaccharide units and comparison with 5. Berge-Lefranc JL, Cartouzou G, Mattei MG, Passage E, Malezet-Desmoulins ´ bovine thyroglobulin. Arch Biochem Biophys. 327:61–70. C, Lissitzky S. 1985 Localization of the thyroglobulin gene by in situ hybrid- 32. Medeiros-Neto G, Targovnik HM, Vassart G. 1993 Defective thyroglobulin ization to human chromosomes. Hum Genet. 69:28 –31. synthesis and secretion causing goiter and hypothyroidism. Endocr Rev. 6. Baas F, van Ommen GJ, Bikker H, Arnberg AC, de Vijlder JJM. 1986 The 14:165–183. human thyroglobulin gene is over 300 kb long and contains introns of up to 33. Lamas L, Anderson PC, Fox JW, Dunn JT. 1989 Consensus sequences for early 64 kb. Nucleic Acids Res. 14:5171–5186. iodination and hormonogenesis in human thyroglobulin. J Biol Chem. 7. Malthiery Y, Lissitzky S. 1987 The primary structure of human thyroglobulin ` 264:13541–13545. deduced from the sequence of its 8448 base complementary DNA. Eur J Bio- 34. de Vijlder JJM, van Voorthuizen WF, van Dijk JE, et al. 1978 Hereditary chem. 165:491– 498. congenital goiter with thyroglobulin deficiency in a breed of goats. Endocri- 8. van de Graaf SAR, Pauws E, de Vijlder JJM, Ris-Stalpers C. 1997 The revised nology. 102:2105–2111. 8307 base pair coding sequence of human thyroglobulin transiently expressed 35. Vono J, Lima N, Knobel M, Medeiros-Neto GA. 1996 The effect of oral in eukaryotic cells. Eur J Endocrinol. 136:508 –515. administration of iodine to patients with goiter and hypothyroidism due to 9. Mendive FM, Vassart G, Targovnik HM. 1997 Identification of a new thy- defective synthesis of thyroglobulin. Thyroid. 6:11–15.
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