A Missense Mutation G2320R in the Thyroglobulin Gene Causes Non ...

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tant Tg that accumulates inside the cell, unable to reach its final destination (2, 3). Normal Tg must first be folded and assembled into its proper tertiary and qua-.
A Missense Mutation G2320R in the Thyroglobulin Gene Causes Nongoitrous Congenital Primary Hypothyroidism in the WIC-rdw Rat

Paul S. Kim*, Ming Ding*, Shekar Menon, Cha-Gyun Jung, Ji-Ming Cheng, Tomomi Miyamoto, Bailing Li, Sen-ichi Furudate, and Takashi Agui Division of Endocrinology (P.S.K., S.M., B.L.) Department of Medicine University of Cincinnati and Veterans Affairs Medical Center Cincinnati, Ohio 45267 Graduate Program in Cell and Molecular Biology (P.S.K., S.M.) Department of Cell Biology University of Cincinnati College of Medicine Cincinnati, Ohio 45267 Center for Experimental Animal Science (M.D., C.-G.J., J.-M.C., T.M., T.A.) Nagoya City University Medical School Nagoya, Aichi 467-8601, Japan Department of Laboratory Animal Science (S.-i.F.) Kitasato University School of Medicine Sagamihara, Kanagawa 228-8555, Japan

A convincing line of evidence is being developed that the congenital nongoitrous hypothyroidism and dwarfism observed in the WIC-rdw rat may indeed be caused by a primary defect in thyroid hormonogenesis. In support of this hypothesis, several recent reports have shown the presence of elevated molecular chaperone levels in the WICrdw thyrocytes, the endoplasmic reticulum of which was markedly dilated, suggesting a defect in intracellular protein transport. Here the studies were undertaken to identify the precise molecular defect in the WIC-rdw rat. First, the genetic linkage analysis revealed that the rdw locus was on rat chromosome 7 and was identical to the thyroglobulin (Tg) gene locus. Moreover, the Tg protein level was reduced in the WIC-rdw thyroid despite a similar level of the Tg gene transcripts that were indistinguishable in their size from the normal. Next, the complete sequencing of the rdw and the normal rat Tg cDNAs revealed a single nucleotide change, G6958C, resulting in a G2320R missense mutation in a highly conserved region of the Tg molecule. Finally, transient expression of the intact Tg cDNA containing the rdw mutation in the COS-7 0888-8809/00/$3.00/0 Molecular Endocrinology 14(12): 1944–1953 Copyright © 2000 by The Endocrine Society Printed in U.S.A.

cells showed no detectable Tg in the secreted media, indicating a severe defect in the export of the mutant Tg. Together, our observations suggest that a missense mutation, G2320R, in the Tg gene is responsible for the rdw mutation in the WIC-rdw rat. (Molecular Endocrinology 14: 1944–1953, 2000)

INTRODUCTION A vast majority of inherited congenital hypothyroid goiter (CHG), characterized by mental retardation and abnormal growth, is caused by a deficiency in one of the components of hormonogenesis in the thyroid, including thyroid peroxidase, sodium iodide symporter, or thyroglobulin (Tg). In humans, the overall incidence of congenital hypothyroidism is ⬃1:3,000– 4,000 newborns, caused either by thyroid dysgenesis (⬃80%) or dyshormonogenesis (⬃20%). Although uncommon, qualitative or quantitative defects of Tg are an established cause of CHG (1). Although the precise molecular mechanism has not been well established in most cases, at least in several reports, a common defect appears to be the presence of misfolded mutant Tg that accumulates inside the cell, unable to reach its final destination (2, 3). Normal Tg must first be folded and assembled into its proper tertiary and quaternary structure in the rough endoplasmic reticulum 1944

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(ER) before it can be exported along the distal secretory pathway, ultimately to an extracellular space known as the colloid lumen, where it is iodinated and stored. Thus, perturbations in the folding of nascent Tg can often lead to defective intracellular transport of Tg (4). During the past, the structural properties of the Tg have been well characterized (5): a 660-kDa glycoprotein that is secreted as a homodimer to serve as the unique peptide backbone on which thyroid hormones are synthesized. As the major secretory product of the thyrocytes, Tg typically accounts for as much as 50% of total protein in the thyroid gland. The WIC-rdw rat, established from a closed colony of Wistar-Imamichi (WIC) rats as a spontaneous mutant exhibiting congenital dwarfism (rdw), is inherited as an autosomal recessive (6). Although the initial reports of reduced circulating levels of both GH and PRL suggested hypopituitarism in the WIC-rdw rat (7–10), elevated TSH levels and the reduced level of T3 and T4 pointed toward a primary defect in thyroid hormone production (11). The latter has been supported by several recent studies which showed the restoration of not only their normal growth but also their normal serum levels of all pituitary hormones after the administration of T4 (8), feeding of extracts of the bovine thyroid (S-i. Furudate, unpublished data), or normal thyroid transplantation (S-i. Furudate, unpublished data). A recent morphological study of WIC-rdw thyrocytes further revealed dilated ER and reduced secretory granules as well as very low levels of Tg in the colloid lumen (12). These and other observations have strongly suggested that the dwarfism is attributable to a primary defect of the thyroid and not of the pituitary. Moreover, it was recently reported (13) that the molecular chaperones were markedly elevated in the WIC-rdw rat thyroid, similar to the cog/cog mouse that was defective in Tg export (4), thus exhibiting many features of an endoplasmic reticulum storage disease [ERSD (3)]. Interestingly, in a dramatic contrast to most human patients and animal models of CHG, histological analysis revealed a surprisingly hypoplastic thyroid gland that was smaller than the normal control despite elevated circulating levels of TSH in the WICrdw rat (11). In the present study, in identifying the gene responsible for the observed phenotype, the rdw locus was mapped to the rat chromosome (Chr) 7 and found to be identical to the Tg gene locus, thus prompting our investigation to focus on the Tg gene. Additional studies revealed that the Tg protein level was reduced in the WIC-rdw thyroid, yet the transcripts of the Tg gene were similar in size and quantity, suggesting a possible defect in the Tg molecule. Consequently, a search for the mutation of the Tg gene revealed a single nucleotide (nt) substitution that cause a Gly to Arg change at a position that is highly conserved in other species including the human, mouse, and bovine. Here we provide solid evidence that a missense mutation in the Tg gene is indeed responsible for the

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nongoitrous congenital primary hypothyroidism in the WIC-rdw rat.

RESULTS Chromosomal Mapping of the rdw and Tg Gene Loci As the first step in identifying the gene responsible for the WIC-rdw phenotype, we started with the chromosomal mapping of the rdw locus using 138 (BN ⫻ WIC-rdw)F1 ⫻ WIC-rdw backcrosses (BC). The rdw locus was mapped to the rat Chr 7, 7.6 centimorgans (cM) downstream of the microsatellite locus D7Rat19 and 6.6 cM upstream of the D7Rat136 (Fig. 1). The rat Tg gene locus has already been assigned to Chr 7 using somatic cell hybrid analysis, although its detailed location was unknown (14). Since it was reported that the mutations in the Tg gene caused Tg deficiency in several animal models, we continued to map the rat Tg gene locus using the same BC genetic panel. Using previously elucidated information on the exon-intron structure of the rat Tg gene (15), Tg gene

Fig. 1. Linkage Mapping of the rdw and Tg Gene Loci in Rat Chr 7 The numbers on the left of the line indicate genetic distance [centimorgans (cM)] between each locus listed on the right.

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introns were sequenced, revealing a G to A substitution at nt 764 in intron 2 of the WIC-rdw rat Tg gene as compared with that of the BN rat. This nt substitution caused loss of a recognition site for the restriction enzyme Cac8I. When the PCR product of the Tg gene intron 2 (1,050 bp) from the BN rat was digested with Cac8I, five DNA fragments (394, 356, 245, 35, and 20 bp) were observed as predicted by the sequence analysis of intron 2, which contained four Cac8I recognition sites. In Fig. 2, lane 2, only three bands (394, 356, and 245 bp) were observed in the BN normal rat sample, since two short fragments (20 and 35 bp) ran off the gel (not shown). However, in the homozygous WIC-rdw rat samples, the band of 245 bp (open arrow) was converted to a 280-bp band (245 ⫹ 35 bp; closed arrow) due to the loss of one of the Cac8I sites (Fig. 2, lane 3). The genotype of the Tg gene was further determined by the presence of the 245-bp and 280-bp bands in all the BC samples (Fig. 2, lanes 4–7). These mapping results indicated that the Tg gene locus was identical to the rdw locus (Fig. 1). The full-length sequence of the rat Tg gene intron 2 has been submitted to GenBank (accession number, AF221623). Examination of Tg Gene Expression in the WIC-rdw Rat Thyroid When compared with the normal control, the expression of the Tg gene was modestly up-regulated in the WIC-rdw rat thyroid. More importantly, as shown in Fig. 3, the transcripts of the Tg gene were normally detected in the WIC-rdw thyroid. The similar size and abundance of the WIC-rdw rat Tg mRNA (⬃8.5 kb) in comparison to the F344 wild-type (F344-wt) normal control further excluded the possibility of a gross gene deletion, a quantitative defect in transcription, or Tg mRNA instability. Next, the steady-state level of the Tg protein in the WIC-rdw rat thyroid was examined by SDS-PAGE and

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Fig. 3. Northern Blotting of the Tg Gene Transcripts in the WIC-rdw and F344-wt Rats mRNAs isolated from the WIC-rdw and F344-wt rat thyroid homogenates were run on 1% agarose gel and analyzed by Northern blot analysis using a Tg cDNA fragment containing the 5⬘-end to detect the full-length Tg gene transcript (⬃8.5 kb). Both the 28S and 18S RNAs (not shown) ran faster than the Tg mRNA. ␤-Actin mRNA levels were also measured on a separate gel that was loaded with equal aliquots of the mRNA extracts.

Coomassie staining (Fig. 4A). The 330-kDa band representing the intact Tg was significantly reduced in the WIC-rdw rat. This band was verified to be the Tg protein by loading the purified human and mouse Tg sample on the same gel (not shown) and by Western blotting using antirat Tg antibody (Fig. 4B). Of note, several lower mol wt bands were significantly elevated in the WIC-rdw rat thyroid, which likely represent GRP94 (Fig. 4A, ⬃p95) BiP/GRP78 (Fig. 4A ⬃p75), and other ER resident proteins (not shown) as reported previously (13). The elevated levels of these ER molecular chaperones, which are essential in assisting in the folding of many nascent polypeptides, in both the heterozygous and homozygous rat thyroids (Fig. 4B, lanes 2 and 3, respectively), further suggested that the WIC-rdw rat appeared to exhibit an ERSD-like feature previously described in the cog/cog mouse (4) and human patients with CHG (16). Together, these observations were consistent with the possibility that the rdw mutation could be a point mutation in the Tg gene as in the cog/cog mouse or several human families (2). Complete Sequencing of the Tg cDNAs of Normal and WIC-rdw Rats

Fig. 2. Determination of the Tg Gene Genotypes in BC Rats Genomic DNAs were amplified by PCR with intron 2specific primers, treated with the restriction enzyme Cac8I, and then subjected to 6% PAGE. Closed and open arrows indicate WIC-rdw (280 bp) and BN (245 bp) alleles, respectively. Lane 1, Hae digests; lane 2, BN rat; lane 3, WIC-rdw rat; lanes 4 and 5, representative BC rats with homozygous WIC-rdw genotype; lanes 6 and 7, representative BC rats with F1 genotype. The band sizes (bp) are listed on the left.

First, incomplete Tg cDNAs were isolated from the two cDNA libraries generated from the F344-wt normal and the WIC-rdw rat thyroids by a plaque-hybridization clone screening method using 967-bp 5⬘-end and 930-bp 3⬘-end rat Tg cDNA fragments as probes. The remaining sequence was obtained from the products of RT-PCR amplification using flanking primer sets that were synthesized based on mouse and human Tg cDNA sequence information (Table 1). From these, the complete sequence of the WIC-rdw rat Tg cDNA was determined (Fig. 5) and compared with that of the F344-wt rat (GenBank accession number, AF221622). Although the full-length sequence information on the

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position 2320 and the surrounding residues are highly conserved among other species (Fig. 7), suggesting that the rdw mutation likely represents a missense mutation associated with a structural and/or functional alteration of the Tg polypeptide and not just a single nt polymorphism (SNP). Highly homologous to the intact acetylcholinesterase (AChE), the C-terminal fifth of the Tg protein, which contains the rdw mutation, was previously hypothesized to contain domain(s) that may be structurally important in the Tg dimerization (20). In support of this hypothesis, G2320 (Fig. 7, arrowhead) appeared to be absolutely conserved not only in other Tgs and AChE but also in other functionally unrelated proteins that share primary structural similarities. Absence of the rdw Mutation in Other Rat Inbred Stains Made possible by the fact that the G6958C nt substitution in the WIC-rdw rat Tg gene caused a loss of the recognition site for the restriction enzyme, NIaIV, the occurrence of this substitution in other rat inbred strains was examined by digesting RT-PCR products (260 bp) containing this region with NIaIV (Fig. 8). In contrast to the RT-PCR product from the WIC-rdw rat Tg, which was the only one resistant to NIaIV, those from other inbred strains including the WIC-⫹/⫹ rat were cleaved into the two expected fragments of 117 and 143 bp (Fig. 8, lanes 2–3 and 5–8). Consistent with its genotype, the WIC-rdw/⫹ rat showed both undigested and digested bands (Fig. 8, lane 9). Fig. 4. Detection of the Rat Tg Protein in F344-wt and WICrdw Rat Thyroids A, Homogenates of F344-wt (10 ␮g), WIC-rdw/⫹ heterozygous (25 ␮g), and WIC-rdw homozygous (50 ␮g) rat thyroids were subjected to reducing 4.5% SDS-PAGE. Protein bands were visualized by Coomassie brilliant blue R-250 staining. Intact Tg bands (330 k) are seen in all three lanes. Approximate molecular sizes are listed on the left (Tg ⬃330 k, myosin ⬃200 k, phosphorylase b ⬃96 k, bovine albumin ⬃66 k). B, The same homogenates were analyzed by reducing 4% SDSPAGE and Western blot using antirat Tg antibody.

human, mouse, and bovine Tg cDNAs are available (17–19), that of the normal rat Tg cDNA has not yet been reported. Therefore, it was necessary to sequence the normal (F344-wt) rat Tg cDNA in its entirety as well. The rat Tg cDNA contained a 8,307-bp open reading frame (ORF), which encoded a protein containing 2,768 amino acids, showing 92.5%, 77.7%, and 75.2% homology with the mouse, human, and bovine Tg cDNA, respectively. The predicted primary structure of the rat Tg showed 90.4%, 73.3%, and 71.0% homology with those of the mouse, human, and bovine Tg, respectively (not shown). As shown in Figs. 5 and 6, there was a single nt substitution, G6958C, that caused a single amino acid substitution, G2320R, in the C-terminal region of the protein. The glycine at

Transient Expression of Full-Length Mouse Tg cDNA Containing the rdw Mutation Using site-directed mutagenesis, an intact normal mouse Tg cDNA (20) was changed to contain the rdw mutation. The COS-7 cells were then transiently transfected with each of the Tg cDNAs that contained either the rdw or the cog mutation and compared with the cells that were transfected with the normal Tg cDNA and the untransfected cells. After overnight chase in serum-free media, the cells were lysed under denaturing conditions and analyzed by reducing SDS-PAGE. When the cellular Tg contents were examined by immunoblotting using anti-Tg antibodies, similar amounts of Tg bands were observed in all the transfected cell lysates but not in the untransfected control (Fig. 9A). In contrast, only the normal Tg was found in the secreted media, suggesting that the rdw mutation prevented the normal export of Tg (Fig. 9B) by rendering the protein incompetent for intracellular transport, very similar to the cog Tg (Fig. 9). As in other models of defective Tg trafficking, the rdw Tg was retained inside the ER in both primary WIC-rdw thyrocytes and COS-7 cells that were transfected with the Tg cDNA containing the rdw mutation (our unpublished data).

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Table 1. RT-PCR Product Used in the Sequencing of the Rat Tg cDNAs Primer Pairs PCR Amplification Region length (bp)

nt nt nt nt nt nt nt nt

832–1799 (967) 1709–2716 (1007) 2629–3797 (1168) 3767–4954 (1187) 4907–5481 (574) 5411–6145 (734) 6075–7075 (1000) 6995–7925 (930)

Sense (5⬘-3⬘)

Antisense (5⬘-3⬘)

5⬘-TACCGGATATTGCAGAGACG-3⬘ -CTCTCCTGGAACTTCCAGAA-CTCAGCCAATACCCAGGTCC-TTCAACAGTGCCAGCTGTTC-TTGCCTGCGTGACTTCTGAC-TGGAAGGAACTCAAGACAGC-CAGACTGGGGATTCAAACAT-CTGCTAACTACAGACTGGGG-

5⬘-CCATCACATCTCCTAAATCT-3⬘ -CAGCCTCATCCACACACCAA-TACAAATCAGTGGCCCTGGT-CCTTCAAGCTGTCCACAGCA-GACCCCATGTCAGAATCTTT-CTCAGAGCCACAGTCCAAAA-GTCCAGTAGCCCCCAGTTGC-CCTGGTAGGCAGAGTAGAAG-

DISCUSSION The mutants exhibiting dwarfism, which was found in a Wistar-Imamichi rat closed colony, inherited as an autosomal recessive was named rdw (rat dwarf) and established as an inbred strain, WIC-rdw. In this study, we showed that the WIC-rdw phenotype was due to a missense mutation in the Tg gene. Several lines of evidence suggested that this mutation was truly responsible for the rdw phenotype. 1) Recombination between the Tg gene and rdw locus was not observed in 138 BC, since the rdw locus was identical to the rat Tg gene locus. 2) In a direct comparison, the only significant difference in the nt sequence between the F344 normal vs. WIC-rdw rat Tg cDNA ORF was the G6958C substitution. The predicted Gly 3 Arg substitution occurred at aa position 2,320, which was strictly conserved not only in other rat and mouse inbred strains but also in other mammalian species including human and bovine. 3) The parental normal rat strain, WIC-⫹/⫹, showed no substitution at codon 2,320, and the WIC-⫹/rdw rats showed heterozygous genotype at codon 2,320. 4) Transient expression of the full-length mouse Tg cDNA containing the rdw mutation, showing the intracellular presence of the intact Tg which was unable to be secreted, provided the confirmation that rdw mutation was indeed responsible for the observed phenotype. Northern blotting and SDS-PAGE analysis of the WIC-rdw rat thyroid homogenates revealed that the transcripts of the mutated Tg gene were not different from those of the wild type with respect to their size and amount. In fact, the Tg mRNA level was modestly elevated in the WIC-rdw thyrocytes, probably reflecting the increased thyroid stimulating effect of the elevated circulating TSH level, yet the amount of the 330-kDa Tg protein was reduced in the WIC-rdw thyroid. Several likely explanations merit consideration. For one, it may be that the missense substitution from a smaller neutral aa, glycine, to a larger basically charged aa, arginine, within a highly conserved domain, may have caused a substantial conformational change in the C-terminal region of Tg protein. As for most other disease-causing mutant proteins, such change often leads to structural instability that renders the protein highly susceptible to aggregation and/or

proteolysis. This was in part supported by the experimental observation that the rdw Tg was quite prone to proteolysis when the thyroid follicles were lysed under nondenaturing conditions, even in the presence of a full battery of lysosomal protease inhibitors (our unpublished data). On the other hand, when the thyroid follicles were lysed in denaturing and reducing buffer at boiling temperature, one of the most potent conditions for inhibiting lysosomal protease activities, a greater amount of the rdw Tg was recovered after SDS-PAGE. To verify this hypothesis, further experiments utilizing limited proteolysis will be helpful. Another plausible explanation for the reduced level of Tg protein in the WIC-rdw thyroid is the inhibition of the translation initiation by the presence of the malfolded proteins in the ER, as a part of the unfolded protein response (UPR). In all eukaryotes, the accumulating malfolded or misfolded proteins in the ER are increasingly bound by several molecular chaperones including BiP/GRP78. Consequently, as the available level of free or unbound BiP/GRP78 falls, a transmembrane kinase known as IRE-1p/ERN-1p is activated, triggering the ER-UPR pathway that results in the transcriptional induction of multiple ER chaperone genes (21). These molecular chaperones then act to reduce the potential harm posed by the misfolded mutant proteins that are prone to aggregation (3). At the same time, the ER-UPR pathway activates another ER resident transmembrane protein kinase or PERK/PEK, which phosphorylates the eukaryotic translation initiation factor or eIF2␣ that eventually leads to the attenuation of translation (22). Together, the elevated levels of ER chaperones and the reduction in the continued synthesis of the misfolded mutant proteins serve to minimize the toxic accumulation of potentially harmful aggregates of misfolded proteins, thereby enhancing the probability of cell survival. Several mutations in the human (2, 23–27), mouse (20), bovine (28), and goat Tg genes (29) have been elucidated at the molecular level. It is interesting to compare the rdw mutation with the recently identified cog mutation in the congenital goiter mouse (20). The cog Tg gene also contained a single nt change that resulted in a missense mutation, L2263P, located near the N-terminal side of the rdw mutation. Both mutations are associated with the marked accumulation of

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Fig. 5. The Complete Primary Structure of the Rat Tg Deduced from Tg cDNA Sequences The complete primary structures of rat Tg deduced from Tg cDNA sequences were obtained for F344-wt normal and WIC-rdw rat, which differed only at position 2,320. The complete rat Tg cDNA encodes a total of 2,768 amino acids. At the N terminus, a 20-residue signal sequence peptide (underlined) is followed by a strictly conserved sequence NIFEY- that contains the hormonogenic Tyr-5. Arg (R) at position 2,320 found in the rdw Tg is shown enlarged in bold.

the chaperone proteins, observed in other ERSDs involving mutant secretory proteins (3). Additional similarities include normal sizes and amounts of the Tg gene transcripts in both mutant thyrocytes, full-length Tg proteins that exhibited increased susceptibility to proteolysis, as well as the decreased synthesis and impairment of intracellular transport of both mutants. Moreover, although not as striking as the conservation of residues flanking the cog mutation, the region surrounding G2320, especially on its C-terminal side, appears to be also well conserved (Fig. 7) not only among Tgs from different species but also among other ho-

mologous proteins. Since the latter, which include neuroligin, a novel neuronal cell surface protein important in cell-cell interactions (30) and AChE, are functionally unrelated to Tg, it may be that the homology stems from their conformational similarities. In this case, substituting a positively charged arginine for a smaller, hydrophobic G2320, which appears to be absolutely conserved (Fig. 7, arrowhead), may lead to structural instability. Confirming such a possibility will require additional studies. Finally, despite many similarities between the rat and mouse models of Tg deficiency, there is one im-

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Fig. 7. Comparison of the Residues Surrounding G2320R Alignment of the WIC-rdw rat, F344 rat, mouse, bovine, and human Tg aa sequences shows that this region is highly conserved. The arrowhead indicates the G2320R mutation found in the WIC-rdw rat and the positions of G2320 (in the normal rat Tg sequence) that is strictly conserved in all species as well as in AChE and other functionally unrelated proteins. Surrounding conserved amino acids are shown in bold (allowing for similar residues, i.e. aspartate for glutamate, threonine for serine, and hydrophobic aa or methionine for alanine) and underlined in the Wic-rdw rat. Abbreviations: Wic-rdw, Tg from the WIC-rdw homozygous rat; NL, neuroligin; CaE, rat carboxylesterase ES-10; Bu ChE, butyryl cholinesterase; T AChE, Torpedo acetylcholinesterase.

Fig. 6. The Identification of the WIC-rdw Mutation The nt sequences surrounding the WIC-rdw mutation (nt nos 6967–6951) show transversion 6,958G⬎C (arrow; shown here in the antiparallel direction) in the WIC-rdw Tg cDNA but not in the WIC-⫹/⫹ Tg cDNA.

portant difference that stands out between the two models: the size of the their thyroid glands. In the cog/cog mouse (and in several human patients with CHG), the constant TSH stimulation that occurs in primary hypothyroidism leads to the development of massive goiter that eventually compensates for the quantitative defect in hormonogenesis. It appears that a very small fraction of the cog Tg, which has been shown to be a temperature-sensitive mutant (4), is able to reach the distal secretory pathway to form thyroid hormones. The absence of this compensatory response in the WIC-rdw rat may in part explain the severity of the dwarfism and infertility in the adult rat, compared with the cog/cog mouse. All previously reported mutations in the Tg gene in other species caused goitrogenesis, yet the rdw mutation was associated with a hypoplastic thyroid gland (11, 12). Although the reason for this phenomenon remains unknown, it is intriguing to consider the possibility that the rdw mutation may be toxic to the host thyrocytes. Hence, comparing the intracellular fates of the cog and the rdw Tgs as well as their interactions with the essential components of the ER-UPR pathway may provide new insights into how mutant proteins exert their

Fig. 8. Detection of a Missense Mutation at nt 6,958 in the Tg Gene The 260-bp Tg cDNA fragments (nt 6,815 3 7,075) containing the location of the rdw mutation were generated using corresponding flanking primers using thyroids removed from six different normal stains of rat (BN, F344-wt, BBDR, WKY, WKAH, and WIC-(⫹/⫹), as well as from the WIC-rdw homozygote and the WIC-rdw/⫹ heterozygote rats. The presence of G6958C mutation renders the 260-bp cDNA resistant to Nia; 260-bp RT-PCR products were digested with Nia, subjected to 6% PAGE, and visualized by ethidium bromide staining.

toxic effects on the host cells. On the contrary, cytotoxic effect of the rdw mutation would be difficult to reconcile with the autosomal recessive nature of the rdw phenotype. Additional experiments are clearly needed to determine the mechanism by which WICrdw heterozygous rat thyrocytes expressing the mutant Tg protein avoid the same fate observed in the homozygous rat. Along this line, ␣1-antitrypsin (AAT) deficiency, which is generally regarded as an autosomal recessive, has been observed as an autosomal

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Mapping of the Rat Tg Gene Locus The Tg gene intron 2 was amplified by PCR with primers 5⬘-AGCAGGATGAATATGTTCCA-3⬘ and 5⬘-ATCCACACACCAGCAAGATT-3⬘. Amplified DNA fragments that were digested with 0.25 U of Cac8I (New England Biolabs, Inc., Beverly, MA) were analyzed in 6% polyacrylamide gels containing 90 mM Tris-borate and 2 mM EDTA. DNA bands were visualized by staining with 0.5 ␮g/ml ethidium bromide solution. Genotypes of the Tg gene were determined by the presence or absence of 280-bp and 245-bp bands (see Fig. 2). Linkage analysis was performed using Map Manager v2.6.5. Detection of the Tg Gene Transcripts Fig. 9. Transient Expression of Tg cDNAs in COS-7 Cells COS-7 cells grown in serum containing media to 70% confluency were transiently transfected with full-length normal, cog, and rdw Tg cDNAs, which were generated by site-directed mutagenesis. Forty eight hours after transfection, the cells were chased in serum-free media for 20 h. Both the cell lysates (A) and the secreted media (B) were analyzed by reducing SDS-PAGE followed by Western blot using anti-Tg antibody.

dominant in some patients as well as in the transgenic animal model (3, 31). Although most of these patients suffer from juvenile pulmonary emphysema, only the patients with a specific mutation in the AAT gene known as the Z-variant are additionally at risk for hepatocellular damage leading to liver cirrhosis. Thus, the susceptibility of each mutant to intracellular degradation may hold the key in determining the potential cellular toxicity of the various mutant proteins. In conclusion, we have shown that the G2320R missense mutation is indeed responsible for the congenital hypothyroidism and dwarfism in the WIC-rdw rat, which will be useful for future investigation of the relationship of Tg protein structure with its stability, transport, and glycosylation mechanisms.

MATERIALS AND METHODS

The Tg gene transcripts were detected by Northern blotting. Poly(A)⫹ RNAs were purified from total RNAs of F344-wt and WIC-rdw rat thyroids using oligo(dT) cellulose type 7 (Amersham Pharmacia Biotech, Buckinghamshire, UK). Aliquots of 2 ␮g of poly(A)⫹ RNA were denatured in 50% formamide, 2.2 M formaldehyde at 65 C for 10 min, electrophoresed in 1% agarose gels containing 2.2 M formaldehyde, and transferred onto Hybond-N⫹ membranes (Amersham Pharmacia Biotech). The poly(A)⫹ RNAs were immobilized on the membranes by heating at 80 C for 2 h. Prehybridization and hybridization of the membranes were performed in buffer containing 50% formamide, 5 ⫻ standard saline citrate solution (SSC), 5 ⫻ Denhardt’s solution, and 0.5% SDS at 45 C for 16 h. The 5⬘-end of the Tg cDNA fragment was amplified by RT-PCR and used as a probe. The probe was labeled with -32P dCTP using a Rediprime Kit (Amersham Pharmacia Biotech). Membranes were washed in buffer containing 0.1 ⫻ SSC and 0.1% SDS and exposed to X-AR film (Kodak, Rochester, NY) at ⫺70 C for 24 h. Detection of the Tg Protein Freshly removed thyroids were homogenized under denaturing condition in solution containing 4% SDS, 2% mercaptoethanol, and 10 mM Tris, pH 6.8, and boiled for 5 min. Different aliquots of thyroid homogenates (⬃50 mg/ml protein) were analyzed by reducing 4 or 4.5% SDS-PAGE. Protein bands were visualized by staining with Coomassie brilliant blue R-250. Parallel samples were also subjected to SDSPAGE and transferred to nitrocellulose before examination for intact Tg by immunoblotting with antirat Tg and antimouse Tg antibodies (4, 20) and secondary antibody conjugated with horseradish peroxidase. ECL chemiluminescence method (Amersham Pharmacia Biotech, Arlington Heights, IL) was used for the band detection. Determination of the Tg cDNA Sequence

Rat Crosses and the Mapping of the rdw Locus Backcrosses (BC) were produced by crossing 138 (BN ⫻ WIC-rdw)F1 ⫻ WIC-rdw rats. Male WIC-rdw rats transplanted with normal thyroids were used for crossing, since WIC-rdw rats are infertile. The rdw phenotypes were identified based on body weights at 8 weeks of age; at this time, the animals were killed and their thyroids and livers were quickly removed for subsequent experiments. DNAs were extracted from the liver and pooled into two groups, ⫹/rdw and rdw/rdw genotypes. PCR was performed first with 60 microsatellite marker primers (Research Genetics, Inc., Huntsville, AL), which had been selected as distributed in all autosomal chromosomes and showed polymorphism between BN and WIC-rdw rats. Since some microsatellite markers localized in Chr 7 showed linkage with the rdw genotype, other microsatellite markers localized in Chr 7 were further examined. Linkage analysis was performed using Map Manager v2.6.5.

The sequences of 5⬘- and 3⬘-ends of the Tg cDNA were determined by sequencing the cDNA clones obtained from the cDNA libraries of the F344 and WIC-rdw rat thyroids. The two cDNA libraries were generated using a ZAP-cDNA Gigapack Gold Cloning Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instruction. The clones were screened by the plaque-hybridization method with probes of 5⬘- and 3⬘end fragments of the rat Tg cDNA, of 967 and 930 bp in size, respectively. The rest of the Tg cDNA sequence was determined by sequencing TA clones containing the rat Tg cDNA fragments, which had been amplified by RT-PCR with the primers shown in Table 1. Primers were synthesized according to the sequences of the murine and human Tg cDNAs. TA cloning was performed using a TA Cloning Kit (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. Sequencing was performed with a DSQ-1000 automatic sequencer (Shimazu, Kyoto, Japan) using a

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primer cycle sequencing kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer’s instructions. Detection of the rdw Mutation by Restriction Enzyme Digestion RT-PCR was performed with total RNAs extracted from thyroids as templates and sense (5⬘-CAACACCTCCTCAAATCAGT-3⬘, nt 6,815–6,835) and antisense (5⬘-GTCCAGTAGCCCCCAGTTGC-3⬘, nt 7,055–7,075) primers. PCR products were digested by incubation with 1 U of NiaIV (New England Biolabs, Inc., Beverly, MA) at 37 C for 16 h and electrophoresed in 6% polyacrylamide gels containing 90 mM Trisborate and 2 mM EDTA. DNA bands were visualized by staining with 0.5 ␮g/ml ethidium bromide solution. Site-Directed Mutagenesis QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to make the G to C point mutation in the full-length normal mouse Tg cDNA (20) in pBK-CMV vector (Stratagene) according to the manufacturer’s instruction. The two primers T1–5⬘-GCT GAC CAT TGA TCG CTC CAT CCT GGC-3⬘ and T2–5⬘-GCC AGG ATG GAG CGA TCA ATG GTC AGC-3⬘, both containing the desired mutation and each complementary to the opposite strands of the vector, were used in a PCR reaction. Four Tg cDNAs were isolated and sequenced to determine the presence of the point mutation. The presence of the rdw mutation, G6958C, in the new Tg cDNA was confirmed by a second sequencing. Transient Transfections of COS-7 Cells COS-7 cell were grown in DMEM containing 10% FBS to approximately 70–80% confluency before transfection with 1 ␮g of each plasmid identified as pBK-CMV-normal Tg, pBK-CMVcog Tg, or pBK-CMV-rdw Tg using Lipofectamine reagent (Life Technologies, Inc.) in serum-free DMEM, according to the instructions. After 5 h incubation, the transfection mixture was washed and replaced with fresh DMEM containing 10% FBS for an additional 48 h. The cells were then chased overnight in serum-free DMEM and lysed in denaturing buffer containing 4% SDS. The secreted medium collected during chase was precipitated with 10% TCA on ice for 1 h before centrifugation at 14,000 ⫻ g for 10 min at 4 C. After washing with 100% ethanol, the obtained pellet was resuspended in denaturing cell lysis buffer. Both the cell lysates and the secreted media were subjected to reducing 4% SDS-PAGE before the Western blot analysis using anti-Tg antibody.

Acknowledgments We thank Drs. Y. Fujii and H. Iwase, Department of the Second Surgery, Nagoya City University Medical School, for the human thyroid tissue and Ms. M. Yasuda, Nagoya City University Medical School, for assistance with genetic linkage analysis of the rdw. Parts of this work were supported by NIH Grant DK-52076 (P.S.K.) and Veterans Affairs Merit Award (P.S.K.).

Received July 21, 2000. Re-revision received September 12, 2000. Accepted September 13, 2000. Address requests for reprints to: T. Agui, Center for Experimental Animal Science, Nagoya City University Medical School, Mizuho-ku, Nagoya, Acihi 467-8601, Japan. E-mail: [email protected]. * These authors contributed equally.

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Call for Papers At the request of Endocrine Society president Dr. Benita Katzenellenbogen, Molecular Endocrinology and the other three Endocrine journals will devote their June 2001 issues to topics in Reproductive Hormones and Human Health. This theme will also be emphasized at the 83rd Annual Meeting of The Endocrine Society in Denver, Colorado, June 20–23. The editors of Molecular Endocrinology are seeking submissions for the special issue. While manuscripts should have a mechanistic focus, we are especially interested in papers that describe new mouse models of human diseases involving reproductive hormones, including mammary, prostate, and other reproductive tract cancers. We would also encourage submission of papers that build on recent developments in functional genomics and structural biology. The deadline for submissions is January 5, 2001. Please indicate explicitly in your cover letter that you wish to have the manuscript considered for the June issue.