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Two Novel Cysteine Substitutions (C1263R and C1995S) of Thyroglobulin Cause a Defect in Intracellular Transport of Thyroglobulin in Patients with Congenital Goiter and the Variant Type of Adenomatous Goiter¹

Department of Clinical Pathology, Dokkyo University School of Medicine (A.H., T.I.), Mibu, Tochigi; the First Department of Internal Medicine, Osaka Medical College (J.T., S.Y.), Takatsuki, Osaka; the Department of Pediatrics, Kitasato University School of Medicine (Y.O., N.M.), Sagamihara, Kanagawa; Kuma Hospital (T.Y., K.K.), Kobe, Hyogo; and Sumitomo Metal Bio-Science, Inc. (Y.K), Tokyo, Japan

Address all correspondence and requests for reprints to: Akira Hishinuma, M.D., Ph.D., Department of Clinical Pathology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We analyzed the thyroglobulin (Tg) gene of 2 unrelated patients with congenital goiter and the Tg gene of 2 siblings with the variant type of adenomatous goiter. The clinical characteristics of the patients with congenital goiter and the variant type of adenomatous goiter were very similar, except for serum Tg levels, which were less than 15 pmol/L in the patients with congenital goiter, but 117–181 pmol/L in the patients with the variant type of adenomatous goiter (normal, 15–50 pmol/L). The tissue content of Tg in the thyroid glands of all 4 patients was reduced at 0.9–3.8% of total protein (normal, 19–40%). The missense mutation C1263R was detected in the 2 unrelated patients with congenital goiter; the pedigree study showed an autosomal recessive pattern of inheritance. In the 2 siblings with the variant type of adenomatous goiter, the missense mutation C1995S was homozygously detected. In the Tg complementary DNA of 110 normal subjects, the allelic frequencies of the C1263R and C1995S mutations were each less than 0.5%. Also in the normal subjects were detected 35 nucleotide polymorphisms, the insertion of 3 nucleotides, and 1 alternative splicing, each of which was not associated with any specific thyroid disease. From these data, the molecular mechanism of the C1263R and C1995S mutations was elucidated. We first analyzed the carbohydrate residues of C1263R Tg and C1995S Tg. Sensitivity to treatment by endoglycosidase H suggests that C1263R Tg and C1995S Tg were retained in the endoplasmic reticulum (ER). Also, the presence of endoglycosidase H-resistant Tg as well as endoglycosidase H-sensitive Tg in the patients with the variant type of adenomatous goiter suggests that a fraction of C1995S Tg was transported to the Golgi and associated with the mildly increased serum Tg levels. Native PAGE and Western blot analysis with anti-Tg antibody showed that C1263R Tg and C1995S Tg form high mol wt aggregates in the ER.

Our results suggest that missense mutations that replace cysteine with either arginine or serine cause an abnormal three-dimensional structure of Tg. Such misfolded Tg polypeptides are retained in the ER as high mol wt aggregates.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
A DEFECT in thyroglobulin (Tg) leads to congenital hypothyroidism, which has an estimated incidence of approximately 1 in 80,000–100,000 newborns (1). We previously reported the results of genetic analysis of a patient with abnormal Tg (2). The patient had severe hypothyroidism with short stature and mental retardation and underwent multiple operations due to continual enlargement of the thyroid gland. The Tg gene of this patient contained a mutation in the acceptor splice site. This caused exon 4 to be missing from the major Tg transcript, resulting in a shorter peptide fragment that lacked the putative donor tyrosine residue involved in the synthesis of T₄. Similar cases of congenital goiter and hypothyroidism due to abnormality of the Tg gene have been reported by other investigators (3, 4). A more recent paper documented that defective intracellular transport of Tg is a feature of congenital hypothyroid goiter (5). We more recently reported a milder case of congenital goiter that was caused by a missense mutation of the Tg gene (6). As this patient was very mildly hypothyroid with the single clinical symptom of goiter, we hypothesized that there are more cases of goiter without established pathogenesis caused by a mutation in the Tg gene.

Approximately 1% of patients with adenomatous goiter who had undergone thyroidectomy were found to have a variant type of adenomatous goiter (7). The clinical characteristics of patients with the variant of adenomatous goiter are similar to those of our previously reported patient (6). Among patients with the variant of adenomatous goiter, the severity of the disease ranged from mild hypothyroidism to euthyroidism (7). The age of onset of enlarged thyroid gland is lower in patients with the variant of adenomatous goiter than in those with the common type of adenomatous goiter. Patients with the variant of adenomatous goiter display a remarkably high level of radioiodine uptake by the thyroid gland and low Tg content in the thyroid gland. This suggests that the etiology of the variant type of adenomatous goiter is an inherited abnormality of the gene that encodes Tg.

The present study undertook sequence analysis of the Tg gene of 2 patients with congenital goiter and 2 patients with the variant type of adenomatous goiter. The sequences of the Tg genes of the immediate family members were also examined to determine the mode of inheritance. The sequences of the Tg genes of the 4 patients were compared with those of 8 patients with other types of thyroid disease. The sequence of the Tg gene of 110 normal subjects were also examined by an allele-specific PCR method to differentiate mutations from normal polymorphisms of the Tg gene.

	Subjects and Methods

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Subjects

Patient A has congenital euthyroid goiter with defective Tg synthesis (8). Patient B has congenital goiter and was born to parents in a consanguineous marriage (6). Patients C and D are sisters, born to parents whose families are unrelated. Thyroid tissue was also obtained from 8 patients with other thyroid diseases, whose diagnoses included Graves’ disease (2 patients), Hashimoto’s thyroiditis (1 patient), thyroid cancer (1 patient), solitary benign adenoma (2 patients), and the common type of adenomatous goiter (2 patients). Thyroid tissue was obtained from these 12 patients during thyroidectomy. In the 110 normal volunteers, the Tg gene was studied from peripheral blood cells.

Tissue Tg content

Thyroid tissue extracts were prepared by homogenizing approximately 20 mg thyroid tissue in 100 µL Tris buffer (10 mmol/L; pH 8.0) that contained a cocktail of protease inhibitors (Complete Protease Inhibitor Cocktail Set, Boehringer Mannheim, Mannheim, Germany). The tissue homogenate was centrifuged at 18,000 x g twice for 30 min each time. The concentration of Tg in the supernatant was measured using a RIA kit (Eiken Chemical Co., Tokyo, Japan). Total protein in the supernatant was determined by the method of Bradford using a protein assay kit (Bio-Rad, Richmond, CA).

Direct sequencing of the Tg complementary DNA (cDNA) of the 12 patients with various thyroid diseases

Total ribonucleic acid was extracted from approximately 30 mg thyroid tissue using the RNeasy Mini kit (Qiagen, Hilden, Germany). RT-PCR and direct sequencing of the RT-PCR products of Tg cDNA were performed as previously reported (6). Briefly, 1 µg total ribonucleic acid was reverse transcribed with reverse transcriptase. The entire Tg cDNA was amplified in 11 segments with overlaps. The RT-PCR products were purified and directly sequenced using forward and reverse primers. The exact condition of RT-PCR and the sequence of the primers have been previously described (6).

Allele-specific PCR

As four homozygous substitutions of nucleotides at positions 2488, 3787, 5983, and 5992, were detected in some of patients A, B, C, and D, but not in the 8 patients with other thyroid diseases, we performed allele-specific PCR in 110 normal subjects to differentiate mutations from normal polymorphisms among these nucleotide substitutions. The primers are listed in Table 1. Genomic DNA from the 110 normal subjects was isolated from peripheral blood cells using the QIAamp Blood Kit (Qiagen). To amplify the genomic fragments that contain nucleotide positions 2488, 5983, and 5992 of the Tg gene, the PCR reaction was carried out using the Expand High-Fidelity PCR System (Boehringer Mannheim) for 35 cycles, which consisted of denaturation at 98 C for 4 s, primer annealing at 55 C for 30 s, and primer extension at 72 C for 30 s, in a Gene Amp 9600 Thermal Cycler (Perkin Elmer, Norwalk, CT). To amplify the genomic fragment that contains nucleotide position 3787 of the Tg gene, the conditions of PCR were the same, except that primer annealing was carried out at 60 C. After the final extension at 72 C for 7 min, the PCR products were electrophoresed on a 1% agarose gel (Seakem GTG agarose, FMC Bioproducts, Rockland, ME). Direct sequencing of the PCR products of each genomic DNA sample confirmed that the correct genomic fragments of the Tg gene were amplified.

Pedigree analysis of the two patients with congenital goiter and the two patients with the variant type of adenomatous goiter, was performed using allele-specific PCR and direct sequencing of genomic DNA. Genomic DNA was isolated from peripheral blood cells obtained from each patient and their immediate family members using a QIAamp blood kit (Qiagen). Allele-specific PCR was performed as described in the previous section. For sequencing of genomic DNA at nucleotide position 3787, the primers 5'-TCT GCA ATG TGC TCA AGA GTG GA-3' and 5'-CGG GCT GTC AGC TCA TCC AA-3' were used to amplify the 1.6-kb genomic fragment. The PCR products were sequenced by the primer 5'-GCC ATG CAG CAG TGC CAA TT-3'. For the nucleotide at position 5983, the primers 5'-CTG AGT AAG AGG GGA AGT GAA 3' and 5'-CCG CAT CGC ACC G-3' were used to amplify the 533-bp genomic fragment; the PCR products were sequenced by the forward primer.

Endoglycosidase H (Endo H) treatment

From each thyroid tissue extract, an aliquot containing 2 µg Tg was digested with 0.3 mU/L Endo H (Boehringer Mannheim) in a buffer that contained 250 mmol/L sodium citrate (pH 5.3), 2.5% SDS, 50 mmol/L ethylenediamine tetraacetic acid, and 5% 2-mercaptoethanol (2-ME) for 15 min at room temperature. An equal volume of an electrophoresis buffer that contained 0.24 mol/L Tris-HCl (pH 8.7), 15% glycerol, 2.5% SDS, and 5% 2-ME, was subsequently added to each sample. Electrophoresis was carried out on a 4–15% gradient polyacrylamide gel using the Phast System (Pharmacia, Uppsala, Sweden). The Tg cDNA of one patient with Graves’ disease did not contain a mutation and served as the control. Some of the gels were subjected to Western blot analysis using anti-Tg antibody, as described in the next section.

Western blot analysis

An aliquot from each thyroid tissue extract containing 0.2 µg Tg was electrophoresed either in a native sample buffer [0.24 mol/L Tris-HCl (pH 8.7) and 15% glycerol] or in a denaturing sample buffer (the native buffer plus 2.5% SDS and 5% 2-ME) on a 4–15% gradient polyacrylamide gel using the Phast System (Pharmacia). Some of the gels were stained with Coomassie brilliant blue; the rest of the gels were transferred onto a nitrocellulose membrane using the semidry blotting method of the Phast System. The membranes were incubated in 3% BSA in Tris saline (150 mmol/L NaCl and 10 mmol/L Tris, pH 7.5), for 2 h. Each membrane was then placed in Tris saline containing mouse monoclonal anti-human Tg antibody (clone B34.1, Biomeda, Foster City, CA). The membranes were then reacted with peroxidase-conjugated secondary antibody and stained with 4-chloro-1-naphthol (Sigma Chemical Co., St. Louis, MO) in the presence of H₂O₂.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical characteristics and tissue Tg content

Thyroid function tests in the two patients with congenital goiter (patients A and B) and the two patients with the variant type of adenomatous goiter (patients C and D) revealed mild hypothyroidism to euthyroidism (Table 2). In patient B, the serum T₄ level was slightly low, whereas the serum TSH level was slightly elevated. The serum Tg concentration was undetectable in patients A and B. The serum Tg concentrations in patients C and D were slightly higher than the normal range, although remarkably lower than those in patients with the common type of adenomatous goiter (7). Antibodies against Tg, thyroperoxidase, and TSH receptor were not detected in any of the subjects. Radioactive iodine uptake by the thyroid gland was remarkably high in all of the patients.

Sequence analysis of Tg cDNA

Results from direct sequencing of the Tg cDNA of the 2 patients with congenital goiter and the 2 patients with the variant type of adenomatous goiter are shown in Table 3. The allelic frequency of each nucleotide substitution is also shown. Allelic frequencies among Caucasians were obtained from the published results (9). Of the 37 nucleotide substitutions in the Tg gene, a homozygous nucleotide substitution at position 3787 was detected in the Tg cDNA of patients A and B, and homozygous substitutions at positions 2488 and 5983 were found in patients C and D (Fig. 1). The nucleotide at position 5992 was homozygously changed in patients A, C, and D. We studied allelic frequencies of these 4 nucleotides in 110 normal subjects by allele-specific PCR to distinguish between mutations and normal polymorphisms. As the nucleotide substitutions at positions 2488 and 5992 were detected in the 110 normal subjects, we concluded that the nucleotide substitutions at positions 2488 and 5992 were normal polymorphisms. Among the 220 copies of the Tg gene in the normal subjects, we could not find a single Tg gene that contained the nucleotide substitutions at positions 3787 and 5983. The nucleotide substitution at position 3787 was specific to the 2 patients with congenital goiter, and that at position 5983 was specific to the 2 patients with the variant type of adenomatous goiter. In patients A and B, cytosine displaced thymine at nucleotide 3787, which caused an amino acid substitution from cysteine to arginine at codon 1263 (C1263R). In patients C and D, a thymine to adenine substitution at nucleotide 5983 resulted in an amino acid substitution from cysteine to serine at codon 1995 (C1995S).

View larger version (38K): [in this window] [in a new window]   Figure 1. Allele-specific PCR analysis of the nucleotides at positions 2488, 3787, 5983, and 5992 in a patient with Graves’ disease and in patients A, B, C, and D. Amplification of the normal allele and the mutant allele are shown in lanes N and M, respectively.

Haplotyping

Pedigree analysis was conducted by the allele-specific PCR method (Fig. 2). The results of the pedigree analysis were confirmed by direct sequencing of the PCR products of the genomic DNA. In the family of patient A, the propositus, who is the younger brother of two siblings, was homozygous for the mutation C1263R. Each of the other family members was heterozygous at this position. Blood samples from some of the family members of patient B were not available for analysis. In the family of patient B, the propositus, the third of five siblings, was homozygous for the mutation C1263R. The other family members were either heterozygous for the mutation or homozygous for the normal allele. We could not study the pattern of inheritance for the C1995S mutation, because blood samples of all of the family members of patients C and D were not available. Allele-specific PCR and sequence analysis showed that patients C and D are homozygous for the mutation in the genomic DNA.

View larger version (21K): [in this window] [in a new window]   Figure 2. Haplotype analysis of the families of patients A, B, C, and D by allele-specific PCR analysis. Individual 4 in the family of patient A and individual 2 in the family of patient B are the propositi who possess the homozygous missense mutation C1263R. The parents of patient B are first cousins. ? denotes individuals for whom data could not be obtained. In allele-specific PCR analysis, amplification of the normal allele and the mutant allele are shown in lanes N and M, respectively. NC, Normal control. Patients C and D are siblings in a family; the genomic DNA of the parents were not available for analysis.

Each thyroid tissue extract was treated with Endo H, which only digests high mannose-type [endoplasmic reticulum (ER)-type] oligosaccharides, and then subjected to SDS-PAGE (Fig. 3). In patients A, B, C, and D, protein bands at 330 kDa exhibited a large shift upon Endo H digestion. In patients C and D, we identified Endo H-resistant (Golgi-type) bands as well as Endo H-sensitive bands. These Endo H-resistant bands were detected even after an extended period (1 h) of Endo H treatment. Western blot analysis by anti-Tg antibody showed that both the Endo H-sensitive and the Endo H-resistant bands were immunoreactive to Tg. In a patient with Graves’ disease who bears no mutations in the Tg gene, Tg was resistant to the treatment with Endo H.

View larger version (50K): [in this window] [in a new window]   Figure 3. Carbohydrate analysis of the Tg of patients A, B, C, and D by treatment with Endo H, followed by SDS-PAGE and Western blot analysis by anti-Tg antibody. The large mol wt shift on Endo H treatment in patients A, B, C, and D shows that the carbohydrate residues are of the high mannose ER type, whereas the small shift in the patient with Graves’ disease (Gr) shows that the carbohydrate residue is of Golgi type. The Tg of patients C and D were partially digested by Endo H.

Native PAGE revealed high mol wt protein aggregates in the stacking gel of the thyroid tissue extract of patients A, B, C, and D (Fig. 4). Such high mol wt protein aggregates were not detected in the tissue of the patient with Graves’ disease, whose Tg cDNA was found not to contain a mutation by direct sequencing. In Western blot analysis using anti-Tg antibody, Tg immunoreactivity was identified in the high mol wt aggregates of the stacking gel as well as in the broad bands of the resolving gel in patients A, B, C and D. The Tg-immunoreactive bands in the resolving gel were more prominent in patients C and D than in patients A and B. In the thyroid tissue extract from the patient with Graves’ disease whose Tg cDNA did not contain a mutation, immunoreactive Tg homodimers were mainly detected. Tg monomers were not detected even by an densitometric analysis. The smaller band at about 540 kDa in the patient with Graves’ disease was a partially cleaved dimeric Tg product due to the presence of a protease-hypersensitive site in Tg (10). In addition to the Tg-immunoreactive broad bands at 660 kDa, the bands at about 70 kDa in patients C and D represented hemoglobin, which has a endogenous red color.

View larger version (49K): [in this window] [in a new window]   Figure 4. Results of native PAGE and Western blot analysis by anti-Tg antibody of thyroid tissue extract obtained from patients A, B, C, and D and a patient with Graves’ disease (Gr). In the patient with Graves’ disease, the major protein bands were Tg homodimers. In patients A, B, C, and D, high mol wt aggregates that were immunoreactive to Tg were detected in the stacking gel; Tg-immunoreactive broad bands were detected in the resolving gel. The Tg-immunoreactive broad bands are more prominent in patients C and D.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In addition to the 2 mutations, the present study revealed many polymorphisms of the Tg gene. Of the 35 normal polymorphisms, 30 are common to both the Japanese and Caucasian populations (9), whereas 5 are specific to the Japanese population. No polymorphism in the Japanese and Caucasian populations is associated with putative N-glycosylation sites. The substitution of tyrosine by histidine at amino acid position 1042 and the insertion of 3 nucleotides CAG were detected in 100% of the chromosomes of the Japanese and the Caucasian populations; these might reflect an error in the earlier report (11). Alternative splicing between nucleotides 5039 and 5231 was detected in the tissue of all 12 patients with various thyroid diseases. Quantitative RT-PCR analysis showed that the spliced form constituted less than 10% of the total RT-PCR products (data not shown). The functional role of the alternatively spliced form of Tg remains to be clarified; such alternative splicings were present in bovine Tg in 2 locations (12, 13).

The mutation C1263R is responsible for the goiter in patients A and B, and the mutation C1995S is responsible for the goiter in patients C and D. These mutations are very rare, each of which has an allelic frequency of less than 0.5%. Haplotyping for the C1263R mutation in the two pedigrees showed an autosomal recessive mode of inheritance. Thus, on the basis of allelic frequency it can be calculated that the incidence of homozygotes of the C1263R mutation is less than 1 in 40,000 newborns. Both of the newly identified mutations changed the cysteine amino acid to another amino acid. The cysteine amino acid is important in the formation of the correct three-dimensional structure of a polypeptide through disulfide bonds. As it has been reported that misfolded proteins are recognized as abnormal and disposed of by a nonlysosomal proteolytic pathway (14), we analyzed the intracellular processing of C1263R Tg and C1995S Tg. This molecular mechanism, which sorts out malfunctional protein products, is called ER quality control (15).

The increased sensitivity of C1263R Tg and C1995S Tg to Endo H treatment suggests that C1263R Tg and C1995S Tg are retained in the ER. In these four patients, pathological findings showed benign proliferation of thyroid epithelial cells and scant colloid in the follicular lumen (data not shown). The C1263R polypeptide was almost completely sensitive to Endo H treatment; this probably results in the undetectable concentration of serum Tg in patients A and B. On the other hand, the C1995S Tg polypeptide was partially resistant to Endo H treatment. The presence of a smaller mol wt shift of the Endo H-resistant bands suggested that treatment of Tg by Endo H was complete. The partial resistance to Endo H treatment suggests that a fraction of C1995S Tg is transported to the Golgi. This may result in the detectable concentration of serum Tg in patients C and D, although the level of serum Tg in patients C and D is not as high as that in patients with the common type of adenomatous goiter (8).

The ER-retained Tg polypeptides formed high mol wt aggregates. Immediately after translation, normal Tg transiently forms high mol wt aggregates, which are mediated by nearly 60 interchain disulfide bonds of the Tg polypeptide and involve molecular chaperones, such as GRP78 and GRP94 (16, 17, 18). As C1263R Tg and C1995S Tg are presumably unable to make the correct disulfide bonds to form Tg monomers, intracellular transport may be blocked, and C1263R Tg and C1995S Tg may be retained in the ER as high mol wt aggregates. Failure in the intracellular transport of C1263R Tg and C1995S Tg underscores the importance of correct disulfide bonds for formation of the correct tertiary structure of Tg and intracellular trafficking. Furthermore, incomplete digestion of C1995S Tg by Endo H suggests that there is a difference in the relative importance of cysteine residues at different locations for the proper folding and intracellular transport of Tg.

The cog/cog mouse (19) as well as humans with congenital goiter and severe hypothyroidism (5) have been reported to have defective intracellular trafficking of Tg. The hereditary dwarf rdw rat, which has been reported to have a reduced content of Tg and an increased accumulation of molecular chaperones in the thyroid gland, may also be another model of defective intracellular transport of Tg (20). From the results of the present study, disorders of Tg trafficking from the ER to the Golgi can be considered as endocrinopathies in the family of endoplasmic reticulum storage diseases (21), which includes central diabetes insipidus (22) and familial hypercholesterolemia (23).

	Acknowledgments

 
The authors thank Miss Masako Saito and Mr. Kazumi Akimoto for technical assistance.

	Footnotes

 
¹ This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture; the Japan Private School Promotion Foundation; and the Ichiro Kanehara Foundation (all to A.H.).

Received August 20, 1998.

Revised November 24, 1998.

Accepted January 19, 1999.

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