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 goiter

We analyzed the thyroglobulin (Tg) gene of 2 unrelated patients with congen ital goiter and the Tg gene of 2 siblings with the variant type of adenomat ous goiter. The clinical characteristics of the patients with congenital go iter and the variant type of adenomatous goiter were very similar, except f or serum Tg levels, which were less than 15 pmol/L in the patients with con genital 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 unrela ted 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 detect ed. in the Tg complementary DNA of 110 normal subjects, the allelic frequen cies of the C1263R and C1995S mutations were each less than 0.5%. Also in t he normal subjects were detected 35 nucleotide polymorphisms, the insertion of 3 nucleotides, and I alternative splicing, each of which was not associ ated with any specific thyroid disease. From these data, the molecular mech anism of the C1263R and C1995S mutations was elucidated. We first analyzed the carbohydrate residues of C1263R Tg and C1995S Tg. Sensitivity to treatm ent by endoglycosidase H suggests that C1263R Tg and C1995S Tg were retaine d in the endoplasmic reticulum (ER). Also, the presence of endoglycosidase H-resistant Tg as well as endoglycosidase H-sensitive Tg in the patients wi th the variant type of adenomatous goiter suggests that a fraction of C1995 S Tg was transported to the Golgi and associated with the mildly increased serum Tg levels. Native PAGE and Western blot analysis with anti-Tg antibod y 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 eith er arginine or serine cause an abnormal three-dimensional structure of Tg. Such misfolded Tg polypeptides are retained in the ER as high mol wt aggreg ates.