Takeshita A, Nagayama Y, Yokoyama N, Ishikawa N, Ito K, Yamashita T, Obara T, Murakami Y, Kuma K, Takamatsu J
First Department of Internal Medicine, Nagasaki University School of Medicine, Japan.
J Clin Endocrinol Metab. 1995 Sep;80(9):2607-11. doi: 10.1210/jcem.80.9.7673402.
Constitutively activating mutations have recently been identified in the thyrotropin receptor (TSHR) of hyperfunctioning thyroid adenomas and familial hyperthyroidism. In the present study, we evaluated the frequency of constitutively activating TSHR mutations in a large series of autonomously functioning thyroid nodules (AFTNs) in Japan. Forty-five AFTNs (38 solitary hyperfunctioning thyroid adenomas and 7 toxic multinodular goiters) were analyzed. Genomic DNA was extracted from paraffin-embedded tissue sections, from which DNA fragments encoding the mutational hot spots of the receptor (the third cytoplasmic loop and the sixth transmembrane segment) were amplified by polymerase chain reaction. In the single-stranded conformation polymorphism (SSCP) analysis, only one hyperfunctioning adenoma (no. 21) displayed a migration abnormality. In sequence analysis, an unusual mutation of alternate three-base deletions at nucleotides 1953-1957 (AAA GAT ACC to AAG TCC), resulting in one amino acid deletion (Asp at 619) and one conservative amino acid substitution (Thr to Ser at 620), was identified in tumor DNA but not in leukocyte DNA of no. 21. Further, the normal sequence in these regions was confirmed in 10 randomly selected samples with normal migrating patterns in SSCP analysis. The functional property of the mutant with delta 619 and T620S (designated TSHR delta 619) was then evaluated with in vitro mutagenesis and transfection studies. Unexpectedly, however, there were no significant differences in TSH binding affinity, and basal and TSH-stimulated levels of cAMP and inositol 1,4,5-triphosphate between the TSHR delta 619 and the wt-TSHR. In conclusion, the incidence of the constitutively activating TSHR mutations in AFTNs appears to be low in Japan. The oncogenic potential of a novel somatic mutant TSHR delta 619 identified in a hyperfunctioning adenoma in this study is at present uncertain because of its intact function.
最近在功能亢进性甲状腺腺瘤和家族性甲状腺功能亢进症的促甲状腺激素受体（TSHR）中发现了组成性激活突变。在本研究中，我们评估了日本大量自主功能性甲状腺结节（AFTN）中组成性激活TSHR突变的频率。分析了45个AFTN（38个孤立性功能亢进性甲状腺腺瘤和7个毒性多结节性甲状腺肿）。从石蜡包埋的组织切片中提取基因组DNA，通过聚合酶链反应扩增编码受体突变热点（第三细胞质环和第六跨膜段）的DNA片段。在单链构象多态性（SSCP）分析中，只有一个功能亢进性腺瘤（21号）显示出迁移异常。在序列分析中，在21号肿瘤DNA中鉴定出核苷酸1953 - 1957处的异常突变，为交替的三个碱基缺失（AAA GAT ACC变为AAG TCC），导致一个氨基酸缺失（619位的天冬氨酸）和一个保守氨基酸取代（620位的苏氨酸变为丝氨酸），但在21号白细胞DNA中未发现。此外，在SSCP分析中随机选择的10个迁移模式正常的样本中，这些区域的正常序列得到了证实。然后通过体外诱变和转染研究评估了具有Δ619和T620S的突变体（命名为TSHRΔ619）的功能特性。然而，出乎意料的是，TSHRΔ619与野生型TSHR之间在促甲状腺激素结合亲和力、基础和促甲状腺激素刺激的环磷酸腺苷（cAMP）及肌醇1,4,5 - 三磷酸水平方面没有显著差异。总之，在日本，AFTN中组成性激活TSHR突变的发生率似乎较低。由于其功能完整，本研究中在一个功能亢进性腺瘤中鉴定出的新型体细胞突变体TSHRΔ619的致癌潜力目前尚不确定。
