Kotani Tomio, Umeki Kazumi, Kawano Jun-ichi, Suganuma Tatsuo, Hishinuma Akira, Ieiri Tamio, Harada Shohei
Department of Laboratory Medicine, Laboratory for Clinical Investigation, Miyazaki Medical College, Kiyotake, Japan.
Clin Endocrinol (Oxf). 2003 Aug;59(2):198-206. doi: 10.1046/j.1365-2265.2003.01823.x.
Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808-13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their father's DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well-conserved amino acids in the peroxidase superfamily, Gly533Cys- and Asp574/Leu575del-TPOs were thought to be affected structurally or functionally. In expression studies using CHO-Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild-type TPO and had enzyme activity, although Gly533Cys-TPO was slightly lower in efficiency of expression and more degenerative than wild-type TPO.
We examined the localization of both mutated TPOs. Gly533Cys-TPO was located on the endoplasmic reticulum (ER) and nuclear envelope but not on the plasma membrane, whereas Asp574/Leu575del-TPO was located not only on the ER and nuclear envelope but also on the plasma membrane, as wild-type TPO. Nevertheless, only one point differed between Asp574/Leu575del- and wild-type TPOs: the mutated TPO was expressed on the plasma membrane surface at less than half the rate of wild-type TPO.
Gly533Cys-TPO synthesized almost no thyroid hormone because of its defective localization on the apical membrane surface of thyrocytes, whereas Asp574/Leu575del-TPO performed thyroid hormone synthesis at a rate of less half that of wild-type TPO. In cotransfection experiments using three combinations of wild-type and G1687T-mRNAs, wild-type and 1808-13del-mRNAs, and G1687T-, 1808-13del-mRNAs, the three kinds of mRNAs were considered to have no influence on cell surface TPO expression of another mRNA when a 50%-maximal amount of each mRNA was transfected. When a larger amount of each mRNA was transfected, the former two combinations showed the level of cell surface TPO expression obtained from the saturating amount of wild-type mRNA, whereas the last combination of mutated mRNAs covered only about half of the expression level.
Defective thyroid hormone production resulting from the abnormal TPOs was at a level that caused latent hypothyroidism when the patients were born. With their growth, thyroid hormone volume gradually became inadequate and their thyroid gland enlarged compensatorily.
三名患有甲状腺肿且伴有潜在至轻度甲状腺功能减退的兄弟姐妹被怀疑存在甲状腺过氧化物酶（TPO）异常。对他们的基因组DNA进行直接测序显示，TPO基因有两个新突变，其中一个是G1687T（甘氨酸533变为半胱氨酸；第9外显子），另一个是1808 - 13del（天冬氨酸574/亮氨酸575缺失；第10外显子）。这两个突变是复合杂合突变，因为前者在其父亲的DNA中为杂合状态被发现，后者在其母亲的DNA中也为杂合状态被发现。由于甘氨酸533和天冬氨酸574/亮氨酸575是过氧化物酶超家族中保守性良好的氨基酸，所以认为甘氨酸533半胱氨酸 - TPO和天冬氨酸574/亮氨酸575缺失 - TPO在结构或功能上受到了影响。在使用CHO - Kl细胞和引入单个突变的mRNA进行的表达研究中，两种突变的TPO蛋白均以与野生型TPO相同的分子大小表达且具有酶活性，尽管甘氨酸533半胱氨酸 - TPO的表达效率略低于野生型TPO且比野生型TPO更易降解。
我们检测了两种突变TPO的定位。甘氨酸533半胱氨酸 - TPO位于内质网（ER）和核膜上，但不在质膜上，而天冬氨酸574/亮氨酸575缺失 - TPO不仅位于ER和核膜上，还像野生型TPO一样位于质膜上。然而，天冬氨酸574/亮氨酸575缺失 - TPO与野生型TPO之间仅存在一点差异：突变的TPO在质膜表面的表达速率不到野生型TPO的一半。
甘氨酸533半胱氨酸 - TPO由于其在甲状腺细胞顶端膜表面的定位缺陷几乎不合成甲状腺激素，而天冬氨酸574/亮氨酸575缺失 - TPO进行甲状腺激素合成的速率不到野生型TPO的一半。在使用野生型和G1687T - mRNA、野生型和1808 - 13del - mRNA以及G1687T - 、1808 - 13del - mRNA三种组合进行的共转染实验中，当每种mRNA转染量达到最大量的50%时，这三种mRNA被认为对另一种mRNA的细胞表面TPO表达没有影响。当转染的每种mRNA量更大时，前两种组合显示出从野生型mRNA饱和量获得的细胞表面TPO表达水平，而最后一种突变mRNA组合的表达水平仅约为其一半。
异常TPO导致的甲状腺激素产生缺陷在患者出生时就处于导致潜在甲状腺功能减退的水平。随着他们的成长，甲状腺激素量逐渐变得不足，甲状腺进行代偿性肿大。
