Veenboer G J, de Vijlder J J
Department of Experimental Pediatric Endocrinology, Academic Hospital University of Amsterdam, The Netherlands.
Endocrinology. 1993 Jan;132(1):377-81. doi: 10.1210/endo.132.1.8380383.
A thyroglobulin (Tg) synthesis defect in Dutch goats causes congenital goiter and hypothyroidism. The disease is inherited in an autosomal recessive way and is linked to restriction fragment length polymorphisms (RFLPs) in the Tg gene. Previous studies showed that Tg mRNA isolated from the goiters was of normal size (8.4 kilobases). Translation of high mol wt polysomal Tg mRNA isolated from goiter in a cell-free rabbit reticulocyte lysate resulted in a single 35,000 mol wt Tg polypeptide. Tg antigens analyzed in T4-arrested goiters were glycosylated and had mol wt of 40,000 and 32,000. The aim of this study was to identify the molecular lesion responsible for this disease. Polysomal Tg mRNA, therefore, was isolated, and cDNA was made using oligonucleotides as primers. This cDNA was multiplied by the polymerase chain reaction and cloned. In comparing the normal and abnormal sequences, we found a C-->G point mutation in exon 8 causing a change from TAC (Tyr)-->TAG (termination signal) at amino acid position 296. This mutation resulted in the appearance of a KpnI restriction site in the goiter DNA. The sequence of Tg mRNA preceding the stop codon was equal for normal and goitrous goats, except for one C-->T mutation in exon 5 which gave a Ser-->Leu transition. The KpnI site introduced by the C-->G point mutation was present in chromosomal DNA of the goitrous goats, making it possible to distinguish goats heterozygous for the defect from normal and goitrous animals. We calculated that the stop codon in exon 8 would result in a Tg polypeptide chain with a mol wt of 39,000, in good agreement with the mol wt of the in vitro and in vivo translation products. In conclusion, the C-->G mutation causing a stop codon in exon 8 is responsible for the Tg synthesis defect in Dutch goats.
荷兰山羊的甲状腺球蛋白（Tg）合成缺陷会导致先天性甲状腺肿和甲状腺功能减退。该疾病以常染色体隐性方式遗传，并与Tg基因中的限制性片段长度多态性（RFLP）相关。先前的研究表明，从甲状腺肿中分离出的Tg mRNA大小正常（8.4千碱基）。在无细胞兔网织红细胞裂解物中，从甲状腺肿中分离出的高分子量多核糖体Tg mRNA的翻译产生了单一的35,000分子量的Tg多肽。在T4停滞的甲状腺肿中分析的Tg抗原被糖基化，分子量为40,000和32,000。本研究的目的是确定导致该疾病的分子损伤。因此，分离出多核糖体Tg mRNA，并使用寡核苷酸作为引物制备cDNA。该cDNA通过聚合酶链反应扩增并克隆。在比较正常和异常序列时，我们发现在外显子8中有一个C→G点突变，导致氨基酸位置296处从TAC（酪氨酸）变为TAG（终止信号）。该突变导致甲状腺肿DNA中出现一个KpnI限制性位点。除了外显子5中的一个C→T突变导致丝氨酸→亮氨酸转变外，正常山羊和甲状腺肿山羊中终止密码子之前的Tg mRNA序列相同。由C→G点突变引入的KpnI位点存在于甲状腺肿山羊的染色体DNA中，从而有可能将缺陷的杂合山羊与正常和甲状腺肿动物区分开来。我们计算出，外显子8中的终止密码子将导致产生分子量为39,000的Tg多肽链，这与体外和体内翻译产物的分子量非常一致。总之，导致外显子8中出现终止密码子的C→G突变是荷兰山羊Tg合成缺陷的原因。
