Molecular mechanism for inherited thyroxine-binding globulin excess and isolated growth hormone deficiency

1.采用双重聚合酶链式反应-高效液相色谱法测定了7个遗传性TBG超标家系(3个日本家系和4个高加索家系)和1个日本散发性家系的TBG基因剂量。本研究评估的8个家系均检测到TBG基因扩增，其中5个家系扩增3倍，2个遗传性家系和1个男孩扩增2倍，被认为是散发性病例。血清总胆红素水平与总胆红素基因剂量相对应。结果：1.基因扩增是遗传性TBG异常的主要机制。4个日本人和1个高加索人家系中的tbg基因扩增也被用染色体和tbg探针进行了FISH评估。尽管在1个日本家系和1个高加索家系中发现了3倍扩增，这与聚合酶链式反应-高效液相色谱分析的结果相一致，但其他3个家系与正常人群没有明显区别。扩增单位的大小可能小于FISH.3的检测下限。为了弄清基因扩增的机制，利用基因组DNA和12种限制性内切酶对4个日本家系的PFLP进行了评估。然而，在所有受试者中都没有检测到RFLP，这表明扩增单位的断裂点存在于12个核苷酸覆盖的52kbp之外。采用等位基因特异性扩增技术对50个表现为TBG完全性或周围性缺陷的日本家系进行了基因筛选。在44个家系中发现CDJ突变(密码子352的核苷酸缺失)，在6个家系中发现PDJ突变(密码子363的核苷酸替换)。因此，这两种突变都被认为出现在日本人的祖先中，并通过创始人效应扩展到后代。对10个CDJ家系和1个PDJ家系的X染色体失活模式进行了分析。在2例女性中发现含有正常tbg基因的X染色体选择性失活。然后，CDJ和PDJ半合子分别被认为表现为TBG-CD，并具有与PDJ雄性相同的TBG值。

1. The gene dosage of TBG was estimated in 7 families with inherited TBG excess (3 Japanese and 4 Caucasian families) and a sporadic Japanese family by using duplex PCR-HPLC method. Amplification of the TBG gene was detected in all 8 families, evaluated in this study, 3 fold in 5 families and 2 fold in 2 inherited families and a boy, considered to be a sporadic case. Serum TBG values were corresponded to TBG gene dosage. Then gene amplification was shown to be a main mechanism for inherited TBG excess.2. Amplification of the TBG gene was also evaluated in 4 Japanese and one Caucasian families with FISH using chromosomes and a TBG probe. Although, 3 fold amplification, corresponding to the results of PCR-HPLC analysis, was demonstrated in one Japanese and one Caucasian families, other 3 families were shown to be indistinguishable from normal subjects. The size of the amplified unit might be smaller than the detection limit of FISH.3. In order to clarify the mechanism for the gene amplification, PFLPs were evaluated in 4 Japanese families using genomic DNAs and 12 restriction enzymes. Nevertheless, no RFLP was detected in all subjects, demonstrating that a breakpoint of amplified unit exist outside of 52 kbp covered by 12 enzumes.4. Gene screening using allele specific amplification was performed in 50 Japanese families manifesting TBG complete or pertial deficiency. A CDJ mutation (a nucleotide deletion in codon 352) was detected in 44 families and a PDJ mutation (a nucleotide replacement in codon 363) in 6 families. Therefore, both mutations were thought to arise in the ansester of Japanese and to expand into offsprings by the founder effect.5. X chromosome inactivation pattern was analyzed in 10 CDJ and a PDJ families using a PGK-1 gene. Selective inactivation of X chromosome containing a normal TBG gene was detected in 2 famales. Then, a CDJ and a PDJ hemizygote were considered to manifest TBG-CD and to have a TBG value same as PDJ males, respectively.