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

1. 采用双相高效液相色谱法测定了7个遗传TBG过量家族（3个日本家族和4个高加索家族）和1个散发性日本家族的TBG基因剂量。本研究评估的8个家族均检测到TBG基因扩增，其中5个家族3倍，2个遗传家族2倍，1例男孩为散发病例。血清TBG值与TBG基因剂量相对应。结果表明，基因扩增是遗传性TBG过量的主要机制。利用染色体和TBG探针对4个日本和1个白种人FISH家族的TBG基因扩增进行了评估。虽然在1个日本人和1个高加索人家族中显示了3倍扩增，与PCR-HPLC分析结果相对应，但其他3个家族与正常受试者无法区分。扩增的单位可能小于fish的检出限。为了明确基因扩增的机制，利用基因组dna和12种限制性内切酶对4个日本家庭的PFLPs进行了评估。然而，在所有受试者中均未检测到RFLP，这表明在被12个酶覆盖的52 kbp之外存在扩增单元的断点。采用等位基因特异性扩增技术对50个表现为TBG完全或部分缺陷的日本家庭进行了基因筛选。在44个家族中检测到CDJ突变（密码子352核苷酸缺失），在6个家族中检测到PDJ突变（密码子363核苷酸替换）。因此，这两种突变都被认为是在日本人的祖先中产生的，并通过奠基人效应扩展到后代中。利用PGK-1基因分析了10个CDJ和1个PDJ家族的X染色体失活模式。在2例女性中检测到含有正常TBG基因的X染色体选择性失活。然后，CDJ和PDJ半合子被认为分别表现为TBG- cd和TBG值与PDJ男性相同。

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.