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

1. 本研究新增3例TBG过量家族，1例家族性，2例散发。双相PCR-HPLC法检测到1例家族性和1例散发性病例中TBG基因扩增，1例散发性病例中未检测到TBG基因扩增，目前正在寻找TBG基因的分子缺陷。采用双链PCR- HPLC法对8个遗传性TBG过量家族（4个日本家族和4个高加索家族）和3个散发性日本家族的TBG基因量进行了测定。10/11个家族中检测到TBG基因扩增，5个家族中检测到3倍扩增，3个遗传家族中检测到2倍扩增，2例散发病例中检测到2倍扩增。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。2例散发性病例均出现2倍扩增，认为基因扩增由2倍增加到3倍是由于染色体的不均匀交叉。利用染色体和TBG探针对6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5个家族与正常受试者无法区分。扩增单位的大小可能小于fish的检出限。为了明确基因扩增的机制，我们利用基因组dna和12种限制性内切酶对5个日本家庭的RFLPs进行了评估。然而，在所有受试者中均未检测到RFLP，这表明扩增单元的断点存在于被12种酶覆盖的52 kbp之外。Cosmid文库是由正常和男性TBG过量合并孤立生长激素缺乏的受试者的基因组dna构建的。然后，通过染色体行走法分别获得正常组75 kbp和患病组82 kbp的DNA片段。到目前为止，两名受试者之间没有发现差异。

1. In this study, new 3 TBG excess families were analyzed, one familial and 2 sporadic cases. Amplification of the TBG gene was detected in 1 familial and 1 sporadic case by using duplex PCR-HPLC method but not in 1 sporadic case, in which a search for the molecular defect is in progress.2. The gene dosage of TBG was estimated in 8 families with inherited TBG excess (4 Japanese and 4 Caucasian families) and 3 sporadic Japanese family by using duplex PCR- HPLC method. Amplification of the TBG gene was detected in 10/11 families, 3 fold in 5 families and 2 fold in 3 inherited families and 2 sporadic cases. Serum TBG values were corresponded to TBG gene dosage. Then gene amplification was shown to be a main mechanism for inherited TBG excess. As 2 fold amplification was shown in both of 2 sporadic cases, the gene amplification was considered to increase from 2 to 3 fold by a unequal crossing over of the chromosome.3. Amplification of the TBG gene was also evaluated in 6 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 1 Japanese and 1 Caucasian family, other 5 families were shown to be indistinguishable from normal subjects. The size of the amplified unit might be smaller than the detection limit of FISH.4. In order to clarify the mechanism for the gene amplification, RFLPs were evaluated in 5 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 enzymes. Cosmid libraries were constructed from genomic DNAs of a normal and a male TBG excess subject complicated with isolated GH deficiency. Then, DNA fragments of 75 kbp in the normal and 82 kbp in the affected subject were obtained by chromosome walking. No differences were so far detected between 2 subjects.