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

1.本研究分析了新发现的3个TBG超常家系，其中1例为家族性，2例为散发性。结论：1.应用双重聚合酶链式反应-高效液相色谱法在1例家族性和1例散发病例中检测到TBG基因的扩增，但在1例散发病例中未检测到该基因的扩增，目前正在进行分子缺陷的寻找。采用双重聚合酶链式反应-高效液相色谱法对8个遗传性TBG超标家系(4个日本家系和4个高加索家系)和3个散发性日本家系的TBG基因剂量进行了估算。11个家系中有10个家系扩增，5个家系扩增3倍，3个遗传性家系扩增2倍，2例散发病例扩增。血清总胆红素水平与总胆红素基因剂量相对应。基因扩增被证明是遗传性TBG过剩的主要机制。由于2例散发病例均有2倍扩增，基因扩增被认为是通过染色体的不等同交换而从2倍增加到3倍。6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。在1个日本家系和1个高加索家系中，除1个日本家系和1个高加索家系有3倍扩增外，其余5个家系与正常对照无明显差异。扩增单位的大小可能小于FISH.4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5个日本家系的RFLP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被12种酶覆盖的52kbp之外。COSMID文库是从一名正常和一名合并单纯性GH缺乏的男性TBG过度受试者的基因组DNA中构建的。然后，通过染色体步移获得正常人75kbp和病变者82kbp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.