| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
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.
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