|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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.