| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Kunimitsu Yamaguchi University, Allied Health Science, Professor, 医療技術短期大学部, 教授 (50035250)
OHBA Yuzo Yamaguchi University, Medicine, Professor, 医学部, 教授 (10035199)
|
|---|
| Research Abstract |
Direct cloning using Polymerase Chain Reaction (PCR) and simple determination of base mutation employing biotin-labeled oligonucleotide probes were developed in the first year. Using these methods in the next two years 39 Japanese families with beta-thalassemia (beta-thal) were analyzed in the following strategy : (1) A mutation was determined by direct cloning and subsequent sequence analysis. (2) A pair of biotinylated oligonucleotide probes, specific to normal and thalassemic sequences were synthesized. They were reacted to PCR-amplified DNAs of other patients on a nitrocellulose membrane, which was called "dot-blot hybridization". Thus all the patients were surveyed and easily identified for the same thalassemic mutation. The results for the 39 families were : (1)-31 cap A ->G in 3 families, (2) initiation codon ATG ->GTG in 3, (3) initiation codon ATG->AGG in one (Korean family), (4) codon 24 GGT->GGA in one, (5) codon 26 GAG->AAG(Hb E) in one, (6) IVS-I-130 G->C in one, (7) codon
… More
41-42 TTCTTT->TT in 7, (8) codon 90 GAG->TAG in 8, (9) IVS-II-1 G->A in 3, (10) IVS-II-654 C->T in 2, (11) IVS-II-848 in one. (12) codon 110 CTG->CCG in 2, (13) codon 121 GAA ->TAA in 2, (14) codon 127-128 CAGGCT->CCT in 4. Thus 14 kinds of beta-thal mutants were discovered in 39 Japanese (including one Korean) families. These Japanese mutants comprise 20% of the beta-thal mutations reported in the world. It is evident that the simple methods described above enabled us to disclose the genetic abnormalities of Japanese beta-thals in such a short time. These Japanese beta-thal mutations vary (14 types). Among them six are unique to the Japanese. These mutants, therefore, seem to be "neutral" in the evolution. Five are common to Chinese, which might contribute to the investigation on Japanese genetics.
The direct cloning was evaluated using many clones analyzed by us. As the study for other inherited disorders gene analyses of Hb H diseases in three Japanese families were performed, and amino acid replacement in Hb Hirosaki, a highly unstable variant, were determined by a base sequences analysis. Less
|
