Development of Genetic Diagnosis of Congenital Metabolic Disorders -Gene Amplification and Determination of Base Sequences by Synthetic Probes-

在第一年开发了使用聚合酶链反应（PCR）的直接克隆和使用生物素标记的寡核苷酸探针的碱基突变的简单测定。在接下来的两年中，使用这些方法，在以下策略中分析了39个日本β-地中海贫血（β-地贫）家系：（1）通过直接克隆和随后的序列分析确定突变。(2)合成了一对特异于正常序列和地中海贫血序列的生物素化寡核苷酸探针。它们与硝酸纤维素膜上其他患者的PCR扩增DNA反应，这被称为“斑点杂交”。因此，所有患者都接受了调查，并很容易确定相同的地中海贫血突变。39个家系的结果为：（1）-31capA → G 3个，（2）起始密码子ATG → GTG 3个，（3）起始密码子ATG → AGG 1个（韩国家系），（4）密码子24 GGT → GGA 1个，（5）密码子26 GAG → AAG（Hb E）1个，（6）IVS-I-130 G → C 1个，（7）密码子24 GGT → GGA 1个，（8）密码子26 GAG → AAG（Hb E）1个。 关于我们 41 - 42 TTCTTT-> TT在7中，（8）密码子90 GAG-> TAG在8中，（9）IVS-II-1 G-> A在3中，（10）IVS-II-654 C-> T在2中，（11）IVS-II-848在1中。(12)（13）密码子121 GAA-> TAA;（14）密码子127 - 128 CAGGCT-> CCT。在日本39个家系（包括1个韩国家系）中发现14种β地中海贫血突变体。这些日本突变体占世界上报告的β-地中海突变的20%。显然，上述简单的方法使我们能够在如此短的时间内揭示日本β-突变体的遗传异常。这些日本人的β-地中海突变各不相同（14种）。其中有六种是日本人独有的。因此，这些突变体在进化中似乎是“中性的”。其中5个是中国人共有的，这可能有助于日本遗传学的研究。作为其他遗传性疾病的研究，对三个日本家庭的Hb H疾病进行了基因分析，并通过碱基序列分析确定了高度不稳定变体Hb广崎中的氨基酸替换。少

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