Rapid Detection of Known Mutations and Its Application to Carrie Testing

已知突变的快速检测及其在Carrie检测中的应用

• 批准号: 06557046
• 负责人: NARISAWA Kuniaki
• 金额: $ 8.06万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557046/
• 关键词: Cloning; Mutation; Allele Specific PCR; Multiplex ASPCR; Holocarboxylase Synthetase Deficiency; GSD; PKU; Metylmalonic Acidemia; フェニルケトン尿症; 変異検出法; STR; 保因者診断; 非ケトーシス型高グリシン血症; 遺伝病; 遺伝子変異; short tandem repeat; 多型診断; アレル特異的PCR

Present study carried out the following projects ;1. Cloning of holocarboxylase synthetase (HCS) geneDeficiency of HCS causes biotin-responsive multiple carboxylase. We have choned the human HCScDNA,which shows homology to BirA and maps to chromosome 21q22.1.2. Mutation studies from various genetic disorders :1) Mutation analysis in 6 Japanese patients revealed the misssense mutation of T997C,G1935A and one base deletion (G1067). These mutations together account for about 83% of Japanese HCS alleles. 2) Mutations of GSD patients ; R83H,P257L,R170X,g727t and IVS 1nt-a, together account for 98% of Japanese GSD mutant alleles.The g727t splicing mutation was the most common mutation among the Japanese patients. 3) Mutaions of PKU patients ; R413P,IVS-4nt-1, R111X,Y204C,R243Q,R252W,R241C,R278I and IVS-9, account for 67% of Japanese PKU mutant alleles. 4) Mutaions of PKU patients ; R243Q,R413P,IVS-4, R365X,R111X,R261Q,Y204C,account for 61% of Chinese PKU mutant alleles. 5) Mutations of Japanese methylmalonic acidemia ; G425T and 769DELTACA.6) Mutations of Japanese CPT II deficient Patients ; F352C,V368I and M647V.7) Mutation of Japanese familial ALS ; H46R.3. Development of multiplex allele specific PCR ; We have applied ASPCR methodology to the detection of mutations in PAH gene. Single ASPCR tests have been developed for 12 mutations found in Japan and China. ASPCR reactions for 3 or 4 mutaions have been multiplexed. The two multiplex tests will detect the presence of the R111X,R261Q and Y204C mutations and the R243Q,R413P,IVS 4nt-1 and Y356X mutations, respectively.

本研究开展了以下项目；1．全羧化酶合成酶 (HCS) 基因的克隆 HCS 缺陷会导致生物素响应性多重羧化酶。我们已经克隆了人类HCScDNA，它与BirA具有同源性，并定位到染色体21q22.1.2。各种遗传性疾病的突变研究：1）6名日本患者的突变分析揭示了T997C、G1935A和一碱基缺失（G1067）的错义突变。这些突变合计约占日本 HCS 等位基因的 83%。 2）GSD患者的突变； R83H、P257L、R170X、g727t和IVS 1nt-a合计占日本GSD突变等位基因的98%。g727t剪接突变是日本患者中最常见的突变。 3）PKU患者突变； R413P、IVS-4nt-1、R111X、Y204C、R243Q、R252W、R241C、R278I 和 IVS-9 占日本 PKU 突变等位基因的 67%。 4）PKU患者突变； R243Q、R413P、IVS-4、R365X、R111X、R261Q、Y204C，占中国PKU突变等位基因的61%。 5）日本甲基丙二酸血症突变； G425T 和 769DELTACA.6) 日本 CPT II 缺陷患者的突变； F352C、V368I 和 M647V.7) 日本家族性 ALS 突变； H46R.3。多重等位基因特异性 PCR 的发展；我们应用 ASPCR 方法来检测 PAH 基因的突变。单一 ASPCR 测试已针对在日本和中国发现的 12 种突变开发出来。 3 或 4 个突变的 ASPCR 反应已被多重化。这两项多重检测将分别检测 R111X、R261Q 和 Y204C 突变以及 R243Q、R413P、IVS 4nt-1 和 Y356X 突变的存在。

项目成果

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Ikeda, H.等人：“二氢蝶啶还原酶缺乏症的分子分析：日本患者中两种新突变的鉴定。”

Suzuki,Y.et al.: "Enzymatic diagnosis of holocarboxylase synthetase deficiency using apocarboxyl carrier protein as a substrate." Clinica Chemica Acta. 251. 41-52 (1996)

Suzuki,Y.et al.：“使用脱辅基羧基载体蛋白作为底物对全羧化酶合成酶缺陷进行酶法诊断。”

Suzuki.Y.et al: "Enzymatic diagnosis of holocarboxylase synthetase deficiency using apo-carboxyl carrler protein as a substrate." Clinica Chemica Acta. 251. 41-52 (1996)

Suzuki.Y.et al：“使用脱辅基羧基 carrler 蛋白作为底物对全羧化酶合成酶缺陷进行酶法诊断。”

Suzuki,Y.et al.: "Isolation and characterization of mutations human holocarboxylase synthetase cDNA." Nature Genetics. 8. 122-128 (1994)

Suzuki,Y.et al.：“人全羧化酶合成酶 cDNA 突变的分离和表征。”

Aoki,Y.et al.: "Characterization of mutant holocarboxylase synthetase (IICS) : a Km was not elevated in a patient with HCS deficiency." Pediatric Research. (in press). (1997)

Aoki,Y.et al.：“突变型全羧化酶合成酶 (IICS) 的表征：HCS 缺乏症患者的 Km 没有升高。”