先天性アンチトロンビンIII欠損症の遺伝子解析

先天性抗凝血酶III缺乏症的遗传分析

• 批准号: 07770873
• 负责人: 中原 祥文
• 金额: $ 0.58万
• 依托单位: Kyoto Prefectural University of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Encouragement of Young Scientists (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07770873/
• 关键词: antithrombin; gene; deficiency; thrombosis; mutation

先天性血栓症発症要因として重視される,先天性アンチトロンビン欠損症20家系を対象に末梢血白血球より,genomic DNAを抽出し,(1)RFLP:Pst-1,EcoR-1,Hind-IIIによるdigestionによりATc-DNA probeを用いてsouthern hybridizationを施行.partialあるいはwhole gene deletion等を検討.(2)PCR:AT遺伝子の各exonを5'末端をbiotinで標識したreverse primerおよびbiotin非標識foward primerによりPCR法によりflanking領域を含めて増幅.(3)Heteroduplex analysis:各PCR産物をheat reaction(95℃,3minutes)後,徐々に(20・30minutes)coolingし,heteroduplexを形成.Hydrolink Gel Solutionを用いたelectrophoresisによりmutant strandの有無を検討.(4)Direct Sequencing:PCR産物をstreptavidinで被覆されたMagnetic beadsを用いて固相にて回収後,アルカリ変性にてsenseおよびantisenseの一本鎖DNAをそれぞれ分離,調整.各一本鎖DNAを鋳型としてSequenase Ver.2.0を用いたdirect sequencing法にて塩基配列を決定.No.1(typeI);exon5,codon 362(TAT to T,2bp deletion,heterozygote).No.2(type I);exon4,codon 310(GAA to GA,1bp deletion,heterozygote).No.3(type II,AT-Osaka1);exon6,codon 407 CCT(Pro)to ACT(Thr)missense mutation,heterozygote.No.4(type II,AT-Osaka 2);exon6,codon 393 CGT(Arg)to CAT(His)missense mutation,heterozygote.以上に加え,No.5(type I);exon3A,codon 129 CGT(Arg)to TGA(stop codon)nonsense mutation,heterozygote.No.6(type I);exon4,codon 260 TAT(Tyr)to TAA(stop codon)nonsense mutation,heterozygoteを検出した.これらの疾患発症に関わる分子生物学的背景が明らかにされた.

20 families with congenital thrombosis were studied for peripheral blood leukocyte deletion and genomic DNA extraction. RFLP:Pst-1,EcoR-1,Hind-III were used for southern hybridization. (2)PCR:AT gene exon 5'terminal biotin marker reverse primer biotin non-marker foward primer PCR flanking field contains increasing amplitude. (3)Heteroduplex analysis: After heat reaction(95℃,3minutes), the PCR products were cooled for (20·30minutes) and heteroduplex was formed.Hydrolink Gel Solution was used to detect the presence or absence of mutant strand in electrophoresis. (4)Direct Sequencing:PCR products streptavidin coated Magnetic beads were separated and adjusted by solid-phase PCR. DNA Sequencing Ver.2.0-Use direct sequencing method to determine base alignment. No.1 (typeI);exon5,codon 362 (TAT to T,2bp deletion,heterozygote).No.2(type I);exon4,codon 310 (GAA to GA,1bp deletion,heterozygote).No.3 (type II,AT-Osaka1);exon6,codon 407 CCT(Pro)to ACT(Thr)missense mutation,heterozygote.No.4 (type II,AT-Osaka 2);exon6,codon 393 CGT(Arg)to CAT(His)missense mutation,heterozygote.在上, No.5 (type I);exon3A,codon 129 CGT(Arg)to TGA (stop codon)nonsense mutation, heterozygote.No.6 (type I);exon4,codon 260 TAT(Tyr)to TAA(stop codon)nonsense mutation,heterozygote. The molecular biological background of the disease is clear.

项目成果

T.Hayashi,Y.Nakahara: "Antithrombin Aomori:Identification of a point mutation resulting in Arg 393-His substitution." Acta Medica et Biologica. 43. 157-163 (1995)

T.Hayashi、Y.Nakahara：“抗凝血酶 Aomori：鉴定导致 Arg 393-His 取代的点突变。”