Single-cell analysis for mitochondrial genetic disorder disease

线粒体遗传病的单细胞分析

• 批准号: 07670891
• 负责人: KOBAYASHI Yoko
• 金额: $ 1.09万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670891/
• 关键词: LHON; MELAS; DNA mitochondirial; Mitochondrial genetics; Polymerase chain reaction; mitochondrial DNA; mitochondrial myopathy; heteroplasmy; PCR; DNA, -Mitochondrial-genetics; Point-Mutation; Mitochondrial-Myopathies-genetic; MELAS-Syndrome-ge

We have examined the distribution of mutant mtDNA molecules in single cells from a patient with Leber hereditary optic neuropathy (LHON) and MELAS (mitochondrial encephalopathy, myopathy, lactic acidosis and stroke-like episodes). The patient was a 46-year-old unaffected male in a large pedigree with LHON.Previous analysis of DNA from the total blood buffy coat had shown that -80 % of the mtDNA molecules carried the 11778 LHON mutation and that 20 % had the normal sequence at this point. Single lymphocytes were isolated using a micromanipulation method. Each isolated cell was used for two step PCR amplification and restriction digestion. Twenty-five samples each containing a single cell were analyzed. Sixteen cells had completely mutant mtDNA,and five cells had completely normal mtDNA (intracellular homoplasmy). Four cells contained a mixture of both. Twenty one of the 25 cells had segregated to either homoplasmic mutant or homoplasmic normal mtDNA.mtDNA heteroplasmy in peripheral lymphocytes in this patient therefore was intercellular to a large extent. However, almost all cells which showed 95 % normal mitochondrial genes in MELAS rapidly changed to the cells involved mutant genes in the homoplasmic fashion after cell culture.

我们研究了Leber遗传性视神经病变（LHON）和MELAS（线粒体脑病、肌病、乳酸酸中毒和卒中样发作）患者单细胞中突变mtDNA分子的分布。该患者是一个患有LHON的大型家系中的46岁未受影响的男性。来自总血液血沉棕黄层的DNA的先前分析显示，约80%的mtDNA分子携带11778 LHON突变，20%在该点具有正常序列。使用显微操作方法分离单个淋巴细胞。每个分离的细胞用于两步PCR扩增和限制性消化。分析了25个样品，每个样品含有单个细胞。16个细胞有完全突变的mtDNA，5个细胞有完全正常的mtDNA（细胞内同质性）。四个细胞含有两者的混合物。25个细胞中有21个分离为同质突变型或同质正常型mtDNA，因此该患者外周血淋巴细胞mtDNA异质性在很大程度上是细胞间的。然而，几乎所有在MELAS中显示95%正常线粒体基因的细胞在细胞培养后迅速转变为以同质方式涉及突变基因的细胞。

项目成果

Yoko Kobayashi, et al.: "A point mutation in the mitochondrial tRNA Leu (UUR) gene in MELAS (mitochondrial mycpathy, encephalopathy, loctic acidosis and stroke-like episodes)." Biochem Biophys Res Commun. 168. 816-822 (1990)

Yoko Kobayashi 等人：“MELAS（线粒体肌病、脑病、乳酸性酸中毒和中风样发作）中线粒体 tRNA Leu (UUR) 基因的点突变。”

Yoko Kobayashi, et al.: "The mutant genes in mitochondrial myopathy, encephaiopathy, lactic acidosis and stoke-like episodes were setectively amplified through generatic." J lnher Metab Dis. 15. 803-808 (1992)

Yoko Kobayashi等人：“线粒体肌病、脑病、乳酸性酸中毒和中风样发作中的突变基因通过遗传选择性扩增。”

小林葉子: "Pearson marrow pancraas 症候群" 小児内科. 4. 939-943 (1992)

小林洋子：“皮尔逊骨髓胰综合征”小儿内科 4. 939-943 (1992)。

Yoko Kobayashi, atal.: "Single-cell analysis of intercellular heteroplasmy of mt DNA in Leber nereditary optic neuropathy." Am J Hum Genet. 55. 206-209 (1994)

Yoko Kobayashi 等人：“Leber 神经性视神经病变 mt DNA 细胞间异质性的单细胞分析”。

Yoko Kobayashi, et al.: "Respiration-deficient cells are caused by a single point mutation in the mitochondrial tRNA-Leu(UUR) gene in mitochondrial myopathy, encephalooathry, lactic acidosis and strokelike episodes (MELAS)." Am J Hum Genet. 49. 590-599 (1

Yoko Kobayashi 等人：“呼吸缺陷细胞是由线粒体肌病、脑病、乳酸性酸中毒和中风样发作 (MELAS) 中线粒体 tRNA-Leu (UUR) 基因的单点突变引起的。”