The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure
The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure
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DOI：
10.1086/301680
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发表时间：
1998-01-01
影响因子：
9.8
通讯作者：
Patel, PI
中科院分区：
文献类型：
--
作者：
Bidichandani, SI;Ashizawa, T;Patel, PI
Friedreich共济失调(FRDA)是一种常染色体隐性遗传性神经退行性疾病，是最常见的遗传性共济失调。绝大多数患者都是X25基因第一内含子中多态GAA三联体重复序列异常扩张的纯合子，X25基因编码线粒体蛋白Frataxin。FRDA中的细胞退化可能是由线粒体功能障碍引起的，可能是由于铁的异常积累，就像在缺乏Frataxin同系物的酵母细胞中观察到的那样。通过核糖核酸酶保护试验，我们发现扩增纯合子的患者有明显的成熟X25基因缺失。GAA内含子三联体的扩展导致X25mRNA减少的机制(S)目前尚不清楚。没有发现扩展的内含子1异常剪接的证据。使用从FRDA患者克隆的重复序列，我们表明GAA重复本身以一种长度依赖的方式干扰体外转录，对原核和真核酶都是如此。当尝试合成富含GAA的转录本时，这种干扰在转录的生理方向上最为明显。这些结果与观察到的三联重复长度与发病年龄呈负相关是一致的。使用体外化学探测策略，我们还表明GAA三联体重复采用了一种不寻常的DNA结构，表现为对四氧化三、羟胺和焦碳酸二乙酯的高反应性。这些结果提出了这样一种可能性，即GAA三联体重复扩增可能导致一种不寻常但稳定的DNA结构，该结构干扰转录，最终导致细胞缺乏Frataxin。
Friedreich ataxia (FRDA), an autosomal recessive, neurodegenerative disease is the most common inherited ataxia. The vast majority of patients are homozygous for an abnormal expansion of a polymorphic GAA triplet repeat in the first intron of the X25 gene, which encodes a mitochondrial protein, frataxin. Cellular degeneration in FRDA may be caused by mitochondrial dysfunction, possibly due to abnormal iron accumulation, as observed in yeast cells deficient for a frataxin homologue. Using RNase protection assays, we have shown that patients homozygous for the expansion have a marked deficiency of mature X25 mRNA. The mechanism(s) by which the intronic GAA triplet expansion results in this reduction of X25 mRNA is presently unknown. No evidence was found for abnormal splicing of the expanded intron 1. Using cloned repeat sequences from FRDA patients, we show that the GAA repeat per se interferes with in vitro transcription in a length-dependent manner, with both prokaryotic and eukaryotic enzymes. This interference was most pronounced in the physiological orientation of transcription, when synthesis of the GAA-rich transcript was attempted. These results are consistent with the observed negative correlation between triplet-repeat length and the age at onset of disease. Using in vitro chemical probing strategies, we also show that the GAA triplet repeat adopts an unusual DNA structure, demonstrated by hyperreactivity to osmium tetroxide, hydroxylamine, and diethyl pyrocarbonate. These results raise the possibility that the GAA triplet-repeat expansion may result in an unusual yet stable DNA structure that interferes with transcription, ultimately leading to a cellular deficiency of frataxin.