THE GAA TRIPLET-REPEAT EXPANSION IN FRIEDREICH ATAXIA INTERFERES WITHTRANSCRIPTION AND MAY BE ASSOCIATED WITH AN UNUSUAL DNA-STRUCTURE

Friedreich ataxia (FRDA), an autosomal recessive, neurodegenerative di sease is the most common inherited ataxia. The vast majority of patien ts are homozygous for an abnormal expansion of a polymorphic GAA tripl et repeat in the first intron of the X25 gene, which encodes a mitocho ndrial protein, frataxin. Cellular degeneration in FRDA may be caused by mitochondrial dysfunction, possibly due to abnormal iron accumulati on, as observed in yeast cells deficient for a frataxin homologue. Usi ng RNase protection assays, we have shown that patients homozygous for the expansion have a marked deficiency of mature X25 mRNA. The mechan ism(s) by which the intronic GAA triplet expansion results in this red uction of X25 mRNA is presently unknown. No evidence was found for abn ormal splicing of the expanded intron 1. Using cloned repeat sequences from FRDA patients, we show that the GAA repeat per se interferes wit h in vitro transcription in a length-dependent manner, with both proka ryotic and eukaryotic enzymes. This interference was most pronounced i n the physiological orientation of transcription, when synthesis of th e GAA-rich transcript was attempted. These results are consistent with the observed negative correlation between triplet-repeat length and t he age at onset of disease. Using in vitro chemical probing strategies , we also show that the GAA triplet repeat adopts an unusual DNA struc ture, demonstrated by hyperreactivity to osmium tetroxide, hydroxylami ne, and diethyl pyrocarbonate. These results raise the possibility tha t 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.