Friedreich ataxia (FRDA) and Fragile X syndrome (FXS) are among 40 diseases 
associated with expansion of repeated sequences (TREDs). Although their 
molecular pathology is not well understood, formation of repressive chromatin 
and unusual DNA structures over repeat regions were proposed to play a role. Our 
study now shows that RNA/DNA hybrids (R-loops) form in patient cells on expanded 
repeats of endogenous FXN and FMR1 genes, associated with FRDA and FXS. These 
transcription-dependent R-loops are stable, co-localise with repressive H3K9me2 
chromatin mark and impede RNA Polymerase II transcription in patient cells. We 
investigated the interplay between repressive chromatin marks and R-loops on the 
FXN gene. We show that decrease in repressive H3K9me2 chromatin mark has no 
effect on R-loop levels. Importantly, increasing R-loop levels by treatment with 
DNA topoisomerase inhibitor camptothecin leads to up-regulation of repressive 
chromatin marks, resulting in FXN transcriptional silencing. This provides a 
direct molecular link between R-loops and the pathology of TREDs, suggesting 
that R-loops act as an initial trigger to promote FXN and FMR1 silencing. Thus 
R-loops represent a common feature of nucleotide expansion disorders and provide 
a new target for therapeutic interventions.