repeat expansion creates the unstable folate-sensitive fragile site FRA9A.

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and , is linked to multiple diseases. (GGGGCC)n expansions (Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immunostimulatory or damaged DNA is unknown. Here, we show Exp in pre-symptomatic and amyotrophic lateral sclerosis-frontotemporal dementia patient cells and brains cause the folate-sensitive chromosomal fragile site, FRA9A. FRA9A centers on >33 kb of as highly compacted chromatin embedded in an 8.2 Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one Exp patient contained a highly rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic Exp repeat instability and chromosomal fragility are sensitive to folate deficiency. Age-dependent repeat instability, chromosomal fragility and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic Exp mice, implicating Exp as the source. Our results highlight unappreciated effects of expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere.