<i>C9orf72</i> repeat expansion creates the unstable folate-sensitive fragile site FRA9A

Abstract

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and <i>C9orf72</i>, is linked to multiple diseases. <i>C9orf72</i> (GGGGCC)n expansions (<i>C9orf72</i>Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. <i>C9orf72</i>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 <i>C9orf72</i>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 <i>C9orf72</i> 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. <i>C9orf72</i>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 <i>C9orf72</i>Exp patient contained a highly rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic <i>C9orf72</i>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 <i>C9orf72</i>Exp mice, implicating <i>C9orf72</i>Exp as the source. Our results highlight unappreciated effects of <i>C9orf72</i> expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere.