C9orf72 expansion creates the unstable folate-sensitive fragile site FRA9A

Abstract

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions ( C9orf72 Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72 Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown. Here, we show C9orf72 Exp in pre-symptomatic and ALS-FTD patient cells and brains cause the folate-sensitive chromosomal fragile site, FRA9A. FRA9A centers on >33kb of C9orf72 as highly-compacted chromatin embedded in an 8.2Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72 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 C9orf72 Exp patient contained highly-rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72 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 C9orf72 Exp mice, implicating C9orf72 Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere.