C9orf72 repeat expansion creates the unstable folate-sensitive fragile site FRA9A

Mila Mirceta; Monika H M Schmidt; Natalie Shum; Tanya K Prasolava; Bryanna Meikle; Stella Lanni; Mohiuddin Mohiuddin; Paul M McKeever; Ming Zhang; Minggao Liang; Ilse van der Werf; Stefaan Scheers; Patrick A Dion; Peixiang Wang; Michael D Wilson; Theresa Abell; Elliot A Philips; Łukasz J Sznajder; Maurice S Swanson; Mustafa Mehkary; Mahreen Khan; Katsuyuki Yokoi; Christine Jung; Pieter J de Jong; Catherine H Freudenreich; Philip McGoldrick; Ryan K C Yuen; Agessandro Abrahão; Julia Keith; Lorne Zinman; Janice Robertson; Ekaterina Rogaeva; Guy A Rouleau; R Frank Kooy; Christopher E Pearson

doi:10.1093/narmme/ugae019

C9orf72 repeat expansion creates the unstable folate-sensitive fragile site FRA9A

NAR Mol Med. 2024 Nov 12;1(4):ugae019. doi: 10.1093/narmme/ugae019. eCollection 2024 Oct.

Authors

Mila Mirceta^{1

2}, Monika H M Schmidt^{1

2}, Natalie Shum^{1

2}, Tanya K Prasolava¹, Bryanna Meikle^{1

2}, Stella Lanni¹, Mohiuddin Mohiuddin¹, Paul M McKeever³, Ming Zhang^{3

4

5

6}, Minggao Liang^{1

2}, Ilse van der Werf⁷, Stefaan Scheers⁷, Patrick A Dion^{8

9}, Peixiang Wang¹, Michael D Wilson^{1

2}, Theresa Abell¹⁰, Elliot A Philips¹⁰, Łukasz J Sznajder^{11

12}, Maurice S Swanson¹¹, Mustafa Mehkary^{1

2}, Mahreen Khan^{1

2}, Katsuyuki Yokoi¹, Christine Jung¹³, Pieter J de Jong¹³, Catherine H Freudenreich¹⁰, Philip McGoldrick³, Ryan K C Yuen^{1

2}, Agessandro Abrahão¹⁴, Julia Keith¹⁴, Lorne Zinman¹⁴, Janice Robertson³, Ekaterina Rogaeva³, Guy A Rouleau^{8

9

15}, R Frank Kooy⁷, Christopher E Pearson^{1

2}

Affiliations

¹ Program of Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada.
² Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON, M3S 1A8, Canada.
³ Tanz Centre for Research of Neurodegenerative Diseases, University of Toronto, 60 Leonard Avenue, Toronto, M5T 2S8, Canada.
⁴ The First Rehabilitation Hospital of Shanghai, Department of Medical Genetics, School of Medicine, Tongji University, Shanghai, 200090, China.
⁵ State Key Laboratory of Cardiology and Medical Innovation Center, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, China.
⁶ Advanced Study, Tongji University, Shanghai, 200092, China.
⁷ Department of Medical Genetics, University of Antwerp, Belgium.
⁸ Montreal Neurological Institute-Hospital, McGill University, 3801 University Avenue, Montreal, Quebec, H3A 2B4, Canada.
⁹ Department of Neurology and Neurosurgery, McGill University, 3801 University Avenue, Montreal, Quebec, H3A 2B4, Canada.
¹⁰ Department of Biology, Tufts University, 200 Boston Avenue, Medford, MA 02155, USA.
¹¹ Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, College of Medicine, University of Florida, 2033 Mowry Road, Gainesville, FL 32610-3610, USA.
¹² Department of Chemistry and Biochemistry, University of Nevada, 4003-4505 South Maryland Parkway, Las Vegas, NV 89154, USA.
¹³ BACPAC Resource Center, Children's Hospital Oakland Research Institute, 25129 NE 42nd Pl, Redmond, WA 98053, USA.
¹⁴ Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, North York, Toronto, ON, M4N 3M5, Canada.
¹⁵ Department of Human Genetics, McGill University, 3801 University Avenue, Montreal, Quebec, H3A 2B4, Canada.

Abstract

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions (C9orf72Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72Exp 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 C9orf72Exp 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 C9orf72 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. C9orf72Exp 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 C9orf72Exp patient contained a highly rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72Exp 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 C9orf72Exp mice, implicating C9orf72Exp 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.