Length, methylation, and purity of expanded C9orf72 repeat across diseases, tissues, and cell populations

/ABSTRACT We seek to elucidate the phenotypic heterogeneity seen in people with an expanded repeat in C9orf72, the most frequent genetic cause of two devastating neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Remarkably, even within families, individuals can develop ALS, FTD, or both diseases. Although symptoms may appear when someone is about 20 years of age, others are 90 years of age. Similarly, survival after symptom onset ranges from several months to over 30 years. Presently, this substantial phenotypic heterogeneity remains largely unexplained. The need for a deeper understanding of this intriguing expansion is further substantiated by the recent failure of antisense oligonucleotide trials targeting C9orf72 transcripts. The C9orf72 repeat expansion is basically ill defined, even though it was discovered over a decade ago. In fact, a well-established pathogenic threshold is still lacking, resulting in uninterpretable size estimates of up to about one hundred repeats. This knowledge gap can probably be attributed to technical limitations of methods commonly used to characterize this expansion. Importantly, sequencing advances now enable an in- depth assessment of expanded repeats. As such, in our present application, we propose to employ a cutting- edge targeted long-read DNA sequencing technology to accurately obtain the repeat length, methylation profile, and sequence content of the C9orf72 expansion. We postulate that variability in these characteristics of the expansion might serve as disease modifiers, contributing to the reported phenotypic heterogeneity. To this end, we will include longitudinally collected ante-mortem blood specimens from C9orf72 expansion carriers who are symptomatic and belong to the ALS/FTD spectrum, pre-symptomatic, or phenoconverters (Aim 1). By capturing the length, methylation, and purity of their expansion at a single-molecule level, we can uncover vital differences between groups and changes over time, which might precede the occurrence of symptoms. Additionally, we will assess post-mortem brain specimens from C9orf72 expansion carriers who received a neuropathological diagnosis of ALS or frontotemporal lobar degeneration (FTLD; Aim 2). Because we will use cell sorting to specifically investigate major cell populations in multiple brain regions, we are equipped to find differences between diseases, regions, and cell types. Thus, our thorough assessment of the C9orf72 repeat expansion in a precious collection of ante-mortem and post-mortem specimens using a pioneering sequencing technology may aid in resolving this unusual expansion, paving the way for tailored treatment strategies. Project Number: 1R01NS144153-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Marka Van Blitterswijk | Institution: MAYO CLINIC JACKSONVILLE, JACKSONVILLE, FL | Award Amount: $627,408 | Activity Code: R01 | Study Section: Clinical Neurodegeneration Translational Neuroscience Study Section[CNTN] View on NIH RePORTER: https://reporter.nih.gov/project-details/11362877