Applying targeted long-read sequencing to assess an expanded repeat in C9orf72

A repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) represents the most common genetic cause of two fatal neurodegenerative diseases: frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To determine the length of this repeat expansion, we used No-Amp sequencing (Pacific Biosciences [PacBio]), a targeted amplification-free long-read sequencing technology. In total, we examined 28 fully characterized C9orf72 expansion carriers for whom cerebellar tissue was available. We obtained more than 3500 circular consensus sequencing (CCS) reads, including 814 reads that spanned the C9orf72 repeat expansion. Repeat lengths obtained using No-Amp sequencing significantly correlated with those obtained using Southern blotting. Importantly, we demonstrated that smaller expansions are associated with prolonged survival after onset. Moreover, we revealed that smaller expansions associate with higher levels of transcripts containing the expansion and of dipeptide repeat (DPR) proteins, a hallmark of C9orf72-linked diseases. These findings suggest that, during transcription, it might be easier to cover small expansions than long expansions, which could explain why we observed elevated levels of expansion-containing transcripts in patients with smaller expansions. Since these expansion-containing transcripts can serve as templates for repeat-associated non-ATG (RAN) translation, this could account for the increased levels of DPR proteins we detected. Investigation of the expansion itself uncovered that its GC-content is high (100%). The expansion appears to be relatively pure, while interruptions are only occasionally encountered (<5%). In conclusion, we have shown that No-Amp sequencing is a powerful tool to examine the length and purity of repeat expansions, which enabled us to identify relevant clinico-pathological associations for C9orf72-related diseases.

Learning Objectives:

1. Learn about methods that can be employed to detect and size C9orf72 repeat expansions

2. Understand that targeted long-read sequencing accurately measures the C9orf72 repeat length and presence of interruptions