Targeted long-read sequencing to quantify methylation of the C9orf72 repeat expansion.

Area of Science:

• Genetics
• Epigenetics
• Neuroscience

Background:

• The C9orf72 gene harbors a hexanucleotide repeat expansion linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
• Previous research quantified repeat expansion length but lacked insights into epigenetic modifications like methylation.

Purpose of the Study:

• To characterize C9orf72 repeat expansions using an amplification-free, long-read sequencing method.
• To quantify methylation levels within the C9orf72 repeat expansion.
• To assess repeat length and purity in individuals with C9orf72 expansions.

Main Methods:

• Targeted, amplification-free long-read sequencing of DNA from 27 individuals with C9orf72 repeat expansions.
• Analysis of on-target reads to determine methylation status, proportion of methylated CpGs, repeat length, and purity.
• Statistical analysis to correlate methylation levels with repeat length, age, and disease onset.

Main Results:

• The C9orf72 repeat expansion is methylated, with CpG methylation proportions ranging from 13% to 66%.
• Expanded alleles exhibit significantly higher methylation than wild-type alleles (P < 0.0001).
• Methylation levels positively correlate with repeat expansion length (P < 0.0001), age at collection (P < 0.0001), and age at disease onset (P = 0.020).
• Repeat lengths up to 4,088 repeats (~25 kb) were detected, with minimal interruptions in blood DNA.

Conclusions:

• This study demonstrates a robust method for quantifying methylation in C9orf72 repeat expansions.
• The findings reveal significant variability in methylation levels among individuals with C9orf72 expansions.
• Quantifiable methylation differences and clinical associations highlight the role of epigenetics in C9orf72-related neurodegenerative diseases.