Validated assays for the quantification of C9orf72 human pathology

Area of Science:

• Neurogenetics
• Molecular Biology
• Biomarker Development

Background:

• The C9orf72 gene repeat expansion is a primary genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS).
• Reliable tools to monitor C9orf72 gene, RNA, and protein are lacking, hindering research and clinical applications.
• Validated assays are crucial for understanding C9orf72 pathobiology and developing effective therapies.

Purpose of the Study:

• To develop and validate assays for quantifying C9orf72 pathobiology at DNA, RNA, and protein levels.
• To establish reliable methods for measuring repeat expansions and C9orf72 products in FTD and ALS research.
• To support discovery research and clinical trials for C9orf72-related neurodegenerative disorders.

Main Methods:

• Single-molecule sequencing was employed to accurately measure C9orf72 repeat expansions and locus changes.
• Droplet digital PCR (ddPCR) assays were developed to quantify C9orf72 transcript variants.
• Western blot and MSD immunoassay were used to validate antibodies for C9orf72 and dipeptide repeat proteins (DPRs).
• Knock-out human induced pluripotent stem cell (iPSC) lines served as controls for assay validation.

Main Results:

• Single-molecule sequencing accurately sized and phased C9orf72 repeat expansions and detected locus alterations.
• Validated ddPCR assays quantified major C9orf72 transcript variants.
• Four commercially available antibodies were validated for C9orf72 protein quantification via Western blot; none were suitable for immunocytochemistry.
• Two antibody combinations showed increased signal for poly-GA and poly-GP DPRs in patient iPSC-motor neurons, validated in human and mouse brain tissues.

Conclusions:

• Validated DNA, RNA, and protein assays provide reliable tools for C9orf72 pathobiology assessment.
• These assays are applicable to both fundamental research and clinical trials in FTD/ALS.
• The developed methods advance the study of C9orf72-associated neurodegenerative diseases.