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Validated assays for the quantification of C9orf72 human pathology.

ABSTRACT: A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to the C9orf72 locus in what has been a traditionally hard to sequence genomic region. This is of particular value to sizing and phasing the repeat expansion and determining changes to the gene locus after gene editing. We developed ddPCR assays to quantify two major C9orf72 transcript variants, which we validated by selective excision of their distinct transcriptional start sites. Using validated knock-out human iPSC lines, we validated 4 commercially available antibodies (of 9 tested) that were specific for C9orf72 protein quantification by Western blot, but none were specific for immunocytochemistry. We tested 15 combinations of antibodies against dipeptide repeat proteins (DPRs) across 66 concentrations using MSD immunoassay, and found two (against poly-GA and poly-GP) that yielded a 1.5-fold or greater signal increase in patient iPSC-motor neurons compared to knock-out control, and validated them in human postmortem and transgenic mouse brain tissue. Our validated DNA, RNA and protein assays are applicable to discovery research as well as clinical trials.

SUBMITTER: Salomonsson SE

PROVIDER: S-EPMC10774390 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Validated assays for the quantification of C9orf72 human pathology.

Salomonsson S E SE Maltos A M AM Gill K K Aladesuyi Arogundade O O Brown K A KA Sachdev A A Sckaff M M Lam K J K KJK Fisher I J IJ Chouhan R S RS Van Laar V S VS Marley C B CB McLaughlin I I Bankiewicz K S KS Tsai Y-C YC Conklin B R BR Clelland C D CD

Scientific reports 20240108 1

A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to ...[more]

PMID: 38191789

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Project description:An expanded GGGGCC hexanucleotide repeat in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration associated with TDP-43 pathology (FTLD-TDP). In addition to TDP-43-positive neuronal and glial inclusions, C9ORF72-linked FTLD-TDP has characteristic TDP-43-negative neuronal cytoplasmic and intranuclear inclusions as well as dystrophic neurites in the hippocampus and cerebellum. These lesions are immunopositive for ubiquitin and ubiquitin-binding proteins, such as sequestosome-1/p62 and ubiquilin-2. Studies examining the frequency of the C9ORF72 mutation in clinically probable Alzheimer's disease (AD) have found a small proportion of AD cases with the mutation. This prompted us to systematically explore the frequency of Alzheimer-type pathology in a series of 17 FTLD-TDP cases with mutations in C9ORF72 (FTLD-C9ORF72). We identified four cases with sufficient Alzheimer-type pathology to meet criteria for intermediate-to-high-likelihood AD. We compared AD pathology in the 17 FTLD-C9ORF72 to 13 cases of FTLD-TDP linked to mutations in the gene for progranulin (FTLD-GRN) and 36 cases of sporadic FTLD (sFTLD). FTLD-C9ORF72 cases had higher Braak neurofibrillary tangle stage than FTLD-GRN. Increased tau pathology in FTLD-C9ORF72 was assessed with thioflavin-S fluorescent microscopy-based neurofibrillary tangle counts and with image analysis of tau burden in temporal cortex and hippocampus. FTLD-C9ORF72 had significantly more neurofibrillary tangles and higher tau burden compared with FTLD-GRN. The differences were most marked in limbic regions. On the other hand, sFTLD and FTLD-C9ORF72 had a similar burden of tau pathology. These results suggest FTLD-C9ORF72 has increased propensity for tau pathology compared to FTLD-GRN, but not sFTLD. The accumulation of tau as well as lesions immunoreactive for ubiquitin and ubiquitin-binding proteins (p62 and ubiquilin-2) suggests that mutations in C9ORF72 may involve disrupted protein degradation that favors accumulation of multiple different proteins.

Dataset Information

Validated assays for the quantification of C9orf72 human pathology.

Publications

Validated assays for the quantification of C9orf72 human pathology.

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