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 pathobiolo...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2024-01-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-50667-3 |
| _version_ | 1797355861190901760 |
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| author | S. E. Salomonsson A. M. Maltos K. Gill O. Aladesuyi Arogundade K. A. Brown A. Sachdev M. Sckaff K. J. K. Lam I. J. Fisher R. S. Chouhan V. S. Van Laar C. B. Marley I. McLaughlin K. S. Bankiewicz Y.-C. Tsai B. R. Conklin C. D. Clelland |
| author_facet | S. E. Salomonsson A. M. Maltos K. Gill O. Aladesuyi Arogundade K. A. Brown A. Sachdev M. Sckaff K. J. K. Lam I. J. Fisher R. S. Chouhan V. S. Van Laar C. B. Marley I. McLaughlin K. S. Bankiewicz Y.-C. Tsai B. R. Conklin C. D. Clelland |
| author_sort | S. E. Salomonsson |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T14:17:14Z |
| format | Article |
| id | doaj.art-782f10f759d84e6aa3c479b903db9782 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-08T14:17:14Z |
| publishDate | 2024-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-782f10f759d84e6aa3c479b903db97822024-01-14T12:18:28ZengNature PortfolioScientific Reports2045-23222024-01-0114111310.1038/s41598-023-50667-3Validated assays for the quantification of C9orf72 human pathologyS. E. Salomonsson0A. M. Maltos1K. Gill2O. Aladesuyi Arogundade3K. A. Brown4A. Sachdev5M. Sckaff6K. J. K. Lam7I. J. Fisher8R. S. Chouhan9V. S. Van Laar10C. B. Marley11I. McLaughlin12K. S. Bankiewicz13Y.-C. Tsai14B. R. Conklin15C. D. Clelland16Weill Institute for Neurosciences, University of California San FranciscoGladstone InstitutesWeill Institute for Neurosciences, University of California San FranciscoWeill Institute for Neurosciences, University of California San FranciscoWeill Institute for Neurosciences, University of California San FranciscoGladstone InstitutesWeill Institute for Neurosciences, University of California San FranciscoWeill Institute for Neurosciences, University of California San FranciscoWeill Institute for Neurosciences, University of California San FranciscoWeill Institute for Neurosciences, University of California San FranciscoDepartment of Neurological Surgery, The Ohio State UniversityGladstone InstitutesPacific BiosciencesDepartment of Neurological Surgery, The Ohio State UniversityPacific BiosciencesGladstone InstitutesWeill Institute for Neurosciences, University of California San FranciscoAbstract 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.https://doi.org/10.1038/s41598-023-50667-3 |
| spellingShingle | S. E. Salomonsson A. M. Maltos K. Gill O. Aladesuyi Arogundade K. A. Brown A. Sachdev M. Sckaff K. J. K. Lam I. J. Fisher R. S. Chouhan V. S. Van Laar C. B. Marley I. McLaughlin K. S. Bankiewicz Y.-C. Tsai B. R. Conklin C. D. Clelland Validated assays for the quantification of C9orf72 human pathology Scientific Reports |
| title | Validated assays for the quantification of C9orf72 human pathology |
| title_full | Validated assays for the quantification of C9orf72 human pathology |
| title_fullStr | Validated assays for the quantification of C9orf72 human pathology |
| title_full_unstemmed | Validated assays for the quantification of C9orf72 human pathology |
| title_short | Validated assays for the quantification of C9orf72 human pathology |
| title_sort | validated assays for the quantification of c9orf72 human pathology |
| url | https://doi.org/10.1038/s41598-023-50667-3 |
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