Abstract
There is increasing evidence for the role of mosaicism due to somatic mutations, including copy number variants (CNVs), in disorders of the nervous system. Targeted assessment of low levels of somatic CNVs can be performed using fluorescent in situ hybridization (FISH). Combining this technique with immunofluorescence in frozen brain sections allows the determination of whether somatic CNVs occur in cells with specific disease and cell-type markers. We present a protocol focused on somatic SNCA CNVs and aggregates of the encoded alpha-synuclein protein. This is particularly relevant to Parkinson’s disease and other synucleinopathies, but could be adapted as required.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
D’Gama AM, Walsh CA (2018) Somatic mosaicism and neurodevelopmental disease. Nat Neurosci 21:1504–1514
McConnell MJ et al (2017) Intersection of diverse neuronal genomes and neuropsychiatric disease: the brain somatic mosaicism network. Science 356:eaal1641
Leija-Salazar M, Piette C, Proukakis C (2018) Review: somatic mutations in neurodegeneration. Neuropathol Appl Neurobiol 44:267–285
Lodato MA, Walsh CA (2019) Genome aging: somatic mutation in the brain links age-related decline with disease and nominates pathogenic mechanisms. Hum Mol Genet 28:R197–R206
Proukakis C (2020) Somatic mutations in neurodegeneration: an update. Neurobiol Dis 144:105021
Rohrback S et al (2018) Genomic mosaicism in the developing and adult brain. Dev Neurobiol 78:1026–1048
Yuan G-C et al (2017) Challenges and emerging directions in single-cell analysis. Genome Biol 18:84
Chronister WD, McConnell MJ (2019) Neurons with complex karyotypes are rare in aged human neocortex. Cell Rep 26:825–835.e7
Rehen SK et al (2001) Chromosomal variations in neurons of the developing and adult mammalian nervous system. Proc Natl Acad Sci U S A 98:13361–13366
Book A et al (2018) A meta-analysis of α-synuclein multiplication in familiar parkinsonism. Front Neurol 11:1021
Proukakis C, Houlden H, Schapira AH (2013) Somatic alpha-synuclein mutations in Parkinson’s disease: hypothesis and preliminary data. Mov Disord 28:705–712
Mokretar K et al (2018) Somatic copy number of gains of a-synuclein (SNCA) in Parkinson’s disease and multiple system atrophy brains. Brain 141:2419–2431
Perez-Rodriguez D et al (2019) Investigation of somatic CNVs in brains of synucleinopathy cases using targeted SNCA analysis and single-cell sequencing. Acta Neuropathol Commun 7:219
Andriani AG, Castagna C (2017) FISH analysis of aging-associated aneuploidy in neurons and nonneuronal brain cells. In: Frade JM, Gage FH (eds) Genomic mosaicism in neurons and other cell types. Neuromethods, vol 131. Humana Press, New York, pp 271–298
Liehr T et al (2017) The standard FISH procedure. In: Lieher T (ed) Fluorescence in situ hybridization (FISH). Springer protocols handbooks. Springer, Berlin, Heidelberg, pp 109–118
Lewis RJ (2004) Sax’s dangerous properties of industrial material. Wiley, Hoboken
O’Connor SJM et al (2020) Practical application of fluorescent in situ hybridization techniques in clinical diagnostic laboratories. In: Nielsen BS, Jones J (eds) in situ hybridization protocols. Methods in molecular biology 2148. Humana Press, New York, pp 35–70
Jolly S et al (2019) Single-cell quantification of mRNA expression in the human brain. Sci Rep 9:12353
Chen WT et al (2020) Spatial transcriptomics and in situ sequencing to study Alzheimer’s disease. Cell 182:976–991
Hanna MG, Patanowitz L (2017) Why is digital pathology lagging behind surgical pathology. Cancer Cytopathol 125:519–520
Maynard KR et al (2020) Dotdotdot: an automated approach to quantify multiplex single fluorescent in situ hybridization (smFISH) images in complex tissues. Nucleic Acid Res 48:e66
Bankhead P et al (2017) QuPath: open source software for digital pathology image analysis. Sci Rep 7:16878
Acknowledgments
Dr. Proukakis is grateful to the Michael J. Fox Foundation for Parkinson’s Research and the Multiple System Atrophy Trust for funding which allowed the development and application of this protocol. Tissue samples were provided by the Queen Square Brain Bank, which is supported by the Reta Lila Weston Institute for Neurological Studies and the Medical Research Council UK, and the Parkinson’s UK Tissue Bank, which is funded by Parkinson’s UK, a charity registered in England and Wales (258197) and in Scotland (SC037554).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Garcia-Segura, M.E., Perez-Rodriguez, D., Proukakis, C. (2022). Combined Fluorescent In Situ Hybridization (FISH) and Immunofluorescence for the Targeted Detection of Somatic Copy Number Variants in Synucleinopathies. In: Proukakis, C. (eds) Genomic Structural Variants in Nervous System Disorders. Neuromethods, vol 182. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2357-2_12
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2357-2_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2356-5
Online ISBN: 978-1-0716-2357-2
eBook Packages: Springer Protocols