Comprehending Neurodegeneration: How a Routine Genetic Analysis Tool is Helping Researchers Probe Neurodegenerative Conditions

Abstract

To treat neurodegenerative diseases such as Parkinson’s and Alzheimer’s, we need a better understanding of how they progress. But unfortunately, the mysteries behind many of these debilitating conditions continue to elude us. In this post, we’ll look at how researchers are using a genetic analysis technique staple to most research labs – fluorescent capillary electrophoresis (CE) – to make progress in the field.

Neurodegenerative diseases will be a significant health challenge in the coming decades. According to the World Health Organization, one in three people will develop a neurological disorder (which includes neurodegenerative diseases) at some point in their life, making them the leading cause of disability and the second leading cause of death (1). In the United States, 5 million people suffer from Alzheimer’s disease (AD), 1 million from Parkinson’s disease (PD), 400,000 from multiple sclerosis (MS), 30,000 from Huntington’s disease (HD), and 30,000 from amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) (2).

Among genomic analysis methods to study neurodegeneration, fluorescent capillary electrophoresis (CE), an integral part of Sanger sequencing and fragment analysis workflows, stands out in its simplicity and high sensitivity. Single-base resolution can be achieved with small sample volumes using rapid sample-to-analysis workflows. Here we look at some examples where researchers leveraged this technology to shed light on the mechanisms underlying neurodegeneration.

Sanger sequencing of IPSCs derived from a PD patient to create a human brain organoid model

Parkinson’s disease is the second most prevalent neurodegenerative disorder, affecting over 8.5 million people worldwide (3). PD patients’ brains contain pathological inclusions called Lewy bodies, which predominantly consist of the α-synuclein protein encoded by SNCA (4). However, most studies probing α-synuclein pathology rely on 2D cell culture models, which often fail to recapitulate the complexity of the disease. To address this issue, Mohamed et al. created 3D midbrain organoids from human induced pluripotent stem cells derived from a patient with an SNCA gene triplication and genome-edited isogenic control cells (5).

To authenticate the iPSC lines, the researchers performed Sanger sequencing of SNCA regions using the Applied Biosystems SeqStudio Genetic Analyzer (5). Sanger sequencing remains a popular method for obtaining DNA sequence information, particularly for cost-effective sequencing of single genes/samples, verifying mutations and cloned inserts, and analyzing long fragments. Watch the step-by-step video to see how easy it is to set it up in your lab.

Accurate sizing of CAG repeats in Huntington’s disease

Although less prevalent than PD, Huntington’s disease is just as debilitating. Mostly inherited, it is caused by an expanded CAG repeat in the huntingtin (HTT) gene, translating into polyglutamine repeats. CAG-repeat length in HTT is inversely correlated with the age of onset of the disease. In addition, genetic alterations in other parts of the genome can also affect the age of onset. Goold et al. showed how one such genetic modifier – FAN1, a nuclease involved in DNA interstrand cross-link repair – alters the progression of HD. They showed that higher expression of the modifier was associated with delayed onset and slower disease progression (6).

To accurately size the CAG repeats in the various cell lines developed by the research team, they performed PCR-based fragment analysis using the ABI 3730 Genetic Analyzer and the GeneMapper software. Figure 2 shows a representative trace. The tallest peak (marked in red), representing the most common value while accounting for normal variations, is taken as the CAG repeat size for a given sample (6).

Accurate detection of allele lengths in Alzheimer’s disease-associated gene polymorphism

Alzheimer’s disease is the most common type of dementia in the elderly. Although we don’t yet understand all the mechanisms underlying the disease and have disease-modifying therapies, we have made notable progress in the past years. For example, the latest phase 3 clinical trial results using the monoclonal antibody lecanemab to reduce amyloid levels and, thereby, slow disease progression is being hailed as a potential breakthrough (7).

The apolipoprotein E-4 (APOE-E4) allele is a strong risk factor for AD. Furthermore, genome-wide association studies show that several other genetic polymorphisms around the locus also strongly correlate with the disease (8). An example is the polymorphism of the TOMM40 gene – poly-T 523 polymorphism– located adjacent to APOE, which was shown to be associated with the age of onset of AD. However, there are conflicting reports in the literature regarding this association. The authors, Linnertz et al., leveraged capillary electrophoresis using the ABI 3730 DNA Analyzer and GeneMapper software to develop a standardized 523 genotyping assay for accurately detecting allele lengths (9).

Learn more about how Applied Biosystems solutions can help you gain deeper insights into neurodegenerative conditions at thermofisher.com/abcomplexdisease.

References

1. WHO: https://www.who.int/news/item/09-08-2022-launch-of-first-who-position-paper-on-optimizing-brain-health-across-life  Accessed November 30, 2022.
2. Harvard NeuroDiscovery Center: https://neurodiscovery.harvard.edu/challenge Accessed November 30, 2022.
3. WHO: https://www.who.int/news-room/fact-sheets/detail/parkinson-disease Accessed November 30, 2022.
4. Lee A, Gilbert RM. Epidemiology of Parkinson Disease. Neurol Clin. 2016 Nov;34(4):955-965. doi: 10.1016/j.ncl.2016.06.012. Epub 2016 Aug 18.
5. Mohamed NV, Sirois J, Ramamurthy J, et al. Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy. Brain Commun. 2021;3(4):fcab223. Published 2021 Sep 25. doi:10.1093/braincomms/fcab223
6. Goold R, Flower M, Moss DH, Medway C, Wood-Kaczmar A, Andre R, Farshim P, Bates GP, Holmans P, Jones L, Tabrizi SJ. FAN1 modifies Huntington’s disease progression by stabilizing the expanded HTT CAG repeat. Hum Mol Genet. 2019 Feb 15;28(4):650-661. doi: 10.1093/hmg/ddy375.
7. BIOGEN Press Release: https://investors.biogen.com/news-releases/news-release-details/eisai-presents-full-results-lecanemab-phase-3-confirmatory
8. Guerreiro RJ, Hardy J. TOMM40 association with Alzheimer disease: tales of APOE and linkage disequilibrium. Arch Neurol. 2012 Oct;69(10):1243-4. doi: 10.1001/archneurol.2012.1935.
9. Linnertz C, Saunders AM, Lutz MW, Crenshaw DM, Grossman I, Burns DK, Whitfield KE, Hauser MA, McCarthy JJ, Ulmer M, Allingham R, Welsh-Bohmer KA, Roses AD, Chiba-Falek O. Characterization of the poly-T variant in the TOMM40 gene in diverse populations. PLoS One. 2012;7(2):e30994. doi: 10.1371/journal.pone.0030994. Epub 2012 Feb 16.