Variants of uncertain significance in neurodegenerative disease (II)
This is the second article in the series of “Variants of uncertain significance in neurodegenerative disease (II)”. This series mainly concentrates on the content of one review article, “ Emerging genetic complexity and rare genetic variants in neurodegenerative brain diseases” (1).
The first article explained briefly what variants of uncertain significance (VUS) are, as well as the current understanding of pathophysiology and genetic epidemiology of the dour diseases of focus: Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this second article, we will explore more about VUS modes of action in these four diseases, VUS application in medical research, and possible solution for better utilization of current underused VUS.
VUS: modes of action and their influence on phenotypes
Numerous rare VUSs have been identified with known and unknown mechanisms. It is proposed in the article that rare VUS can contribute to disease pathology with high penetrance, as shown in Fig 1. Moreover, interpretation of pathogenic mutations is always not straightforward regarding disease onset, severity and gene penetrance. VUS may work as a genetic modifier, influencing these phenotypes. As noted in the article, “Carriers of the same pathogenic mutation often show a wide range of variation in disease onset age and in clinical phenotype”. Finally, findings of different mutations within the same biological pathways have led to the concept of oligogenic inheritance.
In the following two sections, Table 2 and Table 3 summarizes two sections in the review article, “Possible modes of action of rare variants in known genes: the penetrance spectrum” and “Variable expression: age-related reduced penetrance and genetic modifiers”, respectively.
Novel gene discovery without adequate functional study would not lead to robust understanding of the gene. For example, in the ATP-binding cassette sub-family A member 7 gene (ABCA7), both wild type and rare variants are reported to affect AD risk. Yet due to the lack of functional analysis, whether this gene is high-penetrant is still debatable. Hence, in the following section, methods to understand functions of genes will be introduced.
Enhancing the understanding and utilization of VUS: biotechnology and VUS application
Induced pluripotent stem cells (iPSCs), also known as “clinical trial in a dish”, have shown promise in mimicking phenotypes and pathologies (2). iPSCs are one branch in human pluripotent stem cells (hPSCs), which can self-renew in vitro indefinitely and also maintain the ability to differentiate into any cell types of human bodies (3). Genome editing technologies (e.g., CRISPR/Cas) has been applied with studies of iPSCs (4). In a study researching PD pathophysiology (5), two iPSC lines were generated from two patients: one was homozygous LRRK2 p.G2019S mutation, the other is SNCA triplication. The findings from the study showed that these iPSC-derived DA neurons exhibited early phenotypes linked to PD. The cell line of LRRK2 p.G2019S mutation demonstrated disrupted mechanisms, such as aggregation of α-synuclein, mitochondrial transport, and lysosomal autophagy. The study serves as a good model that iPSCs can be used as potential models for disease diagnostics. iPSCs as a disease model are particularly valuable in NBD, since the pathophysiology is always too complicated for animal models to fully recapitulate. A list of publications of iPSCs for disease modelling is available in one review paper (6). Another application of iPSCs is to serve for target identification. For example, a library that included 1258 pharmaceutical compounds was applied to iPSC-derived AD neurons, and Aβ42/40 ratio was used as output. After screening the compounds, 27 potential compounds were left, and finally, the combination of three drugs (bromocriptine, cromolyn and topiramate) were identified capable of reducing Aβ42/40 ratio (7).
From 2D to 3D, we are able to capture more complicated disease mechanisms. Three-dimensional (3D) brain organoids derived from human PSCs (hPSCs) and iPSCs can recapitulate the brain’s 3D arrangements, provide new opportunities to explore disease pathogenesis and serve as a bridge of the translational gap between animal models and clinical trials. Patient-derived brain organoids have already been used to study insights into molecular and genetic mechanisms in several brain diseases, including AD (8). Integration of genome editing technologies, hPSCs and 3D brain organoids is promising to lead to novel findings regarding molecular pathogenesis and therapeutic candidates of diseases, especially NBD. In a study (9), familial AD patient-derived 3D brain organoids were derived from early-onset AD patients carrying an APP duplication. Reduced amyloid and tau aggregates was found with the treatment of β- and γ-secretase inhibitors.
In summary, VUS can be studied in iPSC and 3D brain organoids technology, as well as be utilized as a drug target for lead discovery. Moreover, the utilization of omics tool is important in understanding the protein structure change derived from related mutations.
Clinical applications of VUS are utmost important in NBD treatment. Despite improvement in the precision of genetic testing, understanding VUS enables researchers to design more homogeneous patient groups, reducing confounding factors of drug efficacy in clinical trials and medical practice. This is crucial since the current therapeutic choices for AD, PD, ALS and FTD are deemed to exert variable responses and side effects. And some side effects greatly diminish patients’ life quality. For example, a high proportion of individuals with PD treated with dopamine-replacement therapy develop abnormal involuntary movements, L-DOPA-induced dyskinesia (LID) (10). Recently, Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU), an adaptive platform trial testing multiple drugs to slow or prevent the progression of Alzheimer’s disease in autosomal dominant Alzheimer’s disease (ADAD) families, designed and executed a pioneering prevention trial in AD in 2013. This trial is still recruiting and available on ClinicalTrials.gov (https://www.clinicaltrials.gov/ct2/show/NCT01760005?cond=DIAN-TU+Trial&draw=2&rank=1). In brief, mutation positive and negative subjects are recruited, and further divided into two study arms, with treatment drugs of gantenerumab or solanezumab, respectively. Primary outcome of the study is to compare cognitive efficacy between mutation-positive and mutation-negative individuals in two study arms. From bench to bedside, understanding VUS gives more delicate and holistic patient care, by enabling understanding of the fundamental individual difference.
What else can be done: take oncology as a good model as VUS research
VUS, as its name implies, means uncertain significance. Since these variants are so less, that international collaborative work must be done to accelerate the research. In 2013, the Clinical Genome Resource (ClinGen) project was launched to create a global central resource that defines the clinical validity, the pathogenicity and the clinical usefulness of the genomic information. Two databases were then established by this project, ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and ClinVar Miner (https://clinvarminer.genetics.utah.edu/). These databases serve as international archive of variant-condition interpretations hosted by the National Center for Biotechnology Information (NCBI).
Oncology is one of the most active fields in researching genetics. VUS classification in oncology has gained public attention (11). Breast cancer Information Core (BIC), an open access, online mutation database for breast cancer susceptibility genes, was established in 1995, in order to achieve standardized classification of breast cancer genes. Given the growing number of BRCA1 and BRCA2 VUS, Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) international consortium was established in 2009. ENIGMA is an international initiative to evaluate risk and clinical significance of variants in BRCA1 and BRCA2 genes (12). They provide criteria for assessing variant significance and promote data sharing of large-scale projects with variant annotations.
In fact, in the field of NBD, some international databases exist. Alzheimer Disease and Frontotemporal Dementia Mutation Database was founded in 2018, aiming at collecting a comprehensive list of rare variants related to AD and FTD, including APP, PSEN1, PSEN2, TREM2 and MAPT. For PD, Parkinson Disease Mutation Database is also online for use. Nevertheless, one thing can be learned from oncology community is the establishment of working groups such as IARC Unclassified Genetic Variants Working Group. Working groups give opportunities for experts to communicate and together, systematically tackle with problems, such as harmonized standards of classification of VUS.
Conclusion
In the review article, several opportunities as well as challenges have been proposed. Despite still unknown variants and genes, the international collaboration will be needed to establish standards for VUS in NBD, and also to collect VUS from every corner of the world, to make the uncertain, certain. Furthermore, WGS is predicted to be the standard diagnostic tool in medical genetic testing within 5 years. It is expected that experts will encounter a lot more VUS by that time. Data of VUS combined with functional analysis, such as omics tool, iPSCs and 3D brain organoids will lead to better understanding of disease mechanism, therapeutics development and personalized care.
Reference
1. Perrone F, Cacace R, van der Zee J, Van Broeckhoven C. Emerging genetic complexity and rare genetic variants in neurodegenerative brain diseases. Genome Med. 2021;13(1):59.
2. Blinova K, Schocken D, Patel D, Daluwatte C, Vicente J, Wu JC, et al. Clinical Trial in a Dish: Personalized Stem Cell-Derived Cardiomyocyte Assay Compared With Clinical Trial Results for Two QT-Prolonging Drugs. (1752–8062 (Electronic)).
3. Zhu Z, Huangfu D. Human pluripotent stem cells: an emerging model in developmental biology. Development. 2013;140(4):705–17.
4. Adli M. The CRISPR tool kit for genome editing and beyond. Nature Communications. 2018;9(1):1911.
5. Byers B, Lee Hl Fau — Reijo Pera R, Reijo Pera R. Modeling Parkinson’s disease using induced pluripotent stem cells. (1534–6293 (Electronic)).
6. Chang CY, Ting HC, Liu CA, Su HL, Chiou TW, Lin SZ, et al. Induced Pluripotent Stem Cell (iPSC)-Based Neurodegenerative Disease Models for Phenotype Recapitulation and Drug Screening. Molecules. 2020;25(8).
7. Kondo T, Imamura K, Funayama M, Tsukita K, Miyake M, Ohta A, et al. iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid β Combination for Alzheimer’s Disease. (2211–1247 (Electronic)).
8. Lee CT, Bendriem RM, Wu WW, Shen RF. 3D brain Organoids derived from pluripotent stem cells: promising experimental models for brain development and neurodegenerative disorders. (1423–0127 (Electronic)).
9. Raja WK, Mungenast AE, Lin YT, Ko T, Abdurrob F, Seo J, et al. Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer’s Disease Phenotypes. (1932–6203 (Electronic)).
10. Johnston TH, Lacoste AMB, Visanji NP, Lang AE, Fox SH, Brotchie JM. Repurposing drugs to treat l-DOPA-induced dyskinesia in Parkinson’s disease. Neuropharmacology. 2019;147:11–27.
11. Federici G, Soddu S. Variants of uncertain significance in the era of high-throughput genome sequencing: a lesson from breast and ovary cancers. J Exp Clin Cancer Res. 2020;39(1):46.
12. Spurdle AB, Healey S Fau — Devereau A, Devereau A Fau — Hogervorst FBL, Hogervorst Fb Fau — Monteiro ANA, Monteiro An Fau — Nathanson KL, Nathanson Kl Fau — Radice P, et al. ENIGMA — evidence-based network for the interpretation of germline mutant alleles: an international initiative to evaluate risk and clinical significance associated with sequence variation in BRCA1 and BRCA2 genes. (1098–1004 (Electronic)).
