Abstract
Cardiovascular diseases are leading global causes of death and disability, often presenting as interrelated phenotypes of atherosclerotic vascular disease, heart failure and arrhythmias. Cardiovascular diseases arise from interactions between environmental factors and predisposing genotypes and include common Mendelian lipid disorders, cardiomyopathies and arrhythmia syndromes. The identification of a pathogenic variant through genetic testing can inform disease diagnosis, risk prediction, treatment and family screening. However, a major roadblock in genomic medicine is that for many variants, especially missense variants, we lack sufficient evidence to enable a definitive classification, and therefore these variants are deemed as ‘variants of uncertain significance’. In this Review, we describe how multiplexed assays of variant effects can enable the functional assessment of nearly all coding variants in a target sequence, potentially offering a proactive approach to identifying the functional significance of gene variants that are observed later in a patient. We discuss validation, including the role of in silico variant effect predictors, and how multiplexed experimental methods are informing cardiovascular disease biology and ultimately resolving the problem of variants of uncertain significance at scale.
Key points
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Although genetic testing is increasingly used in the clinical management of inherited cardiovascular disorders, most of the identified variants are classified as ‘variants of uncertain significance’.
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Multiplexed assays of variant effects can be used to assess the function of nearly all coding variants in a target sequence, providing proactive evidence for variants observed in patients.
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In silico variant effect predictors are becoming increasingly accurate and can provide predicted variant effects for nearly every variant in the genome.
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Multiplexed assays of variant effects and variant effect predictors provide complementary data to inform cardiovascular disease biology and to resolve the problem of variant classification as variants of uncertain significance.
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Data availability
The data used for the analyses included in this manuscript are from the All of Us Research Program’s Controlled Tier Dataset version 8, available to authorized users on the Researcher Workbench via https://www.workbench.researchallofus.org. The UK Biobank data set is available by application via https://www.ukbiobank.ac.uk/enable-your-research/apply-for-access.
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Acknowledgements
This work was supported by NIH grants HL149826, HL164675, HL168059, GM150465, HG010904, HL160863, HG010461 and HG011989. The authors gratefully acknowledge UK Biobank and All of Us participants for their contributions, without whom this research would not have been possible. The authors also thank the UK BioBank and the NIH All of Us Research Program for making available the participant data examined in this study.
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A.M.G., D.R.T., V.N.P., B.M.K., A.G.C., C.A.M., E.A.A., F.P.R. and D.M.R. wrote the article. All authors researched data for the article, contributed substantially to discussion of the content and reviewed and/or edited the manuscript before submission.
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A.M.G. is a consultant for BioMarin. V.N.P. is a consultant and Scientific Advisory Board member for Lexeo Therapeutics, clinical adviser for Constantiam Biosciences, consultant and conducts sponsored research for BioMarin and minor consultant for Nuevocor and Borealis. C.A.M. is founder of Atman Health and Tanaist Bio; adviser for Adrestia, Affinia, Alliance of Genomic Resources, Astellas, BioSymetrics, Dewpoint, Foresite Labs, EOM and Nuevocor; non-executive director of Life MD, TMA Precision Medicine and Tanaist Bio; owns stock from BioSymetrics, Bodyport, Life MD and TMA Precision Medicine; and has provided in-kind collaborative support to Astra Zeneca, Quest Diagnostics and Apple. E.A.A. is a founder of Candela, Deepcell, Parameter Health, Personalis, Saturnus Bio and Svexa; is an adviser for SequenceBio, Foresite Labs, Pacific Biosciences and Versant Ventures; is a non-executive director of AstraZeneca and Svexa; owns stock from Pacific Biosciences and AstraZeneca; and has provided in-kind collaborative support to Cache, Cellsonics, Illumina, Oxford Nanopore and Pacific Biosciences. F.P.R. is an adviser for and shareholder of Constantiam Biosciences. The other authors declare no competing interests.
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Peer review information
Nature Reviews Cardiology thanks Jodie Ingles, Roddy Walsh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Informed consent and ethics approval
The authors affirm that human research participants provided informed consent for use of their data via the UK Biobank and All of Us projects, and data sets were analysed in accordance with the associated data use agreements. The transfer of human data was approved and overseen by the UK Biobank Ethics Advisory Committee. The analyses in this manuscript were performed in alignment with the ethical principles outlined in the All of Us Policy on the Ethical Conduct of Research.
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Related links
All of Us: https://allofus.nih.gov/
ClinGen: https://clinicalgenome.org/
ClinVar: https://www.ncbi.nlm.nih.gov/clinvar/
gnomAD: https://gnomad.broadinstitute.org/
MaveDB: https://www.mavedb.org/
UK Biobank: https://www.ukbiobank.ac.uk/
Glossary
- ClinGen
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An NIH-supported effort that curates the relationships between genetic variants and disease susceptibility.
- ClinVar
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A public NIH database of genomic variants and their clinical interpretations.
- Deep sequencing
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DNA sequencing at sufficient depth to enable detection of rare variants and quantification of variant frequency.
- Genome Aggregation Database
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(gnomAD). A publicly available database that, in 2025, listed sequence variants in 730,947 exome sequences and 76,215 whole-genome sequences in ancestrally diverse populations.
- Intrinsically disordered regions
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Protein segments that lack a fixed 3D structure and often mediate flexible interactions between protein domains.
- Landing pad
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A genomic site engineered to allow consistent integration and expression of DNA constructs.
- Massively parallel reporter assays
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(MPRAs). Method to test the regulatory activity of thousands of DNA sequences simultaneously using barcoded reporters.
- Mendelian cardiovascular conditions
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Inherited disorders mainly caused by variants in a single gene that affect the heart, blood vessels or lipid metabolism; for example, familial hypercholesterolaemia, hypertrophic cardiomyopathy and long QT syndrome.
- Polygenic scores
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An estimate of disease risk based on the additive effect of many common genetic variants identified by genome-wide association studies of disease risk.
- Splice-disrupting variants
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Variants that alter RNA splicing, potentially affecting gene expression or protein structure.
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Cite this article
Glazer, A.M., Tabet, D.R., Parikh, V.N. et al. Creating an atlas of variant effects to resolve variants of uncertain significance and guide cardiovascular medicine. Nat Rev Cardiol (2025). https://doi.org/10.1038/s41569-025-01201-7
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DOI: https://doi.org/10.1038/s41569-025-01201-7
