From Genetic Testing to Functional Validation: How In Vivo Models Clarify Variant Pathogenicity in Precision Medicine

Precision medicine has dramatically improved our ability to identify genetic variants associated with disease. However, identifying a variant is not the same as understanding its clinical relevance. As sequencing technologies become more accessible, clinicians and researchers face a fundamental question: Is this genetic variant pathogenic? 

Bridging the gap between genetic detection and biological interpretation requires functional evidence capable of linking genotype to phenotype: Functional genetics.

From Genetic Testing to Genetic Diagnosis: Where Molecular Detection Falls Short

Genetic testing for disease, such as Mendelian and rare disorders, often falls short of providing a definitive molecular diagnosis, with diagnostic rates of 30-50%, despite advances such as exome sequencing (ES), according to 2023 data (Wojcik et al. 2023).

Limitations include technical challenges in detecting certain variant types, such as structural variants, intronic changes, and repeat expansions, as well as an incomplete understanding of variant pathogenicity and genotype-phenotype correlations. Genome sequencing (GS), especially long-read sequencing, improves the detection of variants missed by ES by covering difficult genomic regions and identifying complex rearrangements.

Emerging multi-omics approaches integrating transcriptomic, epigenomic, proteomic, and metabolomic data show promise for better variant interpretation but are not yet widely implemented clinically. Challenges also arise from the complexity of test selection, interpretation by non-genetics clinicians, and the need for ongoing reanalysis and data sharing to resolve unsolved cases.

The Variant of Unknown Significance (VUS) Challenge in Precision Medicine

Variants of uncertain significance (VUS) are common in genetic disease diagnosis. VUS are genetic alterations identified through sequencing that cannot be classified as benign or pathogenic due to insufficient evidence. This complicates clinical care, delaying or misdirecting treatment decisions. It also creates uncertainty and vulnerability among patients, families, and healthcare professionals, causing psychological distress. 

Efforts to find the significance of  VUS include gathering more clinical and experimental evidence, family-based segregation studies, improved computational prediction models using protein-specific data, and periodic reanalysis as new information becomes available. Despite advances, many VUS remain unresolved due to the vast diversity of rare variants and limitations in current knowledge, making them an ongoing challenge in precision medicine. 

Functional Genomics Strategies to Establish Variant Pathogenicity

Functional genomics strategies are essential for establishing the pathogenicity of genetic variants, especially VUS, providing scientifically-based information for clinical intervention. Not to mention the empowerment of patients and caregivers.   

CRISPR/Cas9-based knock-in cellular models allow precise introduction of specific variants to assess their biochemical and cellular effects, providing reliable functional evidence to distinguish pathogenic from benign variants in inherited disorders. Functional genomics platforms using human induced pluripotent stem cells (iPSCs) combined with transcriptomic and physiological assays have been developed to study disease-relevant cell types, such as cardiomyocytes, revealing variant-specific functional consequences that inform reclassification of VUS (Pettinato et al. 2020). 

Nevertheless, cellular models do not provide a basis to replicate the complex interactions of a living organism. Even sophisticated cellular models cannot fully capture systemic factors such as immune responses, metabolism, and inter-organ communication that modulate the impact of VUS in whole organisms. Zebrafish go one step beyond. 

Zebrafish as an In Vivo Model for Gene Variant Testing

Zebrafish represent an in vivo model to establish the pathogenicity of genetic variants due to their genetic similarity to humans, rapid development, transparency, and suitability for large-scale functional studies. They enable precise modeling of human disease variants using genome editing tools like CRISPR/Cas9, allowing to observe phenotypic consequences such as developmental defects, organ-specific abnormalities, or physiological changes that reflect variant pathogenicity in a living organism. 

Zebrafish are less expensive than mammalian models to breed and sustain, and show earlier signs of physiological alterations, which enables clinical decisions to be made within a time frame congruent with pathophysiology. Their use to guide clinical decisions is becoming increasingly established. 

Some examples of their use to establish variant pathogenicity include: 

• Sheppard et al. used a zebrafish model to evaluate a potential heritable thoracic aortic disease in a 55-year-old patient. They replicated a VUS in SMAD3 by injecting mRNA into a transgenic zebrafish line with labelled endothelial cells and demonstrated that the variable was pathogenic. This information modified the patient’s treatment, which would not have been considered without the information regarding the pathogenicity of the variant based on the zebrafish study. 

• Stringer et al. used a zebrafish spinal muscular atrophy (SMA) model to functionally evaluate two SMN1 VUS identified in newborn infants detected through screening programs. They injected mRNA encoding each patient variant into SMN-deficient zebrafish embryos and showed that both variants rescued morphology, motor function, and survival defects, demonstrating that the variants were non-pathogenic (Figure 1). This functional evidence-informed clinical decision-making led physicians to withhold early treatment, avoiding unnecessary intervention and saving more than $2 million per patient while both children remained asymptomatic during follow-up. 

Figure 1. (A) Schematic timeline of infant development highlighting the clinical/timeframe dilemma for detection and confirmation of SMN1-VUS pathogenicity for early-onset SMA (Type I/II). (B) Zebrafish functional/complementation assays can quickly provide valuable information on VUS-pathogenicity in less than 3 months, supporting clinicians in their decision process and in a clinically helpful timeframe. This rapid testing framework is applicable not only to spinal muscular atrophy (SMA) but also to a wide range of pediatric diseases, offering significant benefits in clinical practice. Source: Stringer BW et al. Clinical relevance of zebrafish for gene variants testing. Proof-of-principle with SMN1/SMA. EMBO Mol Med. 2026 Jan;18(1):41-54.

• Several studies summarized by Laranjeira et al. used zebrafish morpholino knockdown and mRNA rescue experiments to functionally evaluate VUS identified in patients with mitochondrial diseases. Researchers reproduced loss-of-function phenotypes in zebrafish embryos and then co-injected mRNA carrying either the wild-type or patient variant. While wild-type mRNA restored normal development, mRNA containing the patient variants frequently failed to rescue the phenotype, demonstrating pathogenicity. These functional results enabled reclassification of previously uncertain variants and supported clinical interpretation by providing experimental evidence beyond genomic prediction alone. 

These examples clearly highlight how zebrafish provide a versatile platform for functional genomics that bridges the gap between genetic variant discovery and clinical interpretation by enabling direct assessment of variant effects in a whole-organism context. 

Turning this approach into reliable experimental evidence requires robust genome editing capabilities and standardized workflows for generating and validating genetic models. At ZeClinics, our advanced zebrafish genome engineering technologies enable precise genetic modification to reproduce human variants and study their biological consequences in vivo. We are CRISPR/Cas9 gene editing experts, officially licensed under the Broad Institute of MIT/Harvard. Importantly, we can couple the generation of precise genetic perturbations with advanced phenotypic capabilities. We operate innovative image acquisition equipment to allow zebrafish imaging in an HTS manner, and we have developed several image analysis tools, often powered with deep learning algorithms, to accurately quantify changes in morphology, cardiac function, or behaviour, to name some potential phenotypic readouts.   

If you are exploringthe generation of genetic models to study the role of genes in the context of disease or understand disease mechanisms in vivo, let’s talk. 

Source

Burke W, Parens E, Chung WK, Berger SM, Appelbaum PS. The Challenge of Genetic Variants of Uncertain Clinical Significance: A Narrative Review. Ann Intern Med. 2022 Jul;175(7):994-1000. doi: 10.7326/M21-4109.

Laranjeira M, Oliveira JMA, Santorelli FM, Marchese M, Nogueira C. Morpholino Knockdown in Zebrafish: A Tool to Investigate the Functional Impact of Variants of Unknown Significance in Mitochondrial Diseases. Neuromolecular Med. 2025 Oct 11;27(1):69. doi: 10.1007/s12017-025-08890-w.

Pettinato AM, Ladha FA, Mellert DJ, Legere N, Cohn R, Romano R, Thakar K, Chen YS, Hinson JT. Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants. Circulation. 2020 Dec 8;142(23):2262-2275. doi: 10.1161/CIRCULATIONAHA.120.047999. 

Prendergast A, Sheppard MB, Famulski JK, Nicoli S, Mukherjee S, Sips P, Elefteriades JA. Modeling thoracic aortic genetic variants in the zebrafish: useful for predicting clinical pathogenicity? Front Cardiovasc Med. 2025 Feb 19;12:1480407. doi: 10.3389/fcvm.2025.1480407.

Sheppard MB, Smith JD, Bergmann LL, Famulski JK. Novel SMAD3 variant identified in a patient with familial aortopathy modeled using a zebrafish embryo assay. Front Cardiovasc Med. 2023 Feb 28;10:1103784. doi: 10.3389/fcvm.2023.1103784.

Stringer BW, Zhang Y, Taghipour-Sheshdeh A, Goh S, Kölbel H, Farrar MA, Wirth B, Giacomotto J. Clinical relevance of zebrafish for gene variants testing. Proof-of-principle with SMN1/SMA. EMBO Mol Med. 2026 Jan;18(1):41-54. doi: 10.1038/s44321-025-00355-8. 

Wojcik MH, Reuter CM, Marwaha S, Mahmoud M, Duyzend MH, Barseghyan H, Yuan B, Boone PM, Groopman EE, Délot EC, Jain D, Sanchis-Juan A; Genomics Research to Elucidate the Genetics of Rare Diseases (GREGoR) Consortium; Starita LM, Talkowski M, Montgomery SB, Bamshad MJ, Chong JX, Wheeler MT, Berger SI, O'Donnell-Luria A, Sedlazeck FJ, Miller DE. Beyond the exome: What's next in diagnostic testing for Mendelian conditions. Am J Hum Genet. 2023 Aug 3;110(8):1229-1248. doi: 10.1016/j.ajhg.2023.06.009.

By Javier Terriente

Javier is the co-founder of ZeClinics and ZeCardio Therapeutics, two biotech firms specializing in zebrafish-based preclinical drug discovery for cardiovascular, neural, and toxicology applications. He combines scientific leadership with business acumen, having successfully driven fundraising efforts and strategic partnerships.

Currently leading scientific efforts at ZeCardioTx (and formerly CSO at ZeClinics), Javier also serves on the Board of Directors of AseBio, where he advocates for industry collaboration. His academic background includes a PhD in Molecular Biology and a Marie Curie Fellowship. Recognized as an expert in zebrafish models, he has published extensively and has supervised five industrial PhD theses.