The Undiagnosed Diseases Network brings together experts across the USA, combining clinical care and research to find diagnoses for undiagnosed patients. Zebrafish are extremely valuable disease models in this research, as they reduce the cost and speed up the process of identifying therapeutic targets. In this blog we highlight one rare disease whose mechanism was successfully identified using zebrafish.
Despite considerable advances in the biomedical sciences over the past few decades with respect to human disease diagnosis and treatment, many people still suffer from undiagnosed diseases (approximately 1 in 13 people in the US alone). Faced with such staggering statistics, the National Institutes of Health Common Fund established the Undiagnosed Diseases Network (UDN)5. Since its formation in 2008, the UDN has brought together medical scientists from across the United States to improve the rate of diagnosis and care of patients with undiagnosed diseases by conducting and sharing research2. You can learn more about the Undiagnosed Disease Network here.
Importance of Zebrafish studies in the UDN
Zebrafish (Danio rerio) are one of three model organisms the UDN uses to investigate rare diseases, along with the nematode Caenorhabditis elegans and fruit flies (Drosophila melanogaster) (figure 2). All three model organisms bring their own unique advantages for modeling human diseases, including ease of genetic manipulation, small size, high fecundity, and rapid development. Zebrafish in particular are ideal organisms for genetic manipulation because their transparency throughout early development and rapid external development allows for direct observation and manipulation of specific tissues and processes. Additionally, they can be housed at relatively high density compared to other vertebrate models, and possess a high degree of conservation of human disease-associated genes (~70-80%)6.
Zebrafish play a crucial role in the UDN network as genetic model organisms, complementing the other models used by serving as a more evolutionarily related model of human disease than C. elegans or fruit flies in many instances. For example, Zebrafish have an obvious advantage over C. elegans and fruit flies for modeling human diseases involving bone development, as zebrafish are vertebrates and thereby provide more physiologically relevant insights for human disease than their invertebrate model counterparts. This in turn can mean that the findings from zebrafish studies can be more easily translated to higher order vertebrate/mammalian models.
At InVivo Biosystems, we composed a fun, and informative Zebrafish CV for you to learn more about the model organism, and highlighting its importance for studying human diseases (figure 1). There are several successful ongoing research studies associated with zebrafish that are helping vastly in diagnosing and treating rare diseases, such as hereditary pediatric diseases6, Hirschsprung disease1, Diamond-Blackfan anemia1, and bone disorders, etc..
Advantages of using the zebrafish model system14 in rare diseases:
- Able to verify causality of candidate disease variants (for example: Hirschsprung disease).
- Provide better understanding of the rare disease pathophysiology (for example: Diamond-Blackfan anaemia).
- Capable of functional annotation of the disease associated genes in zebrafish.
- Capable of discovering novel mediated pathways in vertebrate bone formation (for example: bone disorders).
- Allows for large-scale phenotypic screening.
Figure 2: Model organisms such as worms, flies, zebrafish, etc. can be used to optimize the diagnostic measures, to study the disease mechanisms, and are used to identify potential therapeutic targets for the treatment of rare diseases. Image available at the original source: https://undiagnosed.hms.harvard.edu/research/model-organisms-phase-ii/.
Different zebrafish disease models1 can be generated using:
- Random mutagenesis
- Targeted gene-editing using CRISPR/cas9 technology
Specific rare disease case scenario using Zebrafish
One example of Zebrafish being used to identify the molecular basis of a disorder is the case of Saul – wilson syndrome (SWS). This syndrome is a form of primordial dwarfism, characterized by skeletal abnormalities, short stature, hearing loss, cataracts, and developmental delays, but normal cognition (figure 4)3.
Figure 4: Radiographs depicting skeletal features of individuals with Saul-Wilson syndrome showing the individuals with growth deformities. Image available at the original source: https://doi.org/10.1038/s41436-019-0737-14.
The syndrome was first defined in 1990, and since then there have only been 15 cases reported worldwide. Since this disorder is so rare, those suffering from SWS have spent their life searching for unanswered questions about the cause, and potential treatment, of their condition. Setting out to find a genetic basis, researchers scoured the genomes of 14 individuals with SWS, ultimately finding that they all shared a common variant in the COG4 gene (p.Gly516Arg). This variant was classified as a heterozygous de novo variant, because it was only found on one copy of the gene (heterozygous), and arose spontaneously (de novo).
To further characterize the functional consequences of this variant, the researchers utilized CRISPR/Cas9 gene editing to generate zebrafish cog4 mutants . The zebrafish mutants showed inner ear, growth, and skeletal defects3, mimicking many aspects of human SWS, and establishing that COG4 is necessary for normal skeletogenesis, and cartilage architecture (Figure 5). Taken together, these findings established a role for cog4 mutants in inner ear development and skeletogenic deformities, associated with defective Golgi structure and/or function.Learn more about CRISPR/cas9 gene editing
Figure 5: Morphogenesis and Function of the Inner Ear Are Abnormal in cog4-Deficient Zebrafish. (B) mutant larvae show altered morphology of semicircular canals, indicated by red asterisks. (C-F) Phalloidin staining of the second posterior neuromast in cog4 sibling (E) and cog4 mutant (F) larvae – mutants had reduced numbers of hair bundles & reduced response to auditory stimuli.
Many scientists were involved in this research, one of whom explained the significance of this finding on other conditions, saying that, “with the advent of genetic sequencing, we are learning of more conditions that are caused by de novo changes, including autism and epilepsy. Additionally, we are finding more disorders that are caused by heterozygous mutations.” And that, studying such cases, “could provide insights into additional mysterious conditions.”
This research shows how zebrafish can help make strides in the field of undiagnosed disorders; specifically, discovering that a heterozygous COG4 substitution serves as the molecular basis of Saul-Wilson syndrome, and more broadly, adding to our understanding of the roles of COG4 in protein production and stability which may prove valuable insights in other undiagnosed conditions.
In conclusion, zebrafish are a powerful model for studying disease mechanisms and identifying potential therapeutic targets, especially in the field of rare and undiagnosed diseases. These model organisms have successfully been used by the UDN to identify the mutations in UDN participants which contribute to or underly numerous rare diseases, and indeed the UDN continues to serve as an impressive model of what is possible when cross-species modeling and cross-institution collaborations are effectively leveraged in the pursuit of advancing our understanding of human health and disease.
Both zebrafish as models, and the UDN as an organization, are relatively new additions to the field of medical sciences, but they have already accelerated rare disease gene discovery, helping to learn more about rare and common diseases, and presenting a promising future for patients and their families.
- Adamson KI, Sheridan E, Grierson AJUse of zebrafish models to investigate rare human diseaseJournal of Medical Genetics 2018;55:641-649.
- Doss A. (last updated 2020, Feb 24), Undiagnosed Diseases Network NHGRI “Undiagnosed Diseases Network (UDN)”. Available at https://www.genome.gov/Funded-Programs-Projects/Undiagnosed-Diseases-Network#:~:text=In%202008%2C%20the%20NIH%20Undiagnosed,treatment%20for%20patients%20with%20unknown
- Ferreira, Carlos R et al. “A Recurrent De Novo Heterozygous COG4 Substitution Leads to Saul-Wilson Syndrome, Disrupted Vesicular Trafficking, and Altered Proteoglycan Glycosylation.” American journal of human genetics vol. 103,4 (2018): 553-567. doi:10.1016/j.ajhg.2018.09.003
- Ferreira, C.R., Zein, W.M., Huryn, L.A. et al. Defining the clinical phenotype of Saul-Wilson syndrome. Genet Med 22, 857-866 (2020). https://doi.org/10.1038/s41436-019-0737-1
- Texas Children’s Hospital “The Undiagnosed Diseases Network (UDN)”. Available at https://nri.texaschildrens.org/udn
- Varga, M.; Ralbovszki, D.; Balogh, E.; Hamar, R.; Keszthelyi, M.; Tory, K. Zebrafish Models of Rare Hereditary Pediatric Diseases. Diseases 2018, 6, 43. https://doi.org/10.3390/diseases6020043