Rare diseases, even though they are individually rare, have a great effect on public health due to the high number of individuals affected by these diseases. The most common definition of rare diseases is that they are diseases with a prevalence of less than one out of 2,000 people. Various sources indicate that the number of rare diseases identified globally is somewhere between 7,000 and 10,000 [1]. These diseases are often genetic in nature, with around 80% having a genetic basis. In the United States, rare disease is a term that is used to describe a disorder that affects fewer than 200,000 people [2]. In the US, it is estimated that rare diseases combined are known to affect around 25 to 30 million Americans [3]. These data have shown the occurrence and effect of rare diseases on a significant portion of the population. While the exact cause of many rare diseases remains unknown, scientific breakthroughs in disease research have often stemmed from studies on these uncommon conditions. Rare diseases are associated with the difficulties that arise from the limited knowledge about them and the lack of adequate treatment options. The uniqueness of rare diseases, which is characterized with different symptoms and progression, makes it really hard to come up with general treatments, which will be effective for all patients. This complexity is also spread to the diagnosis process, because of the low awareness of these conditions among healthcare professionals, the delay and misdiagnosis are possible.

With the advancement of CRISPR/Cas9 genome editing technology, the modeling of rare diseases using animal and cell systems has become more accessible. Although model organisms like mice and zebrafish have been used for a long time in scientific research for the purpose of understanding disease mechanisms and developing treatments, zebrafish have become more popular than mice as an alternative for several reasons [4]. The embryos of these species are transparent and therefore it is very easy to observe the developmental processes in real-time, which is especially useful in studying how diseases affect embryonic development. It is often difficult to confidently ascribe a disease to specific genetic variants, especially when rare mutations only affect a small number of patients. Nevertheless, zebrafish have their own benefits. Mutant zebrafish lines can be rapidly produced with the CRISPR/Cas9 technology and their large clutch sizes (often 200 or more) allow for comparisons with genetically well-controlled siblings. Close similarities between human and zebrafish phenotypes strongly support the causal role of a candidate gene. Unlike in mice, where certain gene mutations result in prenatal lethality, zebrafish embryos can still develop, thereby allowing the observation of developmental effects of gene mutations up to mid-larval stages when major phases of brain development are mostly completed [5]. On the other hand, zebrafish are cheap and reproduce fast which makes it possible to perform large-scale genetic screens. The fact that they are genetically similar to humans makes them an excellent model for studying genetic disorders. Furthermore, zebrafish have the capacity to regenerate a wide range of tissues, which helps to understand tissue repair and regeneration in pathological conditions. Overall, the unique attributes of zebrafish make them a powerful model for disease research, offering the potential to uncover valuable insights into human health.

The zebrafish (Danio rerio), which is a genetically accessible vertebrate with the transparent embryos that develop externally, has become a valuable model organism to study various biological processes including neuroscience (Fig. 1) [6]. In the last couple of decades, zebrafish has become an increasingly popular model organism used in scientific research. This small freshwater fish, which ranges from 3 to 4 cm in length and is only alive for about 2 years, has a number of distinct advantages over other vertebrate model organisms [7]. A particularly interesting area where zebrafish have been shown to be of great significance is in the study of rare neurological disorders. The use of zebrafish as an animal model for studying rare neurological disorders offers several advantages. To begin with, zebrafish have a high degree of genetic similarity with humans as they have 70% of human genes and 82% of human disease-related genes as their orthologs [8]. This conservation of protein-coding genes enables researchers to examine the consequences of individual gene mutations related to rare neurological disorders in zebrafish. Besides, the external development of the zebrafish embryos and their optical transparency make it possible for researchers to directly observe and analyze the neuronal development and organization in real-time, which enable them to have a deeper understanding of the mechanisms underlying the neurological diseases [9]. This combined with the small size of the embryo, larva and adult zebrafish, they are suitable for the screening of potential neuroactive substances.

It is quite remarkable that there are anatomical and physiological signaling similarities between the zebrafish and the human nervous system. Zebrafish brain is composed of the forebrain (telencephalon), midbrain (optic tectum) and hindbrain (cerebellum). It has the same cell types as humans, including astrocytes, oligodendrocytes, microglia, cerebellar Purkinje cells, myelin, and motor neurons. The studies that have been done on spinal nerve patterning, neural differentiation, and vertebrate network development in adult zebrafish have shown that they are similar to higher vertebrates. These features of zebrafish make them a popular model for the validation of candidate disease genes and the study of molecular mechanisms and pathophysiology of neurological diseases [10]. The majority of brain regions affected in human patients can be easily located in zebrafish, which allows the study of disease-related changes in brain architecture and the assessment of cell populations with high functional similarity. The neuroanatomy mapping between zebrafish and mammals is not easy due to the lack of complete brain atlases in zebrafish, especially in young fish. In most parts of the zebrafish brain, neurons are not organized with clear nuclear structures, which makes annotation difficult. Homology within vertebrate species is determined by the factors of connectivity, developmental origin, gene expression, and function. The difficulties notwithstanding, the digital atlases by the zebrafish community are useful in making precise comparisons with the latest brain annotations. Moreover, additionally, previously unknown myelinated bundle structure in adult zebrafish brain has been suggested as a counterpart of the deep cerebellar nuclei or DCN in mammals recently [11].

In order to fully utilize the potential of zebrafish as a model organism for studying rare neurological disorders, human genetics studies play a key role. Human genetics studies are a powerful tool that gives us the opportunity to understand the genetic basis of neurological disorders in humans. This research is able to highlight specific gene mutations and variants that are linked to rare neurological disorders in humans [12]. Through the comparison of the genetic information from human patients with zebrafish, researchers can identify the genetic mutations that are conserved across the two species. Through this process, the creation of zebrafish models that accurately mimic the genetic abnormalities in humans, which is a very powerful tool for studying the pathogenesis and underlying mechanisms of these disorders, is made possible [13,14].

Zebrafish play a unique role in stratifying the effects of gene mutations, particularly in major disorders like autism, intellectual disability, schizophrenia, epilepsy and other diseases [15,16]. The fact that the sheer number of causal genes linked to these disorders has led to a push to stratify mutations according to similar phenotypic effects is a clear indication of this. Nevertheless, the heterogeneity of symptoms and severity within these disorders has made diagnosis problematic and the testing of new treatments in homogeneous patient groups difficult. To address this, researchers aim to define patient subgroups based on genotypes that produce shared disease phenotypes through common neurobiological mechanisms, which could improve treatment strategies and aid in the search for new therapies. Despite the vast amount of genetic information available, specific gene variants are often only described in a handful of patients, limiting the ability to use patient phenotypes for disease stratification. Zebrafish, with relevant gene mutations, phenotyping at a detailed level can help overcome this problem. Zebrafish are an ideal model for these studies due to their ability to conduct targeted gene mutations and phenotyping with medium throughput compared to rodent models. The use of zebrafish phenotypes that have been compared with targeted mutations has allowed researchers to group patient mutations in accordance with their phenotypic effects [17,18].

In fact, in most of the common central nervous system (CNS) diseases, the single gene mutations do not fully explain the disorder. Recent advances have shifted the focus to the identification of single nucleotide polymorphisms (SNPs) associated with disease risk, which suggests that genetic risk is complex, involving changes in multiple processes at the cellular level and interaction with environmental factors. Nevertheless, the identification of these risk variants is complex as most of them are located outside the coding regions of the genome and may affect gene expression in a complicated manner. Furthermore, disease risk variants may act in a non-autonomous way or by changing synaptic transmission, which is hard to be studied in cell culture. The investigation of these variants requires an intact vertebrate brain. Zebrafish allow for a high-throughput analysis of the functions and interactions of the genes implicated in these diseases in an intact vertebrate brain [5].

With the increasing interest in rare neurological disorders, various rare diseases have been modeled using zebrafish for better understanding. However, in this review, we briefly introduce several successful cases of rare disease research in our laboratory (Table 1).

1. Kallmann syndrome which is a condition modeled in zebrafish, particularly related to the wdr11 gene

In zebrafish, mutations in the ortholog of the human WDR11 gene are used to study the effects on the development and function of the nervous system. In humans, mutations in WDR11 are associated with Kallmann syndrome, which is characterized by delayed puberty and an impaired sense of smell due to idiopathic hypogonadotropic hypogonadism. In the zebrafish model, the expression of wdr11 and emx1, along with their protein interactions, was suggested during early CNS development [19].

2. Potocki-Shaffer syndrome is a rare contiguous genetic disorder which has been studied with zebrafish models

This syndrome is characterized by different distinguishing features including skeletal anomalies, eye anomalies, multiple exostoses, craniofacial anomalies, and intellectual disability which is caused by interstitial deletion of the p11. 2 bands of chromosome 11. Zebrafish models have been used to explore the role of genes like EXT2, which is associated with multiple exostoses, and ALX4, which is connected to parietal foramina. Additionally, the interruptions in PHF21A at 11p11. 2 in humans have been linked to abnormal craniofacial and intellectual development, and scientists have used zebrafish to investigate the developmental importance of its ortholog phf21a, including its expression pattern and effects in loss- and gain-of-function experiments. This study reveals the molecular and developmental pathways that contribute to the symptoms observed in Potocki–Shaffer syndrome [20].

3. Miles-Carpenter syndrome (ZC4H2 associated rare disorders, ZARD) is an X-linked intellectual disability (XLID) syndrome containing features like short stature in men, exotropia, microcephaly, pinched fingers, long hands, wagging feet, spasticity, and severe intellectual disability

That is due to the mutations on the X chromosome. When a gene is present on the X chromosome, but not on the Y chromosome, it is said to be X-linked. Zebrafish models of Miles–Carpenter syndrome have been the subject of research, and this has led to a better understanding of the syndrome. Specifically, mutations in the ZC4H2 gene, identified through exome sequencing, are associated with the condition. The zebrafish model with a homozygous zc4h2 knockout (KO) mutation shows characteristics such as abnormal swimming, increased twitching, and motor hyperactivity, which are similar to what is seen in human patients having ZC4H2 mutations. Analysis of cell-type-specific markers showed a specific loss of GABAergic interneurons in the brain and spinal cord, likely arising from mis-specification of neural progenitors. Thus, the zebrafish model is a powerful tool in the dissection of the underlying cellular mechanisms of interneuronopathy and movement disorders related to Miles–Carpenter syndrome [21].

4. Down syndrome and autism (DYRK1A)

Dual Specificity Tyrosine Phosphorylation Regulated Kinase (DYRK1A), one of the DYRK family gene members, has been proven to be a vital factor for cell proliferation, differentiation and survival during neurogenesis [22]. DYRK1A maps to the Down syndrome critical region at 21q22. Mutations in DYRK1A have been reported to cause microcephaly associated with either intellectual disability or autism. To investigate the molecular pathways that are altered in microcephaly and autism, a dyrk1aa mutant zebrafish model was made using TALEN technology. In the larval stage the dyrk1aa mutant zebrafish did not show any differences in expression of sox2 or neurog1, which are markers of neural stem cells and neuronal determination, respectively. In this manner, they showed a similar reaction to visual stimuli as the wild type. In adult mutant zebrafish, there was no observable difference in body length or general morphology when compared to the wild type, but the smaller brain was confirmed by dissection. Besides, the adult KO zebrafish presented anxiolytic behavior and impaired social interaction and social cohesion in behavioral assays [23].

5. The 12q14.1 deletion syndrome (SAM2, Samdori-2) is characterized by dysregulation of emotions, including fear and anxiety, which are fundamental behavioral phenomena that are crucial for fitness in all creatures

These responses are regulated by different neuro-modulators and habenula, which is an area in the brain that is associated with addiction and mood disorders. Emotional dysregulation may show itself in extreme behavioral issues and problems with social interactions, as in bipolar disorder, attention deficit hyperactivity disorder (ADHD) and post-traumatic stress disorder (PTSD). sam2 mutant zebrafish model was designed and validated fear and anxiety behaviors, including thigmotaxis, freezing, or erratic movement. They also found that purified SAM2 protein increased inhibitory postsynaptic transmission to corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus, involved in stress and anxiety responses. Furthermore, a human homologue of SAM2 was identified, refining a candidate gene region containing SAM2 among 21 annotated genes associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome [24].

6. In a study of XLID syndrome (FAM50A), which is associated with growth retardation and facial deformations, the occurrence of this disease is more common in males because the enrichment of genes causes XLID disorders

Being found out in 1999, the syndrome manifests itself in eye defects, post-natal growth retardation and epilepsy. Researchers found the FAM50A gene variant in the ultra-rare category which was linked to the Armfield syndrome phenotype. Using the zebrafish model, they investigated FAM50A function, correlated it with the Armfield XLID clinical spectrum, and tested variant pathogenicity. Knocking out fam50a in zebrafish resulted in physical abnormalities resembling those in humans with Armfield XLID syndrome, including dysmorphic facial features and delayed branchial arch patterning [25]. The fam50a zebrafish model was also used to study Guion–Almeida type mandibulofacial dysostosis caused by mutations in EFTUD2.

7. Leukodystrophy with vanishing white matter is also called childhood ataxia with central nervous system hypomyelination

However, the exact incidence of vanishing white matter (VWM) is not known. It is seen to mostly affect children but it can also occur in people of any age, from infants to adults. The disease is characterized by cerebellar ataxia and stress-sensitivity, which may cause the disease to develop or progress rapidly, and in some cases, lead to death. VWM is caused by mutations in the subunits of the eukaryotic translation initiation factor, EIF2B1, EIF2B2, EIF2B3, EIF2B4 or EIF2B5 [7]. The protein eIF2B, vital for synthesizing all other proteins in the body and regulating their production rate, is ubiquitously present. However, in VWM disease, characterized by leukodystrophy, cystic degeneration, astrogliosis, increased white matter sparsity, and malformation of astrocytes and oligodendroglial cells, eIF2B fails to function properly. Zebrafish model of EIF2B subunits has been developed, specifically EIF2B3, which is important for early CNS myelination. An EIF2B3 mutant zebrafish was made using CRISPR mutagenesis which showed key human phenotypes, including defective myelin gene expression and glial cell differentiation. Additionally, novel EIF2B3 variants, such as L168P, have been identified in a Korean patient with VWM-like symptoms [26].

8. Ubiquitin-related rare diseases, such as Angelman syndrome, often involve developmental delays and intellectual disability

Functional characterization of the ubiquitin system-related gene UBE2H using zebrafish has shown that ube2h is crucial for normal brain development, as its depletion activates the ATM-p53 signaling pathway and leads to apoptosis in differentiated neural cells. A de novo missense variant in UBE2H (c.449C>T; p.Thr150Met), identified in a pediatric patient with global developmental delay, was mimicked in zebrafish, similarly disrupting normal ube2h function and confirming its vital role in brain development [27].

9. XLID, whether syndromic or non-syndromic, is associated with multiple genes on the X chromosome

ZFX, located on Xp22.11, encodes a transcription factor involved in various processes such as oncogenesis and development. However, germline variants of ZFX associated with disease have not been characterized previously. The ZFX gene is found to be related to a neurodevelopmental disorder which is characterized by a recurring pattern of facial features that is also termed as facial gestalt. The research focuses on the clinical characteristics and congenital abnormalities in 18 individuals with the germline ZFX variants, underscoring the importance of the diagnosis and the understanding of the disease. The clinical findings in the affected individual were developmental delay/intellectual disability, behavioral aberrations, hypotonia, and congenital anomalies. All subjects shared the features of the face that were overlapping and repetitious, such as the thickening and broadening of the eyebrows, variations in the shape of the face, outer eye abnormalities, the smooth and/or long philtrum, and ear abnormalities. Zebrafish were used to model truncating loss-of-function variants of the ZFX gene to characterize neurocognitive behavioral changes. It has been then analyzed the zfx mRNA expression in wild-type and knockout zebrafish, conducted behavioral tests. The zfx KO zebrafish did not exhibit major morphological changes, although they showed behavioral alterations potentially indicating changes in anxiety levels and anxiolytic response [28].

10. ZNF536 is a gene that was discovered to be associated with the risk of neuropsychiatric disorders such as schizophrenia, autism, and possibly others

However, the specific pathways and how ZNF536 affects patients are not yet completely understood, but studies indicate that genetic variants of ZNF536, including SNPs and rare protein-truncating variants, could contribute to these disorders. The zebrafish study was carried out by knocking out the znf536 gene and as a result, the observed phenotypes were reduced social interaction and anxiety-like behavior, and the brain anatomical changes, especially in the cerebellum.

Interestingly, in this study an unknown myelinated bundle structure in the adult zebrafish brain was discovered. The myelinated structure of the novel was located between the corpus cerebelli and the valvula cerebelli (Va) and was situated bilaterally within the Va of the znf536 KO fish. When compared the knockout zebrafish with their wild-type siblings, it was found that the myelinated structure was reduced and disorganized in the KO fish. We suggest this structure as a counterpart of the deep cerebellar nuclei or DCN in mammals. This implies that the function of znf536 could be involved in the development or maintenance of DCN-like structures in the cerebellum, which could be associated with the behaviors and neuroanatomical abnormalities in znf536 KO fish [11].

11. SRPK3 and TTN digenic mutations were identified in patients with early-onset skeletal muscle myopathy

The disease onset was in childhood or earlier, characterized by poor motor performance. And myopathy was slowly progressive, but most patients remained ambulatory at their last assessment. This pattern of muscle weakness was mainly proximal and axial, more significant in the lower limbs when compared to the upper limbs. Skeletal muscle biopsies were subjected to histopathological analysis which showed myopathic changes, such as increased nuclei internalization, core-like structures, and predominance of type I fibers. Zebrafish mutants were created with a mutation in the SRPK3 and in the TTN genes, which resulted in skeletal muscle myopathy [29]. Additionally, through experiments involving zebrafish models, it was found that ‘monogenic’ mutations in SRPK3 are associated with the phenotype seen in intellectual disabilities. Furthermore, they demonstrated that defects in smooth pursuit eye movements which are critical for visual attention and/or perception, could be related to learning problems and intellectual disabilities [30].

Zebrafish models, which use the genes related to rare-disease mentioned above, provide a promising platform for rapid drug screening and therapeutic development. In addition, human genetics studies can also be used for finding out the potential therapeutic targets for these disorders. Through the knowledge of the genetic mutations that are associated with rare neurological disorders in humans, researchers can create specific experiments in zebrafish to test the potential therapeutic strategies. Through this, they can evaluate the effectiveness and safety of various treatments and interventions, and eventually develop new therapies and personalized medicine strategies for patients with rare neurological disorders.

Nevertheless, zebrafish have some limitations, particularly in the respiratory and reproductive systems, which might not always be similar to humans. Furthermore, the molecular mechanisms between zebrafish and humans can be different in gene expression, protein modification, anatomy, physiology, or behavior. Moreover, the screening of water-insoluble drugs is difficult because of the aquatic environment of zebrafish [7].

Zebrafish is a good animal model for the investigation of rare neurological disorders including the finding of novel therapeutic targets, because the orthologs of human neurological disease-associated genes are well conserved in zebrafish and a real-time observation of the neuronal development stages is possible in zebrafish. The progress of fundamental research into zebrafish neural pathways is opening the way to test whether a zebrafish model can be generated for a particular disease. With the help of genome engineering technology including DNA editing technology (CRISPR/Cas9) and next-generation DNA sequencing, it is now possible to create a precise genetic alteration in zebrafish that exactly mimics the pathogenic mutations observed in human patients. Thus, through the use of conserved genetic information between zebrafish and humans, researchers can study important genetic mutations described above in this paper and in the development of diagnostic techniques and therapeutic approaches.

None.

This work was supported by the National Research Foundation of Korea (NRF) grant (RS-2024-00349650).

Conception and design: DWD. Acquisition of data: DWD, TIC, TYK, KHL. Drafting the article: DWD, CHK. Critical revision of the article: DWD, YL, CHK. Final approval of the version to be published: YL, CHK.