Search

PURPOSE: Somatic variants in tumor necrosis factor receptor-associated factor 7 (TRAF7) cause meningioma, while germline variants have recently been identified in seven patients with developmental delay and cardiac, facial, and digital anomalies. We aimed to define the clinical and mutational spectrum associated with TRAF7 germline variants in a large series of patients, and to determine the molecular effects of the variants through transcriptomic analysis of patient fibroblasts.METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts.RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts.CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies.

When considering relationships between genotype and phenotype we frequently ignore the fact that the genome of a typical animal, notably including that of a fish and a human, harbors a huge amount of foreign DNA. Some of it, including the DNA of “autonomous” transposable elements, can spontaneously mobilize to occupy new chromosomal sites and take on new functions, presenting a challenge to the host organism and also possibly introducing new fuel for evolutionary change. Transposable elements are useful for introducing transgenes, integrating them into host genomes with high efficiency. Transgenesis has become very widespread in biological research, and in our society at large. This year the governments of both Canada and the United States have approved the first use of ‘genetically engineered’ animals in food production, Atlantic salmon,Salmo salar. With the recent advent of amazing gene-editing technology, there is no doubt that the transgene industry will grow explosively in the coming years. The biology of transgenes needs to be included in our understanding of the genome. It is in this spirit that we have investigated an unexpected and novel phenotypic effect of the chromosomally integrated transgenefli1a-F-hsp70l:Gal4VP16. We examine larvalfras1mutant zebrafish (Danio rerio). Gal4VP16 is a potent transcriptional activator, and already well known for toxicity and mediating unusual transcriptional effects. In the presence of the transgene, phenotypes in the neural crest-derived craniofacial skeleton, notably fusions and shape changes associated with loss of functionfras1mutations, are made more severe, as we quantify by scoring phenotypic penetrance, the fraction of mutants expressing the trait. A very interesting feature is that the enhancements are highly specific forfras1mutant phenotypes – occurring in the apparent absence of more wide-spread changes. Except for the features due to thefras1mutation, the transgene-bearing larvae appear generally healthy and to be developing normally. The transgene behaves as a genetic partial dominant: A single copy is sufficient for the enhancements, yet, for some traits, two copies may exert a stronger effect. We made new strains bearing independent insertions of thefli1a-F-hsp70l:Gal4VP16transgene in new locations in the genome, and observed increased severities of the same phenotypes as observed for the original insertion. This finding suggests that sequences within the transgene, e.g. Gal4VP16, are responsible for the enhancements, rather than effect on neighboring host sequences (such as an insertional mutation). The specificity, and biological action underlying the traits, are subjects of considerable interest for further investigation, as we discuss. Our findings show that work with transgenes needs to be undertaken with caution and attention to detail.

The semicircular canals in the inner ear sense angular acceleration. In zebrafish, the semicircular canals develop from epithelial projections that grow toward each other and fuse to form pillars. The growth of the epithelial projections is driven by the production and secretion of extracellular matrix components by the epithelium. The conserved oligomeric Golgi 4 protein, Cog4, functions in retrograde vesicle transport within the Golgi and mutations can lead to sensory neural hearing loss. In zebrafish cog4 mutants, the inner ear is smaller and the number of hair cells is reduced. Here, we show that formation of the pillars is delayed and that secretion of extracellular matrix components (ECM) is impaired in cog4(−/−) mutants. These results show that Cog4 is required for secretion of ECM molecules essential to drive the growth of the epithelial projections and thus regulates morphogenesis of the semicircular canals.

Hypomyelination, a neurological condition characterized by decreased production of myelin sheets by glial cells, often has no known etiology. Elucidating the genetic causes of hypomyelination provides a better understanding of myelination, as well as means to diagnose, council, and treat patients. Here, we present evidence that YIPPEE LIKE 3 (YPEL3), a gene whose developmental role was previously unknown, is required for central and peripheral glial cell development. We identified a child with a constellation of clinical features including cerebral hypomyelination, abnormal peripheral nerve conduction, hypotonia, areflexia, and hypertrophic peripheral nerves. Exome and genome sequencing revealed a de novo mutation that creates a frameshift in the open reading frame of YPEL3, leading to an early stop codon. We used zebrafish as a model system to validate that YPEL3 mutations are causative of neuropathy. We found that ypel3 is expressed in the zebrafish central and peripheral nervous system. Using CRISPR/Cas9 technology, we created zebrafish mutants carrying a genomic lesion similar to that of the patient. Our analysis revealed that Ypel3 is required for development of oligodendrocyte precursor cells, timely exit of the perineurial glial precursors from the central nervous system (CNS), formation of the perineurium, and Schwann cell maturation. Consistent with these observations, zebrafish ypel3 mutants have metabolomic signatures characteristic of oligodendrocyte and Schwann cell differentiation defects, show decreased levels of Myelin basic protein in the central and peripheral nervous system, and develop defasciculated peripheral nerves. Locomotion defects were observed in adult zebrafish ypel3 mutants. These studies demonstrate that Ypel3 is a novel gene required for perineurial cell development and glial myelination., Author summary The study of congenital rare diseases allows the identification of new gene variants involved in development. Genetic and sequence analysis of a patient with a novel complex phenotype including hypomyelination, enlarged peripheral nerves, and motor defects implicated a de novo mutation in the YPEL3 gene as potentially causative. YPEL3 had not previously been associated with disease. Using CRISPR technology, we created a zebrafish model carrying a DNA lesion similar to that of the affected patient. Homozygous mutant embryos display weak and variable peripheral nerve phenotypes and are viable. Because ypel3 transcripts are present from fertilization, we generated maternal zygotic mutants. Analysis of these mutants revealed hypomyelination, defective axonal wrapping, and swimming defects. Time-lapse video lineage analysis showed that ypel3 is required for proper development of oligodendrocytes, Schwann cells, and perineurial cells. These results identify YPEL3 as a new regulator of glial cell development and support the conclusion that the de novo YPEL3 mutation is responsible for the patient’s disease.

The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.

Eye and inner ear diseases are the most common sensory impairments that greatly impact quality of life. Zebrafish have been intensively employed to understand the fundamental mechanisms underlying eye and inner ear development. The zebrafish visual and vestibulo-acoustic systems are very similar to these in humans, and although not yet mature, they are functional by 5 days post-fertilization (dpf). In this chapter, we show how the zebrafish has significantly contributed to the field of biomedical research and how researchers, by establishing disease models and meticulously characterizing their phenotypes, have taken the first steps toward therapies. We review here models for (1) eye diseases, (2) ear diseases, and (3) syndromes affecting eye and/or ear. The use of new genome editing technologies and high-throughput screening systems should increase considerably the speed at which knowledge from zebrafish disease models is acquired, opening avenues for better diagnostics, treatments, and therapies.

SUMMARY Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C): one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients.

SUMMARY Morphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes., Graphical Abstract, In Brief Deafness and vestibular areflexia in Usher syndrome (USH) are due to defective assembly of USH proteins into macromolecular complexes. Blanco-Sánchez et al. show that Grxcr1 negatively regulates the assembly of Ush1c and Ush1ga into complexes and that its activity is essential for correct mechanoreceptor morphology.

Mechanisms underlying the morphogenesis of skeletal shape are largely not understood, mainly because the ability to study the dynamic processes of cellular behavior giving rise to cartilage and bone in living embryos has been limited. In recent years, techniques have emerged that make the study of tissue formation possible in vivo (Beis and Stainier, 2006; Field et al., 2003; Koster and Fraser, 2004), suggesting that skeletal morphogenesis can be similarly studied. The zebrafish is an ideal model organism for studying skeletal development because embryos and larvae are small and transparent, enabling the study of organogenesis in the living organism. Zebrafish cartilage and bone elements develop early and have distinct morphologies, and the genetic mechanisms underlying skeletal formation are shared with other vertebrates (Yelick and Schilling, 2002). Transgenic lines are especially valuable for analysis of living embryos, including time-lapse confocal microscopy (Cooper et al., 2005; Glickman et al., 2003; Smith et al., 2008). Transgenic fish lines have already been produced that express GFP in cells that give rise to, amongst other tissues, cartilage elements in the head including Tg(−1252sox10:GFP)ba5 (Dutton et al., 2008) and Tg(foxp2-enhancerA:EGFP)zc42 (Bonkowsky et al., 2008). A transgenic line expressing an observable marker of osteoblasts would help us explore osteoblast behavior specifically during the formation of intramembranous bony elements, which have no cartilaginous precursor, and for studying the induction of osteoblasts in perichondrium during endochondral ossification. Sp7 is a zinc-finger-containing transcription factor expressed in osteoblasts and not chondrocytes, making it an excellent marker for studying osteoblasts (Nakashima et al., 2002). Recently, the promoter of sp7 has been shown to drive mCherry in osteoblasts of medaka fish allowing for the analysis of osteoblast behavior in the forming skeleton of this species (Renn and Winkler, 2009). This medaka sp7 regulatory sequence has been used to drive fluorescent marker expression in zebrafish (Hammond and Schulte-Merker, 2009; Spoorendonk et al., 2008), however, there exists no transgenic line using the regulatory region of sp7 in zebrafish to drive a fluorescent marker in zebrafish. We used BAC-mediated transgenesis to drive EGFP under the control of sequence upstream of sp7 in a zebrafish BAC. In the case of zebrafish sp7, we do not know the regulatory elements necessary for gene transcription. Therefore, an advantage of using BACs for transgenesis is that they often contain large genomic clones that include the essential regulatory elements of a gene of interest. The presence of large inserts of a zebrafish-specific sequence makes it likely that the BAC contains sequence essential to drive a transgene in an expression pattern consistent with the endogenous gene. We injected the BAC into embryos to generate stable transgenic lines expressing GFP in osteoblasts. We show here the native expression pattern of sp7 by in situ hybridization in whole-mounts and sections, and compare it to the expression of GFP in the Tg(sp7:EGFP)b1212 transgenic line. We found that GFP expression reproduces endogenous sp7 gene expression, indicating that this line will be an excellent tool for future study of the dynamic behavior of cells that create the skeleton.