Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G>T, both reside in exon 13. Skipping of exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in iPSC-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection, and induced a detectable level of exon skipping until at least 6 months post injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

Most components of the thyroid system in bony fish have been described and characterized, with the notable exception of thyroid hormone membrane transporters. We have cloned, sequenced, and expressed the zebrafish solute carrier Slc16a2 (also named monocarboxylate transporter Mct8) cDNA and established its role as a thyroid hormone transport protein. The cloned cDNA shares 56–57% homology with its mammalian orthologs. The 526-amino-acid sequence contains 12 predicted transmembrane domains. An intracellular N-terminal PEST domain, thought to be involved in proteolytic processing of the protein, is present in the zebrafish sequence. Measured at initial rate and at the body/rearing temperature of zebrafish (26 C), T3 uptake by zebrafish Slc16a2 is a saturable process with a calculated Michaelis-Menten constant of 0.8 μM T3. The rate of T3 uptake is temperature dependent and Na+ independent. Interestingly, at 26 C, zebrafish Slc16a2 does not transport T4. This implies that at a normal body temperature in zebrafish, Slc16a2 protein is predominantly involved in T3 uptake. When measured at 37 C, zebrafish Slc16a2 transports T4 in a Na+-independent manner. In adult zebrafish, the Slc16a2 gene is highly expressed in brain, gills, pancreas, liver, pituitary, heart, kidney, and gut. Beginning from the midblastula stage, Slc16a2 is also expressed during zebrafish early development, the highest expression levels occurring 48 h after fertilization. This is the first direct evidence for thyroid hormone membrane transporters in fish. We suggest that Slc16a2 plays a key role in the local availability of T3 in adult tissues as well as during the completion of morphogenesis of primary organ systems.

Background. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5′-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available. Methods. We combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening. Results. We identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system. Conclusion. This study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients. Funding. Society for Inborn Errors of Metabolism for Netherlands and Belgium (ESN), United for Metabolic Diseases (UMD), Stofwisselkracht, Radboud University, Canadian Institutes of Health Research, Dutch Research Council (NWO), and the European Research Council (ERC).

With growing incidence of bone disorders (such as osteoporosis), the demand for fast and reliable alternatives to existing screens based on mammalian bone is increasing. Cell culture-based models that currently are being used, have limited value in the study of cell-cell and cell-matrix interactions. The dermal bone of fish scales offers an unprecedented and attractive model to study bone physiology, both in vivo and in vitro. The elasmoid scale of teleost fishes is of particular interest as it could stand as a model for direct bone formation as found in mammals and it has many advantageous traits: scales carry both bone-forming and bone-degrading cell populations, attached on their natural substrate, such that interactions between cell populations remain intact. The scale contains a collagen type-I matrix impregnated with apatites. Scales come in the hundreds per fish, are easily harvested and can be exposed in vitro for direct study of bone regulatory compounds. Scales are transparent allowing real-time visualization of cellular activities on either side of the scale. Removed scales are replaced quickly and the regeneration process strongly resembles osteogenesis as seen in early bone development. These advantages warrant more fundamental research on scales as models in biomedical bone research. We here address the state-of-the-art on development, structure and physiology of fish scales.