Zebrafish as a Model Organism for TTC37-Related Trichohepatoenteric Syndrome: Generation and Characterization of a New Mutant Line

Background. Trichohepatoenteric syndrome (THES) is a rare multisystem autosomal recessive disorder primarily characterized by life-threatening enteropathy and liver involvement. While mutations in the TTC37 gene are recognized as the main cause of THES, very limited data are available regarding the pathophysiological role of TTC37, mainly due to an absence of in vivo models of disease. Aim. The aim of this research was to develop an in vivo ttc37 knockout zebrafish model of THES to replicate the abnormalities observed in human patients, thereby providing insights into the molecular mechanisms underlying the disease with particular regard to the pathogenesis of intestinal and liver involvement. Methods. We studied TTC37 expression in zebrafish to evaluate its suitability as a model for THES. Then, using CRISPR/CAS9 mutagenesis we developed a ttc37-/- knockout zebrafish line. Validation of the ttc37-/- line was confirmed through Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR), which measured ttc37 expression in intestinal and liver tissues. Subsequently, we evaluated intestinal and liver histology using hematoxylin and eosin (H&E) staining on samples obtained from juvenile ttc37-/- zebrafish at 35 days post- fertilization (dpf). Finally, we used RT-qPCR to evaluate stat3, il-10, AdipoR1, and AdipoR2 mRNA expression levels in intestinal and liver tissues comparing wild-type ttc37+/+ and knockout ttc37-/- zebrafish. Results. TTC37 is highly expressed in wild-type (WT) zebrafish during development. We successfully generated a ttc37-/- knockout zebrafish line featuring a 4-nucleotide deletion, resulting in a functional knockout. RT-qPCR revealed a significant reduction in ttc37 expression in the intestine (P<.0001) and in the liver (P<.001) of ttc37-/- mutants compared to wild type, validating the zebrafish line. Histological analysis revealed both intestinal (degradation and disruption of the fold-villus structures, goblet cell hyperplasia, and abnormal epithelial stratification) and liver (hepatic steatosis) abnormalities. In addition, stat3 mRNA expression levels was significantly downregulated in both the intestine and the liver (P<.05) of ttc37-/- mutants compared to ttc37+/+. Similarly, il-10 and AdipoR2 mRNA levels were reduced in the liver of ttc37-/- zebrafish. Conclusions. Our ttc37-/- knock-out zebrafish is the first disease model of THES and represents a novel and valuable tool for studying the syndrome, providing preliminary but promising insights into the molecular and pathological mechanisms of the disease. Our findings suggest that the downregulation of stat3, il-10, and AdipoR2 may play a critical role in the pathogenesis of THES. Further analyses are required to clarify the full molecular pathogenesis of the syndrome.