Identification of the functional role of TGDS and the effects of mutations in the pathogenic mechanisms of Catel-Manzke Syndrome

Catel-Manzke syndrome (CMS) is a rare recessive autosomal disease that affects mainly the skeletal system. It is characterized by specific malformations, such as micrognathia, glossoptosis, cleft palate, collectively known as the Pierre-Robin sequence (PRS), and clinodactyly of the index finger. Additionally, patients often present other bone and heart abnormalities. This condition is caused by homozygous and compound heterozygous mutations of TGDS gene. However, the function of this gene in vertebrates is currently unknown, and the pathogenic mechanisms behind the syndrome remain unclear. While TGDS orthologs in prokaryotes, lower eukaryotes, and plants are involved in the NDP-L-rhamnose synthesis, catalyzing the first dehydration step, it is currently not known if this pathway exists in higher animals. In our study, we have identified and characterized tgds gene from zebrafish, and successfully expressed the recombinant protein in E. coli. The protein catalyzes UDP-D-glucose (UDP-D-Glc) dehydration, leading to the formation of UDP-4-keto-6-deoxy-D-glucose. We also obtained Tgds mutants with the same substitutions observed in Catel-Manzke patients, which displayed lower catalytic activity and stability. Studies using qRT-PCR, RNAseq and in situ hybridization (ISH) have shown that tgds is a maternal gene, with the highest expression levels in the zygote. After gastrulation, a second wave of expression occurs in specific locations, such as the brain, optic capsule, and pharyngeal cartilage. Furthermore, a preliminary CRISPR-Cas9 experiment was conducted to generate zebrafish tgds knock –out model. In particular, it was noted that some morphotypes involved the craniofacial structure and the architecture of the notochord. These findings suggest that transgenic zebrafish can represent a valuable model for the study of CMS pathogenesis. Moreover, the demonstration of the Tgds catalytic activity raises important questions about its physiological function in vertebrates.