Abstract

NSD1 (Nuclear receptor SET Domain-containing 1) gene maps on human chromosome 5 and encodes for a N-methyltransferase involved in histone H3 methylation at lysine 36.  Genetic alterations in NSD1 gene are detected with high frequency in diagnosed cases of Sotos syndrome, which is characterized by prenatal and infantile overgrowth, facial dysmorphism, intellectual impairment as well as autistic traits.

In order to characterize the effects of NSD1 haploinsufficiency in humans, we used the teleost zebrafish (Danio rerio). In zebrafish NSD1 gene had an evolutionary duplication, from which two orthologues derive, nsd1a and nsd1b, whose expression was characterized by real-time PCR during early developmental stages.

We recently generated a stable loss of function (LoF) mutant zebrafish line in the NSD1 orthologous gene nsd1a, by exploiting CRISPR/Cas9 gene editing technique. Interestingly, the nsd1a mutant larvae are characterized by craniofacial malformations including a prominent jaw, that interestingly resembles a common feature of individuals affected by Sotos syndrome.

 In nsd1a mutant larvae we observed reduced levels of acetylated histone H3 at Lysine 36, the target of human NSD1 enzyme, suggesting a possible conservation of its methyltransferase activity between humans and zebrafish. We are focusing now on a possible impairment of social behaviour of adult nsd1a mutants that may resemble the autistic features of the patients by performing behavioural test on larvae and adult zebrafish. In addition, the creation of stable mutants in the other ortholog nsd1b is ongoing.

In parallel, to modulate the levels of nsd1a and nsd1b mRNA in the early stages of zebrafish embryonic development, we are setting up an approach based on the Cas13d, an endonuclease that targets RNA, determining its subsequent degradation.

Since the first experimental evidence we obtained are supporting the hypothesis that zebrafish can be used as a suitable disease-model to study Sotos syndrome, we are now trying to set up the CRISPR/Cas9-derived strategy of base editing, optimized for zebrafish (zAncBE4max), to create the same mutations found in individuals affected by Sotos syndrome. Firstly, we performed proof of concept experiments to demonstrate the functionality of the base editor by targeting the tyrosinase (tyr) gene, encoding for an enzyme involved in eumelanin synthesis, to check embryo depigmentation as readout.

The mutant model of NSD1 obtained in zebrafish would be able to recapitulate the phenotypic and molecular characteristics of human Sotos syndrome. The future perspective is to exploit this model organism for the identification of new therapeutic approaches, hopefully suited on the molecular characteristics of individual patients.