Rare disease is one that affects no more than 1 person in 2,000, existing between 6,000 and 8,000 different. Patients face problems such as long delay in diagnosis and ineffective/inadequate treatments. Taking advantage of powerful zebrafish model, we propose to generate rare disease models to confirm pathogenic variants and to develop more effective therapeutic strategies. A clinical case of a 7-year-old boy with a rare disease diagnosed as psychomotor retardation, with greater impairment of language and behavior, has been associated with a de novo deletion of ~ 0.2 Mb in the JARID2 gene. JARID2 is a cofactor of Polycomb repressive complex 2 (PRC2), an essential epigenetic regulator which deposits H3K27me3 to silence transcription, and that plays a key role in stem cell differentiation, embryonic development and hematopoiesis.

Zebrafish fertilized eggs/larvae of wildtype and transgenic lines lck:GFP (green fluorescence lymphocytes). Single guide RNA (gRNA) directed to target sequences in the coding regions of jarid2a and jarid2b. Microinjection of CRISPR/Cas9 system of eggs at one-cell stage. Images analysis using a Leica MZ16F fluorescence stereo microscope equipped with fluorescent filters and ImageJ. RNAseq analysis was performed in peripheral blood leukocytes (PBLs; Novogen). Immortalized B cells from healthy subject and patient. HEK 293 cell line. PCR using specific primers for JARID2 and cDNA from patient and healthy subject PBLs and immortalized B cells. Immunoprecipitation and Western Blot. Next-Generation and Nanopore Sequencing. Statistical analysis by one way ANOVA with Tukey post-test. p ≤ 0,05, using GraphPadPrism 7 software.

Consistent with the characterization of the mutation by Nanopore Sequencing, a JARID2 transcript lacking exons 2 to 6 was observed in patient cells, predicting a 107 KDa protein (vs. the 140 KDa wild type protein). The overexpression of this patient isoform in HEK cells showed that it is not capable of interacting with core proteins of the PRC2 complex, while it was not able to modulate the expression of target genes in immortalized B lymphocytes. Moreover, a dramatic gene dysregulation in peripheral blood leukocytes from the patient was observed. In zebrafish, thymus development was impaired by jarid2 disruption, and overexpression of both wildtype and patient isoform were able to rescue this phenotype. In addition, F1 generation of jarid2-deficient zebrafish line has been obtained.

The JARID2-patient isoform loses interaction with the PRC2 complex, so ongoing studies are focused on understanding the role of this protein. Jarid2 deficiency shows a huge deregulation of gene expression in the patient leukocytes and a fundamental role of this gene in hematopoiesis in zebrafish. A jarid2 deficient mutant zebrafish line is being successfully generated by CRISPR/Cas9 system.