Comunicación

ZEBRAFISH AVATARS FOR RARE DISEASES REVEAL A CRITICAL ROLE OF JARID2 IN HEMATOPOIESIS

Autores:

Isabel Cabas Sánchez1, Diana García Moreno1, Francisco Javier Martínez Morcillo1, Alfonsa García Ayala1, MARIA LUISA CAYUELA FUENTES2, Victoriano Mulero Méndez1

Afiliaciones:

(1) INMUNIDAD, INFLAMACIÓN Y CÁNCER, IMIB-Arrixaca, España
(2) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB-Arrixaca, España

Comunicación:

Antecedentes:

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 6-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.

Métodos:

Zebrafish fertilized eggs/larvae of wildtype and transgenic lines lyz:DsRED2nz50 (red fluorescence neutrophils), cd41:GFP (green fluorescence hematopoietic stem and progenitor cells and thrombocytes) 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). PCR using specific primers for JARID2 and cDNA from patient and healthy subject PBLs and immortalized B cells. Next-Generation Sequencing. Statistical analysis by one way ANOVA with Tukey post-test. p ≤ 0,05, using GraphPadPrism 7 software.

Resultados:

In zebrafish, jarid2a and jarid2b showed a 52,8% and 57,7 % of identity with human JARID2, respectively. By using CRISPR/Cas9 system, some promising founders of a jarid2-deficient zebrafish line have been obtained. When the impact of jarid2 deficiency was evaluated in F0-larvae, preliminary data showed that the number of CD41 positive cells (hematopoietic stem and progenitor cells and thrombocytes) was reduced, while those of neutrophils increased. Despite the patient does not show any significant immunological alteration, it has been observed that thymus development in zebrafish was impaired by jarid2 disruption. Consistently, a dramatic deregulation (more than 2600 differentially expressed genes) has been observed in peripheral blood leukocytes from the patient. Genetic studies demonstrated that the deletion resulted in an aberrant JARID2 transcript lacking exons 2 to 6, predicting a 107 KDa protein (vs. the 140 KDa wild type protein).

Conclusiones:

A jarid2 deficient mutant zebrafish line is being successfully generated by CRISPR/Cas9 system. Disruption of jarid2 in F0-larvae reveal a critical role of this genes in hematopoiesis. Moreover, a huge deregulation caused by its deficiency was observed in patient leukocytes. Studies are in progress to understand the role of the patient JARID2 protein and its role in hematopoiesis.


Dirección

Campus de Ciencias de la Salud
Carretera Buenavista s/n, 30120 El Palmar
Murcia, España

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