AN IN VIVO FUNCTIONAL ASSAY TO CHARACTERIZE HUMAN STAT5B GENETIC VARIANTS DURING ZEBRAFISH DEVELOPMENT

LANDI, ESTEFANÍA; KARABATAS, LILIANA; RODRIGUEZ GOMEZ, TOMAS; SCAGLIA, PAULA; RAMÍREZ, LAURA; KESELMAN, ANA; BRASLAVSKY, DÉBORA; SANGUINETI, NORA; PENNISI, PATRICIA; REY, RODOLFO A; BERGADÁ, IGNACIO; JASPER, HÉCTOR G; DOMENÉ, HORACIO M; PLAZAS, PAOLA V; DOMENE, SABINA

Congreso; International Meeting of Pediatric Endocrinology held, IMPE 2023; 2023

Human growth is highly dependent on the GH-IGF-I axis. GH binding to GH receptor activates janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) pathway which stimulates transcription of IGF1, IGFBP3, and IGFALS. Although STAT5b deficiency was established as an autosomal recessive disorder, heterozygous dominant-negative STAT5B variants were reported in patients with less severe growth deficit and milder immune dysfunction.Our aim was to develop an in vivo functional assay in zebrafish to characterize the pathogenicity of three STAT5B human variants (p.A630P, p.Q474R and p.K632N). Previous in vitro assays showed that p.A630P is less well expressed and barely phosphorylated, p.Q474R is expressed, phosphorylated and able to translocate into the nucleus but lacks transcriptional activity, and p.K632N is expressed but not phosphorylated upon GH stimulation. We developed an overexpression assay using zebrafish as a biosensor. Overexpression experiments were performed microinjecting 200 picograms of each construct-derived mRNA for the wildtype (WT) and mutant variants into zebrafish embryos at the 1-cell stage and assessing overt embryonic developmental defects as surrogate outcomes at 72 hours post fertilization. The variants were introduced into the full length STAT5B cDNA clone by site-directed mutagenesis. To generate mRNA, cDNAs were linearized and transcribed with Mmessage Mmachine T7 Transcription Kit.Interestingly, overexpression of human STAT5B mRNAs led to developmental malformations in zebrafish embryos. Overexpression of p.A630P led to an increased number of embryos with pericardial edema (36/113=32%), cyclopia (15/113=13%), and bent spine (20/113=18%) compared with WT STAT5B (pericardial edema 15/102=15%, cyclopia 2/102=2%, bent spine 15/102 embryos=15%). Overexpression of p.Q474R or p.K632N resulted in an increased number of embryos with pericardial edema (59/108=55%; 48/89=54%), cyclopia (32/108=30%; 27/89=30%), and bent spine (66/108=61%; 24/89=27%). Overexpression of p.Q474R or p.K632N led to embryos with a significant reduction of body length compared to WT STAT5B (a 17.3% and a 12.7% reduction respectively) (p