MON-716 A Novel Human Heterozygous STAT5B Variant Leads to Impaired Growth and Developmental Defects in Zebrafish Embryos

Abstract

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of GH signaling in somatic growth. Autosomic recessive human mutations in STAT5B lead to severe growth retardation associated to immune dysregulation. On the other hand, some heterozygous STAT5B mutations have been associated to a milder form of the disease. We have identified a heterozygous novel STAT5B mutation by Whole Exome Sequencing (WES) in a 2.2-year-old boy who presented proportionate short stature (height -2.77 SDS) with mild immune dysregulation. He also had normal GH response to provocative tests, low IGF-I levels, and a limited response to IGF generation test. This variant is located within the highly conserved SH2 domain responsible for recognizing and interacting with tyrosine-phosphorylated target peptides. The aim of our study was to evaluate the functional consequences of this novel heterozygous human STAT5B variant (K632N), using the zebrafish as a biosensor system, to determine its pathogenicity. To do this, we performed overexpression experiments microinjecting construct-derived mRNA for the wildtype (WT) and mutant variant into zebrafish embryos at the 1-cell stage and assessed the consequences at 72 hours post fertilization (hpf). The missense variant was introduced into the full length STAT5B cDNA clone (Origene) by site-directed mutagenesis. To generate mRNA, WT and mutant forms of STAT5B cDNAs were linearized by digestion with XhoI, purified and subsequently transcribed with Mmessage Mmachine T7 Transcription Kit. Zebrafish embryos microinjected with 100 and 200 pg of mutant mRNA show a dose dependent significant reduction of body length at 72 hpf compared to those microinjected with the same dose of WT mRNA (p<0.001). Body length reduction with 100 pg of mutant mRNA was 4%, while with 200 pg was 12.7% (p<0.001). In addition, a significant number of embryos injected with mutant mRNA show developmental defects including pericardial edema, bent spine, and cyclopia compared to those injected with WT mRNA (p<0.001). In the case of pericardial edema, the number of affected embryos increased significantly with the mutant mRNA dose (p<0.005). In conclusion, our study was able to evidence the pathogenic nature of the STAT5B K632N variant since it leads to growth and developmental defects in zebrafish embryos. The zebrafish, and its conserved GH-IGF-I axis, constitutes an ideal in vivo model for characterizing the functional effect of genetic variants in ortholog human genes.