SUN-038 Role Of The Satb 1 Gene In Growth Across Species: Findings In A Cohort Of Patients With Short Stature And In A Knockout Zebrafish Model

Abstract

Background: Short stature is a common pediatric disorder, affecting 3% of the population; however, the identification of its cause is limited by its largely variable clinical presentation and genetic heterogeneity. Identification of novel genetic causes of short stature is of great importance to customize the therapeutic approach of this entity. The especial AT-rich sequence binding protein1 gene (SATB1) encodes a chromatin organizer with important roles in cell growth and immunosuppression. SATB1 is highly expressed in the pituitary gland, where it regulates the differentiation of PIT1 positive cells. Indeed, the conditional knockout of Satb1 in the mouse pituitary decreases growth hormone expression, leading to reduced growth. Aim: To investigate the putative role of SATB1 in growth in a cohort of individuals with short stature and in a zebrafish knockout (KO) model. Methods: We obtained germline DNA samples from a cohort of 287 Brazilian patients diagnosed with short stature (height <2 SDS for age and sex), including 52 patients small for gestational age (SGA), 5 cases with growth hormone deficiency (GHD), 10 cases with syndromic short stature and 220 cases with idiopathic short stature. All cases were screened for SATB1 copy number variations (CNVs) using droplet digital PCR, and 190 cases were screened for mutations via Sanger sequencing. In silico predictions were retrieved from the Varsome browser. All variants were annotated to RefSeq NM_002971.5. Using CRISPR-Cas9, we generated two lines of zebrafish satb1 bearing frameshift mutations; both lines were crossed to obtain full KOs. Growth was monitored from 3 to 12 weeks post-fertilization (wpf) and expression of pituitary hormones was analyzed at 6 wpf by quantitative PCR. Results: No variants predicted as damaging were found in the short stature cohort. Nevertheless, variants of uncertain significance (VUS, mostly intronic) were found in 11% of our cohort, in which 57% of the individuals that carry a VUS presented familial short stature while 43% were non-familial. Interestingly, the variant c.515+112G>A was identified in 3/33 (9.1%) of SGA patients, but only in 2/143 (1.4%) of ISS patients. No CNVs were observed. In the zebrafish model, satb1 knockout led to significantly smaller size at 3 wpf, compared with heterozygous and wild type controls (median standard length: 6.6, 7.4, and 7.8 cm, and mean weight: 2.7, 4.3, and 4.6 mg, respectively, P<0.05 for all comparisons). At 6 wpf, we identified a trend for lower gh1 expression in the KOs vs. heterozygous zebrafish (relative quantification: 0.68 vs. 1.79, P=0.06). Conclusion: Our results support a role for SATB1 in regulating growth hormone production across species. Although loss of SATB1 function could theoretically lead to growth hormone deficiency in humans, defects in this gene do not seem to play a crucial role in the height of individuals with short stature.