Loss-of-phosphorylation of IKZF1 results in gain-of-function associated with immune dysregulation.

Abstract

Immune dysregulation often presents as autoimmunity, inflammation, and/or lymphoproliferation. Several germline genetic defects have been associated with immune dysregulation; they include heterozygous gain-of-function (GOF) mutations in IKZF1, an essential transcription factor for hematopoiesis containing zinc finger domains (ZFs). However, in a large percentage of patients, the genetic origin of their immunedysregulation remains undetermined. A family with 2 members presenting immune dysregulation signs was studied to identify the genetic cause of their disease. Whole exome sequencing, analysis of immunologic parameters, and functional assays (including Western blotting, electrophoretic mobility shift assay during the cell cycle, and T_H cell differentiation) were performed. The 2 patients carried a novel heterozygous mutation in IKZF1 (IKZF1^T398M). IKZF1 heterozygous mutations have previously been shown to be responsible for several distinct human immunologic diseases by directly affecting the ability of ZFs to bind to DNA or to dimerize. Herein, we showed that the IKZF1^T398M, which is outside the ZFs, caused impaired phosphorylation of IKZF1, resulting in enhanced DNA-binding ability at the S phase of the cell cycle, reduction of the G1-S phase transition, and decreased proliferation. Confirming these data, similar functional alterations were observed with IKZF1^T398A, but not with IKZF1^T398D, mimicking dephosphorylation and phosphorylation, respectively. In T lymphocytes, expression of IKZF1^T398M led to T_H cell differentiation skewed toward T_H2 cells. Thus, our data indicate that IKZF1^T398M behaves as a GOF variant underlying immune dysregulation. Disturbed IKZF1 phosphorylation represents a novel GOF mechanism (GOF by loss of phosphorylation (termed as GOF-LOP) associated with immune dysregulation, highlighting the regulatory role of IKZF1 during cell cycle progression through phosphorylation.