Pathogenic germline IKZF1 variant alters hematopoietic gene expression profiles

Abstract

IKZF1 encodes Ikaros, a zinc finger-containing transcription factor crucial to the development of the hematopoietic system. Germline pathogenic variants in IKZF1 have been reported in patients with acute lymphocytic leukemia and immunodeficiency syndromes. Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by erythroid hypoplasia, associated with a spectrum of congenital anomalies and an elevated risk of certain cancers. DBA is usually caused by heterozygous pathogenic variants in genes that function in ribosomal biogenesis; however, in many cases the genetic etiology is unknown. We identified a germline IKZF1 variant, rs757907717 C > T, in identical twins with DBA-like features and autoimmune gastrointestinal disease. rs757907717 C > T results in a p.R381C amino acid change in the IKZF1 Ik-x isoform (p.R423C on isoform Ik-1), which we show is associated with altered global gene expression and perturbation of transcriptional networks involved in hematopoietic system development. These data suggest that this missense substitution caused a DBA-like syndrome in this family because of alterations in hematopoiesis, including dysregulation of networks essential for normal erythropoiesis and the immune system.

Trial registration: ClinicalTrials.gov NCT00027274.

Keywords: congenital hypoplastic anemia.

© 2021 Brodie et al.; Published by Cold Spring Harbor Laboratory Press.

Figures

Figure 1.

Pedigree of NCI-40 and bone marrow characteristics of the proband. (A) Pedigree illustrating clinical features and carrier status of tested family members. Clinical features of NCI-40-1 (proband) and NCI-40-2 are described in detail in the text. Their mother, NCI-40-4, had a history of anemia and is heterozygous for the p.R381C variant. Her sister, the children's maternal aunt, NCI-40-6, had a history of aplastic anemia and thyroid cancer but did not have genetic testing. (WT) Wild-type, p.R381C (IKZF1 isoform Ik-x rs757907717 C > T) heterozygote, (6) number of siblings. (B–F) NCI-40-1 evaluation at 8 yr of age. (B) Hematoxylin and eosin (H&E) stain of bone marrow core biopsy at 40× magnification shows maturing erythroid islands, adequate myeloid:erythroid (M:E) ratio, and mild eosinophilia. (C) Bone marrow core biopsy at 100×. (D) Bone marrow aspirate smear at 50× magnification with progressively maturing trilineage hematopoiesis and no dysplastic changes or increase in blasts. (E) Stainable histiocytic iron (blue) in bone marrow. (F) Normal peripheral blood smear. (G–K) NCI-40-2 evaluation at 8 yr of age. (G) Core biopsy H&E stain at 40× magnification shows higher cellularity than his twin brother, has focal increase in megakaryocytes, maturing erythroid islands, adequate M:E ratio, and mild eosinophilia. (H) Bone marrow core biopsy at 100×. (I) Bone marrow aspirate smear at 50× magnification has progressively maturing trilineage hematopoiesis and no dysplastic changes or increase in blasts. (J) Reduced iron staining. (K) Normal peripheral blood smear with some platelet clumps (50× magnification).

Figure 2.

Protein sequence conservation and structural prediction suggests IKZF1 p.R381C is pathogenic. (A) The four functional isoforms of IKZF1 depicted with predicted Zn finger, DNA binding domains, and location of the variant p.R423C in isoform Ik-1. The variant location is p.R381C in isoform Ik-x. Isoform Ik-x is the isoform used in the described biochemical experiments in this manuscript. Note that the figure is not drawn to scale. (B) M-Coffee alignment reveals strict conservation of p.R381 (blue arrow and box) across orthologs from evolutionarily divergent species. (C) “OpenPredictProtein-effect of point mutation” algorithm predicts any changes at the p.R381C position (blue arrow) to be highly deleterious. (D) Jpred server prediction of secondary structure indicates that a R → C mutation (top vs. bottom panel, blue arrow heads) at p.R423 (equivalent to p.R381C in isoform Ik-x) would result in a loss of a B-sheet and potentially alter secondary structures up to 18 amino acid residues upstream (broad green arrows represent predicted B-sheets; red box indicates potential helical structure). (E) Phosphosite Plus analysis of IKZF1 Ik-1 isoform indicates post-translational modification sites near the p.R423 residue. Of note are p.S427-phos, p.K429-Ub, and p.K429-SUMO.

Figure 3.

Increased IKZF1 protein levels are associated with the p.R381C variant. Epstein–Barr virus (EBV)-transformed lymphoblasts with the IKZF1 p.R381C variant show increased IKZF1 protein levels by immunoblotting compared with wild type (WT) control and an unrelated individual with IKZF1 p.A3D variant. Signal densities quantified by ImageJ.

Figure 4.

IKZF1 p.R381C alters hematopoietic gene expression. RNA from stable cell lines expressing wild-type or p.R381C IKZF1 was extracted and sequenced on Illumina Novaseq S2 platform. Differentially expressed gene lists identified by RNA-seq analysis were queried in Ingenuity Pathway Analysis (QIAGEN) software. (A) Differentially expressed genes identified by RNA-seq form a network containing factors with functions critical to differentiation, proliferation, and development of the hematological system. (B) IKZF1 plays a central role in hematopoiesis, lymphoid, myeloid, and megakaryocytic proliferation and differentiation, leukemia, and erythroid biology. (C) Top functions of network presented in A. The full table is presented in Supplemental Table S3.

Figure 5.

Differentially expressed genes validated by quantitative polymerase chain reaction (qPCR) and immunoblot. (A) RNA-seq log2 fold change, standard error, and adjusted P-value for selected genes present as nodes in network analysis. (B) Targets identified as critical nodes in network analysis were selected for validation by qPCR. TaqMan probes were utilized to perform qPCR. Relative quantity values by ΔΔ−CT between wild-type (WT) and mutant (MUT) samples (n = 3 biological replicates, n = 3 technical replicates) normalized to β-actin and GAPDH are reported. Genes highlighted by the rectangle are included in immunoblot analysis. IKZF3/Aiolos signal was undetected after 40 cycles in WT samples but displayed a value of ∼32 CT in MUT samples. SNAI2 amplified in WT samples but was undetected in MUT samples after 40 cycles. (C) Targets were further validated by immunoblot. Immunoblots were quantified using density analysis in ImageJ software.