A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia

Abstract

The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders whose hallmarks are ineffective erythropoiesis, hemolysis, and morphological abnormalities of erythroblasts in bone marrow. We have identified a missense mutation in KLF1 of patients with a hitherto unclassified CDA. KLF1 is an erythroid transcription factor, and extensive studies in mouse models have shown that it plays a critical role in the expression of globin genes, but also in the expression of a wide spectrum of genes potentially essential for erythropoiesis. The unique features of this CDA confirm the key role of KLF1 during human erythroid differentiation. Furthermore, we show that the mutation has a dominant-negative effect on KLF1 transcriptional activity and unexpectedly abolishes the expression of the water channel AQP1 and the adhesion molecule CD44. Thus, the study of this disease-causing mutation in KLF1 provides further insights into the roles of this transcription factor during erythropoiesis in humans.

Figure 1

Analysis of the Peripheral Blood of CDA Patient ME Shows Unique Abnormalities (A) Peripheral blood smears from the patient (right panels; sample taken on 10/28/2008) and a control (left panel; sample taken at the same time) stained with May-Grünwald Giemsa. Note the large number of circulating erythroblasts (purple nuclei) as well as poikilocytosis, anisocytosis and fragmented erythrocytes in the patient. The scale bars represent 40 μm. (B) Study of the number of enucleated cells during in vitro erythroid differentiation of CD34⁺ cells from the patient (purple; sample taken on 10/25/2005) or a control donor (blue). Note the markedly reduced enucleation capacity of the patient's cell culture. (C) Immunoblot analysis of CD44, CD55, AQP1, and p55 in erythrocyte membrane lysates from the patient (CDA; sample taken on 6/7/2004), his healthy mother (C2), or a random control (C1). Note the combined deficiency of CD44 and AQP1 in the patient's erythrocytes. (D) Flow-cytometry analysis of CD44 on mature erythrocytes (left panels) and granulocytes (right panels) from the patient (bottom panels; sample taken on 1/5/2010) and a control (top panels; sample taken at the same time). Note the erythroid-specific deficiency of CD44 in the patient. For the analysis of erythrocytes, whole blood samples were costained with fluorochrome-conjugated anti-CD44 (black histogram) or isotype control antibody (white histogram) and anti-CD71, and the mature erythrocytes were gated on FSS, SCC, and CD71⁻ so that the reticulocytes and erythroblasts would be eliminated; for the analysis of granulocytes, nucleated blood cells were first isolated by hypotonic erythrocyte lysis, then costained as above and directly gated on FSC and SSC. (E) Isoelectric focusing analysis of the different hemoglobin (Hb) variants in the patient (CDA; sample taken on 10/28/2008) and two controls (C1 and C2). Note the atypical globin expression in the patient; he exhibited very high levels of fetal Hb (α₂γ₂ tetramer, 37.3%; normal range, less that 1%) as well as embryonic Hb Portland (ζ₂γ₂ tetramer, 2.9%; normal range, absent) as ascertained by reverse-phase liquid chromatography; adult HbA and HbA2 were at 55.5% (α₂β₂ tetramer, normal range: 90%–100%) and 1.2% (α₂δ₂ tetramer, normal range: 2%–3%), respectively. Extensive sequencing of patient ME's globin loci detected no gross abnormalities but a heterozygous mutation in the α1-globin gene (c.62_63insT, p.His21fsX36), which could not be responsible for the profound β-globin locus dysregulation and was indeed present in his healthy paternal aunt. Of note, patient SF was a carrier of a 4 bp deletion in the promoter of ^Aγ-globin gene, as was her healthy father. All analyses presented herein were performed on blood samples taken from patient ME after splenectomy and at least 6 months after transfusion.

Figure 2

Identification of the KLF1 Mutation Associated with this CDA and Structural Analysis of the Variant Transcription Factor (A) A detail of KLF1 sequencing in patient ME, his unaffected mother and sister, and unrelated patient SF show the same heterozygous KLF1 mutation in both CDA patients. The experimental sequences were aligned with the NCBI reference sequence of KLF1 (NG_013087.1). Genomic DNA samples from patient ME's father and patient SF's parents were not available. (B) A diagram shows the structure of KLF1 (based on NG_013087.1) and its products and highlights the localization of the mutation c.973G>A, p.Glu325Lys (E325K) was found in patients ME and SF. KLF1 consists of three exons (boxes; black represents coding regions, and gray represents untranslated regions) and encodes a 362 amino acid peptide with a proline-rich domain at the amino-terminus (the transactivation domain is in brown) and three C₂H₂-type zinc fingers (ZF) at the carboxy-terminus (the DNA-binding domain is in green). The pathogenic mutation is located in the third exon and encodes a single amino acid change in the second zinc finger of the transcription factor. (C) A sequence alignment of the second zinc finger of KLF1 from various mammalian species shows the conservation of a glutamate at amino acid position 325 (red box). The two cysteines and the two histidines contacting Zn²⁺ are indicated in bold, and the three conserved residues contacting the DNA are indicated by stars. (D) A modeling structure of wild-type (left) and variant E325K (right) zinc-finger domain of KLF1 shows the enhanced electrostatic interaction between the E325K variant and the DNA backbone. The change of glutamate to lysine at position 325 exerts a double effect by reversing the side-chain charge from negative to positive and extending the side-chain length toward the negatively charged DNA backbone; the putative hydrogen bond created by the E325K variant is shown as a yellow dashed line. The bottom panels show the overall structure of the protein-DNA complexes, as well as the electrostatic potential surfaces (negative is in red, positive is in blue) of wild-type and variant proteins. The top panels show a close-up view of a cartoon representation of the region around residue 325, highlighting as sticks the side chain of residue 325 (orange) and the closest nucleotide (light blue); of note, residue 325 is not oriented toward the nucleotide base but toward the phosphate.

Figure 3

Characterization of the Effect of the E325K Variant on KLF1 Function (A) Immunoblot analysis of transfected K-562 cells shows that the E325K variant does not affect the stability of KLF1. Constructs encoding Flag-tagged KLF1 wild-type (WT), variant E325K, or empty vector were transfected in K-562 cells, and protein expression was analyzed after 24 hr by immunoblot with anti-Flag and anti-GAPDH (loading control). (B) Immunofluorescence analysis of transfected K-562 cells shows that the E325K variant does not affect the nuclear localization of KLF1. K-562 cells transfected as in (a) were analyzed after 24 hr by immunofluorescence with anti-Flag (green) along with propidium iodide for DNA staining (red). No anti-Flag staining was detected in K-562 cells transfected with the empty vector (data not shown). (C) An AQP1 promoter-reporter assay in K-562 cells shows that the E325K variant affects the transcriptional activity of KLF1. K-562 cells were cotransfected with an AQP1 promoter-Photinus luciferase (Pluc) construct and a HSV-TK promoter-Renilla luciferase (Rluc) construct for normalization, along with 2 μg of the indicated KLF1 constructs, and the luciferase activities were analyzed after 24 hr; the results are shown as means ± SD (n = 3) of Pluc activity normalized by Rluc activity. (D) A CD44 promoter-reporter assay in K-562 cells shows that the E325K variant has a dominant-negative effect on the transcription activity of KLF1. Not only does KLF1 E325K have a markedly reduced transcriptional activity, but it is also able to impede the transcriptional activity of coexpressed KLF1 wild-type. K-562 cells were cotransfected with a CD44 promoter-Pluc construct and a HSV-TK promoter-Rluc construct for normalization, along with 2 or 1 μg of the KLF1 constructs as indicated, and the luciferase activities were analyzed after 24 hr; the results are shown as means ± SD (n = 3) of Pluc activity normalized by Rluc activity.