Case Report of Erythroid Transcription Factor EKLF Mutation Causing a Rare Form of Congenital Dyserythropoetic Anemia in a Patient of Taiwanese Origin

Abstract 2154

Congenital dyserythropoietic anemias (CDA) are a rare, inherited form of blood disorders characterized by dyserythropoiesis in the bone marrow, anemia, jaundice and splenomegaly. There are three major types of CDAs, although rarer variants have been identified. We describe a patient with an unusual type of CDA, of which only four other cases have been reported. Our patient had severe hemolytic anemia, increased fetal hemoglobin and abnormal bone marrow pathology inconsistent with previously described forms of CDA. On further study he was also found to have a mutation in KLF1, the gene encoding Erythroid-Kruppel like growth factor (EKLF). Here we describe the full clinical characteristics of our patient and define the diagnostic clinical features of this new variant of CDA by comparing with one of the previously reported patients designated as ME in the table.

EKLF is an erythroid specific transcription factor that is essential for b-globin expression, the switch from fetal to adult globin and definitive erythropoiesis (Siatecka, M and Bieker, J. Blood prepublished May 2011). Various mutations in KLF1 have been identified, some causing the benign In(Lu) type of Lu blood group phenotype. Recently, a missense, dominant-negative KLF1 mutation was reported, c.973G>A, which resulted in a previously unidentified type of CDA (Singleton et al. ASH Abstract 162, 2009; Arnaud et al. Am J Hum Genet 2010,87:721-727).

The G-to-A transition in exon 3 of KLF1 results in the substitution of glutamate 325 by a lysine (E325K) in the second zinc finger. The mutated area of the zinc finger was found to be essential for binding of EKLF to DNA motifs causing a profound dysregulation of globin gene expression. The mutation was found to have a dominant-negative effect on the transcriptional activity of EKLF, thus making the heterozygous patients symptomatic.

Our patient, JL, is an 8 year old male Taiwanese immigrant found to have hyperbilirubinemia and anemia at birth. He is a developmentally normal child with height 10^th centile, weight in the 25^th centile and spleen palpable to his suprapubic area. He has chronic hemolytic anemia, with baseline hemoglobin 7–9 g/dL, MCV 83 fL and RDW 22% and reticulocyte count 16%. Peripheral blood smear shows marked anisopoikilocytosis, schistocytes, mild polychromasia and nucleated RBCs, many with double nuclei. Bone marrow aspirate revealed a hypercellular marrow with erythroid hyperplasia and dyserythropoiesis. Electron microscopy analysis of the bone marrow showed rare immature erythroid cells with marked heterochromatin. Several cells showed a peripheral double membrane of the cytoplasm and there was rare invagination of nuclear membrane with intranuclear precipitated material.

JL received blood transfusions every 2 months for the first 3 years of his life while living in Taiwan. Since moving to the U.S. in 2003, he has only received 2 transfusions secondary to aplastic crisis. Osmotic fragility testing showed mildly increased increased fragility. Hemoglobin electrophoresis revealed an elevated fetal hemoglobin level of 42%. Gene analysis for alpha or beta globin mutations was negative. RBC enzyme testing revealed an ADA of 6.1 and decreased FFK. Due to the unique combination of anemia, elevated fetal hemoglobin, and bone marrow morphology suggestive of, but not fully diagnostic for CDA I, II or III, we tested his EKLF gene and identified the heterozygous E325K mutation. JL's two siblings, mother and father have normal hemoglobin levels and peripheral blood smears. They also have normal In(Lu) blood group phenotype. Since the E325K mutation is dominant-negative, his phenotypically normal family members were not tested for the mutation.

Four other patients have also been identified as having an E325K mutation in their EKLF gene. The patients had severe hemolytic anemia, elevated fetal hemoglobin, and bone marrow morphology showing dyserythropoiesis. Patients' erythrocytes also had low CD44 and water channel AQP1 expression, which is known to be regulated by EKLF. One patient was described with multiple congenital anomalies: hepatomegaly, micropenis, hypospadias, enlarged fontanel and hypertelorism. Taken together, the key clinical characteristics of this rare CDA are: severe normocytic anemia, highly elevated Hb F, presence of nucleated RBCs in the peripheral blood, erythroid hyperplasia with limited dyserythropoiesis in the bone marrow, splenomegaly, and growth delay.