A New Case of KLF1 G973A Mutation and Congenital Dyserythropoeitic Anemia (CDA)- Further Definition of Emerging New Syndrome and Possible Association with Gonadal Dysgenesis

Abstract ²¹⁰¹

Arnaud et al^a  recently reported on two children with an as yet unclassified CDA, characterized by hemolytic anemia with high fetal hemoglobin (HbF), presence of embryonic hemoglobin Portland (HbPort) and absence on red blood cells (RBC) of CD44, an adhesion molecule, aquaporin 1 (AQP1) and the linked In(Lu) and Colton blood groups. The underlying molecular defect involves the erythroid transcription factor KLF1 with a mutation in zinc finger 2 at position 973 of Guanine to Adenine. The G973A mutation results in substitution of glutamate to lysine at position 325 in the transcribed protein. The CDA results from a dominant negative effect of KLF1 E325K on the gamma-globin repressor BCL11A, leading to the persistence of fetal erythropoiesis, high Hb F and HbPort. The absence of CD44, AQP1 and deficiency of certain cytoskeletal proteins in the RBC indicate that KLF1 is necessary for the terminal differentiation of RBC. The presence of CD44 on the leucocytes in these cases would appear to suggest an erythroid restrictive effect of the mutation. Of note, the child in whom the E325K mutation was first identified, a 46XY male, was described to have micropenis and hypospadias; the hemolytic anemia was severe and required splenectomy. The original case was that of phenotypic female with 46XX and the clinical course was much milder.

We have identified a new case (a caucasian child) with the KLF1 E325K mutation. Comparison with the clinical findings in the two published cases indicates that KLF1 E325K mutation may cause profound developmental disturbances in organs beyond erythroid development, particularly of the gonads. Our patient, now 5 yrs old, was followed with an undiagnosed hemolytic anemia, which started with fetal hydrops at 23 weeks gestation, requiring intrauterine transfusions. Because of the presence of spherocytes on the blood smear and history of renal stones, a mutant band 3 was suspected; however direct nucleotide sequence analysis of the promoter and coding region exons of the band 3 gene from amplified genomic DNA of the proband was normal. Splenectomy was done at 4-years-of-age and the emerging picture was one of extreme normoblastemia, with the presence of both spherocytes and massively enlarged erythrocytes. Osmotic gradient ektacytometry showed reduced erythrocyte deformability with a population of swollen erythrocytes. Hemoglobin analyses revealed a HbF of31% and, HgPort (isoelectric focusing done by James Hoyer at Mayo Laboratories). Flow cytometry demonstrated that CD44 was not expressed on patient's erythrocytes but was expressed on lymphocytes. AQP1 was absent on western blots of RBC ghosts. Nucleotide sequence analysis of the KLF1 gene identified the KLF1 E325K mutation in lymphocyte DNA from the patient, but not in her parents. In addition to the severe anemia, the child who is 46XY, has female external genitalia. Ultrasonography demonstrated a vagina and uterus. Surgically excised streak gonads showed no cellular elements. Array comparative genomic hybridization studies did not reveal any copy number variations in male sex determining genes, but identified polymorphic deletion of estrogen metabolizing UGT2B17.

Together these 3 cases suggest that KLF1 E325K may cause more severe anemia and gonadal dysgenesis in 46XY fetuses while the disease is considerably milder in 46XX individuals. Both of the 46 XY cases also had fetal hydrops and were salvaged with transfusions in utero, suggesting that KLF1 mutations may cause early loss of male fetuses. Given that KFL1 interacts with more than 1000 proteins,^b,c  it is likely that KLF1 is important in the developmental regulation of non erythroid genes including male sex determining genes.