Compound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin

Abstract

Brief Reporthaematologica | 2011; 96 (5) 767 IntroductionPersistent expression of fetal hemoglobin (HbF) is of great clinical relevance given its role in the amelioration of the phenotype of beta-thalassemia and sickle cell anemia. Several studies have identified genes and genetic variants controlling HbF levels in adults (HBG1/HBG2, HBS1-MYB and BCL11A) able to improve the severity of the two major beta-hemoglobinopathies, beta thalassemia and sickle cell anemia. [1][2][3][4] Recently a nonsense mutation in the KLF1 gene, which ablates the DNA binding domain of this key erythroid transcriptional regulator, has been reported in a large Maltese family with hereditary persistence of HbF (HPFH). 5,6 Haploinsufficiency of KLF1 expression has been considered to be responsible for HPFH.In the Sardinian family described here, we found a marked increase of HbF only in compound heterozygotes for two KLF1 mutations and we did not confirm the KLF1 haploinsufficiency as a cause of HPFH. Moreover, we report, for the first time in humans, very high levels of zinc protoporphyrin associated with KLF1 mutations. Design and MethodsWe studied a Sardinian family with HPFH. Blood samples were obtained after informed consent. Hematologic and biochemical analyses were performed according to standard procedures. Zinc protoporphyrin in RBC was determined with ZPP hematofluorometer (AVIV Biomedical, Lakewood, NJ, USA) and blood protoporphyrin IX with a fluorometric method.Genomic DNA was obtained from peripheral blood by standard methods.Mutation analysis was performed by PCR amplification and DNA sequence analysis of the KLF1 gene using previously described primers.6 Genotyping of individual SNPs in the HBS1L-MYB (rs9399137) and BCL11A (rs11886868) loci was performed using Taqman genotyping assay (Applied Biosystem, Warrington, UK). Alpha globin and bilirubin UDP-glucuronosyltransferase (UGT1A1) gene genotyping was carried out as previously described. 7,8 The site directed mutation in K332Q in the KLF1 cDNA was obtained with the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA).Band shift, supershift, Western blots and transactivation analysisCompound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin ABSTRACT were performed as previously described. 9 The intensities of the KLF1 shifted bands were determined with the ImageQuant software after gel autoradiography on phosphoscreen and acquisition with PhosphoImager Storm 840 (GE Healthcare).The study was approved by the institutional review board of the hospital (ASL8 Ethics Commitee).

Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin

“…23 In the present reporter assays in K-562 cells, the transcriptional activities of the γ-globin promoter were unaffected, because these cells do not express BCL11A. High levels of zinc protoporphyrin were present only in compound heterozygotes with two KLF1 mutations in trans, as previously described in a Sardinian HPFH family, 13 although the combination of mutations in this study differed from the Sardinian variants c.809C4A (p. (Ser270Ter)) and c.994A4C (p.(Lys332Gln)). Surprisingly, compound heterozygotes of the KLF1 mutations c.809C4A (p. (Ser270Ter)) and c.994A4C (p.(Lys332Gln)), both of which affect the ZF domain, did not produce severe anemia or a β-thalassemia phenotype, suggesting that the c.994A4C (p.(Lys332Gln)) mutation has little impact on the transactivation capacity of KLF1.…”

“…KLF1 mutations can lead to loss or gain-of-function, giving rise to distinct phenotypic states, including CDA IV, CNSHA, and mild anemia, and induce increases in HbF level. [12][13][14] In contrast, the patients in this study exhibited the thalassemia phenotypes and unusual changes associated with KLF1 loss-of-function mutations. For instance, the microcytic hypochromic anemia with poikiloblast and abnormal heterochromatin organization in erythroblasts are not observed in CDA IV.…”

“…Surprisingly, compound heterozygotes of the KLF1 mutations c.809C4A (p. (Ser270Ter)) and c.994A4C (p.(Lys332Gln)), both of which affect the ZF domain, did not produce severe anemia or a β-thalassemia phenotype, suggesting that the c.994A4C (p.(Lys332Gln)) mutation has little impact on the transactivation capacity of KLF1. 13,17 Furthermore, the HS2 − γ prom/ Renilla − β prom/Firefly reporter assay demonstrated that the compound c.994A4C (p.(Lys332Gln)) mutations in the Sardinian family had less effect than the mutations described in the present study. (Supplementary Figure S4).…”

“…Compound heterozygotes for KLF1 mutations are associated with both benign and disease phenotypes. In one Sardinian family, compound heterozygosity for mutations in KLF1 was associated with mild anemia and increased fetal hemoglobin (HbF) and RBC protoporphyrin, 13 and a recent report described eight compound heterozygotes for mutations in KLF1 among patients presenting with severe, transfusion-dependent hemolytic anemia known as chronic nonspherocytic hemolytic anemia (CNSHA). In that study, most of the cases had co-inherited α or β thalassemia mutations and it was therefore not possible to clearly discern their hematological status.…”

Erythropoiesis and globin switching in compound Klf1::Bcl11a mutant mice

“…16,17 Furthermore, Klf1 knockout embryos carrying a human b-globin locus transgene fail to activate the human b-globin gene, while the g-globin genes are fully expressed. 18,19 Mutations in human KLF1 are associated with a spectrum of phenotypes, such as the In(Lu) blood group, 20 zinc protoporphyria, 21 increased HbA2, 22 congenital dyserythropoietic anemia (CDA), 23 and hereditary persistence fetal hemoglobin (HPFH). 13 Analysis of the HPFH phenotype has led to the proposal that KLF1 has a dual role in g-globin suppression through its preferential activation of the b-globin gene and as a key activator of expression of the BCL11A repressor protein.…”