Identification and Functional Analysis of Known and New Mutations in the Transcription Factor KLF1 Linked with β-Thalassemia-like Phenotypes

SIMPLE SUMMARY: A key regulator of erythropoiesis is the erythroid transcriptional factor Krüppel-like factor 1 (KLF1). Increased fetal hemoglobin (HbF) and hemoglobin A2 (HbA2) levels have been associated with KLF1 haploinsufficiency mutations and have been shown to mitigate the severity of β-thalassemia. In our study, two novel KLF1 haploinsufficiency mutations that result in enhanced fetal-globin gene expression, C94X and P173fxP236, are described and functionally characterized. Furthermore, our analysis also shows that some in cis KLF1 mutation combinations may aggravate the phenotype of β-thalassemia. Further understanding of the multiple functions of KLF1 in erythropoiesis is achieved by this study, which also emphasizes the possibility that a subset of KLF1 mutations may be responsible for the severity of the thalassemia phenotype. These findings support the relevance of KLF1 screening for genetic counseling and the efficacy of programs for hemoglobinopathies prevention screening. ABSTRACT: The erythroid transcriptional factor Krüppel-like factor 1 (KLF1) is a master regulator of erythropoiesis. Mutations that cause KLF1 haploinsufficiency have been linked to increased fetal hemoglobin (HbF) and hemoglobin A(2) (HbA(2)) levels with ameliorative effects on the severity of β-thalassemia. With the aim of determining if KLF1 gene variations might play a role in the modulation of β-thalassemia, in this study we screened 17 subjects showing a β-thalassemia-like phenotype with a slight or marked increase in HbA(2) and HbF levels. Overall, seven KLF1 gene variants were identified, of which two were novel. Functional studies were performed in K562 cells to clarify the pathogenic significance of these mutations. Our study confirmed the ameliorative effect on the thalassemia phenotype for some of these variants but also raised the notion that certain mutations may have deteriorating effects by increasing KLF1 expression levels or enhancing its transcriptional activity. Our results indicate that functional studies are required to evaluate the possible effects of KLF1 mutations, particularly in the case of the co-existence of two or more mutations that could differently contribute to KLF1 expression or transcriptional activity and consequently to the thalassemia phenotype.