Abstract
Beta-hemoglobinopathies become prominent after birth due to a switch from γ-globin to the mutated β-globin. Haploinsufficiency for the erythroid specific indispensable transcription factor Krueppel-like factor 1 (KLF1) is associated with high persistence of fetal hemoglobin (HPFH). The In(Lu) phenotype, characterized by low to undetectable Lutheran blood group expression is caused by mutations within KLF1 gene. Here we screened a blood donor cohort of 55 Lutheran weak or negative donors for KLF1 variants and evaluated their effect on KLF1 target gene expression. To discriminate between weak and negative Lutheran expression, a flow cytometry (FCM) assay was developed to detect Lu antigen expression. The Lu(a−b−) (negative) donor group, showing a significant decreased CD44 (Indian blood group) expression, also showed increased HbF and HbA2 levels, with one individual expressing HbF as high as 5%. KLF1 exons and promoter sequencing revealed variants in 80% of the Lutheran negative donors. Thirteen different variants plus one high frequency SNP (c.304 T > C) were identified of which 6 were novel. In primary erythroblasts, knockdown of endogenous KLF1 resulted in decreased CD44, Lu and increased HbF expression, while KLF1 over-expressing cells were comparable to wild type (WT). In line with the pleiotropic effects of KLF1 during erythropoiesis, distinct KLF1 mutants expressed in erythroblasts display different abilities to rescue CD44 and Lu expression and/or to affect fetal (HbF) or adult (HbA) hemoglobin expression. With this study we identified novel KLF1 variants to be include into blood group typing analysis. In addition, we provide further insights into the regulation of genes by KLF1.
Highlights
Blood group phenotyping of donors is crucial to provide matched blood to anemic patients to prevent transfusion reactions and alloimmunization
Krueppel-like factor 1 (KLF1) variants can cause a multitude of phenotypes, some cause benign loss of specific blood groups while others lead to high persistence of fetal hemoglobin (HPFH) or the inherently dominant disease, congenital dyserythropoiesis type IV (CDA IV)
To investigate if HPFH is a common feature of In(Lu) individuals, or whether this is determined by specific variants and to identify novel variants in KLF1, a cohort of In(Lu) donors was investigated
Summary
Blood group phenotyping of donors is crucial to provide matched blood to anemic patients to prevent transfusion reactions and alloimmunization. Anti-Lu3 (previously known as anti-Luab) are rare antibodies found in alloimmunised individuals with the recessive type of Lu(a–b–) ([Lunull phenotype], caused by variants in the BCAM gene). These individuals should only receive Lu(a − b −). Lu negative donors with KLF1 variants displayed low levels of CD44 and in some cases, increased HbF (α2γ2), HbA2 (α2δ2) and lower HbA1 levels These effects on CD44, Hemoglobin Beta (HBB) and BCAM RNA expression on in vitro cultured In(Lu) erythroblast have been described before[2] as the result of decreased binding to the promoters of these g enes[12]. The data indicates that the pleiotropic effects on globin and RBC membrane protein expression are KLF1-variant class specific
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