Novel variants in Krueppel Like Factor 1 that cause Persistence of Fetal Hemoglobin in In(Lu) individuals

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. These KLF1 variants often lead to KLF1 haploinsufficiency. We screened a donor cohort of 55 Lutheran weak or negative donors for KLF1 variants. To discriminate between weak and negative Lutheran expression, a flow cytometry (FCM) assay was developed to detect Lu polymorphisms. The Lu(a-b-) (negative) donor group, showing a significant decreased CD44 (Indian blood group) expression, also showed increased HbF and HbA2 levels, with outliers expressing >5% HbF. KLF1 exons and promoter sequencing revealed variants in 80% of the Lutheran negative donors. Thirteen different variants plus one high frequency SNP (c.304T>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.

Identification of a genomic DNA sequence that quantitatively modulates KLF1 expression in differentiating human hematopoietic cells

“…Our discovery that focused deletion of the ETV6 site was not sufficient to reliably decrease KLF1 levels, but that a larger deletion was required, makes the dual Cpf1 approach a defined way to achieve our aim of quantitatively knocking down KLF1 levels two-fold. Such a robust design may also avoid genetic modifier issues whereby there is an extensive level of variation seen in induction of HbF levels in patients containing the same monoallelic KLF1 mutation (e.g., (Eernstman et al, 2021)).…”