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. 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.

Identification of Lutheran Blood Groups and Genetic Variants within KLF1 among Thai Blood Donors

“…Interestingly, the KLF1 variant alleles found in two donors are characterised by combinations of the above mentioned polymorphisms in cis , as c.[304T>C, 1001C>G] and c.[304T>C, 519_525dupCGGCGCC]. Although the effects of missense variant c.304T>C have been reported to cause the In(Lu) phenotype in the early publication, this variant has been shown by three groups to have no impact on Lu a and Lu b expression [ 26 , 31 , 32 ]. Helias et al [ 26 ] showed c.304T>C is relatively frequent in general population and thus could not be responsible for the rare In(Lu) phenotype.…”

“…The LU (now BCAM) gene is located on chromosome 19 at 19q13. 32 and is organised into 15 exons distributed over approximately 12.5 kb. The Lu a and Lu b antigens have codominant allelic relationships and represent a single nucleotide variant (SNV), rs28399653, c.230A>G (p. His77Arg) in exon 3 of the BCAM gene [7,8].…”

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

“…In recent years, it has emerged that KLF1 contributes to control adult and fetal globin gene expression. Accordingly, several reports have revealed that heterozygosity for loss-of-function mutations in KLF1 can ameliorate the severity of hemoglobinopathies by leading to persistent fetal globin gene expression in adult life (HPFH) [ 13 , 24 , 52 , 53 , 54 ]. Therefore, besides providing crucial information about the molecular processes that regulate the transcriptional regulation of the human fetal globin genes, these conditions have gained considerable clinical interest, given the potential therapeutic role of increased HbF levels in hemoglobinopathies.…”

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

“…A perplexing observation in the field is the extensive variation in HbF levels seen in individuals containing the same monoallelic KLF1 mutation, even within the same family pedigree 16 , 29 , 95 . This was also seen in our own studies (comparison of the dual gRNA data between Figs.…”

“…3 and 4 ). This is thought to be due to variable penetrance and microvariation of KLF1 expression as contributed by additional mutations on the remaining WT allele 76 , 95 . Our data suggest that the overall architecture of the KLF1 genomic region is buffered against localized changes having a substantive effect on its expression.…”