HGNC Approved Gene Symbol:KLF1
Cytogenetic location:19p13.13 Genomic coordinates(GRCh38) :19:12,884,422-12,887,201 (from NCBI)
| Location | Phenotype View Clinical Synopses | Phenotype MIM number | Inheritance | Phenotype mapping key |
|---|---|---|---|---|
| 19p13.13 | [Hereditary persistence of fetal hemoglobin] | 613566 | AD | 3 |
| Anemia, congenital dyserythropoietic, type IVb | 620969 | AR | 3 | |
| Anemia, dyserythropoietic congenital, type IVa | 613673 | AD | 3 | |
| Blood group--Lutheran inhibitor | 111150 | AD | 3 |
The KLF1 gene encodes a transcription factor that is a master regulator of terminal erythroid differentiation by regulating several essential pathways in erythropoiesis, including the switch from fetal and embryonic hemoglobin (Hb) to adult Hb (summary byViprakasit et al., 2014).
KLF1 directs high-level expression of the adult beta-globin (HBB;141900) promoter by binding to its CACCC element (Bieker, 1996). KLF1 also acts as a transcription factor for the BCAM protein (612773) (responsible for the Lutheran (Lu) blood group;111200) as well as for other proteins expressed on erythroid cells (summary byHelias et al., 2013).
Bieker (1996) isolated KLF1, which is the human homolog of murine Eklf. The predicted KLF1 protein contains 3 zinc fingers that share more than 90% sequence similarity with, and are predicted to bind the same target sequence as, mouse Eklf. The rest of the protein is proline-rich and retains approximately 70% sequence similarity to the mouse gene. Human KLF1 is expressed in bone marrow and erythroleukemic cells lines but not in myeloid or lymphoid cell lines.
Van Ree et al. (1997) cloned KLF1 from a bone marrow cDNA library. The predicted 362-amino acid human protein is 69% identical to that of mouse Eklf. Northern blot analysis revealed expression exclusively in erythropoietic tissues (fetal liver and adult bone marrow).
Bieker (1996) determined that the KLF1 gene is contained within 3 kb of genomic DNA, and its coding region is interrupted by 2 introns whose locations are conserved with the murine gene.
Van Ree et al. (1997) mapped the KLF1 gene to chromosome 19p13.13-p13.12 by fluorescence in situ hybridization.
Nuez et al. (1995) andPerkins et al. (1995) used homologous recombination in embryonic stem cells to inactivate the mouse Eklf gene and demonstrated defective hematopoiesis. The Eklf gene was originally isolated from mouse erythroid cell RNA by differential screening and was shown to be erythroid-specific, although a lower level of Eklf was found in mast cell lines. Eklf contains 3 zinc fingers homologous to those found in the Kruppel family of transcription factors (see165220). Because it binds to the sequence CCACACCCT, EKLF was suspected to affect erythroid development through its ability to bind to the CAC box in the promoter of the beta-globin gene (HBB;141900). The mutation in this element leads to reduced beta-globin expression, and it appears to mediate the effect of the globin locus control region on the promoter. From study of transgenic mice heterozygous for a lacZ reporter sequence within the EKLF gene,Nuez et al. (1995) found that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-deficient mice appeared normal during the embryonic stage of hematopoiesis in the yolk sac, but developed a fatal anemia during early fetal life when hematopoiesis shifted to the fetal liver. Enucleated erythrocytes were formed, but these did not contain the proper amount of hemoglobin.Perkins et al. (1995) pointed out that the anemia developing during fetal liver erythropoiesis has the molecular and hematologic features of beta-globin deficiency found in beta-thalassemia. Although it is expressed at all stages, EKLF is not required for yolk sac erythropoiesis, erythroid commitment, or expression of other potential target genes. Its stage-specific and beta-globin gene-specific requirement suggests that EKLF may facilitate completion of the fetal-to-adult (hemoglobin gamma to beta) switch in humans.
EKLF is necessary for stage-specific expression of the human beta-globin gene.Armstrong et al. (1998) showed that EKLF requires a SWI/SNF-related chromatin remodeling complex, EKLF coactivator remodeling complex-1 (ERC1), to generate a DNase I hypersensitive, transcriptionally active beta-globin promoter on chromatin templates in vitro. ERC1 contains BRG1, BAF170 (601734), BAF155 (601732), and INI1 (601607) homologs of yeast SWI/SNF subunits, as well as a subunit unique to higher eukaryotes, BAF57 (603111), which is critical for chromatin remodeling and transcription with EKLF. Thus, a member of the SWI/SNF family acts directly in transcriptional activation and may regulate subsets of genes by selectively interacting with specific DNA-binding proteins.
Drissen et al. (2004) noted that, when actively expressed, the cis-regulatory elements of the beta-globin locus are in proximity in the nuclear space, forming a compartment termed the active chromatin hub (ACH).Drissen et al. (2004) found that an ACH formed at the beta-globin locus in cells from Eklf -/- mouse fetal liver, but that it was only a substructure and not the complete ACH. Further analysis showed that Eklf was directly involved in spatial organization of the beta-globin locus. The findings suggested that Eklf plays an essential role in the 3-dimensional organization of the beta-globin locus.
Using cultured erythroid progenitors derived from Eklf -/- mice,Drissen et al. (2005) showed that Eklf was required for in vitro differentiation of erythroid progenitors. RT-PCR analysis revealed reduced expression of genes involved in heme synthesis in Eklf -/- erythroid cells. Further analyses identified putative Eklf-regulated target genes, including Ahsp (605821) and Epb49 (125305). Eklf positively regulated expression of Ahsp, an abundant erythroid cell-specific protein that plays a role in hemoglobin metabolism, by forming a stable complex with free alpha-globin, in both definitive and primitive erythroid cells. Ahsp expression was downregulated in Eklf -/- erythroid cells. Epb49, a protein related to membrane stability, was the most differentially expressed membrane-specific gene in wildtype liver compared with Eklf -/- liver, and expression of Epb49 in erythroid cells was strictly dependent on the presence of Eklf.
Singleton et al. (2008) found that loss-of-function mutations in the KLF1 gene result in the dominantly inherited Lutheran-negative In(Lu) red blood cell phenotype (INLU;111150). In(Lu) was originally postulated to result from inheritance of a gene that inhibited or suppressed the Lutheran antigen gene (BCAM;612773) (Gibson, 1976). The findings ofSingleton et al. (2008) indicated that the lack of expression of the Lu antigen in this phenotype results from decreased transcription of erythroid-specific genes associated with red blood cell maturation.
By cell sorting analysis,Pilon et al. (2008) showed that the fetal liver of Eklf -/- mouse embryos did not contain terminally differentiating erythroid cells. Instead, Eklf -/- fetal liver cells had increased numbers of erythroid colony-forming cells, as erythropoiesis was blocked between the R2 and R3 stages. Transcriptional profiling identified significant perturbation of a network of genes involved in cell-cycle regulation, with the critical regulator of the cell cycle, E2f2 (600426), at a hub. E2f2 expression was markedly decreased in Eklf -/- cells, impairing cell-cycle progression from G1 into S phase in erythroid progenitor and precursor cells. Further analysis identified E2f2 as a direct target of Eklf in erythroid progenitor cells. Eklf occupied binding sites in the E2f2 promoter located in a region of Eklf-dependent DNase I sensitivity in early erythroid progenitor cells.
Schoenfelder et al. (2010) found that mouse Hbb and Hba (141800) associate with hundreds of active genes from nearly all chromosomes in nuclear foci that they called 'transcription factories.' The 2 globin genes preferentially associated with a specific and partially overlapping subset of active genes.Schoenfelder et al. (2010) also noted that expression of the Hbb locus is strongly dependent upon Klf1, while expression of the Hba locus is only partially dependent on Klf1. Immunofluorescence analysis of mouse erythroid cells showed that most Klf1 localized to the cytoplasm and that nuclear Klf1 was present in discrete sites that overlapped with RNAII foci. Klf1 knockout in mouse erythroid cells specifically disrupted the association of Klf1-regulated genes within the Hbb-associated network. Klf1 knockout more weakly disrupted interactions within the specific Hba network.Schoenfelder et al. (2010) showed that KLF1-regulated genes share KLF1-containing transcription factories and that KLF1 is required for the clustering of these coregulated genes. They concluded that transcriptional regulation involves a complex 3-dimensional network rather than factors acting on single genes in isolation.
Borg et al. (2010) demonstrated that KLF1 binds to and activates the promoter region of the BCL11A gene (606557), which is a repressor of the fetal hemoglobin genes HBG1 (142200) and HBG2 (142250). Chromatin immunoprecipitation (ChIP) assay of human erythroid progenitors from adult peripheral blood showed strong binding of KLF1 to the BCL11A promoter, whereas such binding was not observed in human fetal liver erythroid progenitors. These findings indicated that KLF1 acts as a dual regulator of fetal-to-adult globin switching in humans by acting as a preferential activator of the HBB gene and by activating expression of BCL11A, which in turn represses the HBG1x and HBG2 genes.
Arnaud et al. (2010) found that KLF1 plays a role in the expression of the water channel AQP1 (107776) and the adhesion molecule CD44 (107269) on erythroid cells.
Lutheran Red Blood Cell Group
Singleton et al. (2008) identified 9 different heterozygous loss-of-function mutations in the KLF1 gene (see, e.g.,600599.0001-600599.0004) in 21 of 24 persons with the dominant In(Lu) phenotype (INLU;111150). The individuals had no reported pathology, indicating that 1 functional KLF1 allele is sufficient to sustain human erythropoiesis. KLF1 mutations were not identified in 37 controls.
In red blood cell samples from 10 probands with the dominant In(Lu) phenotype,Helias et al. (2013) identified 10 different heterozygous loss-of-function mutations in the KLF1 gene (see, e.g.,600599.0007-600599.0009). Flow cytometric analysis indicated that the red blood cells from these individuals had some weak expression of the Lu(b) antigen and low expression of CD44 (107269). In addition, these individuals had increased levels of fetal hemoglobin (HbF) (mean of 2.14%) compared to controls (mean less than 1.0%), and slightly increased levels of HbA2 (141850). Finally, 9 In(Lu) individuals who were heterozygous for the P1 allele (607922.0007) did not express the P1 antigen (see111400), whereas 1 who was homozygous for the P1 allele expressed only weak P1. These findings suggested that the expression of P1 is suppressed in the In(Lu) blood type.Helias et al. (2013) concluded that the KLF1 haploinsufficiency has different effects on the expression of different erythroid proteins, likely reflecting the variable dependence of their respective genes on the KLF1 transcription factor.
Hereditary Persistence of Fetal Hemoglobin, KLF1-Related
In affected members of a Maltese family with hereditary persistence of fetal hemoglobin (613566),Borg et al. (2010) identified a heterozygous mutation in the KLF1 gene (K288X;600599.0005). In vitro functional expression assays showed that loss of KLF1 function resulted in decreased BCL11A expression and increased expression of the fetal hemoglobin genes HBG1 and HBG2.
Congenital Dyserythropoietic Anemia IVa
In 2 unrelated patients with congenital dyserythropoietic anemia IVa (CDAN4A;613673), one of whom was reported byWickramasinghe et al. (1991),Arnaud et al. (2010) identified a heterozygous de novo mutation in the KLF1 gene (E325K;600599.0006). The findings indicated that the KLF1 gene plays a critical role in the regulation of several genes during erythropoiesis, and that dysregulation of certain gene targets can result in dyserythropoiesis.
Congenital Dyserythropoietic Anemia IVb
In 8 unrelated patients with congenital dyserythropoietic anemia IVb (CDAN4B;620969),Viprakasit et al. (2014) identified compound heterozygous mutations in the KLF1 gene (see, e.g.,600599.0010-600599.0014). The mutations, which were found by direct sequencing of the KLF1 gene, segregated with the disorder in the families. Mutation types included missense, nonsense, frameshift, and splice site; no patient had 2 nonsense or frameshift mutations, suggesting that this would be embryonic lethal. Four unrelated patients (P5-P8) had the same genotype (A298P,600599.0010 and a frameshift,600599.0011). All patients had decreased PKLR (609712) levels, suggesting that the mutations interfered with KLF1 binding to the erythroid promoter of PKLR, and all had the rare In(Lu) blood group phenotype. The hematologic phenotypes in the patients likely resulted from the effects of KLF1 on the globin genes, the CD44 gene (107269), and the PKLR gene, which causes CNSHA2 (266200). The patients had variable abnormal patterns of globin synthesis with increased levels of fetal hemoglobin (HbF) and detectable levels of embryonic globins. In the single case (P2) that could be analyzed, the level of the KLF1 downstream target BCL11A (606557) mRNA was reduced. Fourteen of 15 heterozygous parents had increased HbF.
In a boy, born of unrelated Australian parents, with CDAN4B,Magor et al. (2015) identified compound heterozygous mutations in the KLF1 gene: a nonsense mutation (W30X;600599.0015) and a frameshift (600599.0001). Each mutation was inherited from a parent, both of whom had elevated HbF. Transcriptome analysis of patient PBMCs showed decreased BCL11A and SOX6 (607257) compared to controls. Further detailed transcriptome analysis suggested that KLF1 plays a role in the regulation of genes in multiple pathways, including the regulation of hemoglobin switching from fetal and embryonic to adult, red cell development, blood groups, cell cycle and mitosis, assembly of the cytoskeleton, hemoglobin assembly, cell signaling, and autophagy. The findings suggested that KLF1 acts indirectly through other transcription factors to repress embryonic and fetal globin genes in adults. Direct functional studies of the variants were not performed.
In a female infant, born of unrelated Chinese parents, with CDAN4B presenting as hydrops fetalis,Lee et al. (2016) identified compound heterozygous mutations in the KLF1 gene: a frameshift (600599.0011) and a missense (P338T;600599.0016). Each parent was heterozygous for 1 of the mutations. Functional studies of the variants were not performed, but the authors postulated that the P338T variant may have some residual function.
Wijgerde et al. (1996) produced a strain of Eklf-knockout mice, embryos of which expressed the epsilon- and gamma-globin genes normally. Gamma- and beta-globins were expressed with altered ratios in heterozygous knockout mouse fetal liver. Homozygous knockout mouse fetuses had no beta-globin transcription and had coincident changes in chromatin structure at the beta promoter.Wijgerde et al. (1996) proposed that EKLF stabilizes the interaction between the globin locus control region and the beta-globin gene. In addition, they considered these findings to provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by alteration of the frequency and/or duration of transcriptional periods of a gene, rather than by a change in the rate of transcription.
Zhou et al. (2010) found that transgenic mice homozygous for a deletion of the 50-bp HS1 enhancer (EHS1) in the Klf1 gene had greatly increased gene expression ratios of mouse epsilon-y2-globin/beta-globin and BAC-derived human gamma-globin/beta-globin in the liver at embryonic day 14.5. Adult erythroid progenitors isolated from the mutant mice showed markedly reduced Bcl11a expression, suggesting that Klf1 regulates Bcl11a expression. ChIP analysis showed that wildtype Klf1 binds to a CACCC box in the promoter region of Bcl11a. Studies in adult human progenitor blood cells showed that knockdown of KLF1 resulted in decreased BCL11A expression and upregulation of gamma-globin genes, similar to the mouse studies. The findings indicated that developmental stage-specific changes in KLF1 abundance mediate the competitive interactions of globin gene expression.
Lyon (1983,1986) described an ethylnitrosourea-induced mouse mutation, neonatal anemia (Nan), resulting in a semidominant hemolytic anemia that shares several features of hereditary spherocytosis (HS; see182900). Nan was mapped to mouse chromosome 8.Siatecka et al. (2010) identified the Nan mutation as a glu339-to-asp (E339D) substitution in the second C2H2 zinc finger (ZF2) motif of Eklf. E339 is absolutely conserved across the entire mouse and human KLF family and across EKLF proteins from different species. The substitution, which is conservative, did not affect Eklf protein expression, but it abrogated binding of mutant Eklf to a subset of Eklf target promoters containing a thymidine in the middle position of the Eklf-binding motif. This altered binding specificity of mutant Eklf resulted in distorted Eklf-dependent gene expression and abnormal residual embryonic beta-h1 globin expression in Nan heterozygous mice. The Nan mutation was more severe than Eklf deletion, as homozygous Nan mutant mice died at an earlier embryonic time point than Eklf -/- embryos. Furthermore, heterozygous Nan mice showed severe anemia, whereas Eklf +/- mice were indistinguishable from wildtype.
Independently,Heruth et al. (2010) identified an E339D mutation in Klf1 as the cause of HS in the Nan mouse model. The mutation is located in ZF2 of Klf1, affects a glutamic acid highly conserved among all mammalian members of the KLF family, and disrupts a motif required for interactions of ZF2 with its DNA targets. The authors verified the causative nature of the E339D mutation using an allelic test cross between Nan +/- and Klf1 +/- mice. Protein homology modeling predicted that the Klf1 E339D mutant protein would bind to CACCC elements in target genes more tightly than wildtype, suggesting that the E339D mutant protein could be a competitive inhibitor of wildtype Klf1.
In 8 individuals of English descent with the dominant In(Lu) blood phenotype (INLU;111150),Singleton et al. (2008) identified a heterozygous 1-bp duplication (c.954dupG) in exon 3 of the KLF1 gene, resulting in a frameshift and premature termination (Arg319GlufsTer34). The substitution was predicted to render the transcription factor nonfunctional. The individuals had no reported pathology, indicating that 1 functional KLF1 allele is sufficient to sustain human erythropoiesis.
For discussion of the c.954dupG in the KLF1 gene that was found in compound heterozygous state in a patient with congenital dyserythropoietic anemia, type IVb (CDAN4B;620969) byMagor et al. (2015), see600599.0015. This patient inherited the c.954dupG from a parent who had elevated fetal hemoglobin (HbF) (HBFQTL6;613566), and who had the In(Lu) serologic phenotype.
In 6 individuals of Spanish descent with the dominant In(Lu) blood phenotype (INLU;111150),Singleton et al. (2008) identified a heterozygous 1-bp deletion (c.569delC) in exon 2 of the KLF1 gene, resulting in a frameshift and premature termination (Pro190LeufsTer47). The substitution was predicted to render the transcription factor nonfunctional. The individuals had no reported pathology, indicating that 1 functional KLF1 allele is sufficient to sustain human erythropoiesis.
In an individual with the dominant In(Lu) blood phenotype (INLU;111150),Singleton et al. (2008) identified a heterozygous c.874A-T transversion in the KLF1 gene, resulting in a lys292-to-ter (K292X) substitution. The substitution was predicted to result in premature termination and a lack of all zinc finger domains.
In an individual with the dominant In(Lu) blood phenotype (INLU;111150),Singleton et al. (2008) identified a heterozygous c.895C-T transition in the KLF1 gene, resulting in a his299-to-tyr (H299Y) substitution. The substitution was predicted to result in diminished zinc binding.
In affected members of a Maltese family with hereditary persistence of fetal hemoglobin (HBFQTL6;613566),Borg et al. (2010) identified a heterozygous A-to-T transversion in the KLF1 gene, resulting in a lys288-to-ter (K288X) substitution, which was not found in 400 controls. The mutation was predicted to ablate the complete zinc finger domain and abrogate DNA binding. The truncated protein was not detected in patient cells, suggesting nonsense-mediated mRNA decay. The proband was ascertained because of mild hypochromatic microcytic indices, but no other phenotypic abnormalities were described. Gene expression profiles showed that mutation carriers had decreased expression of the fetal globin repressor BCL11A (606557), and upregulation of the fetal hemoglobin genes HBG1 (142200) and HBG2 (142250). Knockdown of KLF1 in control cells caused similar changes in gene expression, and further expression studies excluded a dominant-negative effect of the K288X mutant protein.Borg et al. (2010) concluded that haploinsufficiency for KLF1 is a cause of HPFH.
In 2 unrelated patients with congenital dyserythropoietic anemia type IVa (CDAN4A;613673),Arnaud et al. (2010) identified a heterozygous de novo c.973G-A transition in exon 3 of the KLF1 gene, resulting in a glu325-to-lys (E325K) substitution. One of the patients had previously been reported byWickramasinghe et al. (1991). The phenotype was characterized by hydrops and severe anemia at birth, ineffective erythropoiesis, nucleated peripheral red blood cells, and absence of expression of CD44 (107269) and AQP1 (107776) on erythrocytes. Both patients also showed increased fetal hemoglobin. The E325K mutation occurred in a conserved residue in the second zinc finger domain, and structural modeling predicted that the mutation would stabilize the bond between KLF1 and DNA target sequences. Expression studies in human erythroid cells showed that the mutant E325K protein had similar expression and nuclear localization as the wildtype protein. However, the mutant protein showed markedly decreased transcriptional activity toward CD44 and AQP1 compared to wildtype, consistent with the clinical findings. The mutant KLF1 protein also showed a dominant-negative effect. The findings indicated that the KLF1 gene plays a critical role in the regulation of several genes during erythropoiesis, and that dysregulation of certain gene targets can result in dyserythropoiesis.
Variant Function
By in vitro functional expression studies,Helias et al. (2013) demonstrated that the E325K mutant KLF1 protein retained transactivation activity for the BCAM (612773) promoter as well as, or ever better than, wildtype KLF1. The findings were consistent with the fact that CDAN4 is not associated with reduced levels of BCAM on red blood cells.
Using an in vitro selection strategy followed by validation,Kulczynska et al. (2020) identified the preferred DNA-binding site of mouse Klf1 with an E339K mutation, which is equivalent to the human E325K mutation. Like wildtype Klf1, the Klf1 E339K mutant interacted with a 9-nucleotide binding site. However, unlike wildtype, it only bound to a motif containing G in the fifth position, and there was high degeneracy at the 3-prime end of the motif. The binding sites for wildtype and Klf1 E339K mutant were mutually exclusive, and E339K did not bind the wildtype consensus sequence. Luciferase reporter assays in K562 cells confirmed that the Klf1 E339K mutant bound and activated the novel consensus sequence. Quantitative analysis of endogenous levels of Klf1 target genes in K562 cells expressing the E339K mutant showed that the mutant protein recognized and activated variant DNA-binding sites in some target genes, compensating for any drop in expression of wildtype Klf1. The authors concluded that consensus binding sites for E339K are uniquely recognized, bound, and transcriptionally functional endogenously in cells.
In a blood sample derived from a patient with the dominant In(Lu) blood phenotype (INLU;111150),Helias et al. (2013) identified a heterozygous c.977T-G transversion in the KLF1 gene, resulting in a leu326-to-arg (L326R) substitution in the zinc finger domain. In vitro functional expression studies indicated that the mutant protein resulted in reduced transcriptional activity of BCAM (612773) compared to wildtype KLF1, consistent with a loss of function.
In a blood sample derived from a patient with the dominant In(Lu) blood phenotype (INLU;111150),Helias et al. (2013) identified a heterozygous c.1071C-A transversion in the KLF1 gene, resulting in a his357-to-gln (H357Q) substitution in the zinc finger domain. In vitro functional expression studies indicated that the mutant protein resulted in reduced transcriptional activity of BCAM (612773) compared to wildtype KLF1, consistent with a loss of function.
In a mother and son with the dominant In(Lu) blood phenotype (INLU;111150),Helias et al. (2013) identified a heterozygous c.591C-G transversion in the KLF1 gene, resulting in a tyr197-to-ter (Y197X) substitution. Flow cytometric analysis of patient blood cells showed weak expression of the Lu(b) antigen and low expression of CD44 (107269). Both individuals also had increased levels of fetal hemoglobin (HbF) (3.5% and 3.7%, respectively) compared to non-KLF1 mutation family members (0.9-1.1% HbF).
In 4 unrelated patients (P5, P6, P7, and P8) with congenital dyserythropoietic anemia type IVb (CDAN4B;620969),Viprakasit et al. (2014) identified compound heterozygous mutations in exon 2 of the KLF1 gene: a c.892G-C transversion, resulting in an ala298-to-pro (A298P) substitution at a highly conserved residue in the ZF1 domain, and a 7-bp insertion (c.525_526insCGGCGCC;600599.0011), resulting in a frameshift and premature termination (Gly176ArgfsTer179) in the transactivating domain. Another patient (P3) was compound heterozygous for A298P and a -154C-T transition 5-prime of the initiating ATG codon (600599.0012), and another (P4) was compound heterozygous for A298P and a c.172C-T transition in exon 2, resulting in a gln58-to-ter (Q58X;600599.0014) substitution in the transactivating domain.
For discussion of the 7-bp insertion (c.525_526insCGGCGCC) in the KLF1 gene, resulting in a frameshift and premature termination (Gly176ArgfsTer179), that was found in compound heterozygous state in 4 unrelated patients with congenital dyserythropoietic anemia type IVb (CDAN4B;620969) byViprakasit et al. (2014), see600599.0010.Viprakasit et al. (2014) identified another CDAN4B patient (P2) who was compound heterozygous for the 7-bp insertion and a missense mutation in the KLF1 gene (R301H;600599.0013). The parent from whom P2 inherited the 7-bp insertion had increased HbF (HBFQTL6;613566).
In a Chinese infant with CDAN4B presenting as hydrops fetalis,Lee et al. (2016) identified compound heterozygosity for the 7-bp insertion and a missense mutation (P338T;600599.0016) in the KLF1 gene.
For discussion of the -154C-T transition in the KLF1 gene that was found in compound heterozygous state in a patient (P3) with congenital dyserythropoietic anemia type IVb (CDAN4B;620969) byViprakasit et al. (2014), see600599.0010.
In a 12-year-old boy (P2) with congenital dyserythropoietic anemia type IVb (CDAN4B;620969),Viprakasit et al. (2014) identified compound heterozygous mutations in exon 2 of the KLF1 gene: a c.902G-A transition, resulting in an arg301-to-his (R301H) substitution at a conserved residue in the ZF1 domain, and a 7-bp insertion (c.525_526insCGGCGCC;600599.0011). Erythroid cell samples from this patient showed decreased levels of BCL11A (606557). Each parent carried one of the mutant alleles; both had increased HbF (HBFQTL6;613566).
For discussion of the c.172C-T transition in exon 2 of the KLF1 gene, resulting in a gln58-to-ter (Q58X) substitution in the transactivating domain, that was found in compound heterozygous state in a patient (P4) with congenital dyserythropoietic anemia type IVb (CDAN4B;620969) byViprakasit et al. (2014), see600599.0010.
In a boy, born of unrelated Australian parents, with congenital dyserythropoietic anemia type IVb (CDAN4B;620969)Magor et al. (2015) identified compound heterozygous mutations in the KLF1 gene: a trp30-to-ter (W30X) substitution in exon 2, inherited from the father, and a frameshift (Arg319GlufsTer34;600599.0001), inherited from the mother. Both parents had mildly elevated fetal Hb (HBFQTL6;613566). The maternal frameshift KLF1 allele was represented at only 15% of total mRNA, suggesting some degree of nonsense-mediated mRNA decay; no functional protein was present from the paternal nonsense allele. Transcriptome analysis of patient PBMCs showed decreased BCL11A and SOX6 compared to controls. Further detailed transcriptome analysis suggested that KLF1 plays a role in the regulation of genes in multiple pathways, including embryonic and fetal globin, globin gene switching, red cell development, blood groups, cell cycle and mitosis, assembly of the cytoskeleton, hemoglobin assembly, cell signaling, and autophagy. Direct functional studies of the variants were not performed.
In a female infant, born of unrelated Chinese parents, with congenital dyserythropoietic anemia type IVb (CDAN4B;620969) presenting as hydrops fetalis,Lee et al. (2016) identified compound heterozygous mutations in the KLF1 gene: a c.1012C-A transversion, resulting in a pro338-to-thr (P338T) substitution, and a 7-bp insertion, resulting in a frameshift and premature termination (Gly176ArgfsTer179;600599.0011). Each parent was heterozygous for 1 of the variants. Functional studies of the variants were not performed, but the authors postulated that the P338T variant may have some residual function.
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Alternative titles; symbols
HGNC Approved Gene Symbol: KLF1
SNOMEDCT: 115824003, 719453009;
Cytogenetic location: 19p13.13 Genomic coordinates(GRCh38) : 19:12,884,422-12,887,201(from NCBI)
| Location | Phenotype | Phenotype MIM number | Inheritance | Phenotype mapping key |
|---|---|---|---|---|
| 19p13.13 | [Hereditary persistence of fetal hemoglobin] | 613566 | Autosomal dominant | 3 |
| Anemia, congenital dyserythropoietic, type IVb | 620969 | Autosomal recessive | 3 | |
| Anemia, dyserythropoietic congenital, type IVa | 613673 | Autosomal dominant | 3 | |
| Blood group--Lutheran inhibitor | 111150 | Autosomal dominant | 3 |
The KLF1 gene encodes a transcription factor that is a master regulator of terminal erythroid differentiation by regulating several essential pathways in erythropoiesis, including the switch from fetal and embryonic hemoglobin (Hb) to adult Hb (summary by Viprakasit et al., 2014).
KLF1 directs high-level expression of the adult beta-globin (HBB; 141900) promoter by binding to its CACCC element (Bieker, 1996). KLF1 also acts as a transcription factor for the BCAM protein (612773) (responsible for the Lutheran (Lu) blood group; 111200) as well as for other proteins expressed on erythroid cells (summary by Helias et al., 2013).
Bieker (1996) isolated KLF1, which is the human homolog of murine Eklf. The predicted KLF1 protein contains 3 zinc fingers that share more than 90% sequence similarity with, and are predicted to bind the same target sequence as, mouse Eklf. The rest of the protein is proline-rich and retains approximately 70% sequence similarity to the mouse gene. Human KLF1 is expressed in bone marrow and erythroleukemic cells lines but not in myeloid or lymphoid cell lines.
Van Ree et al. (1997) cloned KLF1 from a bone marrow cDNA library. The predicted 362-amino acid human protein is 69% identical to that of mouse Eklf. Northern blot analysis revealed expression exclusively in erythropoietic tissues (fetal liver and adult bone marrow).
Bieker (1996) determined that the KLF1 gene is contained within 3 kb of genomic DNA, and its coding region is interrupted by 2 introns whose locations are conserved with the murine gene.
Van Ree et al. (1997) mapped the KLF1 gene to chromosome 19p13.13-p13.12 by fluorescence in situ hybridization.
Nuez et al. (1995) and Perkins et al. (1995) used homologous recombination in embryonic stem cells to inactivate the mouse Eklf gene and demonstrated defective hematopoiesis. The Eklf gene was originally isolated from mouse erythroid cell RNA by differential screening and was shown to be erythroid-specific, although a lower level of Eklf was found in mast cell lines. Eklf contains 3 zinc fingers homologous to those found in the Kruppel family of transcription factors (see 165220). Because it binds to the sequence CCACACCCT, EKLF was suspected to affect erythroid development through its ability to bind to the CAC box in the promoter of the beta-globin gene (HBB; 141900). The mutation in this element leads to reduced beta-globin expression, and it appears to mediate the effect of the globin locus control region on the promoter. From study of transgenic mice heterozygous for a lacZ reporter sequence within the EKLF gene, Nuez et al. (1995) found that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-deficient mice appeared normal during the embryonic stage of hematopoiesis in the yolk sac, but developed a fatal anemia during early fetal life when hematopoiesis shifted to the fetal liver. Enucleated erythrocytes were formed, but these did not contain the proper amount of hemoglobin. Perkins et al. (1995) pointed out that the anemia developing during fetal liver erythropoiesis has the molecular and hematologic features of beta-globin deficiency found in beta-thalassemia. Although it is expressed at all stages, EKLF is not required for yolk sac erythropoiesis, erythroid commitment, or expression of other potential target genes. Its stage-specific and beta-globin gene-specific requirement suggests that EKLF may facilitate completion of the fetal-to-adult (hemoglobin gamma to beta) switch in humans.
EKLF is necessary for stage-specific expression of the human beta-globin gene. Armstrong et al. (1998) showed that EKLF requires a SWI/SNF-related chromatin remodeling complex, EKLF coactivator remodeling complex-1 (ERC1), to generate a DNase I hypersensitive, transcriptionally active beta-globin promoter on chromatin templates in vitro. ERC1 contains BRG1, BAF170 (601734), BAF155 (601732), and INI1 (601607) homologs of yeast SWI/SNF subunits, as well as a subunit unique to higher eukaryotes, BAF57 (603111), which is critical for chromatin remodeling and transcription with EKLF. Thus, a member of the SWI/SNF family acts directly in transcriptional activation and may regulate subsets of genes by selectively interacting with specific DNA-binding proteins.
Drissen et al. (2004) noted that, when actively expressed, the cis-regulatory elements of the beta-globin locus are in proximity in the nuclear space, forming a compartment termed the active chromatin hub (ACH). Drissen et al. (2004) found that an ACH formed at the beta-globin locus in cells from Eklf -/- mouse fetal liver, but that it was only a substructure and not the complete ACH. Further analysis showed that Eklf was directly involved in spatial organization of the beta-globin locus. The findings suggested that Eklf plays an essential role in the 3-dimensional organization of the beta-globin locus.
Using cultured erythroid progenitors derived from Eklf -/- mice, Drissen et al. (2005) showed that Eklf was required for in vitro differentiation of erythroid progenitors. RT-PCR analysis revealed reduced expression of genes involved in heme synthesis in Eklf -/- erythroid cells. Further analyses identified putative Eklf-regulated target genes, including Ahsp (605821) and Epb49 (125305). Eklf positively regulated expression of Ahsp, an abundant erythroid cell-specific protein that plays a role in hemoglobin metabolism, by forming a stable complex with free alpha-globin, in both definitive and primitive erythroid cells. Ahsp expression was downregulated in Eklf -/- erythroid cells. Epb49, a protein related to membrane stability, was the most differentially expressed membrane-specific gene in wildtype liver compared with Eklf -/- liver, and expression of Epb49 in erythroid cells was strictly dependent on the presence of Eklf.
Singleton et al. (2008) found that loss-of-function mutations in the KLF1 gene result in the dominantly inherited Lutheran-negative In(Lu) red blood cell phenotype (INLU; 111150). In(Lu) was originally postulated to result from inheritance of a gene that inhibited or suppressed the Lutheran antigen gene (BCAM; 612773) (Gibson, 1976). The findings of Singleton et al. (2008) indicated that the lack of expression of the Lu antigen in this phenotype results from decreased transcription of erythroid-specific genes associated with red blood cell maturation.
By cell sorting analysis, Pilon et al. (2008) showed that the fetal liver of Eklf -/- mouse embryos did not contain terminally differentiating erythroid cells. Instead, Eklf -/- fetal liver cells had increased numbers of erythroid colony-forming cells, as erythropoiesis was blocked between the R2 and R3 stages. Transcriptional profiling identified significant perturbation of a network of genes involved in cell-cycle regulation, with the critical regulator of the cell cycle, E2f2 (600426), at a hub. E2f2 expression was markedly decreased in Eklf -/- cells, impairing cell-cycle progression from G1 into S phase in erythroid progenitor and precursor cells. Further analysis identified E2f2 as a direct target of Eklf in erythroid progenitor cells. Eklf occupied binding sites in the E2f2 promoter located in a region of Eklf-dependent DNase I sensitivity in early erythroid progenitor cells.
Schoenfelder et al. (2010) found that mouse Hbb and Hba (141800) associate with hundreds of active genes from nearly all chromosomes in nuclear foci that they called 'transcription factories.' The 2 globin genes preferentially associated with a specific and partially overlapping subset of active genes. Schoenfelder et al. (2010) also noted that expression of the Hbb locus is strongly dependent upon Klf1, while expression of the Hba locus is only partially dependent on Klf1. Immunofluorescence analysis of mouse erythroid cells showed that most Klf1 localized to the cytoplasm and that nuclear Klf1 was present in discrete sites that overlapped with RNAII foci. Klf1 knockout in mouse erythroid cells specifically disrupted the association of Klf1-regulated genes within the Hbb-associated network. Klf1 knockout more weakly disrupted interactions within the specific Hba network. Schoenfelder et al. (2010) showed that KLF1-regulated genes share KLF1-containing transcription factories and that KLF1 is required for the clustering of these coregulated genes. They concluded that transcriptional regulation involves a complex 3-dimensional network rather than factors acting on single genes in isolation.
Borg et al. (2010) demonstrated that KLF1 binds to and activates the promoter region of the BCL11A gene (606557), which is a repressor of the fetal hemoglobin genes HBG1 (142200) and HBG2 (142250). Chromatin immunoprecipitation (ChIP) assay of human erythroid progenitors from adult peripheral blood showed strong binding of KLF1 to the BCL11A promoter, whereas such binding was not observed in human fetal liver erythroid progenitors. These findings indicated that KLF1 acts as a dual regulator of fetal-to-adult globin switching in humans by acting as a preferential activator of the HBB gene and by activating expression of BCL11A, which in turn represses the HBG1x and HBG2 genes.
Arnaud et al. (2010) found that KLF1 plays a role in the expression of the water channel AQP1 (107776) and the adhesion molecule CD44 (107269) on erythroid cells.
Lutheran Red Blood Cell Group
Singleton et al. (2008) identified 9 different heterozygous loss-of-function mutations in the KLF1 gene (see, e.g., 600599.0001-600599.0004) in 21 of 24 persons with the dominant In(Lu) phenotype (INLU; 111150). The individuals had no reported pathology, indicating that 1 functional KLF1 allele is sufficient to sustain human erythropoiesis. KLF1 mutations were not identified in 37 controls.
In red blood cell samples from 10 probands with the dominant In(Lu) phenotype, Helias et al. (2013) identified 10 different heterozygous loss-of-function mutations in the KLF1 gene (see, e.g., 600599.0007-600599.0009). Flow cytometric analysis indicated that the red blood cells from these individuals had some weak expression of the Lu(b) antigen and low expression of CD44 (107269). In addition, these individuals had increased levels of fetal hemoglobin (HbF) (mean of 2.14%) compared to controls (mean less than 1.0%), and slightly increased levels of HbA2 (141850). Finally, 9 In(Lu) individuals who were heterozygous for the P1 allele (607922.0007) did not express the P1 antigen (see 111400), whereas 1 who was homozygous for the P1 allele expressed only weak P1. These findings suggested that the expression of P1 is suppressed in the In(Lu) blood type. Helias et al. (2013) concluded that the KLF1 haploinsufficiency has different effects on the expression of different erythroid proteins, likely reflecting the variable dependence of their respective genes on the KLF1 transcription factor.
Hereditary Persistence of Fetal Hemoglobin, KLF1-Related
In affected members of a Maltese family with hereditary persistence of fetal hemoglobin (613566), Borg et al. (2010) identified a heterozygous mutation in the KLF1 gene (K288X; 600599.0005). In vitro functional expression assays showed that loss of KLF1 function resulted in decreased BCL11A expression and increased expression of the fetal hemoglobin genes HBG1 and HBG2.
Congenital Dyserythropoietic Anemia IVa
In 2 unrelated patients with congenital dyserythropoietic anemia IVa (CDAN4A; 613673), one of whom was reported by Wickramasinghe et al. (1991), Arnaud et al. (2010) identified a heterozygous de novo mutation in the KLF1 gene (E325K; 600599.0006). The findings indicated that the KLF1 gene plays a critical role in the regulation of several genes during erythropoiesis, and that dysregulation of certain gene targets can result in dyserythropoiesis.
Congenital Dyserythropoietic Anemia IVb
In 8 unrelated patients with congenital dyserythropoietic anemia IVb (CDAN4B; 620969), Viprakasit et al. (2014) identified compound heterozygous mutations in the KLF1 gene (see, e.g., 600599.0010-600599.0014). The mutations, which were found by direct sequencing of the KLF1 gene, segregated with the disorder in the families. Mutation types included missense, nonsense, frameshift, and splice site; no patient had 2 nonsense or frameshift mutations, suggesting that this would be embryonic lethal. Four unrelated patients (P5-P8) had the same genotype (A298P, 600599.0010 and a frameshift, 600599.0011). All patients had decreased PKLR (609712) levels, suggesting that the mutations interfered with KLF1 binding to the erythroid promoter of PKLR, and all had the rare In(Lu) blood group phenotype. The hematologic phenotypes in the patients likely resulted from the effects of KLF1 on the globin genes, the CD44 gene (107269), and the PKLR gene, which causes CNSHA2 (266200). The patients had variable abnormal patterns of globin synthesis with increased levels of fetal hemoglobin (HbF) and detectable levels of embryonic globins. In the single case (P2) that could be analyzed, the level of the KLF1 downstream target BCL11A (606557) mRNA was reduced. Fourteen of 15 heterozygous parents had increased HbF.
In a boy, born of unrelated Australian parents, with CDAN4B, Magor et al. (2015) identified compound heterozygous mutations in the KLF1 gene: a nonsense mutation (W30X; 600599.0015) and a frameshift (600599.0001). Each mutation was inherited from a parent, both of whom had elevated HbF. Transcriptome analysis of patient PBMCs showed decreased BCL11A and SOX6 (607257) compared to controls. Further detailed transcriptome analysis suggested that KLF1 plays a role in the regulation of genes in multiple pathways, including the regulation of hemoglobin switching from fetal and embryonic to adult, red cell development, blood groups, cell cycle and mitosis, assembly of the cytoskeleton, hemoglobin assembly, cell signaling, and autophagy. The findings suggested that KLF1 acts indirectly through other transcription factors to repress embryonic and fetal globin genes in adults. Direct functional studies of the variants were not performed.
In a female infant, born of unrelated Chinese parents, with CDAN4B presenting as hydrops fetalis, Lee et al. (2016) identified compound heterozygous mutations in the KLF1 gene: a frameshift (600599.0011) and a missense (P338T; 600599.0016). Each parent was heterozygous for 1 of the mutations. Functional studies of the variants were not performed, but the authors postulated that the P338T variant may have some residual function.
Wijgerde et al. (1996) produced a strain of Eklf-knockout mice, embryos of which expressed the epsilon- and gamma-globin genes normally. Gamma- and beta-globins were expressed with altered ratios in heterozygous knockout mouse fetal liver. Homozygous knockout mouse fetuses had no beta-globin transcription and had coincident changes in chromatin structure at the beta promoter. Wijgerde et al. (1996) proposed that EKLF stabilizes the interaction between the globin locus control region and the beta-globin gene. In addition, they considered these findings to provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by alteration of the frequency and/or duration of transcriptional periods of a gene, rather than by a change in the rate of transcription.
Zhou et al. (2010) found that transgenic mice homozygous for a deletion of the 50-bp HS1 enhancer (EHS1) in the Klf1 gene had greatly increased gene expression ratios of mouse epsilon-y2-globin/beta-globin and BAC-derived human gamma-globin/beta-globin in the liver at embryonic day 14.5. Adult erythroid progenitors isolated from the mutant mice showed markedly reduced Bcl11a expression, suggesting that Klf1 regulates Bcl11a expression. ChIP analysis showed that wildtype Klf1 binds to a CACCC box in the promoter region of Bcl11a. Studies in adult human progenitor blood cells showed that knockdown of KLF1 resulted in decreased BCL11A expression and upregulation of gamma-globin genes, similar to the mouse studies. The findings indicated that developmental stage-specific changes in KLF1 abundance mediate the competitive interactions of globin gene expression.
Lyon (1983, 1986) described an ethylnitrosourea-induced mouse mutation, neonatal anemia (Nan), resulting in a semidominant hemolytic anemia that shares several features of hereditary spherocytosis (HS; see 182900). Nan was mapped to mouse chromosome 8. Siatecka et al. (2010) identified the Nan mutation as a glu339-to-asp (E339D) substitution in the second C2H2 zinc finger (ZF2) motif of Eklf. E339 is absolutely conserved across the entire mouse and human KLF family and across EKLF proteins from different species. The substitution, which is conservative, did not affect Eklf protein expression, but it abrogated binding of mutant Eklf to a subset of Eklf target promoters containing a thymidine in the middle position of the Eklf-binding motif. This altered binding specificity of mutant Eklf resulted in distorted Eklf-dependent gene expression and abnormal residual embryonic beta-h1 globin expression in Nan heterozygous mice. The Nan mutation was more severe than Eklf deletion, as homozygous Nan mutant mice died at an earlier embryonic time point than Eklf -/- embryos. Furthermore, heterozygous Nan mice showed severe anemia, whereas Eklf +/- mice were indistinguishable from wildtype.
Independently, Heruth et al. (2010) identified an E339D mutation in Klf1 as the cause of HS in the Nan mouse model. The mutation is located in ZF2 of Klf1, affects a glutamic acid highly conserved among all mammalian members of the KLF family, and disrupts a motif required for interactions of ZF2 with its DNA targets. The authors verified the causative nature of the E339D mutation using an allelic test cross between Nan +/- and Klf1 +/- mice. Protein homology modeling predicted that the Klf1 E339D mutant protein would bind to CACCC elements in target genes more tightly than wildtype, suggesting that the E339D mutant protein could be a competitive inhibitor of wildtype Klf1.
In 8 individuals of English descent with the dominant In(Lu) blood phenotype (INLU; 111150), Singleton et al. (2008) identified a heterozygous 1-bp duplication (c.954dupG) in exon 3 of the KLF1 gene, resulting in a frameshift and premature termination (Arg319GlufsTer34). The substitution was predicted to render the transcription factor nonfunctional. The individuals had no reported pathology, indicating that 1 functional KLF1 allele is sufficient to sustain human erythropoiesis.
For discussion of the c.954dupG in the KLF1 gene that was found in compound heterozygous state in a patient with congenital dyserythropoietic anemia, type IVb (CDAN4B; 620969) by Magor et al. (2015), see 600599.0015. This patient inherited the c.954dupG from a parent who had elevated fetal hemoglobin (HbF) (HBFQTL6; 613566), and who had the In(Lu) serologic phenotype.
In 6 individuals of Spanish descent with the dominant In(Lu) blood phenotype (INLU; 111150), Singleton et al. (2008) identified a heterozygous 1-bp deletion (c.569delC) in exon 2 of the KLF1 gene, resulting in a frameshift and premature termination (Pro190LeufsTer47). The substitution was predicted to render the transcription factor nonfunctional. The individuals had no reported pathology, indicating that 1 functional KLF1 allele is sufficient to sustain human erythropoiesis.
In an individual with the dominant In(Lu) blood phenotype (INLU; 111150), Singleton et al. (2008) identified a heterozygous c.874A-T transversion in the KLF1 gene, resulting in a lys292-to-ter (K292X) substitution. The substitution was predicted to result in premature termination and a lack of all zinc finger domains.
In an individual with the dominant In(Lu) blood phenotype (INLU; 111150), Singleton et al. (2008) identified a heterozygous c.895C-T transition in the KLF1 gene, resulting in a his299-to-tyr (H299Y) substitution. The substitution was predicted to result in diminished zinc binding.
In affected members of a Maltese family with hereditary persistence of fetal hemoglobin (HBFQTL6; 613566), Borg et al. (2010) identified a heterozygous A-to-T transversion in the KLF1 gene, resulting in a lys288-to-ter (K288X) substitution, which was not found in 400 controls. The mutation was predicted to ablate the complete zinc finger domain and abrogate DNA binding. The truncated protein was not detected in patient cells, suggesting nonsense-mediated mRNA decay. The proband was ascertained because of mild hypochromatic microcytic indices, but no other phenotypic abnormalities were described. Gene expression profiles showed that mutation carriers had decreased expression of the fetal globin repressor BCL11A (606557), and upregulation of the fetal hemoglobin genes HBG1 (142200) and HBG2 (142250). Knockdown of KLF1 in control cells caused similar changes in gene expression, and further expression studies excluded a dominant-negative effect of the K288X mutant protein. Borg et al. (2010) concluded that haploinsufficiency for KLF1 is a cause of HPFH.
In 2 unrelated patients with congenital dyserythropoietic anemia type IVa (CDAN4A; 613673), Arnaud et al. (2010) identified a heterozygous de novo c.973G-A transition in exon 3 of the KLF1 gene, resulting in a glu325-to-lys (E325K) substitution. One of the patients had previously been reported by Wickramasinghe et al. (1991). The phenotype was characterized by hydrops and severe anemia at birth, ineffective erythropoiesis, nucleated peripheral red blood cells, and absence of expression of CD44 (107269) and AQP1 (107776) on erythrocytes. Both patients also showed increased fetal hemoglobin. The E325K mutation occurred in a conserved residue in the second zinc finger domain, and structural modeling predicted that the mutation would stabilize the bond between KLF1 and DNA target sequences. Expression studies in human erythroid cells showed that the mutant E325K protein had similar expression and nuclear localization as the wildtype protein. However, the mutant protein showed markedly decreased transcriptional activity toward CD44 and AQP1 compared to wildtype, consistent with the clinical findings. The mutant KLF1 protein also showed a dominant-negative effect. The findings indicated that the KLF1 gene plays a critical role in the regulation of several genes during erythropoiesis, and that dysregulation of certain gene targets can result in dyserythropoiesis.
Variant Function
By in vitro functional expression studies, Helias et al. (2013) demonstrated that the E325K mutant KLF1 protein retained transactivation activity for the BCAM (612773) promoter as well as, or ever better than, wildtype KLF1. The findings were consistent with the fact that CDAN4 is not associated with reduced levels of BCAM on red blood cells.
Using an in vitro selection strategy followed by validation, Kulczynska et al. (2020) identified the preferred DNA-binding site of mouse Klf1 with an E339K mutation, which is equivalent to the human E325K mutation. Like wildtype Klf1, the Klf1 E339K mutant interacted with a 9-nucleotide binding site. However, unlike wildtype, it only bound to a motif containing G in the fifth position, and there was high degeneracy at the 3-prime end of the motif. The binding sites for wildtype and Klf1 E339K mutant were mutually exclusive, and E339K did not bind the wildtype consensus sequence. Luciferase reporter assays in K562 cells confirmed that the Klf1 E339K mutant bound and activated the novel consensus sequence. Quantitative analysis of endogenous levels of Klf1 target genes in K562 cells expressing the E339K mutant showed that the mutant protein recognized and activated variant DNA-binding sites in some target genes, compensating for any drop in expression of wildtype Klf1. The authors concluded that consensus binding sites for E339K are uniquely recognized, bound, and transcriptionally functional endogenously in cells.
In a blood sample derived from a patient with the dominant In(Lu) blood phenotype (INLU; 111150), Helias et al. (2013) identified a heterozygous c.977T-G transversion in the KLF1 gene, resulting in a leu326-to-arg (L326R) substitution in the zinc finger domain. In vitro functional expression studies indicated that the mutant protein resulted in reduced transcriptional activity of BCAM (612773) compared to wildtype KLF1, consistent with a loss of function.
In a blood sample derived from a patient with the dominant In(Lu) blood phenotype (INLU; 111150), Helias et al. (2013) identified a heterozygous c.1071C-A transversion in the KLF1 gene, resulting in a his357-to-gln (H357Q) substitution in the zinc finger domain. In vitro functional expression studies indicated that the mutant protein resulted in reduced transcriptional activity of BCAM (612773) compared to wildtype KLF1, consistent with a loss of function.
In a mother and son with the dominant In(Lu) blood phenotype (INLU; 111150), Helias et al. (2013) identified a heterozygous c.591C-G transversion in the KLF1 gene, resulting in a tyr197-to-ter (Y197X) substitution. Flow cytometric analysis of patient blood cells showed weak expression of the Lu(b) antigen and low expression of CD44 (107269). Both individuals also had increased levels of fetal hemoglobin (HbF) (3.5% and 3.7%, respectively) compared to non-KLF1 mutation family members (0.9-1.1% HbF).
In 4 unrelated patients (P5, P6, P7, and P8) with congenital dyserythropoietic anemia type IVb (CDAN4B; 620969), Viprakasit et al. (2014) identified compound heterozygous mutations in exon 2 of the KLF1 gene: a c.892G-C transversion, resulting in an ala298-to-pro (A298P) substitution at a highly conserved residue in the ZF1 domain, and a 7-bp insertion (c.525_526insCGGCGCC; 600599.0011), resulting in a frameshift and premature termination (Gly176ArgfsTer179) in the transactivating domain. Another patient (P3) was compound heterozygous for A298P and a -154C-T transition 5-prime of the initiating ATG codon (600599.0012), and another (P4) was compound heterozygous for A298P and a c.172C-T transition in exon 2, resulting in a gln58-to-ter (Q58X; 600599.0014) substitution in the transactivating domain.
For discussion of the 7-bp insertion (c.525_526insCGGCGCC) in the KLF1 gene, resulting in a frameshift and premature termination (Gly176ArgfsTer179), that was found in compound heterozygous state in 4 unrelated patients with congenital dyserythropoietic anemia type IVb (CDAN4B; 620969) by Viprakasit et al. (2014), see 600599.0010. Viprakasit et al. (2014) identified another CDAN4B patient (P2) who was compound heterozygous for the 7-bp insertion and a missense mutation in the KLF1 gene (R301H; 600599.0013). The parent from whom P2 inherited the 7-bp insertion had increased HbF (HBFQTL6; 613566).
In a Chinese infant with CDAN4B presenting as hydrops fetalis, Lee et al. (2016) identified compound heterozygosity for the 7-bp insertion and a missense mutation (P338T; 600599.0016) in the KLF1 gene.
For discussion of the -154C-T transition in the KLF1 gene that was found in compound heterozygous state in a patient (P3) with congenital dyserythropoietic anemia type IVb (CDAN4B; 620969) by Viprakasit et al. (2014), see 600599.0010.
In a 12-year-old boy (P2) with congenital dyserythropoietic anemia type IVb (CDAN4B; 620969), Viprakasit et al. (2014) identified compound heterozygous mutations in exon 2 of the KLF1 gene: a c.902G-A transition, resulting in an arg301-to-his (R301H) substitution at a conserved residue in the ZF1 domain, and a 7-bp insertion (c.525_526insCGGCGCC; 600599.0011). Erythroid cell samples from this patient showed decreased levels of BCL11A (606557). Each parent carried one of the mutant alleles; both had increased HbF (HBFQTL6; 613566).
For discussion of the c.172C-T transition in exon 2 of the KLF1 gene, resulting in a gln58-to-ter (Q58X) substitution in the transactivating domain, that was found in compound heterozygous state in a patient (P4) with congenital dyserythropoietic anemia type IVb (CDAN4B; 620969) by Viprakasit et al. (2014), see 600599.0010.
In a boy, born of unrelated Australian parents, with congenital dyserythropoietic anemia type IVb (CDAN4B; 620969) Magor et al. (2015) identified compound heterozygous mutations in the KLF1 gene: a trp30-to-ter (W30X) substitution in exon 2, inherited from the father, and a frameshift (Arg319GlufsTer34; 600599.0001), inherited from the mother. Both parents had mildly elevated fetal Hb (HBFQTL6; 613566). The maternal frameshift KLF1 allele was represented at only 15% of total mRNA, suggesting some degree of nonsense-mediated mRNA decay; no functional protein was present from the paternal nonsense allele. Transcriptome analysis of patient PBMCs showed decreased BCL11A and SOX6 compared to controls. Further detailed transcriptome analysis suggested that KLF1 plays a role in the regulation of genes in multiple pathways, including embryonic and fetal globin, globin gene switching, red cell development, blood groups, cell cycle and mitosis, assembly of the cytoskeleton, hemoglobin assembly, cell signaling, and autophagy. Direct functional studies of the variants were not performed.
In a female infant, born of unrelated Chinese parents, with congenital dyserythropoietic anemia type IVb (CDAN4B; 620969) presenting as hydrops fetalis, Lee et al. (2016) identified compound heterozygous mutations in the KLF1 gene: a c.1012C-A transversion, resulting in a pro338-to-thr (P338T) substitution, and a 7-bp insertion, resulting in a frameshift and premature termination (Gly176ArgfsTer179; 600599.0011). Each parent was heterozygous for 1 of the variants. Functional studies of the variants were not performed, but the authors postulated that the P338T variant may have some residual function.
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