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KLF1 mutation in human anemia
tions of diseased cells, whether derived from individual patients or from a more extended grouping. To surmount this, we took care in our reliance on morphological assess- ment and global expression evaluation and comparisons, particularly given the limitations with respect to flow analysis of surface differentiation markers. Importantly, the observation that our patient’s cells have difficulty in establishing differentiation in culture has been noticed in another patient.13 The only solution to this dilemma is to analyze additional patients, and/or to establish a ready source of cells (e.g. induced pluripotent stem cells from the patient) so that, at least, technical replicates can be more easily generated and analyzed.
As only BCL11A, but not ZBTB7A, levels were affected in the patient samples, it may be surprising that g-globin accounted for up to 90% of total β-like globin in our analysis, given that the present patient’s HbF levels were 42%.5 However, erythroid cells in culture may not exactly mimic the in vivo situation; for example, shRNA knock- down of ZBTB7 in differentiating human erythroblasts led to near 90% g-globin levels.55
KLF1 ablation in the mouse leads to increased megakaryocyte colony-forming potential and gene expression (at the expense of erythropoiesis) as part of its role in regulating bipotential lineage decisions in the MEP.56-60 We do not find a similar increase in expression of megakaryocyte-restricted genes61 (Online Supplementary Figure S2). Although the CDA patient cells have higher levels of FLI1 and PECAM expression, these likely follow from differentiation deficits rather than lineage diver- gence.
Patients who are compound heterozygous for KLF1 mutations present with non-spherocytic hemolytic ane- mia.27 In the present case, the extensive disruption of struc- tural and transport membrane proteins explain the mem- brane fragility and may also account for the low number of cells obtained during differentiation, when the acquisi- tion of the erythroid-specific cell membrane structure is essential for survival.62,63 This is a suboptimal situation that not only explains the hemolysis but also the apparent lack of differentiation seen in the CDA cultures and in the patient, and could also explain the lowered red cell sur- vival.6,8 In other words, rather than resulting solely from a differentiation block, the anemia and apparent hyperpla- sia may follow the physical survival and preferential enrichment of immature cells in the CDA patient.
Mechanistic implications of the E325K substitution
The dominant effect of KLF1-E325K expression follows from mutation of only one allele that is sufficient to pro- duce the altered genetic and cellular properties of the CDA red cell. Our data suggest this follows from two different causes. First, recognition by KLF1-E325K of its normal cognate site is impaired;4,10 indeed, a quantitative reduc- tion at all tested promoters by KLF1-E325 has been observed10 (K Kulczynska, 2019, submitted manuscript). KLF1 is known to interact with transcriptional regulators, his- tone modification proteins, and chromatin remodelers,17 and is critical for formation of the proper 3D chromatin complex and transcription factories at a number of ery- throid target sites.64,65 Accordingly, altered downstream
consequences in the CDA cell may well follow from destabilized or incorrect protein complex formation that interferes with optimal WT activity.10
This effect is likely augmented/intensified by our sec- ond major observation that total KLF1 RNA levels are low. Hypomorphic levels of KLF1 are known to negatively affect expression of only a minority of selected targets, and these effects appear phenotypically benign.19,23-25 However, in the present scenario low expression has been compounded by co-expression of a mutant allele. The sum of these changes is a dramatically dysregulated ery- throid cell with altered physical and expression parame- ters.
It is instructive to compare our data with that from the Nan mouse.31,66 Although the amino acid substitution is different, this mouse is anemic by virtue of intrinsic red cell parameters that are changed, many in a similar way to that of the CDA type IV patient. However, there are two fundamental differences. 1) The most obvious difference is that a substitution of lysine for glutamic acid (in our patient) is not expected to yield the same effect as substi- tution of aspartic acid (in the Nan mouse), as the location of this change is at a critical DNA recognition amino acid in the zinc finger structure. 2) The more subtle corollary of this is that the Nan mutation only affects a subset of its normal 5’CCMCRCCCN3’ target sites (because Nan- KLF1 still recognizes 5’CCMCGCCCN3’); as a result, many KLF1 targets are not affected in the Nan erythroid cell.31 This is less the case in the present situation, as the lysine substitution in KLF1-E325K would not favor recog- nition of a sequence with “R” in the middle position.10,32,48,49
However, one important concept derived from the Nan mouse is directly relevant to the CDA erythroid cell: that any amino acid change at this critical glutamic acid residue (to D or to K) leads to recognition of an abnormal target sequence, and thus ectopic expression of genes normally not present in the red cell.32 Such misexpression in the Nan mouse led to measurable and physiologically effective lev- els of secreted proteins in the serum that contributed to its splenomegaly and anemia.33 We suggest a similar occur- rence here that could contribute to some of the non-ery- throid characteristics (short stature, gonadal dysgenesis) observed in many of the CDA type IV patients. Some of the most highly dysregulated genes described here predict that it might be useful to monitor respiratory and/or autoimmune/inflammatory issues in CDA patients.50,51,67 In this context, it will be of interest to compare RNA expres- sion profiles of as many of the other patients as possible, particularly with respect to gender differences and identi- fication of potential modifier loci that affect the other, non-shared phenotypes of the CDA type IV patients.13
Acknowledgments
This work was supported by National Institutes of Health grant R01 DK046865 to JJB, by the Myeloproliferative Neoplasms Research Consortium to ARM, and by R01 HL134684 to JJB and ARM. We thank Nithya Gnanapragasam, Kaustav Mukherjee, Li Xue, and Giovanni Miglicaccio for discussion throughout the study; Sunita D’Souza for PBMC purification; and Ravi Sachidanandam and Saboor Hekmaty for library preparation and deep sequencing.
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