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Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient

ABSTRACT: Congenital dyserythropoietic anemia (CDA) type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. A molecular understanding of this disease is absent, partly due to its rarity. We expanded erythroid cells from a patient’s peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain the patients’ clinical and phenotypic features and elucidate the dominant character of the mutation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE128718 | GEO | 2019/03/23

REPOSITORIES: GEO

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Project description:Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified, but others remain elusive. Ten of twenty-seven members from a Maltese family presented with HPFH. A genome-wide SNP scan followed by linkage analysis revealed a candidate region on chromosome 19p13.12-13. Sequencing identified a nonsense mutation in the KLF1 gene, p.K288X, ablating the DNA binding domain of this key erythroid transcriptional regulator. Only HPFH family members were heterozygote carriers of this mutation. Expression profiling on primary erythroid progenitors revealed down-regulation of KLF1 target genes in HPFH samples. Functional assays demonstrated that, in addition to its established role in adult globin expression, KLF1 is a critical activator of the BCL11A gene, encoding a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels. To identify differentially expressed genes, RNA was isolated from erythroid progenitors (HEPs) cultured from peripheral blood of four HPFH (KLF1 p.K288X/wt) and four non-HPFH family members (wt/wt) and used for genome-wide expression analysis. A minimum of 1.5E6 HEPs were harvested at day12 of culture and RNA was extracted with Trizol reagent (Sigma) and purified using the RNeasy Mini Kit (Qiagen, Crawley, UK), including an on-column DNaseI digestion, according to the manufacturer’s instructions. RNA yield was determined using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 8-10 micr-g of total RNA was analysed by microarrays using cells from day 12 of culture. Quality of the total RNA samples and the resulting cRNA was assessed on the Bioanalyzer. Fragmented biotinylated cRNA was prepared and 15μg hybridized to HG-U133 plus 2 GeneChips, according to the manufacturer’s protocols (Affymetrix). The data was normalized by Robust Multi-Array average (RMA) algorithm.

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Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient

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