Genetic Heterogeneity of KLF1, a Master Regulator of Erythropoiesis, Revealed an Autosomal Recessive Ψβ-Thalassemia and a Very Strong Promoter In Vivo

The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the early stage genetic programming of erythroid progenitors to promote physiological γ to β globin gene switching. Indeed, we showed that a truncation mutation (p.K288X) in KLF1 that deleted the DNA binding zinc finger domain resulted in marked KLF1 deficiency and a hematological condition that resembled a hereditary persistence of fetal hemoglobin (HbF) or a β thalassemia. Here, we describe five additional families with the same p.K288X mutation but varied hematological and HbF levels together with genetic and phenotypic data on a 600 data-set from the same Maltese population. The data accounted for a strong promoter embedded within a region of genetic heterogeneity of KLF1 that led to a ψβ thalassemia.

Whole genome sequencing on 15 subjects of six families (FamF1 - FamF6) segregating (two) p.K288X frameworks of KLF1 had variable degrees of microcytosis (MCV; 76.1fL -77.4 fL) and HbF levels (HbF 2480 mg/dL - 802mg/dL) due to complex heterozygosity between promoter, coding and truncating mutations in KLF1. Case II-6 of FamF1 with the highest HbF (2480 mg/dL) had 2 promoter and 2 coding mutations in cis and in trans to the p.K288X truncation. Nine (9) KLF1 frameworks (A - I) were derived by transmission disequilibrium analyses of the family data, each assembled from 15 mutations and resulting in 7 genotypes among the families. The p.K288X truncation was found on a second rarer framework. Additional, rarer KLF1 frameworks were found with haploview in the population dataset.

The population dataset was made up of 198 β thalassemia heterozygotes and 400 others from the clinic and the biobank and that had no β globin gene mutations, variable blood counts or hemoglobin profiles or both. They were older than 2 years of age, not pregnant and had normal iron levels. The number of KLF1 mutations differed from 0 in the wild-type framework A to 6 in one of the rare frameworks X. Six mutations were in the promoter region and 6 were in the coding region that defined a “KLF1 Variable Region” 5’ genomic coordinate 12887183 - 12888273, whereas very few were found in the 3’ (genomic coordinate 12884589 – 12884752) that defined the KLF1 “Constant Region” The Constant region has also been evolutionary conserved. It included the zinc finger domain and the proteasome binding site.

The genotype – phenotype correlations and the family data were consistent with an autosomal recessive condition that resembled a β thalassemia, thus a ψβ thalassemia. It differed from a silent thalassemia because the β globin gene sequence was wild type. It provided a diagnosis for families with iron resistant microcytosis and borderline hemoglobin phenotypes suitable for counselling in the clinical setting. It was consistent with the observation among the families regarding the high strength of the KLF1 promoter. Multiple “hits” were necessary to suppress the biosynthesis of KLF1 for hemoglobin switching to escape perinatal suppression. The effect of the 6 promoter mutations were confirmed in vitro with native and induced K562 and Hek293T cell lines.

Presumably, during normal development, the strong promoter served to rapidly drown KLF1 binding sites with KLF1 molecules to direct progenitors to erythropoiesis with the appropriate adult hemoglobin profile in the perinatal period. The diagnosis of the patients with selected genotypes due to compound and double heterozygosities in promoter and coding sequences shall further permit quantification of differential promoter function in vivo.