Hereditary persistence of fetal hemoglobin in two patients with KLF1 haploinsufficiency due to 19p13.2–p13.12/13 deletion
Authors: Abdelhafid Natiq, Philippe A. Lysy, Nynke Gillemans, Rianne Schaap, Abdelaziz Sefiani, Saaid Amzazi, Siham Chafai El‐Alaoui, Ileana Cantú, Bella Banjanin, Kirsten van Lom, Cornelis L. Harteveld, Sjaak Philipsen
Journal: American Journal of Hematology (2016)

To the Editor: Expression of fetal hemoglobin (HbF, composed of two α-globin and two γ-globin chains) is clinically relevant since high HbF levels ameliorate the symptoms of β-thalassemia and sickle cell disease patients. In adults, the expression of HbF is normally reduced to less than 1% of total hemoglobin (Hb). In rare individuals expression of HbF is sustained throughout life, a condition known as hereditary persistence of fetal hemoglobin (HPFH). Such individuals display a wide range of HbF levels, from ∼1% to over 20% of total Hb. This depends on the particular combination of genomic variants carried by HPFH individuals 1. Genome-wide association studies have revealed that variants in the HBB locus, in which all β-like globin genes reside, and in the MYB and BCL11A loci, encoding DNA-binding transcription factors, account for ∼50% of the variability in HbF levels 1. In addition to GWAS studies, genome-wide linkage analysis in a Maltese pedigree revealed that haploinsufficiency for the erythroid transcription factor KLF1 causes HPFH 2. To date, over 65 nonsense, frameshift and missense variants in the KLF1 transcription factor have been reported 3. KLF1 variants have been linked to a broad spectrum of erythroid phenotypes, including diminished expression of blood group antigens such as Lutheran (BCAM) and Indian (CD44), and a consistent but highly variable increase in HbF levels 2, 3. The effects of deletion of the KLF1 gene have not been reported previously. Here we describe erythroid parameters of two independent patients with microdeletion of chromosome 19p13.2p13.12/13 in which the deleted area includes KLF1 4, 5. Patient 1 is a case of Syndromic Craniosynostosis with cryptic 19p13.2-p13.13 deletion 4. Array comparative genomic hybridization (aCGH) revealed a 3 Mbp interstitial deletion on chromosomal bands 19p13.2–19p13.13. The outer boundaries of the breakpoints are chr19:10,274,975-13,308,389 (GRCHh38p.5). This region contains 114 genes/ESTs between the ICAM1 and CACNA1A genes. Patient 2 is a case of 19p13.2-p13.12 deletion 5. aCGH revealed a microdeletion of 1.45 Mb in 19p13.2-p13.12 with breakpoints at genomic positions 12,580,427-14,030,732 (GRCHh38p.5). This region contains 44 genes. Patient 2 displayed psychomotor and language delay, intellectual disability, seizures, hypotonia, skeletal anomalies and facial dysmorphism 5. In both patients, the deletion had been acquired de novo. The region of overlap between the patients covers 728 kb on chr19p13.2-p13.12:12,580,427-13,308,389 (Supporting Information Fig. 1). Hematological analysis has not been reported previously for these patients. At the time of blood collection, Patient 1 was 10 years and 11 months old, and Patient 2 was 4 years and 6 months old. Automated complete blood counts were performed and values outside normal range are indicated in Supplementary Table I. The subtly increased red cell distribution width and immature reticulocyte fraction, and the mild reduction in reticulocyte hemoglobin content are consistent with KLF1 haploinsufficiency 2, 3, as are the slightly increased reticulocyte counts and modestly decreased mean corpuscular hemoglobin content observed only in Patient 2. Reduced expression of CD44, carrying the Indian blood group, is another hallmark of KLF1 haploinsufficiency 2, 3, and flow cytometry analysis revealed that both patients displayed markedly reduced expression of CD44 (Fig. 1A). Expression of CD235a (glycophorin A, GPA) was normal in Patient 1 and slightly reduced in Patient 2 (Fig. 1A). Next, we analyzed hemoglobin by HPLC and expression of globin mRNAs by quantitative S1 nuclease protection assays and RT-qPCR analysis 2. We found that Patient 1 displayed 7.1% HbF (Fig. 1B), this was reflected in γ-globin mRNA expression as measured by quantitative S1 nuclease protection assays (Fig. 1C) and RT-qPCR analysis (Fig. 1D). Patient 2 displayed 16.8% HbF (Fig. 1B), this was also mirrored in γ-globin mRNA expression (Fig. 1C,D). Thus, consistent with KLF1 haploinsufficiency 2, 3, both patients displayed a HPFH phenotype. Potentially, variants in the HBA and HBB loci and in the remaining KLF1 allele could contribute to the high variability in HbF levels between cases with KLF1 haploinsufficiency. We therefore analyzed the remaining KLF1 allele and the HBA and HBB loci (see data supplement for details). Sequencing of the three exons and exon/intron junctions of KLF1 revealed wildtype sequences for both patients. Thus, no variants were detected in the remaining KLF1 allele of the patients. Analysis of HBA and HBB loci revealed that in Patient 1 no variants or deletions were found that could cause α-thalassemia or β-thalassemia, and no large deletions associated with HPFH were present. In the HBG2 promoter homozygosity for HBG2: c.−53-158C > T (−158C > T, XmnI polymorphism, rs7482144) was found. This variant contributes to increased HbF levels under conditions of erythropoietic stress 1. In Patient 2 no variants or deletions were found that could cause α-thalassemia or β-thalassemia, and no large deletions associated with HPFH were present. In the HBG1 promoter a heterozygous 4 bp deletion HBG1:c.−53-225_-53-222delAGCA (e.g. −225_-222delAGCA) was present. This deletion is associated with relatively low expression of the HBG1 gene 6. Finally, we genotyped SNPs in the HBS1L-MYB and BCL11A loci that are associated with variations in HbF levels in adults 1. We observed that the SNP genotypes at the HBS1L-MYB and BCL11A loci were not particularly favorable for high HbF expression. In summary, we report hematological parameters of two unrelated patients with microdeletion of 19p13.2–p13.12/13. The commonly deleted region between the patients encompasses 728 kb and contains 33 protein-coding genes including KLF1. In the two patients reported here, the remaining KLF1 allele encodes a wildtype protein. The HbF levels of 7.1% and 16.8% can therefore not be attributed to the presence of a KLF1 variant on the remaining allele. In a Maltese pedigree in which HPFH was caused by KLF1 haploinsufficiency 2, HbF levels varied from 3.3% to 19.5% between HPFH individuals. Thus, the HbF levels of our two patients fall within this range. There is currently no explanation for this high variability in HbF levels. The two patients reported here are the first in which the entire KLF1 gene is deleted. We note that the common deleted region contains four genes (DNASE2, KLF1, LYL1, PRDX2) with known roles in erythropoiesis, and one (NFIX) that was recently linked to fetal hemoglobin levels in a population study (Supporting Information Fig. 1). It remains to be established whether haploinsufficiency for any of these genes contributes to the erythroid phenotypes of the patients. In addition, HBG1/2 promoter variants may contribute to HbF levels in the context of KLF1 haploinsufficiency. In Patient 1, homozygosity for HBG2:c.−53-158C > T (XmnI polymorphism, rs7482144) was found. A positive correlation between the T allele and HbF levels in the context of KLF1 haploinsufficiency has been previously noted 2. In Patient 2, heterozygosity for a 4 bp deletion in the HBG1 promoter was observed (HBG1:c.−53-225_-53-222delAGCA). However, this deletion is associated with low expression of the HBG1 gene 6. In conclusion, the clinical observations reported here support the notion that attenuation of KLF1 activity could be developed as a therapeutic approach to increase HbF levels in β-hemoglobinopathy patients. In addition, it will be of interest to investigate the potential role in HbF modulation of the other ‘erythroid’ genes deleted in both patients, DNASE2, LYL1, PRDX2 and NFIX. With the advent of CRISPR/Cas9 genome editing technology for the study of human erythropoiesis, systematic analysis of the roles of these genes in human erythroid cells will be the topic of future research in our laboratories. Analysis of two patients with 19p13.2–p13.12/13 deletion. A. Flow cytometry analysis of CD44 and GPA expression. Expression of GPA (CD235a) and CD44 on erythrocytes was assessed by flow cytometry. Expression of GPA is normal in Patient 1 and slightly reduced in Patient 2. CD44 expression is strongly reduced in Patient 1 and Patient 2. The control was an unrelated healthy blood donor. B. HPLC analysis of HbF levels. HbF and HbA2 levels in peripheral blood were determined by HPLC and are show as percentage of total Hb. The control was an unrelated healthy blood donor. C, D. Expression of globin mRNAs. C. Quantitative S1 nuclease protection assays show 7% (Patient 1) and 17% (Patient 2) γ-globin expression of total β-like globin mRNA. D. RT-qPCR analysis of globin mRNA expression in Patient 1 and Patient 2. ct = control, unrelated healthy donor. RNA was isolated from peripheral blood. [Color figure can be viewed at wileyonlinelibrary.com] Abdelhafid Natiq,1,2,† Philippe A. Lysy,3† Nynke Gillemans,4 Rianne Schaap,5 Abdelaziz Sefiani,1,6 Saaid Amzazi,2 Siham Chafai El-Alaoui,1,6 Ileana Cantú,4 Bella Banjanin,4 Kirsten van Lom,7 Cornelis L. Harteveld,5 and Sjaak Philipsen4* 1Département de Génétique Médicale, Institut National d'Hygiène, Rabat, Morocco; 2Faculté des Sciences de Rabat, Université Mohamed V Agdal, Rabat, Morocco; 3Pediatric Endocrinology Unit, Cliniques Universitaires Saint Luc, Université Catholique de Louvain, Brussels, Belgium; 4Erasmus MC Department of Cell Biology, Rotterdam, The Netherlands; 5Department of Clinical Genetics (LDGA), Leiden University Medical Centre, Leiden, The Netherlands; 6Centre de Génomique Humaine, Faculté de Médecine et de Pharmacie, Université Mohammed V, Rabat, Morocco; 7Erasmus MC Department of Hematology, Rotterdam, The Netherlands Additional Supporting Information may be found in the online version of this article. Additional Supporting Information may be found in the online version of this article. Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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