Unravelling the globin gene switch mechanism in patients with hereditary persistence of foetal haemoglobin

Abstract

Haemoglobinopathies are widely recognised as one of the most common monogenic diseases globally, representing a significant global health issue. Hereditary persistence of foetal hemoglobin (HPFH) is a benign genetic condition characterised by continuous production of high levels of foetal hemoglobin (HbF) throughout adulthood, resulting from disrupted globin gene switching. Clinical investigations and molecular findings have demonstrated that the presence of HPFH in conjunction with other haemoglobinopathies reduces the severity of associated symptoms, attributed to elevated levels of HbF. This study focused on three Maltese families, encompassing 11 individuals affected by HPFH due to a truncation mutation (p.K288X) in the KLF1 gene and 11 healthy relatives serving as controls. The primary objective was to gain insights into the underlying genetic and molecular mechanisms involved in the regulation of globin gene switching. Whole genome sequencing (WGS) was performed using DNA extracted from 11 affected individuals and 9 healthy controls. A total of 205 unique variants following a dominant inheritance pattern were identified and found to be present in all affected individuals. All these variants were discovered to be located on chromosome 19 in close proximity to the KLF1 gene. Furthermore, novel variants were discovered in the LMO2 and KLF1 genes, which potentially contribute to the onset of HPFH. A subset analysis focusing on four subjects from Fam F1, exhibiting the highest HbF levels (>3%), revealed variants in the NLRP3 gene and in the RPS9 gene when following autosomal dominant and recessive inheritance patterns, respectively. These variants likely account for the sustained elevation of HbF levels in Fam F1, despite carrying the same KLF1 mutation as other families. Flow cytometry data analysis confirmed the role of KLF1 in the regulation of antigen expression, as individuals with HPFH exhibited reduced levels of BCAM, CD44, and P1 antigens on erythrocytes. Furthermore, analysis of globin gene mRNA expression, revealed that healthy controls had elevated mRNA levels of adult α-, β-globin genes, while individuals with HPFH exhibited higher mRNA levels of the fetal Aγ-globin gene. Proteomic analysis using mass spectrometry (MS) supported these findings and additionally identified 53 proteins significantly correlated with HbF levels in HPFH-affected subjects, suggesting their involvement in globin gene regulation. In conclusion, this study highlights the importance of adapting an integrative approach to understand the molecular mechanisms underlying KLF1 deficiency. The identification of potentially causal variants associated with HPFH, provides valuable insights into the onset of this condition. Further investigations involving functional work to confirm the precise impact of these variants, as well as the role of the identified proteins in the upregulation of HbF levels, can provide a more comprehensive understanding of the underlying genetic architecture of HPFH.