UNASSIGNED: Clinical manifestations of patients with Hemoglobin E/beta-thalassemia vary from mild to severe phenotypes despite exhibiting the same genotype. Studies have partially identified genetic modifiers. We aimed to study the association between rare variants in protein-coding regions and clinical severity in Thai patients.
UNASSIGNED: From April to November 2018, a case-control study was conducted based on clinical information and DNA samples collected from Thai patients with hemoglobin E/beta-thalassemia over the age of four years. Cases were patients with severe symptoms, while patients with mild symptoms acted as controls. Whole exome sequencing and rare variant association study were used to analyze the data.
UNASSIGNED: All 338 unrelated patients were classified into 165 severe and 173 mild cases. Genotypes comprised 81.4% of hemoglobin E/beta-thalassemia, 2.7% of homozygous or compound heterozygous beta-thalassemia, and 0.3% of (δβ)0 thalassemia Hb E while 15.7% of samples were not classified as beta-thalassemia. A novel cis heterozygotes of IVS I-7 (A > T) and codon 26 (G > A) was identified. Six genes (COL4A3, DLK1, FAM186A, PZP, THPO, and TRIM51) showed the strongest associations with severity (observed p-values of <0.05; significance lost after correction for multiplicity). Among known modifiers, KLF1 variants were found in four mild patients and one severe patient.
UNASSIGNED: No rare variants were identified as contributors to the clinical heterogeneity of hemoglobin E/beta-thalassemia. KLF1 mutations are potential genetic modifiers. Studies to identify genetic factors are still important and helpful for predicting severity and developing targeted therapy.

The master erythroid regulator KLF1,plays a pivotal role during erythroid lineage development by regulating the expression of many erythroid genes. Variations in the KLF1 gene are found to be associated with varied erythroid phenotypes. With the aim of determining the role of KLF1 gene variations in HbF induction and their genotype phenotype relationship, in this study, we screened 370 individuals with different hemoglobinopathy condition. Hematological analysis was carried out using automated blood cell counter and Variant II HPLC (Biorad). KLF1 gene mutations were screened using automated DNA sequencing. Expression analysis was carried out using q-RT PCR of KLF1, BCL11A and γ-globin after selective enrichment and culturing of CD 34 +ve cells into an erythroid lineage. Over all 14 KLF1 gene variations were identified, of which six variants were novel. The incidence of KLF1 gene mutations was found to be 8.1%. It was seen that KLF1 mutations contributed in borderline HbA2 levels as 7.6% of our borderline HbA2 cases showed presence of KLF1 variations. It also contributed in induction of HbF levels under stress erythropoietic conditions. Gene expression studies revealed inverse correlation of KLF1, BCL11A (reduced) with γ-globin gene expression (increased) in patients showing KLF1 gene mutations, thus indicating the role of KLF1 gene in regulating the γ-globin gene expression. The identification of genomic variants of the KLF1 may help in determining the functionally active domain of this protein and will facilitate in understanding the wide spectrum of phenotypes generated by these variants.

The Kruppel-like factor 1 (KLF1) gene is an essential transcription factor that is required for the proper maturation of the erythroid cells. Recent studies have reported that KLF1 variations are associated with increased fetal hemoglobin (HbF) levels. Here we report a novel KLF1 gene variation codon 211 A→G (c.632 A>G) in a family who was referred for hemoglobinopathy screening. Both parents were classical β-thalassemia trait (mother: HbA2 4.1%, HbF 8.6%; father: HbA2 5.5%, HbF 0.6%) codon 15 G→A heterozygous, and the child was β-thalassemia homozygous. Because the mother showed a high HbF level, the genetic determinants for raised HbF were screened. We detected a novel KLF1 gene variant in the mother and the child in the heterozygous state. The co-inheritance of this novel KLF1 variant might have increased the HbF levels in the mother and may have ameliorated the clinical manifestations of the 6-year-old untransfused β-thalassemia homozygous child. Identification of KLF1 gene variants may act as a novel target for increasing HbF levels in patients with β-hemoglobinopathies.