KLF1 gene and borderline hemoglobin A2 in Saudi population

Abstract

Introduction: Elevated HbA₂ (hemoglobin A₂) level is considered the most reliable hematological parameter for the detection of β-thalassemia carriers. However, some carriers are difficult to recognize because the level of HbA₂ is not in the distinctive carrier range, i.e. 4.0-6.0%; instead, some carriers have HbA₂ levels between normal and carrier levels, i.e. borderline HbA₂ (HbA₂ = 3.1-3.9%). Studies have shown that variations in the erythroid Krüppel-like factor (KLF1) gene lead to borderline HbA₂ in β-thalassemia carriers from various populations. The incidence of borderline HbA₂ in Saudis is high.

Material and methods: To confirm the influence of variations in KLF1, HBA1, HBA2 and HBB genes for the reduction of the level of HbA₂ in Saudi β-thalassemia carriers, we performed a direct sequence analysis of KLF1, HBA1, HBA2 and HBB genes from 212 healthy Saudis (88 subjects: HbA₂ < 3; 72 subjects: HbA₂ = 3.1 to 3.9; 52 subjects HbA₂ > 4.3).

Results: The presence of the borderline HbA₂ level is not specific to any type of β-thalassemia variation or β⁺-thalassemia variations in Saudis. Two exonic (c.304T>C and c.544T>C) and two 3' untranslated region (3'UTR) (c.*296G>A and c.*277C>G) variations have been identified in the KLF1 gene for the first time from an Arab population. None of these four variations in KLF1 genes are significantly associated with the Saudis with borderline HbA₂. α Globin genotype, -α₂^3.7/α₁α₂, is found to be the most frequent (55.55%) among healthy Saudis with borderline HbA₂ compared with the other groups (HbA₂ < 3 = 20.45%; HbA₂ > 4.3 = 13.51%).

Conclusions: Further studies are necessary to determine the influence of other factors on the presence of borderline HbA₂ in 41.67% of Saudis.

Keywords: HBA1 gene; HBA2 gene; HBB gene; KLF1 gene; Saudi Arabia; borderline HbA2; variations; β-thalassemia carrier.

Figure 1

Sequencing chromatogram of newly identified variations in KLF1 gene. A – Multiple sequence alignment of KLF1 gene sequences, shows varied base pairs on 3′ untranslated region at *296th and at *277th on KLF1 gene sequences. B – Sequencing chromatogram and single base substitution (substituted base is indicated by the colored box) at the *296th (violet box) and at the *277th (nut brown box) positions. Homo variant: Homozygous variant sequence due to substitution at *296th G>A and *277th C>G in both the chromosomes. Hetero variant: Heterozygous variant sequence due to substitution at *296th G>A and *277th C>G in single chromosome resulted in peaks overlapping. Wild type: Wild type sequence of the sense strand. C – Multiple sequence alignment of all sequences, shows variegated base pairs on intronic region at 544th position. D – Sequencing chromatogram and single base substitution (substituted base is indicated by the nut brown box) at 544th position. Homo variant: Homozygous variant sequence due to substitution at 544th T>C in both the chromosomes. Hetero variant: Heterozygous variant sequence due to substitution at 544th T>C in single chromosome resulted in peaks overlapping. Wild type: Wild type sequence of the sense strand. E – Concentration of MCV (fl) in different groups of subjects with KLF1 gene variations. HomoA: Homozygous for NM_006563.3:c.*296G>A and NM_006563.3:c.*277C>G. HeteroA: Heterozygous for NM_006563.3:c.*296G>A and NM_006563.3:c.*277C>G. HomoB: Homozygous for NM_006563.3:c.544T>C. HeteroB: Heterozygous for NM_006563.3:c.544T>C. C: Control. F – Percentage of HbA2 and HbF values in different groups of subjects with KLF1 gene variations. HomoA: Homozygous for NM_006563.3:c.*296G>A and NM_006563.3:c.*277C>G. HeteroA: Heterozygous for NM_006563.3:c.*296G>A and NM_006563.3:c.*277C>G. HomoB: Homozygous for NM_006563.3:c.544T>C. HeteroB: Heterozygous for NM_006563.3:c.544T>C. C: Control

Figure 2

Protein models of wild (left) and variant (right) KLF1 gene. Arrow indicates the mutated region due to the S102P (NM_006563.3:c.304T>C) and F182L (NM_006563.3:c.544T>C) variations in the KLF1 gene. Bottom: Active site of KLF1 protein. Predicted amino acid positions on active site: 159, 168, 187, 206, 210, 211, 214, 215, 218, 233, 235, 237, 242, 246, 249, 290, 292, 295, 299, 302, 320, 322,325,329 and 332. Arrow indicates the link between the amino acid between L187 and P182