eISSN: 1896-9151
ISSN: 1734-1922
Archives of Medical Science
Current issue Archive Manuscripts accepted About the journal Special issues Editorial board Abstracting and indexing Subscription Contact Instructions for authors
SCImago Journal & Country Rank
1/2018
 
Share:
Share:
more
 
 
Basic research

KLF1 gene and borderline hemoglobin A2 in Saudi population

J. Francis Borgio, Sayed AbdulAzeez, Ahmed M. Al-Muslami, Zaki A. Naserullah, Sana Al-Jarrash, Ahmed M. Al-Suliman, Mohammed S. Al-Madan, Amein K. Al-Ali

Arch Med Sci 2018; 14, 1: 230–236
Article file
- KLF1 gene.pdf  [0.29 MB]
Get citation
ENW
EndNote
BIB
JabRef, Mendeley
RIS
Papers, Reference Manager, RefWorks, Zotero
AMA
APA
Chicago
Harvard
MLA
Vancouver
 
 

Introduction

The Al-Qatif and Al-Ahsa regions in the Eastern Province of Saudi Arabia are well known for their high prevalence of sickle cell anemia and β-thalassemia. Patients homozygous for β-thalassemia require long life blood transfusion therapy that imposes a tremendous impact on the health institutes in the area. To reduce the rate of incidence of β-thalassemia major, the health authorities implemented a premarriage screening policy to identify subjects with β-thalassemia trait [1–5]. Elevated HbA2 (hemoglobin A2) level (4% to 6.0%) is considered the most reliable haematological parameter used for identification of patients heterozygous for a β-thalassemia variation [6–9]. However, in some instances the level of HbA2 is not within the range which infers the carrier status of β-thalassemia [5, 7–9]. These carriers usually have a borderline level of HbA2 in the range of 3.1–3.9%, which makes it difficult to infer the presence or absence of the β-thalassemia variation. Therefore, molecular identification of the β-thalassemia carrier variation has been proposed for inclusion in the pre-marital screening program to reduce the incidence of newborns with β-thalassemia major [4, 7–9]. However, screening of a large population using molecular diagnosis is proven to be challenging and expensive especially in rural areas.
Recent studies have shown coinheritance of a variation in the erythroid Krüppel-like factor (EKLF or KLF1) leads to a borderline HbA2 level in β-thalassemia carriers [7–12]. KLF1 is a sequence-specific DNA binding factor, which is encoded by the KLF1 gene. KLF1 is an essential and primary transcription factor for α and β globin genes [11]. Therefore, the present study was conducted to establish the association of variations in the KLF1 gene in our subjects with borderline HbA2 and also to shed some light on the possible effects of this variation in the KLF1 gene on the level of HbA2.

Material and methods

A total of 212 Saudi healthy individuals (88 subjects: HbA2 < 3 or ranging from 0.6% to 3%; 72 subjects: HbA2 = 3.1 to 3.9; 52 subjects HbA2 > 4.3 or ranging from 4.3% to 7.2%) were included in the study (Table I). The study was approved by the Institutional Review Board at the University of Dammam (approval number: IRB-2013-08-030). Written informed consent was obtained from all participants. The clinical data of each subject, including the level of HbA2, were obtained. Genomic DNA was isolated from the blood samples using commercially available genomic DNA (GE health care, UK). The level of HbA2 was measured for all subjects using a Variant II Analyzer (Bio-Rad, USA). All the samples were subjected for the identification of β and α globin gene variations [5, 6, 13].
Direct DNA sequence analysis of the KLF1 (forward primer: 5-TTTATCTGGGAGGGGCATTTTTATAG­GACC-3 and reverse primer: 5-GTGTCCAGCCCGCGATGT-3), HBB, HBA1 and HBA2 genes was performed using a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) as described earlier [4, 5, 12]. Sequence analysis was carried out using DNA sequencing analysis software V5.4 (Applied Biosystems, Foster City, CA, USA) and Variobox [14]. MAFFT (Multiple sequence alignment and NJ/UPGMA phylogeny) version 7 was used for the multiple sequence alignment of the KLF1 gene sequences [15]. Three-dimensional (3D) homology modeling of wild and mutated (NM_006563.3:c.304T>C and NM_006563.3:c.544T>C corresponding to S102P and F182L) KLF1 proteins was carried out as described earlier using different kinds of bioinformatics tools [16, 17].

Statistical analysis

For statistical analysis between the hematological data and the presence of various mutations in the KLF1 gene, Student’s t-test was used using IPM SPSS statistics 19.

Results

The present study was carried out to identify the influence of variations in the KLF1 gene in Saudis with borderline HbA2. Four variations were identified, including two 3 untranslated region or 3UTR NM_006563.3:c.*296G>A and NM_006563.3:c.*277C>G, and 2 exonic NM_006563.3:c.304T>C and NM_006563.3:c.544T>C variations for the first time from the Saudi Arabian population (Figure 1). But none of these variations have shown significance over this borderline HbA2 through significance analysis using Student’s t-test between the group with and without KLF1 gene variations.
The presence of the borderline HbA2 level is not specific to a particular β°-thalassemia variation or β+-thalassemia variations in the Saudi population (Table I). A normal level of HbA2 (< 3) has been observed in healthy Saudis with heterozygous β°-thalassemia variation (n = 2, HBB:c.25_26delAA; n = 22, HBB:c.20A>T), furthermore, the KLF1 gene was found to be variation free in these individuals (Table II). Deep analysis of these subjects revealed the presence of the HBA12 gene in five subjects, which supports an earlier report [13]. KLF1 is a sequence-specific DNA binding factor, which is an essential transcription factor for α and β globin genes [11]. Reports from various populations have shown that there is an association between the variations in KLF1 with borderline HbA2 level in β-thalassemia carriers [7–11]. Therefore, the present study was conducted to establish the association of variations in the KLF1 gene in Saudi subjects with borderline HbA2 and also to shed some light on the possible effects of this variation in the KLF1 gene on the level of HbA2. Sequence analysis of the KLF1 gene from the subjects with HbA2 < 3; HbA2 = 3.1 to 3.9 and HbA2 > 4.3 identified four variations, which were observed in a few samples in all the groups. They were found to be not specific for any of the three groups (Table II). Two 3 untranslated region or 3UTR substitution variations were found at base pair 296 and at 277 (NM_006563.3:c.*296G>A and NM_006563.3:c.*277C>G respectively). Notably, none of the subjects with HbA2 > 4.3 were identified with α globin gene variants (Table I). The variation NM_006563.3:c.304T>C was identified in many Saudis (n = 58 hetero; n = 18 homo) subjects with varied levels of HbA2; hence we could say that this variation is also not specific to borderline HbA2. The detailed study on the α globin genotype revealed that the prevalence of –α23.71α2 is the most frequent (29.06%) among healthy Saudis (Table I). However, the α globin genotype, –α23.71α2 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%).
Effects of the amino acid changes S102P and F182L due to the exonic variations such as NM_006563.3:c.304T>C and NM_006563.3:c.544T>C respectively on the KLF1 protein (PDB ID: 2I3B) were studied by structural modeling using earlier methodology using SWISS MODEL and PROCHECK [18–21]. Three-dimensional (3D) homology modeling of KLF1 protein from the wild and variants revealed that the variants S102P (Å 3.58) and F182L (Å 3.42) show significantly deviated RMSD (Å) compared to the wild type (Å 0) (Figure 2). The total energy values of S102P (2723945 kJ/mol) and F182L (1871 kJ/mol) variegated proteins were deviated from the wild (656 kJ/mol) protein. The interaction between the amino acid (L187) at the active site and studied amino acid (F182) as well as the changes in the RMSD (Å) due to the variation (F182L) show its biological significance (Figure 2). However, we could not identify any notable impact on the phenomes of the subjects with F182L.

Discussion

The allelic association or genotypic association of the NM_006563.3:c.304T>C with the levels of the HbA2 was found to be insignificant (p = 0.9842; odd ratio 1.02 (95% CI: 0.46–2.2). However, studies from Italian and Chinese populations on the level of HbA2 and the influence of variation in KLF1 (K332Q; S270X; p.AlA298Pro, p.Thr334Arg; c.913+1G>A; p.His299Asp; p.Cys341Tyr; p.Glu5Lys) showed prominent changes [22–25]. These variants (K332Q; S270X; p.AlA298Pro, p.Thr334Arg; c.913+1G>A; p.His299Asp; p.Cys341Tyr; p.Glu5Lys) do not exist in the Saudi population; therefore, these variants need not be included in screening.
Detailed analysis of the molecular nature of the α globin (HBA1 and HBA2), β globin (HBB) and KLF1 genes on Saudis with various levels of HbA2 revealed that the KLF1 gene or its variants was found to be not associated with the presence of borderline HbA2. The rate of consanguinity among Saudis is high [26]. Hence, precise identification of individuals with β globin gene variation is necessary for distinguishing couples at risk for having progeny with β-thalassemia major of sickle cell disease, especially considering the level of HbA2 for the identification of β-thalassemia carriers. Hence the identification of the –α23.71α2 and the HBA12 gene on the subjects with borderline HbA2 should be incorporated in the existing premarital screening in Saudi Arabia to eliminate the erroneous diagnosis of β-thalassemia carriers as normal [27]. Enzymes may play a critical role; hence enzyme activities among these subjects can also be considered in future [28]. There is no evidence of the HBD gene variations in the subjects with borderline HbA2 in Saudis. Hence, the  globin gene and its association with the changes in the level of the HbA2 should be studied in detail in Saudi subjects.
The occurrence of hemoglobin disorders is common among Saudis [1–5].
In conclusion, two exonic (NM_006563.3:c.304T>C and NM_006563.3:c.544T>C) and two 3 untranslated region or 3UTR (NM_006563.3:c.*296G>A and NM_006563.3:c.*277C>G) variations have been identified for the first time from an Arab population. None of these four variations are significantly associated with the Saudis with borderline HbA2. α Globin genotype, –α23.71α2 and the HBA12 gene might be associated with borderline HbA2 in Saudis. The real reason for the borderline HbA2 in 44.5% of Saudis is still unclear.

Acknowledgments

The authors would like to extend their gratitude to King Abdulaziz City for Science and Technology (to JFB LGP-35-204), and the National Science, Technology and Innovation Plan (to JFB, grant no: 12-MED 2798-46) Saudi Arabia for the partial funding of this research. We thank Mr. Ranilo. M. Tumbaga, Mr. Horace T. Pacifico, Mrs. Charmine Villanueva; Mrs. Jee Entusiasamo Aquino and Mr. Eswardson Evancelista for their technical expertise.

Conflict of interest

The authors declare no conflict of interest.

References

1. Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis 2010; 5: 13.
2. Akhtar MS, Qaw F, Borgio JF. Spectrum of alpha-thalassemia mutations in transfusion-dependent beta-thalassemia patients from the Eastern Province of Saudi Arabia. Hemoglobin 2013; 37: 65-73.
3. Hamamy HA, Al-Allawi NA, Epidemiological profile of common haemoglobinopathies in Arab countries. J Community Genet 2013; 4: 147-67.
4. Borgio JF. Molecular nature of alpha-globin genes in the Saudi population. Saudi Med J 2015; 36: 1271-6.
5. Borgio JF, AbdulAzeez S, Naserullah ZA, et al. Mutations in the beta-globin gene from a Saudi population: an update. Int J Lab Hematol 2016; 38: e38.
6. Al-Nafie AN, Borgio JF, AbdulAzeez S, et al. Co-inheritance of novel ATRX gene mutation and globin (alpha & beta) gene mutations in transfusion dependent beta-thalassemia patients. Blood Cells Mol Dis 2015; 55: 27-9.
7. Mosca A, Paleari R, Galanello R, et al. IFCC Working Group on Standardization of HbA2. New analytical tools and epidemiological data for the identification of HbA2 borderline subjects in the screening for beta-thalassemia. Bioelectrochemistry 2008; 73: 137-40.
8. Giambona A, Passarello C, Vinciguerra M, et al. Significance of borderline hemoglobin A2 values in an Italian population with a high prevalence of beta-thalassemia. Haematologica 2008; 93: 1380-4.
9. Perseu L, Satta S, Moi P, et al. KLF1 gene mutations cause borderline HbA2. Blood 2011; 118: 4454-8.
10. Tallack MR, Perkins AC, Three fingers on the switch: Krüppel-like factor 1 regulation of gamma-globin to beta-globin gene switching. Curr Opin Hematol 2013; 20: 193-200.
11. Love PE, Warzecha C, Li L. Ldb1 complexes: the new master regulators of erythroid gene transcription. Trends Genet 2014; 30: 1-9.
12. Singleton BK, Burton NM, Green C, et al. Mutations in EKLF/KLF1 form the molecular basis of the rare blood group In (Lu) phenotype. Blood 2008; 112: 2081-8.
13. Borgio JF, AbdulAzeez S, Al-Nafie AN, et al. A novel HBA2 gene conversion in cis or trans: “α12 allele” in a Saudi population. Blood Cells Mol Dis 2014; 53: 199-203.
14. Gaspar P, Lopes P, Oliveira J, et al. Variobox: automatic detection and annotation of human genetic variants. Hum Mutat 2014; 35: 202-7.
15. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013; 30: 772-80.
16. AbdulAzeez Sayed, Francis Borgio J. In-Silico computing of the most deleterious nsSNPs in HBA1 gene. PloS One 2016; 11: e0147702.
17. Borgio JF, Al-Madan MS, AbdulAzeez S. Mutation near the binding interfaces at alpha-hemoglobin stabilizing protein is highly pathogenic. Am J Transl Res 2016; 8: 4224-32.
18. Kiefer F, Arnold K, Künzli M, et al. The SWISS-MODEL Repository and associated resources. Nucl Acids Res 2009; 37: D387-92.
19. Berman HM, Westbrook J, Feng Z. The protein data bank. Nucleic Acids Res 2000; 28: 235-42.
20. Feng L, Gell DA, Zhou S, et al. Molecular mechanism of AHSP-mediated stabilization of alpha-hemoglobin. Cell 2004; 119: 629-40.
21. Laskowski RA, MacArthur MW, Moss DS, et al. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 1993; 26: 283-91.
22. Satta S, Perseu L, Moi P, et al. Compound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin. Haematologica 2011; 96: 767-70.
23. Satta S, Perseu L, Maccioni L, et al. Delayed fetal hemoglobin switching in subjects with KLF1 gene mutation. Blood Cells Mol Dis 2012; 48: 22-4.
24. Liu D, Zhang X, Yu L, et al. KLF1 mutations are relatively more common in a thalassemia endemic region and ameliorate the severity of beta-thalassemia. Blood 2014; 124: 803-11.
25. Yu LH, Liu D, Cai R, et al. Changes in hematological parameters in alpha-thalassemia individuals co-inherited with erythroid Krüppel-like factor mutations. Clin Genet 2015; 88: 56-61.
26. Al-Asmary SM, Kadasah S, Arfin M, et al. Apolipoprotein E polymorphism is associated with susceptibility to schizophrenia among Saudis. Arch Med Sci 2015; 11: 869-76.
27. Guvenc B, Canataroglu A, Unsal C, et al. Beta-globin chain abnormalities with coexisting alpha-thalassemia mutations. Arch Med Sci 2012; 8: 644-9.
28. Okuturlar Y, Gedikbasi A, Akalin N, et al. Serum paraoxonase 1 activity in patients with iron deficiency anemia. Arch Med Sci 2016; 12: 697-703.
Copyright: © 2017 Termedia & Banach. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
Quick links
© 2018 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.
PayU - płatności internetowe