Molecular Analysis of Non-Transfusion Dependent Thalassemia Associated with Hemoglobin E-β-Thalassemia Disease without α-Thalassemia
1 Medical Science Program, Graduate School, Khon Kaen University.
2 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University.
3 Department of Internal Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Received: February 12, 2019
Accepted: May 17, 2019
Mediterr J Hematol Infect Dis 2019, 11(1): e2019038 DOI 10.4084/MJHID.2019.038
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Background: The finding of many Thai Hb E-β0-thalassemia
patients with non-transfusion dependent thalassemia (NTDT) phenotype
without co-inheritance of α-thalassemia has prompted us to investigate
the existence of other genetic modifying factors.
Preliminary study on subjects with a mild form of thalassemia encountered among couple at risk of having fetuses with thalassemia diseases in northeast Thailand has been carried out. The result indicated that four informative SNPs, including rs7482144 in HBG2 gene and rs2297339, rs4895441 & rs9399137 of HBS1L-MYB gene were associated with high Hb F levels in the patients. Further studies on homozygous Hb E identified the rs11886868 additionally in the BCL11A gene and 4 SNPs in the Krüppel-like factor 1 (KLF1) gene (G176AfsX179, T334R, -154 (C-T) and R328H) to be associated with high Hb F level in homozygous Hb E.[15-17] It is likely therefore that these informative SNPs might be important genetic modifying factors among NTDT- Hb E-β0-thal patients. However, data on these SNPs among clinically well-defined cases of NTDT with Hb E-β-thal patients in northeast Thailand is relatively limited. It has been known that co-inheritance of α-thalassemia is associated with a mild phenotype of the Hb E-β-thal disease. However, we have demonstrated previously that among Hb E-β0-thal patients associated with NTDT phenotypes, co-inheritance of β-thalassemia could explain the phenotypic expression only in a few cases. We report in this study, the existence of several genetic modifying SNPs in the HBS1L-MYB, BCL11A, and KLF1 genes among 122 clinically well-defined NTDT Hb E-β-thal patients in northeast Thailand.
Materials and Methods
Hematological and DNA analyses. Hematological parameters were recorded at steady state (no blood transfusion and no fever) using automated blood cell counter (Beckman Coulter Co., Fullerton, California, USA). Hb analysis was done using capillary electrophoresis (Capillarys 2; Sebia, Lisses, France) or high -performance liquid chromatography (Variant II, Bio-Rad Laboratories, Hercules, California, USA). Identification of β-thalassemia and the Hb E mutations found in Thailand was performed in our laboratory using allele-specific PCR assays and DNA sequencing. Identification of α0-thalassemia (SEA and THAI deletions), α+-thalassemia (3.7 and 4.2 kb deletions), Hb Constant Spring and Hb Paksé genes are routinely performed in our laboratory using multiplex gap PCR and allele-specific PCR.
SNP Genotyping. Four KLF1 SNPs including G176AfsX179, -154 (C-T), T334R and R328H were determined using allele-specific PCR assays and DNA sequencing as described.[16,17] Representative gel electrophoresis of these SNPs genotyping was shown in Figure 1. The rs4895441 (G-A) and rs9399137 (T-C) of HBS1L-MYB gene and rs4671393 (A-G) of BCL11A gene were determined using high resolution melting (HRM) analysis on an Illumina Eco Real-Time PCR System (Illumina, CA, USA). Primers G166 (5’ CACAACACTCCAGGGAGGCAG 3’) and G167 (5’ GGAGGCAGGGGGAATCTTAAT 3’) were used to produce an 84 bp fragment for detection of rs4671393 (A-G) of BCL11A gene. The rs4895441 (G-A) of HBS1L-MYB intergenic region was determined on a 157 bp fragment generated using primers G156 (5’ GGGGGTAAGAAGGAAACCAG 3’) and G157 (5’ TCTGAGGGCCTTCGAACTTA 3’). The rs9399137 (T-C) of HBS1L-MYB intergenic region was detected on a 136 bp fragment produced by primers G158 (5’ TCACCTTAAAAGGCGGTATTG 3’) and G159 (5’ TCAGAACTTATCCCAAGATTTTAAC 3’). Representative temperature shifted curves, and corresponding difference curves of these HRM assays were demonstrated in Figure 2. Identification of the Gγ-XmnI of HBG2 gene and rs2297339 (C-T) of the HBS1L-MYB gene was done using PCR-restriction fragment length polymorphism (PCR-RFLP) assay as described.[8,9]
|Figure1. Representative agarose gel electrophoresis for identification of four KLF1 SNPs using allele specific PCR assays including the G176AfsX179 (A), -154 (C-T) and T334R (B), and R328H (C).|
|Figure 2. The temperature shifted curves and difference curves of the three HRM assays for identification of rs4671393 (G-A) in the BCL11A gene and rs4895441 (A-G) & rs9399137 (T-C) of the HBS1L-MYB gene.|
Statistical analysis. The STATA statistical software version 10.0 (StataCorp, Tx, USA.) was used for data analyses. Descriptive statistics, mean and standard deviation, were used to describe all continuous variables, including red blood cell indices and Hb F levels. Multiple regression analysis was applied to demonstrate the effect of various SNPs on Hb F levels. Statistical significance was set at P < 0.05.
|Table 1. Globin genotypes and associated hematological parameters of 122 NTDT subjects with Hb E-β-thalassemia.|
|Table 2. The proportions of SNPs in HBG2, KLF1, BCL11A and HBS1L-MYB genes observed among 122 Thai NTDT patients.|
Among 4 SNPs of the KLF1 gene examined, including the G176AfsX179, T334R, -154 (C-T) and R238H, only T334R was detected. While no R328H, -154 (C-T) and G176AfsX179 was observed, heterozygosity for the T334R was identified in 11 (9.0%) of 122 cases. In contrast, a relatively higher proportion of the rs4671393 (G-A) of the BCL11A, i.e., GG, GA, and AA varieties were detected in 84 (68.8%), 35 (28.7%) and 3 (2.5%) cases, respectively.
For the HBS1L-MYB gene, the proportions of AA, AG and GG of the rs4895441 (A-G) were identified in 91 (74.6%), 30 (24.6%) and 1 (0.8%) cases, respectively. Heterozygosity for the rs9399137 (T-C) was found in 28 (23.0%) cases. The most common SNP in this HBS1L-MYB gene was found to be the rs2297339 (C-T) including CT and TT which were identified in 60 (49.2%) and 46 (37.7%) cases, respectively.
Multiple regression analysis was applied to demonstrate the effect of these SNPs detected on Hb F levels of 122 subjects with Hb E-β-thal (Table 3). As shown in the table, statistical significance (P < 0.001) was observed only on the homozygosity (+/+) of the Gγ-XmnI polymorphism. However, a low proportion of this Gγ-XmnI (+/+) in this group of Thai patients (9 of 122) makes it unlikely to be the sole factor on phenotypic expression of these cases. In fact, we observed that each patient carried at least one of these SNPs. Table 4 listed number of patients carrying 1-5 SNPs observed, and Figure 3 plots the proportions of subjects in correspondence with the number of conferring SNPs in this study. As shown in the figure, while only 12 of 122 cases carried single SNP, the remaining subjects had 2-5 SNPs at different genes, possibly indicating of interaction between these SNPs in the phenotypic modification of the cases.
|Table 3. Effect of SNPs detected on Hb F levels in 122 Hb E-β-thal patients.|
|Table 4. Proportions of patients according to number of carrying SNPs (1-5) observed among 122 Thai NTDT patients with Hb E-β-thalassemia disease.|
|Figure 3. Proportions of subjects with 1-5 SNPs among 122 Thai NTDT patients with Hb E-β-thalassemia disease.|
It has been known that major genetic modifying factor in β-thalassemia disease is a coinheritance of β-thalassemia as this leads to a more balanced in α- and non-α- globin chains ratio. However, this could not explain the phenotypic expression of all cases. Multiple single nucleotide polymorphisms (SNPs) associated with high Hb F expression have been identified in many populations on genes such as the HBG2, BCL11A, HBS1L-MYB, and KLF1 genes.[22-25] The results from our study of 122 Thai NTDT Hb E-β-thalassemia patients without β-thalassemia revealed that all of them carried at least one SNPs in these modifying genes (Table 4). While the majority of them (59 of 122) had two SNPs, the remaining carried one (12 of 122), three (29 of 122), four (18 of 122) or five (4 of 122) SNPs as shown in Figure 3. These 9 genetic modifying SNPs on the Gγ-globin, HBS1L-MYB, BCL11A, and KLF1 genes are known to play important roles in modifying disease severity. Among them, the Gγ-XmnI polymorphism was the most common SNP observed in our patients, i.e., 70.5% in heterozygous and 7.4% in homozygous states. Study in Thai homozygous Hb E has indicated a strong association between this polymorphism and increased Hb F level. We also observed that the Gγ-XmnI (+/+) has a significant effect on the Hb F in Thai NTDT Hb E-β-thalassemia patients, as shown in Table 3. However, the finding of only 9 of 122 cases with homozygotic form (+/+) of this polymorphism (Table 2) might underscore the importance of this SNP in Thai population and point possibly to interaction with other genetic modifiers.
We have previously documented in Thai subjects with homozygous Hb E that four KLF1 SNPs including G176AfsX179, T334R, -154 (C-T) and R328H are associated with increased Hb F expression.[16,17] In this study on 122 Thai NTDT Hb E-β-thalassemia patients, only one of them; the T334R was identified in heterozygote, the frequency of which was 9.0 % (Table 1). Although KLF1 gene has been thought to play an essential role in the clinical modification of the disease severity and homozygous for KLF1 mutation may be associated with mild thalassemia intermedia phenotype, our result on Thai NTDT patients indicates that KLF1 gene alone may play a minimal role in Thai population.
In contrast, a higher proportion of an A allele of the rs4671393 (G-A) polymorphism of the BCL11A gene was detected among 122 Thai NTDT patients i.e., 28.7% in heterozygote form and 2.5% in the homozygote. This rs4671393 (G-A) polymorphism is associated with Hb F variation and clinical events in sickle anemia. As compared to other genes, more prevalence of the G allele of rs4895441 (A-G), the C allele of rs9399137 (T-C) and T allele of rs2297339 (C-T) of the HBS1L-MYB intergenic region were observed among our Thai NTDT patients. This data is consistent with a previous finding for Thai homozygous Hb E. Study on the Mediterranean β-thalassemia intermedia patients has indicated a minor effect of the rs4671393 (G-A) of the BCL11A and the rs4895441 (A-G) & rs9399137 (T-C) of HBS1L-MYB intergenic region on phenotypic expression of the patients.
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