Molecular basis of a high Hb A₂/Hb Fβ-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction with α-globin gene triplication

Introduction

β-hemoglobinopathies are inherited hemoglobin (Hb) disorders caused by mutations in the β-globin gene, resulting in quantitatively reduced (β⁺-thalassemia) or absent (β⁰-thalassemia) β-globin chain synthesis and qualitative β-globin chain defect called structural β-globin chain variant. Although some β-globin gene deletions have been reported, most defects are non-deletional mutants of the β-globin gene (Steinberg et al., 2009). In Thailand, a high prevalence of β-hemoglobinopathies has been described, including 20–30% of Hb E (HBB:c.79G>A) and 3–9% of β-thalassemia (Paiboonsukwong et al., 2022). In northeast Thailand, eight β-thalassemia mutations including CD 41/42 (-TTCT) (HBB:c.126_129delCTTT), CD17 (A-T) (HBB:c.52A>T), NT-28 (A-G) (HBB:c.-78A>G), CD71/72 (+A) (HBB:c.217dupA), IVSI-1 (G-T) (HBB:c.92+1G>T), IVSII-654 (C-T) (HBB:c.316-197C>T), 3.4 kb deletion and IVSI-5 (G-C) (HBB:c.92+5G>C) accounted for 97.4% of the total mutations (Yamsri et al., 2011). Generally, β-thalassemia heterozygote has normal or slightly decreased Hb level and reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) with increased Hb A₂ to around 5.0% and Hb F 0–3%. Homozygote or compound heterozygote of β-thalassemia with Hb E led to β-thalassemia major and Hb E-β-thalassemia diseases, with variable thalassemia phenotype. The β⁺-thalassemia has a milder thalassemia phenotype than the β⁰-thalassemia (Yamsri et al., 2011; Yamsri et al., 2015).

Interestingly, among these common mutations, it was found that the carrier of β-thalassemia with 3.4 kb deletion was associated with higher Hb A₂, Hb F, MCV, and MCH values as compared with those with other mutations (Yamsri et al., 2011). A high Hb F expression may presumably lead to a milder β-thalassemia phenotype in homozygotic or compound heterozygotic forms. This 3.4 kb DNA deletion removes an entire β-globin gene, including its 5′ promoter. In this study, we have examined several genetic single nucleotide polymorphisms (SNPs) modifiers affecting Hb A₂ and Hb F expression in a large cohort of β⁰-thalassemia with 3.4 kb deletion. The molecular basis, and phenotype and genotype interaction observed in both heterozygote and compound heterozygote forms, as well as a hitherto undescribed interaction of the 3.4 kb deletion β⁰-thalassemia with an α-globin gene triplication, are described.

Materials and Methods

DNA sequencing

Direct DNA sequencing was used to determine deletion breakpoints of the β⁰-thalassemia with 3.4 kb deletion generated using primers G9 (5′-TCCCCAGTTAACCTCCTATT-3′) and N1 (5′-ACATATGAGCAAGGTTGTGT-3′) on a specific fragment of 456 base pair (bp). To examine three BCL11A binding motifs (TGGTCA) located between +3 kb 3′of ^Aγ-globin gene and about 1 kb 5′δ-globin gene, the three amplified fragments using primer pairs F29 (5′-CACGAGTCAGCCTTCAGAAA-3′) & G188 (5′- GCAGAGGAGACCAGCATACA-3′) (1,386 bp), F18 (5′-GACATGGACTATTGTTCAATGA-3′) & G191 (5′-AGACAACGGGCTAATCCCTC-3′) (1,286 bp), and F31 (5′-ACCCACATT GGCATTACAC-3′) & G192 (5′-AGAATGTGTTTGTGAGGGAGGA-3′) (1,111 bp) were generated as shown in Fig. 1 and the fragments were sequenced directly. To analyze the whole γ-globin genes, primer F22 (5′-TACTGCGCTGAAACTGTGGC-3′) & γ35 (5′-AGGTAGTTGTTCCCCTTCAA-3′) and SF5 (5′-TTAACGTCTTCAGCCTACAA-3′) & SF6 (5′-CAATCTGCACACTTGAGGGC-3′) were used to amplify DNA fragments of 1,843 and 993 bp, specific for ^Aγ-globin gene, and primers γ4 (5′-GGCCTAAAACC ACAGAGA-3′) & γ34 (5′-AGGTAGTTGTTGTTGTTGCA-3′) and SF3 (5′-GTTTGTGTGTG TGTGAGAGC-3′) & SF4 (5′-TCTTTAGGCATGCGTCAACA-3′) were used to amplify DNA fragments of 1,787 and 1,121 bp of ^Gγ-globin gene, followed by DNA sequencing (Fucharoen, Shimizu & Fukumaki, 1990; Panyasai et al., 2004; Svasti et al., 2007; Singha et al., 2015) [9-12]. DNA sequencing was carried out using Sanger sequencing method on an ABI PRISM™ 3730XL analyzer (Applied Biosystems, Foster City, CA, USA).

Identification of the four bp deletion (-AGCA) of the^Aγ-globin gene promoter

The PCR-RFLP assay was developed to detect four bp deletion (-AGCA) at −225 to −222 of the ^Aγ-globin gene promoter (rs1451276553). Specific amplification of ^Aγ-globin gene promoter was performed using primers F22 and γ35 to produce a fragment of 1,847 bp wild-type or 1,843 bp specific for the four bp deletion. This was confirmed by DNA sequencing and PCR-RFLP assay. The PCR product was digested with a Mse I restriction enzyme (5′-T^▾TAA-3′) (Vivantis Technologies Sdn. Bhd, Selangor Darul Ehsan, Malaysia). After digestion, the 1,021 bp and 167 bp indicate wild type, whereas the 1,184 bp is a specific fragment for the AGCA deletion, as shown in Fig. 2.

Allele-specific PCR assay for identification of rs5006884 polymorphism in the OR51B6 gene

Allele-specific PCR was developed to identify the rs5006884 (NC_000011.10:g.5352021C>T) in the OR51B6 gene on chromosome 11. In this system, primers G197 (5′-AAGACCAGG ATGCAGCAGAT-3′) and G198 (5′-ACCTCGGAGTGACATTGACC-3′) were used to produce an amplified fragment of 544 bp internal control. Specific amplification of the C allele (252 bp) and T allele (331 bp) were obtained using primers G197 & G199 (5′-CCTACTGTCGATCCCATGTAC-3′), and G200 (5′-AGACAGAAAGCATGGGAGAA-3′) & G198, respectively. The PCR reaction mixture (50 µL) contains 50–200 ng of genomic DNA, 200 µM of dNTPs, 30 pmol of each primer, and 1 unit of Taq DNA polymerase (New England Biolab, Inc., USA) in 10 mM Tris–HCl buffer pH 8.3, 50 mM KCl, 3 mM MgCl₂, and 0.01% gelatin. The PCR consists of an initial denaturation at 94 °C for 3 min followed by 30 cycles of amplification at (94 °C for 1 min and 65 °C for 90 s). The PCR product was analyzed on 2% agarose gel electrophoresis and visualized under UV light after ethidium bromide staining. Confirmation of SNP rs5006884 was done using DNA sequencing (Fig. 3).

Results

DNA sequencing of the deletion breakpoint of the β⁰-thalassemia with 3.4 kb deletion encountered in Thai patients identified the deletion as NG_000007.3:g.69825_73314del3488 or NC_000011.10:g.5224302_5227791del3490 (GenBank Accession no. OQ161123), exactly the same with that reported previously in a Chinese patient (He et al., 2018). Table 1 and Fig. 4 demonstrated the hematological parameters associated with the β⁰-thalassemia trait with 3.4 kb deletion (n = 103) compared to those of the β⁰-thalassemia trait with other mutations (n = 259) encountered in our series. As shown in the table, significant higher Hb (11.6 ± 1.7 vs. 11.2 ± 1.6 g/dL), hematocrit (Hct) (36.1 ± 5.2 vs. 34.8 ± 5.2%), MCV (66.3 ± 4.3 vs. 63.2 ± 3.8 fL), MCH (21.3 ± 1.3 vs. 20.2 ± 1.4 pg), Hb A₂ (6.9 ± 0.8 vs. 5.7 ± 0.6%) and Hb F (6.5 ± 3.4 vs. 1.3 ± 0.9%) values were observed for the former group of subjects. These are also the cases for the patients with compound heterozygous β⁰-thalassemia and Hb E. We observed significant higher Hb, Hct, MCV, MCH, and Hb F (48.1 ± 8.3 vs. 32.2 ± 15.6%) but lower Hb A₂ (3.9 ± 1.1 vs. 6.5 ± 1.9%) values for the patients with 3.4 kb deletion (n = 20) as compared to those with other β⁰-thalassemia mutations (n = 125). These results indicate that the 3.4 kb deletion β⁰-thalassemia is associated with high Hb A₂ and Hb F in a heterozygote state and a mild hematological phenotype when found in association with Hb E in a Hb E- β⁰-thalassemia disease. The effect of co-inheritance of α-hemoglobinopathy on the hematological phenotype of heterozygous β-thalassemia with 3.4 kb deletion was summarized in Table 2. Significant elevation of MCV and MCH values and a reduction of Hb F (3.0 ± 3.3%) were observed in heterozygous β-thalassemia with 3.4 kb deletion with heterozygous α⁺-thalassemia (– α/αα, n = 13). Lower Hb F but not Hb A₂ were also observed in double heterozygotes for 3.4 kb deletion β-thalassemia and other deletional forms of α-thalassemia including homozygous α⁺-thalassemia (– α/– α, n = 1), α^o-thalassemia (--/ αα, n = 2) and Hb H disease (--/- α, n = 1). In contrast, we observed that co-inheritance of a non-deletional α-thalassemia, the Hb Constant Spring (α^CSα/αα, n = 7), did not lead to a reduction in Hb F expression, although it did have some hematological improvement. However, no statistical analysis was performed due to the small sample sizes of these α-hemoglobinopathies.

Interestingly, an adult male patient was encountered with a double heterozygote for β-thalassemia with 3.4 kb deletion and an α-globin gene triplication (ααα^anti−3.7/ αα), a hitherto undescribed condition. He had relatively normal Hb and Hct values with reduced MCV & MCH characteristics of a plain β-thalassemia carrier. Elevated Hb A₂ (7.2%) and Hb F (6.8%) were observed. The result indicates that, unlike other β⁰-thalassemia alleles, the interaction of α-globin gene triplication (ααα^anti−3.7/ αα) and a β⁰-thalassemia with 3.4 kb deletion does not lead to a β-thalassemia disease in heterozygotic form.

SNPs were examined in representative subjects to determine if the existence of known Hb F associated SNPs are responsible for the high Hb A₂ and Hb F phenotype in our patients. As shown in Table 3, the majority of the subjects had wild-type sequences for the three SNPs, including deletion of four nucleotides (-AGCA) between −225 to −222 on ^Aγ-globin promotor, rs5006884 (C>T) of the OR51B6 gene, and ^Gγ-Xmn I of ^Gγ-globin gene promoter. In addition, no significant difference in Hb F and other hematological parameters was observed between those of subjects with and without these SNPs. DNA sequencing of the three BCL11A binding motifs (TGGTCA) between the 3′ ^Aγ-globin gene and the 5′ δ-globin gene (Fig. 1) identified no alteration of the nucleotide at the binding motifs and their vicinities. Further sequencing of the whole ^Aγ- and ^Gγ-globin genes in representative subjects identified no mutation.

Discussion

The carrier of β-thalassemia usually accompanies elevated Hb A₂ and normal Hb F levels. Hb A₂ isa product of tetrameric assembly of two α- and two δ-globin chains (α₂δ₂), which is synthesized at a low level (2.5−3.5%) in normal adult individuals. Several genetic and non-genetic conditions are associated with Hb A₂ expression (Singha et al., 2021). Among many different β-thalassemia genes in Thailand, we observed that only the 3.4 kb deletion β⁰-thalassemia is always associated in heterozygotic form with high Hb A₂ (6.9 ± 0.8%) and Hb F (6.5 ± 3.4%) levels. Heterozygosity for other β-thalassemia alleles is usually found with elevated Hb A₂ (5.7 ± 0.6%) but not Hb F (1.3 ± 0.9%) (Yamsri et al., 2010; Yamsri et al., 2011; Yamsri et al., 2015; Yamsri et al., 2022). Hematological characteristics in a large cohort of β-thalassemia carriers with this mutation listed in Table 1 confirmed this phenotype characteristic. This 3.4 kb deletion β⁰-thalassemia has been frequently described among Southeast Asian and Chinese patients. It has rarely been encountered on population of India, Pakistan and Bangladesh (Nadkarni et al., 2019; Khaliq, 2022; Chowdhury, Sultana & Das, 2022). This 3,488 bp deletion β-thalassemia allele removes DNA between positions −125 to +78 of the β-globin gene, eliminating the CACCC, CCAAT, and TATA elements in the β-globin gene promoter. The high Hb F characteristic of this β⁰-thalassemia allele could likely ameliorate the patient’s clinical phenotype. As shown in Table 1, when found in combination with Hb E, it could lead to non-transfusion-dependent thalassemia (NTDT). As compared to other β⁰-thalassemia/Hb E, which is generally associated with severe transfusion-dependent thalassemia (TDT), the patients with β^3.4-thalassemia/Hb E had milder NTDT phenotype with significantly higher Hb, Hct, MCV, MCH, MCHC, and Hb F but lower RDW values. We have also documented recently that the patients with homozygous β⁰-thalassemia of 3.4 kb deletion were presented with mild NTDT phenotype with Hb ranging from 7–9 g/dL and did not require regular blood transfusion (Soontornpanawet, Singha & Fucharoen, 2022). It is noteworthy for a compound Hb E- β-thalassemia with 3.4 kb deletion that the patient had significant higher Hb, Hct, MCV, MCH, and Hb F (48.1 ± 8.3 vs. 32.2 ± 15.6%) but lower Hb A₂ (3.9 ± 1.1 vs. 6.5 ± 1.9%) as compared to those of other β⁰-thalassemia mutations (Table 1). This lower Hb A₂ in Hb E-β⁰-thalassemia with 3.4 kb deletion as compared with Hb E-β⁰-thalassemia with non-deletional mutations is most likely due to the higher Hb F expression. There is an inverse relationship between Hb A₂ and Hb F expression in β-thalassemia. Red blood cells that produce relatively large amount of Hb F will produce less Hb A₂, and vice versa (Singha et al., 2018). It is conceivable that although the 3.4 kb deletion results in β⁰-thalassemia, the increase in Hb F may compensate for the complete absence of Hb A and ameliorate the severity of the disease as seen in our patients.

As shown in Table 2, co-inheritance of the 3.4 kb deletion β⁰-thalassemia and α-thalassemia (-α/αα, α^CSα/αα, -α/-α, --/αα) could further improve the hematological phenotype of the patients due to the balance between α- and non-α-globin chains. Fortunately, in all cases of these double heterozygotes, the Hb A₂ levels are still within the diagnostic range for a β-thalassemia carrier. Interestingly, we have encountered a subject with a hitherto undescribed condition of double heterozygote for the β^{3.4kb deletion} and α-globin gene triplication (ααα^anti−3.7/αα). This subject presented with β-thalassemia trait phenotype without anemia with Hb 13.0 g/dL and Hct 40.0%, Hb A₂ 7.2%, and Hb F 6.8% (Table 2). The heterozygous β-thalassemia with α-globin gene triplication generally presents with thalassemia intermedia phenotype due to more excess α-globin chain and imbalance in α- and β-globin chain synthesis (Steinberg et al., 2009). This might be explained by the fact that, unlike other point mutations, the β^{3.4kb deletion} is associated with increased expression of both δ- and γ-globin genes and could therefore improve the balance between α- and non- α-globin chains. This information indirectly supports that the β⁰-thalassemia with 3.4 kb deletion is a mild β⁰-thalassemia allele. We observed that this high Hb A₂ and Hb F β-thalassemia trait is also the case with the Filipino β-thalassemia deletion occasionally encountered in our setting, although with different extent of Hb F expression. This Filipino β-thalassemia deletion [NG_000007.3:g.66258_184734del118477] was described originally as approximately 45 kb long starting from position -4,279 bp relative to the mRNA cap site of the β-globin gene but with an uncertain 3′ breakpoint. It was later described using gap-PCR and DNA sequencing as 118 kb in length, with the 5′ breakpoint at position −4,279 of mRNA cap site, and the 3′ breakpoint extending to the downstream olfactory receptor (OR) region where it deletes four functional OR genes and two OR pseudogenes including the OR52A1 that contains a γ-globin gene enhancer (Yamsri et al., 2012; Teh et al., 2018; Yasin et al., 2022). Table 4 compared the hematological parameters of β⁰-thalassemia carriers with three different β-thalassemia deletions in our series, including the 3.4 kb deletion (n = 103), the Filipino β⁰-thalassemia (n = 9), and 105 bp β⁰-thalassemia deletion (n = 7), all without α-thalassemia. The 3.4 kb deletion and the Filipino β⁰-thalassemia are associated with high Hb A₂ and high Hb F β-thalassemia trait but not the 105 bp β⁰-thalassemia. In fact, this is not unexpected. The 105 β⁰-thalassemia is caused by a DNA deletion of −24 or −25 to +80 or +81 relative to the cap site of β-globin mRNA. Unlike the 3.4 kb deletion and the Filipino β⁰-thalassemia with 118 kb deletion, this 105 bp deletion does not remove the β-globin gene promoter TATA, CCAAT, and CACCC motifs (Nopparatana et al., 1999).

It has been noted that variation preliminarily in the two SNPs, rs4895441 and rs9399137 in HBS1L-MYB intergenic region, may have a small effect on Hb F expression in this β-thalassemia allele (Tepakhan, Kanjanaopas & Srewaradachpisal, 2020). We have also documented that the G allele of rs4895441 and C allele of rs9399137, alone or in combination with other SNPs, could explain the mild phenotypic expression of NTDT associated with Hb E-β-thalassemia disease (Phanrahan et al., 2019). To understand the high Hb F characteristic of the 3.4 kb deletion β⁰-thalassemia allele, we have examined other SNPs associated with high Hb A₂ & Hb F expression. These included the -AGCA deletion at −225 to −222 of ^Aγ-globin gene promoter, rs5006884 (C>T) on OR51B6 gene, −158^Gγ-Xmn I, BCL11A binding motifs between ^Aγ- and δ-globin genes as well as the whole γ-globin gene sequencing. It has been shown that the four bp deletion (-AGCA) at −225 to −222 of the ^Aγ-globin gene was associated with reduced ^Aγ-globin chain and elevation of ^Gγ chain with slightly increased Hb F level in β-thalassemia subjects (Manca et al., 1991; Ugrin et al., 2016). The rs5006884 (C>T) on OR51B6 gene located upstream of β-globin gene cluster might intervene in the tetramer formation of α-globin chain with β- and δ-globin chains and regulated Hb A₂ level in β-thalassemia carriers (Cyrus et al., 2019). The −158^Gγ-Xmn I polymorphism is associated with high Hb F expression in β-hemoglobinopathies during erythropoietic stress (Thein, 2017). This ^Gγ-Xmn I is almost linked to the +25 (G>A)^Aγ-globin promoter (rs368698783). The polymorphism leads to a decreased Ly-1 antibody reactive clone (LYAR) binding efficiency, which is the repressor of γ-globin genes (Bianchi et al., 2016; Chen et al., 2017). BCL11A maintains silencing of γ-globin expression in adult erythroid cells and direct promoter repression to control the fetal to adult Hb switch in humans. The BCL11A binding motifs (TGGTCA or TGACCA) have been identified on the β-globin gene cluster at the third hypersensitive site (HS3) of the β-LCR, downstream of ^Aγ-globin gene and upstream of δ-globin gene (Sankaran et al., 2008; Sankaran, Xu & Orkin, 2010; Liu et al., 2018). Mutation in these binding motifs may therefore result in increased Hb F expression. As shown in Table 3, we did not observe the association of these SNPs with high Hb F and higher hematological values for the β⁰-thalassemia with 3.4 kb deletion. In fact, most of the cases had wild-type SNPs sequences, and no mutation was found in the BCL11A binding motifs between 3′^Aγ-globin gene and 5′ δ-globin gene and within ^Aγ- and ^Gγ-globin genes of the patients examined. It is, therefore, unlikely that the unusually high Hb A₂ and Hb F β-thalassemia trait of the β⁰-thalassemia with 3.4 kb deletion is caused by the effect of these high Hb F SNPs. However, this might be explained by the fact that removal of the 5′  β-globin promoter in the 3.4 kb deletion may release competition of the β-globin gene for the upstream locus control region (β-LCR), leading to its increased interaction with the γ- and δ-globin genes in cis, increasing their expression resulting in elevation of Hb F and Hb A₂ (Thein, 2018). Recently, it has been documented that disrupting the adult β-globin promoter reactivates γ-globin gene expression by promoter competition model in the Hb switching (Topfer et al., 2022). The 3.4 kb deletion removing β-globin promoter and the entire β-globin gene is therefore associated with elevated Hb F in addition to elevated Hb A₂ in heterozygotic form.

Conclusions

Nonetheless, the high Hb, Hct, MCV, MCH, Hb A₂, and Hb F values in the heterozygote state and a mild hematological phenotype observed in Hb E-β^{3.4kb deletion} disease and double heterozygosity for α-globin gene triplication (ααα^anti−3.7) and β^{3.4kb deletion} support that the β^{3.4kb deletion} is mild β⁰-thalassemia allele and might ameliorate severity when occurring in homozygote or compound heterozygote state. This high Hb A₂ and high Hb F β-thalassemia characteristic could also be a useful marker for the β^{3.4kb deletion} before being further confirmed by PCR analysis. This information related to the 3.4 kb deletion β-thalassemia should be provided to the patient’s family for the appropriate management and genetic counseling. This is especially important information for routine prenatal thalassemia screening in a prevention and control program in the regions.

Supplemental Information