A novel HBD gene mutation associated with normal-range hb A2 in β-thalassemia carriers

Abstract

β-thalassemia is one of the most common single-gene inherited conditions in the world, prevalence of β-thalassaemia in south China is 3–4%,increased Hb A2 level is one of the most important markers of β-thalassemia heterozygous carriers.Interaction of HBD gene defect with β-thalassemia can result in the normal Hb A2 β-thalassemia, potentially leading to a misdiagnosis of β- thalassemia carrier state. This study aimed to identify a novel mutation in HBD gene resulted in normal Hb A2 levels in β-thalassemia carriers, and explore the underlying mechanism of the novel HBD gene mutation using a minigene splicing assay and in vivo validation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00277-026-06892-7.

Dear editor-in-chief

This report identified a novel mutation in HBD (δ-globin) gene as the cause of normal Hb A2 levels in β-thalassemia carriers from a Chinese family, and demonstrated that nonsense-mediated mRNA decay (NMD) was the underlying mechanism.

β-thalassemia is one of the most common single-gene inherited conditions in the world, prevalence of β-thalassaemia in south China is 3–4% [1]. In China, a three-tiered prevention model for β-thalassemia encompassing carrier screening, genetic diagnosis, and prenatal diagnosis for high-risk fetuses. The initial screening of these couples involves a complete blood count (CBC) and hemoglobin electrophoresis. Individuals exhibiting an Hb A2 level > 3.4–3.6%, along with reduced mean corpuscular volume (MCV < 80 fL) and low mean corpuscular hemoglobin (MCH < 27 pg), are referred for genetic testing [2, 3].

Hb A2 results from a tetrameric assembly of two α- and two δ-globin chains (α2δ2). It comprises about 2.5%- 3.5% of total hemoglobin (Hb) in normal individuals [4]. Increased Hb A2 level is one of the most important markers of β-thalassemia heterozygous carriers [4, 5]. But, studies have identified β-thalassemia carriers demonstrating HbA2 levels within the 3.0%-3.5% range, despite this being conventionally considered normal or borderline [6, 7]. In the identification of β-thalassemia carrier, borderline Hb A2 levels are clinically significant because they can lead to underestimation of carrier status and misinterpretation of the results [8].Hb A2 levels may be lowered by factors such as iron deficiency, α-thalassemia trait, and HBD gene mutations, although most of the HBD gene mutation are clinically benign and are not associated with phenotypic abnormalities, interaction of HBD gene defect with β-thalassemia can result in the normal Hb A2 β-thalassemia, potentially leading to misdiagnosis of β- thalassemia carrier state [3, 9].

The family in this study originated from Shenzhen City, Guangdong Province, in southern China. Given the decreased MCV and MCH levels observed in the proband, combined with her husband being a carrier of the -- ^SEA/αα genotype, thalassemia genetic test is warranted. Although the common thalassemia genotype for the proband is β-IVS-II-654/βN, her hemoglobin electrophoresis revealed a Hb A₂ level of 3.1%, which falls within the normal range. To further clarify her genetic information and corresponding hematological phenotype, peripheral blood samples from her parents and umbilical cord blood sample from the newborn were collected after delivery. This study was approval by the Ethical Committee of Shenzhen Maternity and Child Healthcare Hospital and informed consents were obtained from all participants.

Hb analysis was performed using a capillary electrophoresis system (Capillarys 2 Flex Piercing, Sebia, Lisses, France). Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, REF51104, GmbH, Hilden, Germany). Common detection methods for thalassaemia, including multiplex gap-polymerase chain reaction (Gap-PCR) and reverse dot blot (RDB) (Yaneng, China), were used to detect 23 common types of thalassaemia variants. Additionally, whole β- and δ- globin gene DNA sequencing was performed using an ABI PRISMTM 3730XL analyzer. Total RNA was extracted from peripheral blood, first-strand complementary DNA (cDNA) was synthesized from 1 µg of total RNA. The target gene sequence of HBD gene was amplified from the synthesized cDNA via polymerase chain reaction (PCR). The minigene assay was performed as previously described [10], Fragments of wild-type and mutant sequences of HBD gene were cloned into Exon trap Cloning Vector pET01 and expressed accordingly.

Patients with the β-IVS-II-654/βN genotype typically present with elevated Hb A2 levels (> 3.5%) [11]. To explore the cause of normal Hb A₂ in our case, after excluding co-inheritance of α-thalassemia and iron deficiency, we performed Sanger sequencing for the HBD gene. A novel heterozygous intronic mutation (NM_000518.5:c.93-21G > A) was detected in HBD gene (Fig. 1A). To our knowledge, this mutation is not present in HbVar database. Although it has been recorded on the Ithagenes database, no established clinical assertions are available in locus-specific databases.

Fig. 1.

A. Sanger sequencing results of novel mutations in the HBD gene, mutation sites are indicated by red arrows, heterozygous intronic mutation (NM_000518.5:c.93-21G > A) was detected in HBD gene1. B Results of in silico splice prediction tools. Lowercase nucleotides indicate intron 1 sequences, yellow upper case nucleotides indicate exon 1 sequences, green upper case nucleotides indicate exon 2 sequences. Nucleotides marked in red indicate potential splice donor or acceptor sites. The scores calculated for wildtype sequence (WT) and mutated sequence (Mut) using Human Splice AI are displayed below each splice site. A higher score implies greater potential for a splice site. C Results of the minigene splicing assay. WT: Shows the Sanger sequencing result of the amplified product after transfecting cells with the wild-type fragment plasmid. This captures the full workflow (transfection -> culture -> RNA extraction -> RT -> PCR -> sequencing) concisely while making Sanger sequencing result the primary object. The blue underlined portion represents the plasmid vector sequence. The green underlined portion represents the sequence of the second exon of the HBD gene. MUT Shows the result after transfecting cells with the mutant fragment plasmid. Implies the same subsequent workflow as WT, uses consistent phrasing for all color descriptions and the red underlined portion represents the sequence corresponding to retained intron 1 of the HBD gene

The same mutation in the HBD gene, along with the mutation in HBB gene were identified in samples from the proband, the proband’s father, and the proband’s daughter; however, no other family members carried these mutations (Fig S1). According to the principles of inheritance, the δ-globin and β-globin mutations are located in cis configuration in our study. Hematological data for the proband’s newborn daughter were not available; Hb A2 level of both the proband and his father falls within the normal range, moreover, they presented a microcytic hypochromic hematological phenotype indistinguishable from that of pure β⁺-thalassemia.This observation suggests that the novel HBD gene mutation does not modify the anemic status of β⁺-thalassemia carriers when co-inherited with the HBB gene mutation, exerting a sole impact on hemoglobin A2 (HbA2) levels.

In splice AI in silico tools predicted that the AG-creating mutation (Acceptor Gain, score 0.84) would activate an intronic acceptor site (Fig. 1B). Minigene analysis, in which wild-type and mutant HBD gene fragments were cloned into the Exon trap Cloning Vector pET01and expressed, demonstrated intronic retention of 19 bp upstream of Exon 2 (c.92_93ins19). The position of this aberrant splicing event matched the Splice AI prediction (Fig. 1). To further validate the functional impact of the mutation site in vivo based on the minigene results, we performed PCR using two distinct primer sets. Forward primer for Primer 1 binding sites were positioned internal to the 19-bp retained intron sequence, whereas Primer 2 was placed upstream of this insertion. A common reverse primer for both primer sets was positioned to span the junction of Exon 2 and Exon 3 (Fig. 2A). The use of these primer sets allowed us to confirm the presence and proportion of the mutant transcript in vivo. Successful amplification was achieved exclusively with Primer set 1, yielding a fragment of 257 bp (Fig. 2B), whereas no product was obtained with Primer set 2. Sanger sequencing of this amplification product confirmed retention of an intron upstream of Exon 2 (Fig. 2C).

Fig. 2.

Experimental Results of HBD Gene mRNA Intron 1 Retention Validation. A Positions marked F and R in the figure represent the locations of the forward and reverse primers, respectively. B Sanger sequencing results of the amplified fragment show the nucleotide sequence. Retained intronic sequence is visible upstream of the second exon. C Electrophoresis results of the fragment amplified from sample cDNA using the primers shown in panel A, the target band is indicated by the red arrow

The novel mutation created a new canonical GT-AG splice site, this aberrant splicing resulted in the insertion of 19 bp between nucleotides c.92 and c.93 in the HBD mRNA (c.92_93ins19bp), generating a premature termination codon (PTC) that is expected to trigger nonsense-mediated mRNA decay (NMD) mechanisms. NMD is an essential eukaryotic mRNA surveillance mechanism that specifically recognizes and degrades transcripts containing PTCs [12]. Consequently, the diminished availability of the δ-globin chain leads to decreased synthesis of Hemoglobin A2. The efficiency of NMD induced specifically by this mutation has not yet been experimentally validated. More quantitative analyses are needed to test this mechanism.

In conclusion, we identified a novel HBD gene mutation (NM_000518.5:c.93-21G > A) in a Chinese family. Functional validation through a minigene assay demonstrated that the mutation induces partial intron retention, thereby generating a premature termination codon (PTC) and likely triggering NMD, ultimately leading to reduced HbA2 levels. In compound heterozygotes with β-thalassemia, HbA2 levels consistently fell within a borderline range of 2.9–3.5%, which may be attributed to cryptic splice site activation and subsequent intron retention. Our findings underscore that such co-inherited HBD gene mutations can obscure the typical hematological profile of β-thalassemia, posing a risk for underdiagnosis in routine screening. Our findings have important implications for understanding the hematological phenotype, improving clinical diagnosis, and guiding genetic counseling for β-thalassemia.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Lei Zhang, and Lijun Zhang collected samples and performed the research. Lei Zhang, Jingxin Yang, Shan Duan, Chao Liu and Ming Chang designed the study, Xingping Li and Yang Liu analysed the data, Lei Zhang and Ming Chang wrote the paper, Ming Chang, Wenlan Liuand Lijun Zhang supervised the research. All authors reviewed, edited and approved the manuscript.

Funding

This work was supported by Guangdong Medical Science and Technology Research Fund (Grant No.: B2025261), Sanming Project of Medicine in Shenzhen (No. SZSM202311005) and Investigator-initiated research project conducted at Shenzhen Maternity and Child Healthcare Hospital(NO.2024-KY-379).

Data availability

Data is available upon request.

Declarations

Competing interests

The authors declare no competing interests.