Beta Thalassemia Research: Hbb-bs&Hbb-bt DKO Mouse Models

In this edition, we provide an overview of Beta Thalassemia (β-thalassemia), a genetic blood disorder caused by reduced or absent synthesis of the beta globin (β-globin) chain of hemoglobin, and introduce Hbb-bs&Hbb-bt DKO mice as a promising Beta-thalassemia research model for preclinical therapeutics. Beta-thalassemia is one of the global social and public health issues monitored by the World Health Organization (WHO).^[1]

Pathogenic Effects of HBB Gene Mutations

Hemoglobin (Hb) is a protein in red blood cells responsible for transporting oxygen throughout the body. Its structure is primarily composed of two alpha-globin subunits and two beta-globin subunits. Each subunit contains a heme molecule at the center of the hemoglobin molecule. The heme can bind to an iron ion, enabling hemoglobin to carry oxygen.

In normal adults, the β-globin chain is encoded by the HBB gene. Adult hemoglobin (HbA) is composed of two β-globin chains, along with two α-globin chains (encoded by the HBA1 or HBA2 genes), which accounts for about 97% of total hemoglobin.^[2] In patients with Beta-thalassemia, mutations in the HBB gene lead to reduced or absent synthesis of the β-globin chain, resulting in lower hemoglobin levels and causing disorders in red blood cell production, premature red blood cell destruction, and anemia.

Clinical Types of Beta Thalassemia

Beta thalassemia is a hereditary hematological disease caused by mutations in the β-globin gene (HBB) that lead to quantitative reduction or even absence of functional hemoglobin (Hb) subunit beta (beta globin chain) synthesis, clinically known as β-thalassemia intermedia and β-thalassemia major. Those with β-thalassemia minor (also known as β-thalassemia trait) are heterozygous for mutations in the HBB gene and are often asymptomatic or have mild anemia – opposed to the homozygosity or compound heterozygosity required for the severe clinical phenotypes, β-thalassemia intermedia and β-thalassemia major.^[3] Unlike hemoglobinopathies, which result from structural abnormalities in hemoglobin, thalassemias primarily affect hemoglobin production. The deficiency in beta-globin causes an overabundance of alpha-globin chains, contributing to the disease's development.

Molecular Pathogenesis of β-thalassemia

The clinical severity of the β-thalassemia depends on the extent of alpha/non-alpha globin chain imbalance (i.e., ratio of alpha globin chains to beta globin chains). The unassembled alpha globin chains that result from unbalanced alpha/non-alpha globin chain synthesis precipitate in the form of inclusions,which damage erythroid precursors in the bone marrow and the spleen and cause ineffective erythropoiesis. Roughly 350 pathogenic variants of β-thalassemia alleles have been characterized to-date (http://globin.cse.psu.edu) and are functionally classified according to the extent of the reduction of the β-chain output:

• β⁰-thalassemia alleles (complete absence of hemoglobin subunit beta production): resulting from nonsense, frameshift, or (sometimes) splicing variants
• β⁺-thalassemia alleles (reduced output of beta globin chains): resulting from pathogenic variants in introns, the promoter area (either the CACCC or TATA box), the polyadenylation signal, the 5' or 3' untranslated region, or splicing abnormalities
• β⁺⁺-thalassemia alleles (minimal deficit in β chain production): are so mild that they are sometimes ‘silent’, and carriers may not display any evident hematological phenotype

Notably, the clinical severity associated with β⁰ and β⁺ variants is variable and may be modified in the presence of ameliorating genetic factors, such as coinheritance of alpha-thalassemia-associated pathogenic variants in HBA1 or HBA2, which reduce alpha globin expression, thereby decreasing the alpha/non-alpha globin chain imbalance.^[3] 

Now, we will explore the genotype-phenotype correlations for each type of β-Thalassemia in detail.

β-Thalassemia Major

Patients with severe Beta Thalassemia (Beta-Thalassemia Major) carry two copies of defective beta-globin alleles, the homozygous mutations result in the complete or nearly complete absence of β-globin chain production and leads to manifestation of the disease as follows. The mutations cause a reduction or absence of HbA synthesis, which contains the β-globin chain. The absence of the β-globin chain causes an imbalance in the ratio of α-globin to non-α-globin chain synthesis, leading to a relative excess of α-globin chains. These excess α-globin chains form inclusion bodies within red blood cells, causing oxidative damage to the red cell membrane, resulting in red cell destruction and ineffective erythropoiesis in the bone marrow.

β-Thalassemia Intermedia

Patients with intermediate Beta-thalassemia (β-Thalassemia Intermedia) carry certain double heterozygous mutations of β⁺ thalassemia and certain homozygous mutations of atypical thalassemia, or they may have a compound heterozygous state of two different variant hemoglobin production disorders. Their pathophysiological changes fall between those of severe and mild forms.

β-Thalassemia Minor

Patients with mild Beta thalassemia (Beta-Thalassemia Minor) carry one abnormal beta globin gene, making them heterozygous carriers while also leading to a slight reduction in β-globin chain synthesis.  β+ silent variants are associated with consistent residual output of beta globin chains, normal red blood cell indices, and normal or borderline HbA₂. Biallelic β+ variants can be associated with β-thalassemia intermedia or β-thalassemia minor. Their pathological symptoms include milder forms of anemia, but may sometimes be asymptomatic.^[3]

Construction of Hbb Gene Editing Models

The C57BL/6 mouse has two highly similar adult β-globin protein-coding genes, Hbb-bs and Hbb-bt, located adjacent to each other on mouse chromosome 7, each comprising three exons.^[4-5] The Hbb-bs&Hbb-bt DKO mouse model for Beta-thalassemia disease research has been established using gene editing technology to simultaneously knockout (KO) the Hbb-bs and Hbb-bt genes in C57BL/6J mice. This double knockout (DKO) model is homozygous lethal, and the heterozygous mice exhibit typical phenotypic characteristics of severe Beta-thalassemia, including abnormalities in hemoglobin content, red blood cell count, hematocrit, mean corpuscular hemoglobin concentration, red blood cell distribution width, platelet count, spleen size, and red blood cell morphology, but retain fertility. Therefore, heterozygous Hbb-bs&Hbb-bt DKO mice are a promising platform for preclinical research related to Beta-thalassemia and potential therapeutics.

Validation Data for the Hbb-bs&Hbb-bt DKO Model

(1) Growth Curve

Figure 1. Weight Change Curve of Heterozygous Hbb-bs&Hbb-bt DKO Mice and Wild-Type (WT) Mice

Both female and male heterozygous Hbb-bs&Hbb-bt DKO mice exhibit growth conditions that are relatively consistent with those of wild-type (WT) mice.

(2) Survival Curve

Figure 2. Survival Curve of Heterozygous Hbb-bs&Hbb-bt DKO Mice and Wild-Type (WT) Mice

Male heterozygous Hbb-bs&Hbb-bt DKO mice began to exhibit mortality starting at week 13, while female heterozygous Hbb-bs&Hbb-bt DKO mice started to show mortality from week 15.

(3) Complete Blood Count Test

Figure 3. Complete Blood Count Test for 14-Week-Old Male Heterozygous Hbb-bs&Hbb-bt DKO Mice and Wild-Type (WT) Mice

The results of the complete blood count test indicate that, compared to wild-type mice, heterozygous Hbb-bs&Hbb-bt DKO mice have significantly decreased red blood cell count (RBC), hemoglobin content (HGB), and hematocrit (HCT). Mean corpuscular hemoglobin concentration (MCHC) is slightly decreased, while red cell distribution width (RDW) and platelet count (PLT) are significantly increased. These changes in parameters are similar to the clinical phenotype of Beta-thalassemia caused by similar genetic mutation types.

(4) Blood Smear Analysis

Figure 4. Blood Smear Analysis for 14-Week-Old Male Heterozygous Hbb-bs&Hbb-bt DKO Mice and C57BL/J Wild-Type Mice

The blood smear analysis results show that heterozygous Hbb-bs&Hbb-bt DKO mice have an increased proportion of nucleated cells, with an expanded central pallor in red blood cells and the presence of more abnormal, fragmented red blood cells and nucleated red blood cells. In their peripheral blood, various abnormal erythrocyte morphologies are present, including target cells (indicated by yellow arrows), spur cells (red arrows), tear drop cells (green arrows), and fragmented cells (blue arrows). In contrast, the red blood cells of C57BL/J wild-type mice are structurally intact, presenting a biconcave disc shape without significant abnormalities.

Cyagen Blood Disease Related Models

In addition to Beta-thalassemia, Cyagen has developed a range of genetically modified models for preclinical hematology research across a range of blood diseases –especially rare blood disorders– including gene knockouts, gene knock-ins, and point mutations. Furthermore, we can customize or collaboratively develop models based on the requirements of researchers.

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References:

[1]Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis. 2010 May 21;5:11. 

[2]Hardison RC. Evolution of hemoglobin and its genes. Cold Spring Harb Perspect Med. 2012 Dec 1;2(12):a011627.

[3]Langer AL. Beta-Thalassemia. 2000 Sep 28 [Updated 2024 Feb 8]. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2024. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1426/

[4]Zhang F, Zhang B, Wang Y, Jiang R, Liu J, Wei Y, Gao X, Zhu Y, Wang X, Sun M, Kang J, Liu Y, You G, Wei D, Xin J, Bao J, Wang M, Gu Y, Wang Z, Ye J, Guo S, Huang H, Sun Q. An extra-erythrocyte role of haemoglobin body in chondrocyte hypoxia adaption. Nature. 2023 Oct;622(7984):834-841.

[5]Trimborn T, Gribnau J, Grosveld F, Fraser P. Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci. Genes Dev. 1999 Jan 1;13(1):112-24.