World Thalassemia Day | Powerful Tool for Disease Research: Hbb-bs & Hbb-bt DKO Mouse Models

May 8 marks World Thalassemia Day — a timely opportunity to introduce β-thalassemia, a hereditary blood disorder caused by reduced or absent synthesis of the β-globin chains of hemoglobin. Recognized by the World Health Organization (WHO) as a global public health concern, β-thalassemia affects populations worldwide. ^[1]

Pathogenic Mutations in the HBB Gene

Hemoglobin (Hb) is the protein in red blood cells responsible for transporting oxygen throughout the body. Its structure consists of two α-globin subunits and two β-globin subunits. Each subunit contains a heme molecule at its core, which binds to an iron ion, enabling hemoglobin to carry oxygen.

In healthy adults, the β-globin chains are encoded by the HBB gene. Together with two α-globin chains (encoded by either the HBA1 or HBA2 gene), they form adult hemoglobin (HbA), which accounts for approximately 97% of total hemoglobin. ^[2]

In patients with β-thalassemia, mutations in the HBB gene result in reduced or absent synthesis of the β-globin chains. This leads to lower hemoglobin levels and causes ineffective erythropoiesis, premature destruction of red blood cells, and symptoms such as anemia.

Clinical Types of β-Thalassemia

β-thalassemia caused by mutations that completely eliminate the production of β-globin chains is referred to as β⁰-thalassemia, while mutations that partially suppress β-globin chain production are classified as β⁺-thalassemia. ^[3]

Patients with severe (major) β-thalassemia carry either homozygous mutations of β⁰ or β⁺ type, or compound heterozygous mutations of both β⁰ and β⁺ types. These mutations result in the complete or near-complete absence of β-globin chain synthesis, leading to significantly reduced or absent production of hemoglobin A (HbA), which contains β-globin. The lack of β-globin disrupts the balance between α- and non-α-globin chains, causing an excess of unpaired α-globin chains. These unpaired chains precipitate in red blood cells as inclusions, inducing oxidative damage to the cell membrane and resulting in hemolysis and ineffective erythropoiesis.

Patients with mild (minor) β-thalassemia are heterozygous for either a β⁰ or β⁺ mutation. These individuals have a mild reduction in β-globin synthesis and typically present with milder clinical symptoms.

Intermediate (intermedia) β-thalassemia occurs in individuals with certain combinations of β⁺ compound heterozygous mutations or homozygous atypical β-thalassemia mutations. It may also result from compound heterozygosity for two different types of globin synthesis disorders. The pathophysiology of intermediate β-thalassemia lies between that of the major and minor forms. ^[4]

Generation of the Hbb Gene-Edited Mouse Model

In C57BL/6 mice, two highly similar adult β-globin protein-coding genes—Hbb-bs and Hbb-bt—are located adjacent to each other on chromosome 7, and both consist of three exons. ^[5–6] The Hbb-bs & Hbb-bt double knockout (DKO) mouse model (Product No.: C001508) was established by simultaneously knocking out both genes using gene-editing technology, thereby creating a β-thalassemia disease model.

This model is homozygous lethal, while heterozygous mice exhibit classical features of severe β-thalassemia, including abnormalities in hemoglobin concentration, red blood cell (RBC) count, hematocrit, mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count, spleen size, and RBC morphology. Importantly, the heterozygous mice are fertile.

As such, heterozygous Hbb-bs & Hbb-bt DKO mice provide a valuable tool for studying the pathophysiology and potential therapies related to β-thalassemia.

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

(1) Growth Curve

Figure 1. Body Weight Growth Curve of Heterozygous Hbb-bs & Hbb-bt DKO Mice Compared to Wild-Type (WT) Mice

Both female and male heterozygous Hbb-bs & Hbb-bt DKO mice exhibit growth patterns that are generally consistent with those of wild-type mice.

(2) Survival Curve

Figure 2. Survival Curve of Heterozygous Hbb-bs & Hbb-bt DKO Mice Compared to Wild-Type (WT) Mice (♂n=9, ♀n=8)

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

(3) Complete Blood Count (CBC)

Figure 3. Complete Blood Count (CBC) Analysis of 14-Week-Old Male Heterozygous Hbb-bs & Hbb-bt DKO Mice Compared to Wild-Type (WT) Mice

The complete blood count (CBC) results show that, compared to wild-type mice, heterozygous Hbb-bs & Hbb-bt DKO mice exhibit significantly decreased red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit (HCT). The mean corpuscular hemoglobin concentration (MCHC) shows a slight reduction, while red cell distribution width (RDW) and platelet count (PLT) are markedly increased. These hematological changes closely resemble the clinical phenotypes observed in β-thalassemia caused by similar β-globin gene mutations.

(4) Blood Smear Analysis

Figure 4. Blood Smear Analysis of 14-Week-Old Male Heterozygous Hbb-bs & Hbb-bt DKO Mice Compared to C57BL/6J Wild-Type Mice

Blood smear analysis revealed an increased proportion of nucleated cells in heterozygous Hbb-bs & Hbb-bt DKO mice. Their red blood cells exhibited enlarged central pallor along with numerous abnormal forms, including poikilocytes, fragmented erythrocytes, and nucleated red blood cells. Various morphological abnormalities were observed in peripheral blood, such as target cells (yellow arrows), echinocytes (red arrows), teardrop-shaped cells (green arrows), and schistocytes (blue arrows). In contrast, red blood cells from C57BL/6J wild-type mice displayed intact morphology, appearing as biconcave discs with no apparent abnormalities.

Cyagen's Hematological Disease-Related Models

In addition to β-thalassemia, Cyagen has developed a series of gene-edited models targeting various hematological diseases, particularly rare blood disorders. These models include gene knockouts, knock-ins, and point mutations. Furthermore, we offer customized model generation and collaborative development services tailored to researchers' specific needs. If you are interested in learning more or discussing a specific project, feel free to contact us for expert consultation and support.

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]Thein SL. Molecular basis of β thalassemia and potential therapeutic targets. Blood Cells Mol Dis. 2018 May;70:54-65.
[4]Origa R. β-Thalassemia. Genet Med. 2017 Jun;19(6):609-619.
[5]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.
[6]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.