Abstract
The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood–derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies.
Highlights
THE MAJOR HEMOGLOBINOPATHIES sickle cell anemia (SCA) and thalassemia as a group represent the commonest monogenic disorders
The present method is suitable for the detection of the therapeutically relevant HBBT87Q variant[45,52] and would allow post-treatment analyses of corresponding gene addition experiments in the presence of normal HBB, separation and precise quantification of normal and T87Q b-globin chains would benefit from variant-specific optimization of conditions
Whereas absolute quantification will require absolute external standards, relative quantification is instead performed by normalization to an internal reference peak assumed constant followed by calculation of fold-changes compared to a control sample
Summary
THE MAJOR HEMOGLOBINOPATHIES sickle cell anemia (SCA) and thalassemia as a group represent the commonest monogenic disorders. Whereas SCA is caused by a pathologic b-globin HBBE6V variant,[1] a- and b-thalassemia are caused by loss or depletion of the eponymous globin chain and consequential toxic excess of its counterpart.[2,3] Lifelong management with blood transfusions and medication, such as iron chelation to avoid iron overload and hydroxyl urea to reduce transfusion requirements in SCA patients, reduces suffering by anemia, hemolysis, and disease-related severe pleiotropic effects. Such palliative treatment is prohibitively expensive for standard care in low- and middle-income countries, and poses the persistent threat of blood contamination and long-term damage to vital organs.[4,5,6,7,8,9,10] Curative treatment by allogeneic transplantation of hematopoietic stem cells, is only available to the fraction of patients for whom suitable donors can be identified and, despite ongoing improvements, carries a significant risk of treatment-related mortality.[11]
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