Identification of a rare [^G(^AæĦ)⁰] -thalassemia using tandem mass spectrometry

Thalassemias are a group of inherited monogenic disorders characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Delta-beta (delta beta-) thalassemia has large deletions in the beta globin gene cluster involving delta- and beta-globin genes, leading to absent or reduced synthesis of both delta- and beta-globin chains. Here, we used direct globin-chain analysis using tandem mass spectrometry for the diagnosis of delta beta-thalassemia. Two cases from unrelated families were recruited for the study based on clinical and hematological evaluation. Peptides obtained after trypsin digestion of proteins extracted from red blood cell pellets from two affected individuals and their parents were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometric analysis revealed a severe reduction in delta, beta, and A gamma globin proteins with increased (G)gamma globin protein in the affected individuals. The diagnosis of (G)gamma((A)gamma delta beta)(0) -thalassemia in the homozygous state in the affected individuals and in the heterozygous state in the parents was made from our results. The diagnosis was confirmed at the genetic level using multiplex ligation-dependent probe amplification (MLPA). Our findings demonstrate the utility of direct globin protein quantitation using LC-MS/MS to quantify individual globin proteins reflecting changes in globin production. This approach can be utilized for accurate and timely diagnosis of hemoglobinopathies, including rare variants, where existing diagnostic methods provide inconclusive results.