Detection of a thalassemic alpha-chain variant (Hemoglobin Groene Hart) by reversed-phase liquid chromatography

BACKGROUND: Hemoglobin (Hb) Groene Hart [alpha 119 (H2)Pro -> Ser (alpha 1)], also known as Hb Bernalda, is a nondeletional alpha-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The a-thalassemic behavior of this variant has been explained as an impaired interaction between the a-globin chain and the alpha-Hb-stabilizing protein. METHODS: To separate globin chains, we developed a modified reversed-phase liquid chromatography (RPLC) procedure that uses acetonitrile-water solvents containing up to 3 mL/L trifluoroacetic acid. After RPLC, we characterized the isolated globin chains by electrospray ionization (ESI) mass spectrometry (MS) and analyzed their tryptic peptides with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nano-LC-ESI-MS/MS. RESULTS: RPLC detected an abnormal peak with a retention time substantially greater than that of the wildtype alpha(A)-globin chain. We identified this variant as Hb Groene Hart and found it in the hemolysates of 11 unrelated patients (1 homozygote, 9 heterozygotes, and 1 heterozygote associated with the -alpha(3.7) deletion). These patients possessed abnormal hematologic features suggesting an alpha-thalassemia, phenotype. Molecular modeling suggested that the increase in hydrophobicity was due to opening of the GH interhelical segment following replacement of amino acid residue 119 with a nonhelix breaker residue. CONCLUSIONS: This method allows the detection of Hb variants at low concentrations, and adjusting the composition of the organic solvents enables the method to identify Hb variants with large changes in hydrophobicity. (c) 2008 American Association for Clinical Chemistry.