SULM – Schweizerische Union für Labormedizin | Union Suisse de Médecine de Laboratoire | Swiss Union of Laboratory Medicine

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L. Bernasconi1 , R. Herklotz2 , M. Hergersberger2 , A. Huber2

1Molecular Biology Institute, Zentrum für Labormedizin, Kantonsspital Aarau, Switzerland, 2Zentrum für Labormedizin, Kantonsspital Aarau, 5001 Aarau

Haemoglobinopathies are the most common single gene disease world-wide, resulting in either structural Hb variants (abnormal haemoglobin) or unbalanced ratios between α- and β-globin chain synthesis (α- or β-thalassemia). The most frequent genetic defects leading to α-thalassemia are deletions of one (α+) or both (α0) α-globin genes. Rarely also non-deletional pointmutations can be identified in the α-globin locus. β-thalassemia are primarily due to single nucleotide mutations, and so far about 200 different sequence variants were identified affecting the β-globin gene. The clarification of the molecular background of haematological complex cases, such as anaemia of chronic disease (ACD), iron deficiency anaemia (IDA) or combinations thereof is hence essential for their comprehension. Therefore we developed a method for the rapid and accurate genotyping of non-deletional mutations located in the human α1-, α2- and β-globin genes using denaturing HPLC (dHPLC) technique. Overlapping regions located into the α and β-globin genes were amplified by PCR, and DNA homo- and herteroduplexes were generated by denaturation and reannealing of the amplicons. The DNA sequences were then analysed at different partial melting temperatures. In a preliminary evaluation we compared the β-globin dHPLC analysis to the β-globin StripA reverse-hybridisation assay by examining a panel of β-thalassemia patients. In a first analysis of 13 different mutations originally detected by β-Globin Strip, we were able to detect all variants by dHPLC. The examination of 31 additional samples, which had been judged as β-thalassemia on the basis of their high HbA2 value, were subsequently analysed by dHPLC, β-Globin StripA and sequencing. Overall, 11 of 31 (65%) single base mutations were detected by β-Globin StripA, whereas all were detected by dHPLC. We conclude that the sensitivity of dHPLC is significantly superior to that β-strip for the detection of β-thalassemia single base alterations.


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