An Effective Screening Algorithm and a Simple DNA Sequencing Protocol for Molecular Characterization of Rare Alpha-Globin Variants

Background: Hemoglobin (Hb) analysis sometimes yields unidentified Hb variants. However, there have been no validated phenotypic characteristics differentiating between a- and b-variants. Furthermore, amplification of α-globin genes are problematic due to almost identical α1-and α2-gene sequences.

Methods: Cases showing unidentified variants on isoelectric focusing (IEF) or high performance liquid chromatography (HPLC) during 2012-2013 were subjected to direct sequencing of α1-, α2- and b-globin genes. The appropriate cutoff Hb variant percentages were determined using receiver operator characteristic (ROC) analysis. The distinctive phenotypes of a-globin mutations were used to construct an algorithm which was subsequently validated in the other cohort of unknown variants.

Results: The characteristics that can differentiate between subjects with rare a-globin variants (N = 39) and b-variants (N = 24) were the presence of unknown variants of HbA₂ (23.1% sensitivity and 100% specificity) and/or Hb variant levels of < 37% on IEF and < 31% on HPLC (100% sensitivity and 100% specificity). Applying these phenotypes (Figure 1) to another validation cohort (N = 67), a-globin variants could be correctly identified with 100% sensitivity and 97.1% specificity. Among the total of 72 subjects with rare a-globin variants, Hb Hekinan (69.4%) was the most common followed by Hb Q-Thailand (8.3%). Other mutations were very rare. This was the first report of Hb Lansing, Hb Owari, Hb G-Georgia, Hb Port Phillip, Hb I and Hb J-Singapore in Thai population. When iron deficiency anemia was excluded, these α-globin variants demonstrated normal or α⁺-thalassemia phenotypes. The percentages of Hb variants and their phenotypes were determined by the presence of mutations on either α1-globin or α2-globin gene and co-inherited α-globin gene deletions. Notably, some a-globin variants such as Hb Hekinan and Hb G-Georgia showed more severe phenotypes similar to thalassemia intermedia when they were co-inherited with other a- and/or β-globin gene mutations. We hereby reported the Hb Lansing mutation on the α1-globin gene coexisting with a⁺-thalassemia resulting in increased Hb Lansing percentage and hence falsely low oxygen saturation.

Conclusions: This is the largest series of rare α-globin mutations. An unknown Hb variant level is the most effective criteria to suggest α-globin variants. Our proposed simple algorithm and DNA sequencing protocol are effective for characterization of α-globin variants.

Data were shown as mean + standard deviation when sample sizes were of ³3.

HbX-IEF/HPLC: unknown variant on IEF and HPLC, HbXA₂-IEF/HPLC: unknown variant of HbA₂ on IEF and HPLC, ND: not detected, SEA: Southeast Asia deletion