An Effective Screening Algorithm and a Simple DNA Sequencing Protocol for Molecular Characterization of Rare Alpha-Globin Variants
Authors: Uaprasert Noppacharn , Settapiboon Rung , Amornsiriwat Supaporn , Sutcharitchan Pranee , Rojnuckarin Ponlapat 
Published: October 2019

Abstract
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.
Table 1. Hematological parameters and hemoglobin analysis data of rare α-globin variants in Thailand Genotypes (N) Mutation Hb (g/dL) MCV (fL) HbX-IEF/HPLC (%) HbXA2-IEF/HPLC (%) Coexistence with other mutations Hb Hekinan (39) α1 27(B8) Glu>Asp 13.0+1.4 85.7 + 5.7 22.9±6.5/ ND 5.4±0.6 (4)/ND Hb Hekinan (3) α1 27(B8) Glu>Asp 11.8+1.2 68.7 + 3.5 34.8±3.6/ 24.5 ± 3.4 6.9±4.8/3 ± 2.7 SEA deletion Hb Hekinan(6) α1 27(B8) Glu>Asp 12.8+1.3 79.8 + 6.6 16.7±11.1/ 25.4 (1 case) 5.5±2.0/ 4.5 (1 case) Heterozygous HbE Hb Hekinan (1) α1 27(B8) Glu>Asp 8.4 66.0 6.0/ ND ND/ ND Homozygous HbE Hb Hekinan (1) α1 27(B8) Glu>Asp 9.6 88.0 17.0 (Hekinan)/46.8 (Hope) 10.0 (Hekinan)/ 60.0 (Hope) HbHope/HbE Hb Q-Thailand (6) α1 74(EF3) Asp>His 12.1+0.6 77.7 +2.9 35.0 ± 1.0/ 28.7 ±1.0 1.6 (1)/ND Hb Owari (3) α1 121(H4) Val>Met 11.3±0.6 89.7 +5.9 19.7 ± 6.8/ ND ND/ND Hb G-Georgia (1) α2 95(G2) Pro>Leu 12.0 92.0 25.6/ ND 2.0/ND Hb G-Georgia (1) α2 95(G2) Pro>Leu 9.9 75.0 22.4/ ND 3.3/ND Homozygous HbE Hb G-Georgia (1) α2 95(G2) Pro>Leu 7.5 67.0 4.4/ ND 1.2/ ND Codon 17 A>T (Lys>Stop) Hb Westmead (2) α2 122(H5) His>Gln 11.8, 12.7(CB) 71.0, 117.0 (CB) 23.6, 4.7(CB)/ ND ND/ ND Hb Port Phillip (2) α1 91(FG3) Leu>Pro 12.3, 10.6 84.0, 73.0 17.4/ 5.9, 21.3/10.7 1.7/ ND, ND/ND Hb Lansing (1) α1 87(F8) His>Gln 11.9 78.0 7.2/ 2.5 ND/ ND Hb Lansing (1) α1 87(F8) His>Gln 12.6 88.0 14.2/ 5.5 ND/ ND -3.7 kb deletion Hb I (1) α2 16(A14) Lys>Glu 13.1 95.0 28.9/ 28.4 ND/ ND Hb J-Singapore (1) α2 79(EF8) Ala>Gly 15.9 94.0 30.0/ 25.1 ND/ ND Hb J-Buda (1) a1 61(E10) Lys>Asn 15.1 89.0 25.4/ 15.8 ND/ ND Hb Phnom Penh (1) α1 117(GH5)-Ile-α118(H1) 16.0 82.0 30.7/ ND ND/ ND
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

Disclosures
No relevant conflicts of interest to declare.