Hemoglobin Analyses in The Netherlands Reveal More Than 80 Different Variants Including Six Novel Ones

Abstract

More than 20 000 blood samples of individuals living in The Netherlands and suspected of hemolytic anemia or diabetes were analyzed by high resolution cation exchange high performance liquid chromatography (HPLC). Besides common disease-related hemoglobins (Hbs), rare variants were also detected. The variant Hbs were retrospectively analyzed by capillary zone electrophoresis (CZE) and by isoelectric focusing (IEF). For unambiguous identification, the globin genes were sequenced. Most of the 80 Hb variants detected by initial screening on HPLC were also separated by capillary electrophoresis (CE), but a few variants were only detectable with one of these methods. Some variants were unstable, had thalassemic properties or increased oxygen affinity, and some interfered with Hb A₂ measurement, detection of sickle cell Hb or Hb A_1c quantification. Two of the six novel variants, Hb Enschede (HBA2: c.308G > A, p.Ser103Asn) and Hb Weesp (HBA1: c.301C > T, p.Leu101Phe), had no clinical consequences. In contrast, two others appeared clinically significant: Hb Ede (HBB: c.53A > T, p.Lys18Met) caused thalassemia and Hb Waterland (HBB: c.428C > T, pAla143Val) was related to mild polycytemia. Hb A₂-Venlo (HBD: c.193G > A, p.Gly65Ser) and Hb A₂-Rotterdam (HBD: c.38A > C, p.Asn13Thr) interfered with Hb A₂ quantification. This survey shows that HPLC analysis followed by globin gene sequencing of rare variants is an effective method to reveal Hb variants.

• Capillary zone electrophoresis
• hemoglobinopathy
• hemoglobin (Hb) separation
• hemoglobin (Hb) variants
• polycythemia
• thalassemia

Acknowledgements

We are grateful to Sebia Benelux for providing us with a Sebia Capillarys system model 2 including all disposables to analyze our Hb library. We thank Professor Dirk Roos (Laboratory of Blood Cell Research, Sanquin Blood Supply Organization, Amsterdam, The Netherlands) for critically reading the manuscript.