The proband, a 48-year-old Caucasian woman from England, was referred following an abnormal ion exchange HPLC trace on Hb A1c measurement. Ion exchange HPLC of her Hb showed an abnormal peak, amounting to ca. 40% of the total Hb, and eluting between Hb A and Hb A2 (Fig. 1).

Fig. 1. Elution pattern of the hemolysate containing Hb A and Hb Harlequin obtained by cation exchange HPLC on the VARIANT™ system (Bio-Rad Laboratories, Hercules, CA, USA) using the b-Thalassemia short program.

 In contrast, isoelectrofocusing (IEF) and electrophoretic studies, done under the various experimental conditions used in our laboratory, did not reveal the presence of the abnormal Hb. The reversed phase HPLC elution pattern of the globin chains was also normal. ES/MS analysis of the globin indicated that the proband was heterozygous for a β chain variant having a mass increased by 28 Da, which, in the case of an electrophoretically neutral variant, may account for a Lys—>Arg or Ala—>Val change. Red blood cell (RBC) parameters were normal (RBC 4.63 x 1012/L, Hb 13.1 g/dL, PCV 0.409 L/L, MCV 88.0 fL, MCH 28.3 pg). There were no red cell abnormalities on the film and no significant clinical features.
A mixture containing both the normal and the abnormal b chains was separated from the a chains by reversed phase HPLC. The elution pattern of the peptides obtained after aminoethylation and digestion with trypsin is shown in Fig. 2. It revealed the presence of two bT-9 peptides of almost the same size, the abnormal being more hydrophobic than the normal one. Tandem MS analysis of this peptide demonstrated that alanine at position 76 was replaced by a valine (Fig. 3), in agreement with the results of the ES analysis of the chains. This variant, which has not been described before, was named Hb Harlequin for the city where the proband lives. At the DNA level, the presumed mutation is a GCT to GTT change in codon 76.

Fig. 2. Elution pattern of the tryptic digest of an AE mixture of normal and abnormal b chains. The abnormal bT-9 peptide is indicated by the arrow.