Hb CHAMPAGNE [β113(G15)Val->Leu]: A NEW NEUTRAL VARIANT
Wajcman H1, Riou J1, Promé D2, Richelme-David S2, Galactéros F1
*Corresponding Author: Dr. Henri Wajcman, INSERM U 468, Génétique Moléculaire et Physiopathologie, Hôpital Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France; Tel: +33-1-49-81-35-78; Fax: +33-1-48-99-33-45; E-mail: Henri.Wajcman@im3.inserm.fr
page: 23

RESULTS

This variant Hb was found in a 50-year-old Caucasian French man from the region of Champagne who was investigated because of a mild anemia. The RBC parameters were as follows: Hb 12.5 g/dL, RBC 4 x 1012/L, MCV 92 fL, MCH 31.3 pg, reticulocytes 1.1%. Bilirubinemia (9 mmol/L) and haptoglobin levels (1 g/L) were within normal limits. An abnormal Hb that focalized at a position near to that of Hb F, and amounting visually to ca. 40%, was observed. Cation exchange HPLC revealed no abnormal component, hampering a precise measurement of the amount of the variant. The mobility of this Hb, under the different experimental conditions used in our laboratory for presumptive diagnosis of mutants, suggested that it resulted from an exchange between two residues carrying the same electrophoretic charge (Table 1).


Electrospray (ES) analysis of the globin showed a b chain with a mass increased by 15  1 Da, allowing for several possible amino acid exchanges. Reversed phase HPLC on the Lichrospher column revealed an abnormal chain eluting as a hydrophobic shoulder within the b chain peak (retention time = 11.5 versus 10 for normal b? and 20 for a). From these biochemical properties it was proposed that this variant had not been described before.

Peptide analysis done on the tryptic digest of the aminoethylated b chain of the purified abnormal Hb, showed an abnormal bT-12b. This peptide was more hydrophobic than normal, eluting slightly earlier than the bT-13 (Fig. 1). Tandem MS revealed that in this case the valine residue at position 113 was replaced by leu­cine (Fig. 2), accounting for a mass difference of +14 Da. The variant here described was named Hb Champagne or b113(G15) ValLeu.

 

Table 1: Electrophoretic and chromatographic parameters of Hb Champagne compared to those of Hb A and of several b chain variants with a ValLeu substitution

 

 

 

 

Fig. 1. HPLC pattern of the tryptic digest of the aminoethylated b chain of Hb Champagne. Peptide bT-12b is more hydrophobic than normal. Experimental conditions: 0.2 mg tryptic digest was applied on a Vydac C8 column (25 x 0.46 cm) (The Separations Group, Hesperia, CA, USA). Elution was obtained at a flowrate of 1.5 mL/min using a concave curvilinear gradient between solvent A [trifluoroacetic acid (TFA) 0.2% in water] and solvent B (acetonitrile/water/TFA, 70:30:0.2, vol. to vol.) from 0 to 60% B in 60 minutes (corresponding to an increase of acetonitrile concentration from 0 to 42%).

 

 

Fig. 2. MS/MS pattern of the abnormal bT-12b peptide. Starting from the C-terminal, all the ions of the Y series down to Y7 have an identical mass in Hb A and Hb Champagne; conversely, starting from the N-terminal, all the ions of the B series had their mass increased by 14 Da. This demonstrates that the difference of mass is carried by the N-terminal residue of the peptide: valine is thus replaced by leucine.

 




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