Hemoglobin Windsor or β11(A8)Val→Asp: A new Unstable β-Chain Hemoglobin Variant Producing a Hemolytic Anemia

Abstract

A new beta-chain variant, Hemoglobin Windsor [beta 11 (A8)Val----Asp] was discovered in a 9-month-old child who presented with a hemolytic anemia of 59 g/l with an intercurrent viral infection. Her blood film demonstrated fragmented cells, target cells, stipple cells, nucleated red cells, polychromasia and some spherocytes, indicative of acute hemolysis. Hemoglobin electrophoretic studies were requested and a beta-chain variant, constituting 27% of the total hemoglobin, separated towards the anode under alkaline conditions. The variant was unstable, producing numerous "Hb H"-like inclusions and a positive isopropanol stability test. The variant hemoglobin was purified by precipitation. The variant beta-chain was purified by column chromatography and its tryptic peptides fractionated by high performance liquid chromatography. Amino acid analysis and sequence data indicated that the valine at position 11(A8) had been substituted by an aspartic acid residue. This substitution, in the bottom of the heme pocket, has resulted in instability of the hemoglobin molecule and an increase in oxygen affinity. The variant appears to have resulted from a spontaneous mutation as both parents are hematologically normal. A younger sibling is also hematologically normal.

δ-Thalassemic phenotype due to two “novel” δ-globin gene mutations: CD11[GTC → GGC (A8)-HbA2-pylos] and CD 85 [TTT → TCT (F1)-HbA2-etolia]

“…Isoleucine, also a hydrophobic amino acid, exists normally at cd11 of the human ò-and Ö-globin gene; similar changes in cases of Ä-variants as Hb-Washtenaw (cd11,ValÃPhe) or HbHamilton (cd11,ValÃIleu) result either in mild instability (Krishnan et al, 1994) or in a "silent" variant (Wong et al, 1984). By contrast, the substitution of Val to Asp in the Ä-globin gene (Hb-Windsor) (Gilbert et al, 1989) reduces the hydrophobicity of the "basket," resulting in an unstable molecule, "Hb H"-like inclusions, and severe hemolysis. Although a direct parallelism cannot be made in the case of Ç-variants, the substitution of Val by the less hydrophobic Gly, should not change the electrophoretic mobility of the molecule, but should reduce hydrophobicity of the "basket" and may produce a mildly unstable Hb; these considerations are supported by the reduced percentage (1.6%) of HbA 2 , which is similar to that found in the case of the mild (Ç+-type) mutation cd27(GCCÃTCC) (Pirastu et al, 1990).…”

“…Valine occupying the helical position A8 is an internal amino acid and occurs at the base of the heme pocket (Perutz et al, 1968). In the case of Ä-globin gene Val in this position acts as a contact with the H-helix (Gilbert et al, 1989). It is known that the residues lining the heme pocket form a kind of "hydrophobic basket," thus preventing the oxidation of the heme iron to its ferric state.…”

Two novel unstable hemoglobin variants due to in‐frame deletions of key amino acids in the β‐globin chain

“…Conservation analysis reveals that valine is the most conserved residue of these three residues. There is only 1 variant with clinical repercussions for the substitution of this residue: Hb Windsor ( HBB :c.35T>A, p.Val11Asp ) that leads to hemolytic anemia. There are 2 other variants described: Hb Washtenaw ( HBB : c.34G>T, p.Val11Phe ), slightly unstable with decreased affinity for the O 2 , and Hb Hamilton ( HBB :c.34G> A, p.Val11Ileu ) without any particular phenotype associated.…”