Hb Hounslow is an example of a variant which has no charge difference from Hb A and is silent in IEF. However, two other variants that carry the same amino acid exchange (Asn→Tyr), namely Hb Saint Mandé [β102 (G4)] and Hb Aurora [β139(H17)] are both well separated from Hb A by this technique (6). Hb Hounslow was detected because of its behavior on cation exchange HPLC, in which the retention time depends not only upon the difference in charge but also on other interactions with the stationary phase that result from local modifications induced by the mutation. Several variants, which behave like Hb Hounslow, include Hb Alzette [β104(G6)Arg→Lys], Hb Puttelange [β140(H18)Ala→Val] and Hb Rainier [β145(HC2)Tyr→Cys] [6]. Many of the neutral variants described are clinically silent or have minimal hematological consequences in the heterozygous state but some interact with a thalassemia trait, resulting in a thalassemia intermedia phenotype or with a sickle cell trait and favor sickling. When screening for a specific abnormality, such as Hb S or a thalassemic phenotype, one method may be sufficient to reveal the defect, but it is important to be aware that another silent abnormality may coexist and act as a gene modifier. Thus, phenotype screening should include at least two methods that explore different properties in order not to miss a neutral variant. Additional techniques such as reversed phase HPLC and ES-MS may show the presence of a neutral variant when a combination of IEF and cation exchange HPLC fail to reveal it, while hematological arguments would suggest its presence. For example, these two last techniques were required for detecting Hb Zoetermeer [α21(B2)Ala→Ser] [7].

Residue EF4, which is modified in Hb Hounslow, is located externally near to the 2,3-DPG binding pocket. A comparison of more than 1,100 sequences of molecules from the globin family (Hbs, myoglobins, cytoglobins and neuroglobins from various species) showed that this position is mostly occupied by an aspartic acid (35%) or an asparagine (30%) and means that conservation of the size of this residue and a possibility for hydrophilic interaction may be important in evolution [8]. Hb G-Szuhu (Asn→Lys), also clinically silent, is the other b chain variant that involves position EF4 [9].