Congenital Erythrocytosis (CE), also called congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. 3Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and generally results from mutations in the erythropoietin-receptor gene (EPOR).Secondary congenital erythrocytosis arises from conditions which cause tissue hypoxia thus resulting in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (genes HBB, HBA1 and HBA2), decreased production of 2,3-biphosphoglycerate due to mutations in the BPGM gene, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1 and EGLN1). Depending on the affected gene CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo.Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients.With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive internet-based database focusing on the registration of clinical history, hematological, biochemical and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database (LOVD).

Mass spectrometry has a basic limitation when human hemoglobin variants are analyzed, because it cannot resolve two globin chains that differ in mass by <6 Da. Several common beta-chain variants differ by 1 Da from normal and, hence, when present in heterozygotes, are not resolved from the normal beta-chain. Normal and variant chains appear together in the spectrum as a single entity, whose mass is the abundance weighted mean of the two chains. Here we show that such heterozygotes can be detected in 500-fold diluted blood by accurately measuring the mass of the beta-chain using an electrospray ionization quadrupole instrument and the alpha-chain for internal mass calibration. A statistical analysis of the normal beta-chain mass (n = 86) showed that the standard deviation (SD) of the mean was <+/-0.05 Da (<+/-3.2 ppm). Hence, at the 95% confidence level (+/-2 SD), an abnormal alpha- or beta-chain differing by 1 Da from normal should be detectable in a heterozygote provided its abundance is >10% of total alpha- or beta-chains, respectively. Variants whose masses lay between 1 and 4 Da from normal were detected in 19 heterozygotes. Moreover, the proportion of each variant estimated from the mass change correlated with the proportion determined by cation-exchange HPLC. Variants were assigned to the alpha- or beta-chain by combining the sign of the mass change with the polarity change inferred from electrophoretic data. This procedure could be used for screening clinically significant hemoglobin variants.

In this report, we present data to illustrate how human hemoglobin (Hb) variants can be identified by electrospray tandem mass spectrometry (MS/MS) of the intact Hb chains following the one-step dilution of whole blood. MS/MS spectra were recorded on a series of intact beta-chain human Hb variants. The resultant spectra were interpreted, and using the information gleaned from the fragmentation patterns of known variants, two unknown beta-chain variants were characterized solely by this mass spectrometric method. Fragment ions that serve to identify beta-chain variants were identified. The fragmentation patterns of the intact beta-chain [M + 18H]18+ ions showed classical facile cleavages adjacent to acidic residues and N-terminal to proline residues, with Thr50-Pro51 being the most prominent cleavage site. Abundant product ions were formed by peptide bond cleavage in the regions close to the termini of the beta chain, the central region being less well-represented in the MS/MS spectra. Nearly 50% of the beta-chain primary structure could be determined by MS/MS of the intact chain. However, analysis of the Hb variants where mutations have occurred in the inner region (residues 58-111) of the beta globin proved to be difficult and required mass spectrometric analysis of their tryptic peptides for a complete identification.