Hb Lepore Rochester-MN, a novel βδβ double crossover hemoglobin variant

The β-globin gene cluster harbors highly homologous globin genes. Crossover events involving the δ (HBD) and β (HBB) genes result in Lepore (δβ) and anti-Lepore (βδ) hemoglobins (Hbs). Recently, double crossover (βδβ) variants have been reported. Herein, we report βδβ variants identified in our laboratory including the novel Hb Lepore Rochester-MN (LRM).

Blood samples were obtained with Institutional Review Board approval. Protein characterization included cation exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and mass spectrometry (MS). Molecular analysis included HBB Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA).

Two distinct βδβ crossover variants were identified: Novel Hb LRM (2 infants, 1 adult) and Hb Wanjiang. Hb LRM separated on protein studies (HPLC, CE and MS) and showed identical protein characteristics as Hb Lepore-Hollandia, however, it was expressed at a higher percentage. Sanger sequencing characterized the variant as NM_000518.4(HBB):c.28_68delins41 (p.Ser10_Glu23delins14TAVNALWGKVNVDA). Hb Wanjiang protein did not separate from Hb A using routine methods (HPLC, CE and IEF) but was identifiable by MS and DNA sequencing as NM_000518.4(HBB):c.255_264delinsTTTTTCTCAG (p.A87_T88delinsSQ).

The copy neutral incorporation of δ segments into β gene does not worsen the clinical phenotype. Some substitutions may even have a protective effect when coinherited with Hb S. These uncommon double crossover βδβ variants can pose a diagnostic challenge for laboratories as they can be mistaken for other similar variants on protein evaluation. Also, they may require specialized analysis such as MS, Sanger sequencing or NGS. Interpretation can be challenging if comparison to δ-gene is not considered.