Abstract
Introduction: A 26 year old male patient was investigated at Maisonneuve-Rosemont Hospital (Montreal, Canada) for chronic hemolysis. The patient was known for many years to have chronic hemolysis of unknow etiology and splenomegaly. He had previously undergone cholecystectomy. There was no family history of hemolytic anemia. The investigation revealed a normal automated CBC but the blood smear showed polychromatophilia, basophilic stippling and a few degmacytes (bite cells). The serum chemistry analysis revealed an increased LDH and bilirubin and a decreased haptoglobin level. The search for Heinz body was positive after exposure to acetyl phenylhydrazine. Unstable hemoglobin was demonstrated using the isopropanol test, but the heat instability test was negative. The dosages of erythrocyte enzymes (G-6PD, pyruvate kinase, gluthation reductase and acethylcholinesterase) were normal. Hb HPLC analysis was done using a VARIANT II, Bio-Rad. Results demonstrated the presence of normal HbA and an abnormal Hb migrating as Hb Hope at a level of 24% of total hemoglobin.
Methods and Results: Blood DNA sample was obtained to perform automated sequencing of the β-globin gene (HBB). The HBB gene was sequenced using primers designed to amplify coding sequences of the three exons, splice junctions, introns and the promoter region. Five samples (the patient and four controls) were analysed. Sequence were compared to the cDNA sequence of the HBB gene (NM_000518) and from the genomic sequence of the region (AC104389). In the case of the propositus, a 1 nucleotide deletion was detected at codon 142 (nucleotide 480 of GenBank entry NM_000518) in exon 3, causing a reading frameshift. This deletion (TGCCCTGGCC [C/del] ACAAGTATCA) was never detected before and eliminates the regular stop codon (TAA) at position 147. A new stop codon is therefore produced after the addition of 14 residues, identical to those observed in Hbs Saveme, Trento and TAK, all of which result from a similar reading frameshift secondary to a deletion. However, a threonine residu is present at position 142 in the case of the propositus.
Conclusion: Hemoglobin Ville-Marie is an elongated C-terminal hemoglobin variant causing mild compensated hemolytic anemia with some degree of hemoglobin instability. Others analysis are being performed to better characterise this new hemoglobin variant, the results of which will be presented at the meeting.
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