We report a novel haemoglobin (Hb) variant at position [β42 Phe → Cys] found in a Caucasian male infant with severe chronic haemolytic anaemia. He presented at 5 weeks of age with fever and vomiting and was found to have apparent oxygen saturations of 82% in air by pulse oximetry and Hb 6.7 g/dl. Absolute reticulocyte count 353 × 109/l, LDH 872 u/L, bilirubin 23 mmol/L, direct antiglobulin test (DAT) negative. Blood film showed marked anisopoikilocytosis, keratocytes, irregularly contracted cells, basophilic stippling, nucleated red blood cells, and increased polychromasia consistent with haemolysis and oxidative damage. Echocardiogram, electrocardiogram and chest radiograph were normal. Arterial blood gas revealed normal PaO2. Haptoglobins were absent. Required regular blood transfusions until 6 months of age when he achieved a 3 months period without transfusion and could be further investigated. Hb electrophoresis showed a normal pattern. No evidence of glucose 6 phosphate deficiency, pyruvate kinase or 5' pyrimindine nucleotide deficiency. Heinz preparation was positive, with positive heat stability and isopropanol test suggesting the presence of variant haemoglobin. Snap frozen mass spectrometry was performed which demonstrated a low abundance (4%) of a variant haemoglobin. This was further characterised by DNA sequencing of the beta-globin gene which revealed the presence of a novel heterozygous mutation of the beta chain. A single amino acid substitution at the 42 amnio acid of the beta chain was identified resulting in Phenylalanine being replaced by cysteine [β42 Phe → Cys]. Both parents were studied for the mutation and were found to be negative. At 2 years of age the level of variant haemoglobin was 12% and oxygen dissociation curve showed oxygen P50 of 29.5 mmHg (reference range: 29.5 – 32.0) indicating no evidence of altered oxygen affinity. He continues with severe chronic haemolytic anaemia exacerbated by infections and requires a monthly regular transfusion regime.
Phenylalanine in position 42 (the first position of the region between the C and D helices - CD1) of the β chain participates in the contacts with haem. It is a critical amino acid in the haem pocket, maintaining solubility and stability. Substitution of this phenylalanine by cysteine removes a contact with haem leaving a gap at the surface of the haem pocket and results in instability. There are three other Hb variants resulting from substitutions in position 42: Hb Hammersmith, Hb Louisville and Hb Sendagi, substitutions for serine, leucine and valine respectively. Hb Hammersmith being more severe and leading to transfusion dependence in some of the cases, whilst the others are mild to moderate, only requiring transfusions for infective episodes. There are over 800 variant Hb are described with the majority not being clinically significant. Of those which are significant, greater then 95% are due to single amino acid substitutions (as in our case) which changes the structure of the Hb. The clinical abnormalities are attributable to the changes in Hb solubility, stability and capacity to carry and deliver oxygen.
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