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Letters to the Editor |
82Ala
Thr) Substitution Identified in an Infant with Severe Hemolytic Anemia
1 Canterbury Health Laboratories, Christchurch, New Zealand
2 Pathology Department, Christchurch School of Medicine, University of Otago, Christchurch, New Zealand
aAddress correspondence to this author at: PO Box 151, Canterbury Health Laboratories, Christchurch, New Zealand. Fax 64-3-3640545; e-mail steve.brennan{at}chmeds.ac.nz.
To the Editor:
The identification of new hemoglobin mutations and the correlation of structural changes with potential pathology continue to provide insights into how normal structure and function are preserved in the tetrameric molecule.
Hematological investigation of a 6-week-old infant with severe hemolytic anemia revealed a decreased hemoglobin concentration of 53 g/L, a decreased hematocrit of 0.15, and a decreased erythrocyte count of 1.8 x 1012/L. In addition the infant had an increase in reticulocytes (322 x 109/L) and bilirubin (96 µmol/L). Blood films showed a normochromic picture with irregularly shaped cells, elliptocytes, and erythrocyte fragments suggestive of an erythrocyte membrane defect. G6PD and pyruvate kinase concentrations were within reference intervals, and a presumptive diagnosis of hereditary elliptocytosis was made.
Cellulose acetate and citrate agar electrophoresis results were both within reference intervals, as was cation exchange chromatography on the Bio-Rad Variant ß-thalassemia system. Examination of whole lysate by electrospray ionization mass spectrometry on a VG Platform (1) showed that the complement of ß (15 867 Da), 
G (15 995 Da), and A (16 010 Da) chains were within reference intervals for the age of the infant. However, the valley between the Na and K adducts of the 
chain (15 126 Da) contained a new component with a mass increase of 30–33 Da over the normal chain (Fig. 1A
). Reversed-phase HPLC on a C-4 Jupiter column (2) failed to separate out the new component, so DNA sequencing was used to identify the putative mutation.
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The entire coding regions of both the hemoglobin alpha 1 and alpha 2 genes were individually amplified from genomic DNA using the primer pairs 5'-TGG AGG GTG GAG ACG TCC TG-3' with 5'-CCA TGC TGG CAC GTT TCT GA-3', and 5'-TGG AGG GTG GAG ACG TCC TG-3' with 5'-CCA TTG TTG GCA CAT TCC GG-3', respectively, and sequenced on an ABI 3130xl genetic analyzer with Big Dye Terminator v3.1 cycle sequencing chemistry according to the manufacturer’s recommendations. This process revealed heterozygosity for a GCC
ACC transition at codon 82 of the 1 gene, and we have named this new 82AlaThr variant Hb Hagley Park.
Unstable hemoglobinopathies are a well-recognized cause of hemolytic anemia, and the identification of this novel substitution raises the question as to whether it contributed to the hemolytic condition. This question could be answered by stability tests; however, insufficient blood (50 µL) was available for analysis and, in any case, the presence of substantial amounts of HbF, which registers a positive result in these tests, would confound the results. Family studies would also be expected to be informative; however, neither parent was available for analysis.
There are no published reports of any other mutation at position 82, the 3rd residue of the F helix. This short amphipathic helix contains the proximal histidine, and its inner hydrophobic surface makes direct contact with the heme plate. Mutations along this surface result in molecular instability and can cause mild hemolytic anemia (1)(2)(3). The Ala82 side chain is positioned between the inner and outer hydrophilic surface of the helix, and introduction of an additional CH2OH moiety could potentially destabilize the structure. However, alignment of 439 -globin sequences from different species shows that residue 82 is only loosely conserved as alanine, and some 18 species, including mouse and rabbit, have a threonine residue at position 82. Threonine is also found at the F3 position of the human ß chain, supporting the notion that its occurrence in the chain should not cause any major distortion, at least of the helix.
We received a 2nd blood sample when the infant was 18 months old, and again this sample showed evidence of significant hemolysis, with decreased hemoglobin (60 g/L), hematocrit (0.18), and erythrocyte count (2.4 x 1012). The normochromic blood film showed pencil cells, some elliptocytes, and erythrocyte fragments. Mass spectrometry confirmed a complete switch to an adult pattern of hemoglobin synthesis, with no significant amount of chains detected; the abnormal 15 157-Da chain was still present at a level of approximately 18% (Fig. 1
). Isopropanol stability tests showed no precipitation at 30 min, and although a small amount of precipitate formed after the incubation was extended to 45 min, electrospray analysis showed no enrichment of the variant chain. This normal stability suggests that the mutation is benign; making it unlikely that it contributes to the hemolytic condition.
This case highlights the importance of isolating the potential pathological effect of novel hemoglobin mutations when they are identified in association with changes in hemoglobin concentrations. In this case it appears the hemolysis is due to the accompanying, but unrelated, erythrocyte membrane defect.
Acknowledgments
Grant support/funding: None declared.
Financial disclosures: None declared.
Acknowledgments: We gratefully acknowledge the assistance of Jaine Duncan and Vanessa Buchan.
References
87 HisAsn): a new mutation of the proximal histidine identified by electrospray mass spectrometry. Hemoglobin 1997;21:393-403.[ISI][Medline]
[Order article via Infotrieve]PRO]: a new moderately unstable variant. Hemoglobin 1998;22:129-140.[ISI][Medline]
[Order article via Infotrieve]
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