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Letters to the Editor |
91Leu
Phe) Affects Heme Interactions and 1ß2 Contacts
1 Molecular Pathology Laboratory, 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:
Since the discovery of the causative mutation of sickle cell anemia, hemoglobinopathies have become the iconic molecular diseases, with some 900 mutations reported to date (http://globin.cse.psu.edu). Detection and characterization of novel mutations, facilitated by increasingly sensitive analytical techniques, continue to shed light on how molecular structure and function are preserved in the tetrameric molecule. Here we report the identification of a novel 
chain substitution (91Leu
Phe) in a patient who was also heterozygous for the SEA 2-gene deletion.
A full blood count on this 40-year-old woman revealed a microcytic picture with a hemoglobin (Hb) concentration of 103 g/L, a mean cell volume of 63 fL, and a hematocrit of 0.33. These results prompted a hemoglobinopathy/thalassemia screen that revealed a normal cellulose acetate electrophoresis pattern, a marginally positive isopropanol stability test, and values within reference intervals for HbA2 (3.0%), HbF (2%), and reticulocytes. Occasional HbH bodies were seen, and the Bio-Rad Variant cation exchange system for ß-thalasaemia revealed an aberrant component of 35.5% in the HbA2 window.
Examination of whole lysate by electrospray ionization mass spectrometry on a VG Platform (1) indicated a new -chain with a mass increase of 34 Da (Fig. 1A
). Reversed-phase HPLC on a C-4 Jupiter column revealed a hydrophobic shoulder on the -chain peak (not shown), and mass analysis indicated that this trailing edge was enriched in the +34-Da chain. Tryptic mass mapping (1) of these fractions within the range 300–1550 m/z proved uninformative, however.
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The entire coding regions of both the hemoglobin, alpha 1 and hemoglobin, 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 manufacturer’s recommendations. This revealed apparent homozygosity for a CTTTTT transition at codon 91 of the 1 gene. We have named the novel 91LeuPhe substitution "Hb Grey Lynn" after the suburb of Auckland. This replacement of an alkyl leucine side chain by an aromatic phenylalanine is entirely consistent with the observed mass increase of 34 Da, and with both the increase in hydrophobicity and the normal electrophoretic pattern.
The thalassemic blood picture together with the occasional HbH bodies seemed more likely to be attributable to deletional -thalassemia than homozygosity for the novel gene mutation. Further screening for the gene locus for the common 3.7, 4.2, SEA, FIL, MED, and 20.5 deletions (2) did in fact show that this patient was heterozygous for the SEA deletion, which removes both the 1 and 2 genes from 1 allele, leaving only 2 functional genes. This finding explains the comparatively high expression level (40%) of the variant chain.
In an alignment of 449 globin sequences from different species, 91 is absolutely conserved as leucine, and this extraordinary level of conservation suggests an important functional role. X-ray structural analyses show that the alkyl side chain heads internally toward the heme plate, where it comes to within 2.4 Å of the Fe2+-bound proximal histidine (87) and within 3.6 Å of the heme (Fig. 1B
). Its main chain carbonyl group is located on the surface and forms an H-bond with the guanidine group of Arg40 in the adjacent ß2 subunit. On oxygenation, movements of the heme Fe2+ are transmitted through 87His and 91Leu onto the adjacent ß2 subunit to regulate cooperatively and produce the characteristic sigmoidal O2 binding curve (3). As this model correctly predicts, mutation of ß40 Arg also impacts O2 binding; its mutation to Ser in Hb Austin alters both affinity and cooperativity (4).
There are 2 predictable consequences of the novel 91LeuPhe mutation, altered O2 affinity and decreased molecular stability. We were unable to measure O2 affinity, but we were able to demonstrate decreased molecular stability. A small amount of precipitate was collected after extending a standard isopropanol stability test to 40-min incubation. The precipitate was then dissolved in 1% formic acid and analyzed directly by mass spectrometry (Fig. 1A
). This showed selective precipitation of the variant Hb, which was enriched from 40% in lysate to 56% in the precipitate. Only one mutation has been previously reported at this position, 91LeuPro. In this case the less conservative mutation was associated with mild anemia and a more marked instability (5).
This case highlights the importance of investigating abnormal Hbs at biochemical, hematological, and genetic levels to understand the pathological implications of the mutation and the risks it poses to carriers.
Acknowledgments
Grant/funding support: None declared.
Financial disclosures: None declared.
References
87 HisAsn): A new mutation of the proximal histidine identified by electrospray mass spectrometry. Hemoglobin 1997;21:393-403.[Web of Science][Medline]
[Order article via Infotrieve]1ß2 contact region. Arch Biochem Biophys 1977;179:86-94.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]91 (FG3) LeuPro. A new unstable haemoglobin. FEBS Lett 1977;81:115-117.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
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