Tim Chan

Novel hemoglobin α chain elongation resulting from a 15-residue insertion and tandem duplication of the F helix

OBJECTIVES To determine the cause of an unusual hemoglobin pattern with two novel components eluting after HbA(2) on cation exchange HPLC. This variant was detected during HbA1c measurement and was associated with a normal blood count and a positive isopropanol test. DESIGN AND METHOD Whole hemolysate and isopropanol precipitates were analysed by ESI MS, and individual components were purified by reverse phase and cation exchange HPLC. Tryptic peptide mapping of isopropanol precipitates was used to detect the molecular lesion and DNA sequencing was used to characterise the precise rearrangement. RESULTS ESI MS showed a mass increase of 1614Da in 9% of the alpha globin chains and sequence analysis of the alpha2 gene revealed the heterozygous insertion of 45 nucleotides after codon 93. The predicted in phase incretion of ALSALSDLHAHKLRV (+ 1613Da) is a direct repeat of residues alpha79-93 and signature ions from the new peptide were clearly visible in peptide maps of the unstable hemoglobin. CONCLUSION The insertion probably results from replication slippage during DNA synthesis and the 15-residue repeat results in full repetition of the heme-linked F helix. The nature of the inserted sequence explains the molecular instability and the electrophoretic mobility, but not the twin peaks observed on cation exchange HPLC. These components had the same chain composition, (alpha(L) beta), the same number of heme groups per chain, were not in rapid equilibrium with each other, and probably represent hemoglobin species with different conformers of the elongated alpha(L) chain.

A Second Case of Hb Fontainebleau [α21(B2)Ala→Pro] in an Individual with Microcytosis

The identification of a second case of Hb Fontainbleau [α21(B2)Ala→Pro] allowed us to re‐examine its association with microcytosis, explore the effects of the mutation on protein stability and define the mutation at a DNA level. Although slightly unstable, the variant was expressed at 28–29% of the total and was caused by a heterozygous mutation in the α2 gene. There was no evidence for concomitant α-thalassemia (α-thal); both α-globin gene deletion analysis and sequencing of the α-globin locus failed to detect any additional mutations that might explain the relatively high expression level.

Novel sequence insertion in a Mâori patient with transfusion-dependent β-thalassaemia

Although β‐thalassaemia is common throughout the world, it has not been previously described in Polynesia. We report a novel sequence insertion where homozygosity for the defect results in transfusion‐dependent anaemia. The repeated 45 base pair (bp) insertion causes duplication of the start codon and consequent transcription from the original initiation code would be predicted to lead to the production of an irrelevant seven‐residue peptide, while residual translation from the novel initiation site would result in diminished yields of β‐globin and consequent clinical β+‐thalassaemia.

Hb Koya Dora [α142, Term→Ser (TAA>TCA in α2)]: A Rare Mutation of the α2 Gene Stop Codon Associated with α-Thalassemia

Hb Constant Spring [(Hb CS) α142, Term→Gln (TAA>CAA in α2)] and Hb Koya Dora [α142, Term→Ser (TAA>TCA in α2)] both involve mutations of the α2 gene stop codon and while Hb CS is the most frequent cause of nondeletional α-thalassemia (α-thal) in Southeast Asia, Hb Koya Dora is limited to a restricted population from Andhra Pradesh, India. Here we identify a homozygous case of Hb Koya Dora and confirm the structure of the 31 residue α chain extension.

Hb Taradale [β82(EF6)Lys→Arg]: A Novel Mutation at a 2,3-Diphosphoglycerate Binding Site

Hb Taradale [β82(EF6)Lys→Arg] was initially detected as a split Hb A0 peak on Hb A1c monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant β chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide βT-9. DNA sequencing confirmed the presence of a novel β82(EF6)Lys→Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.

Hb Riccarton [α51(CE9)Gly→Ser]: A Variant Arising from a Novel Mutation in the α1 Gene

Hb Riccarton was identified in a young boy under investigation for fatigue and microcytosis. However, the novel α51(CE9)Gly→Ser mutation did not appear to be the cause of the microcytosis as it was also detected in the boys father who had normal red cell indices.

Novel α2 Gene Deletion (c.349_359 del GAGTTCACCCC) Identified in Association with the –α3.7 Deletion

We report a novel α2 gene mutation identified in compound heterozygosity with the common –α3.7 deletion. The mutation (c.349_359 del GAGTTCACCCC), causes a frameshift after codon α115 with the predicted extension of the αchain from 141 to 166 residues. The new polypeptide would be expected to have a mass of 17,463 Da, but no such product was detected in hemolysates, confirming the mutation’s thalassemic phenotype. Mechanistically, the deletion is probably caused by replication slippage during DNA synthesis as the sequence is bracketed by a CC repeat. The lack of protein expression is probably caused by loss of critical binding sites to the chaperone, α Hb stabilizing protein (AHSP).

Hb Papanui [α99(G8)Lys→Arg; HBA2: c.299A>G]: a novel silent substitution interfering in Hb A1c determination.

We report a novel hemoglobin (Hb) mutation [Hb Papanui [α99(G8)Lys→Arg; HBA2: c.299A>G] that was identified in an individual with an inappropriately low Hb A1c value of 2.8% when using an ion exchange method. Although occurring at a well conserved site, the conservative substitution was not associated with any adverse clinical features.

Hb Perpignan [β136(H14)Gly→Ser], A Silent Variant Associated with Normal Hematology

A second case of Hb Perpignan [β136(H14)Gly→Ser] was identified in a Burmese woman living in New Zealand. Although previously detected in France, this is the first formal description of the variant, which is electrophoretically silent and hematologically normal. This presentation as a benign substitution is in keeping with the low level of phylogenetic conservation of the H14 glycine.

Hb Lusaka [α131(H14)Ser→Phe (α1)]: A New Variant Found in a Woman Heterozygous for Hb S [β6(A3)Glu→Val]

Hb Lusaka [a131(H14)Ser!Phe] was detected in association with Hb S [b6(A3)Glu!Val] in a 40-year-old Zambian woman now living in Auckland, New Zealand. Her red cell indices were within the normal range with the following values: Hb 12.2 g dL, PCV 0.37 L L, MCV 80 fL, MCH 27 pg; her Hb F level was also normal at <2.0%. Hb A2, as measured by ion exchange chromatography (VARIANT ; Bio-Rad Laboratories, Hercules, CA, USA), appeared elevated at 4.4%, but with hindsight, this elevation was probably due to co-migration with Hb S1c. The red cells appeared normal and the reticulocyte count (84 10 L), and isopropanol stability were also normal. Cellulose acetate electrophoresis (pH 8.6) indicated an abnormal band running in the Hb S position, and this was quantified at 40% of the total by cation exchange high performance liquid chromatography (HPLC). Electrospray ionization mass spectrometry of whole lysate (1), however, showed two abnormal chains; an abnormal b chain with a mass decrease of 30 Da, and an abnormal a chain with a mass increase of 60 Da (Fig. 1). These variant chains were present at levels of 47 and 20%, respectively, compared to the total b and a chains. The electrophoretically abnormal component of the lysate was purified [Fig. 1 (panel c)] by chromatography on DEAE-Sephadex, and tryptic peptide mapping (1)