Hemoglobin Interlaken in combination with beta thalassemia trait

We report a rare a1 globin gene variant (Hb Interlaken) found in a 63-year-old woman of Italian ancestry living in Buenos Aires Province, Argentina. The variant, a missense mutation at cd15 (GGT → GAT) causing a Gly → Asp amino acid substitution and also known as Hb J Oxford, was found in combination with the common thalassemia trait cd 39 (C→T). The clinical picture of the patient was that of a b-thalassemia trait.


Introduction
In 1964 Liddell first described hemoglobin Interlaken in an English family. 1 Only isolated reports exist in which the same mutation was named Hb J-Oxford or Hb N-Cosenza. 2The variant has thus far been described in combination with b-thalassemia homozygosity and HbS but not in combination with b-thalassemia trait and never before in Argentina, a multi ethnic country with many ethnic components.
Hb Interlaken is a stable a1 chain variant caused by a GAC→CAC transversion at codon 15 of the a1 gene causing a Gly→Asp amino acid substitution (HGVS nomenclature HBA1:c.47G>A). 3

Case Report
We report the identification of Hb Interlaken in a 63-year-old Argentinian woman of Italian ancestry, referred to our laboratory because of a microcytic hypocromic anemia.Hb Interlaken was found in this patient in combination with the common Mediterranean b 0 tha-lassemia trait cd 39 (C→T) (HGVS nomenclature HBB:c.118C>T), the most frequent bthalassemia mutation in Argentina. 4omplete blood count (CBC) was obtained with a Coulter Counter model ACT10 (Beckman Coulter Inc, Brea, CA, USA): Hb (g/L) 11.6, RBC (10 12 /L) 5.88, MCV (fL) 62.4, HCM (pg) 19.7, reticulocytes (%) 1.0.
The separation of the Hb fractions was done on alkaline cellulose acetate electrophoresis (Figure 1A).The presence of a J like minor fraction was suggestive for an a chain variant.Hb A2 measured by elution from electrophoresis followed by spectrophotometric measurement of the absorbance at 415 nm was estimated at 3% while Hb X was 28%.In spite of the normal HbA 2 level, the CBC, the slightly elevated HbF (1.3%) estimated according to Betke et al. 5,6 indicated a possible b-thalassemia trait while the very low mean corpuscolar hemoglobin could be an indication for a b-/a thalassemia combination.Iron parameters were measured as previously described: 7 serum iron 56 ug/dL, total iron-binding capacity 328 ug/dL, transferrin saturation 17% and serum ferritin 4 ng/mL. 8Isopropanol, 9 heat stability and sickle tests were performed and were all negative.
DNA was extracted from peripheral blood sample. 10Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) 11 was used to confirm the presence of the b 0 39 mutation (data not shown).
GAP-PCR were used to detect a 0 deletions -(a)20.5, -MED and a + deletions -a 3.7 anda 4.2 and were all negative.Sequencing of a1 gene revealed a GAC → CAC (Asp→ His) substitution at codon 15, corresponding to Hb Interlaken (Figure 1B).
Even though Hb Interlaken is detectable by electrophoresis a correct characterization of the genotype requires better methods, especially in combination with and a thalassemia.Then more sophisticated systems able to measure Hb fractions more precisely like highperformance liquid chromatography or capillary electrophoresis 13 are needed before molecular analysis.
In our case the estimation of the HbA 2 that should have been elevated was typically normal risking misdiagnosis.The underestimation was due to the fact that part of the delta chain is bound by the mutated a chain and this abnormal HbA 2 fraction migrates on a different spot and is lost for measurement.Therefore the measured HbA2 value (3%) should be augmented by 28% resulting into a 3.84% a still ambiguous but elevated HbA 2 . 6][16] It is the first time that this abnormal hemoglobin is described in our country.The low red blood cell indexes observed in this case are due to co-inheritance of b 0 thalassemia and the underestimation of HbA 2 could be caused