Hemoglobin, 32 (6):592–595, (2008)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260802507964

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LHEM0363-02691532-432XHemoglobin, Vol. 32, No. 6, september 2008: pp. 0–0Hemoglobin

SHORT COMMUNICATION

ASSOCIATION OF MILD AND SEVERELY UNSTABLE a CHAIN

VARIANTS: THE FIRST OBSERVATION OF A COMPOUND

HETEROZYGOTE WITH Hb SETIF [a94(G1)Asp®Tyr (a2)] AND Hb

AGRINIO [a29(B10)Leu®Pro (a2)] IN A GREEK FAMILY

Interaction of Hb Setif and Hb AgrinioV. Douna et al.

Varvara Douna,1 Ioannis Papassotiriou,2 Alexandra Stamoulakatou,3

Anna Metaxotou-Mavrommati,4 Emmanuel Kanavakis,4 and

Joanne Traeger-Synodinos4

1Hematology Laboratory, P. and A. Kyriakou Children’s Hospital, Athens, Greece2Department of Clinical Biochemistry, Aghia Sophia Children’s Hospital, Athens, Greece3Hematology Laboratory, Aghia Sophia Children’s Hospital, Athens, Greece4Medical Genetics, University of Athens, Aghia Sophia Children’s Hospital, Athens, Greece

� Hb Setif is a relatively rare, mildly unstable a2-globin hemoglobin (Hb) variant first describedin an Algerian family, and subsequently in various populations of the Mediterranean region andthe Middle East. Hb Agrinio is a highly unstable variant, classified as a nondeletional a-thalassemia(a-thal) mutation, which, to date, has only been described in Greece and Cyprus. We report here theclinical and hematological findings in a case of Greek origin, who, following DNA analysis, wascharacterized with the unusual interaction of the Hb Setif a2-globin gene variant at codon94 variant, in trans to Hb Agrinio, an a2-globin gene variant at codon 29. The compoundheterozygote proband had only mild anemia with no transfusion requirements and with normalgrowth and development. We also report the laboratory findings in members of his family, highlightingdiagnostic difficulties in the absence of molecular analysis.

Keywords Hb Setif, Hb Agrinio, Unstable hemoglobin (Hb), α-Thalassemia (α-thal)

Hb Setif [α94(G1)Asp→Tyr, GAC > TAC (α2)] is a relatively rare,mildly unstable α chain hemoglobin (Hb) variant, first described in anAlgerian family, and subsequently in various populations of the Mediterra-nean region and the Middle East (1–5). Hb Agrinio [α29(B10)Leu→Pro,

Received 25 February 2008; accepted 5 June, 2008.Address correspondence to Joanne Traeger-Synodinos, D.Phil., Medical Genetics, University of

Athens, “Aghia Sophia” Children’s Hospital, Thivon and Levadias Streets, Athens 11527, Greece; Tel.:+30-210-745-7461; Fax: +30-210-779-5553; E-mail: jtraeger@med.uoa.gr

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Interaction of Hb Setif and Hb Agrinio 593

CTG > CCG (α2)] is a highly unstable variant, classified as a nondeletionalα-thalassemia (α-thal) mutation, which has only been described in Greeceand Cyprus (6–10) to date.

We report here the clinical and hematologic findings in a Greek child,presently 6 years old, who, when he was 4 years old, was referred to theHematology Laboratory of the P. and A. Kyriakou Children’s Hospital,Athens, Greece, from a private laboratory, following the observation of anabnormal Hb fraction detected by cellulose acetate electrophoresis(pH 8.6). His only symptom was a mild anemia, and he showed normalgrowth and development, in the absence of any other clinical findings.

Following his referral to the P. and A. Kyriakou Children’s Hospital, bloodsamples were also taken from his 10-year-old brother and parents. All bloodsamples (10–20 mL) were collected in EDTA. Red blood cell indices weremeasured using a Coulter STKS whole-blood analyzer (Beckman Coulter,Fullerton, CA, USA). Red cell morphology and reticulocyte count was evalu-ated by an experienced hematologist after appropriate staining methods.Hemolysates were analyzed by cellulose acetate electrophoresis (pH 8.6) andby citrate agar electrophoresis (pH 6.2). Cation exchange high performanceliquid chromatography (HPLC) was performed using a VARIANT II™ Hbtesting system with the β-Thalassemia Short Program (Bio-Rad Laboratories,Hercules, CA, USA). Heinz body formation and thermal and isopropanol Hbstability tests were not performed. Serum ferritin levels were measured usingthe IMXsystems Ferritin kit (Abbot Laboratories, Abbott Park, IL, USA).

Genomic DNA was isolated from white blood cells using the QiagenMini Blood DNA kit (Qiagen GmbH, Hilden, Germany), as described previ-ously (11). α-Globin gene mutations were investigated using previouslypolymerase chain reaction (PCR)-based protocols as described (11,12),with additional direct sequencing of selectively amplified α2 genes andanalysis on a Visible Genetics (Ontario, Canada) automatic DNAsequencer. Pathological mutations in the β-globin gene were excluded bydenaturing gradient gel electrophoresis (DGGE) analysis (13).

DNA analysis of the proband characterized two mutations on theα2-globin gene: the GAC>TAC substitution at codon 94, predicted tochange the aspartic acid residue to a tyrosine at position 94 of the α2 chain,thus resulting in Hb Setif, and a single nucleotide substitution at codon 29of the α2-globin gene (CTG>CCG), known to cause the unstable HbAgrinio variant. The father of the proband carried the Hb Setif mutation,whereas the mother carried the Hb Agrinio variant. The proband’s brotherhad also inherited both variant alleles (Table 1).

The hematological findings in the proband and his family are summa-rized in Table 1. In the proband, red cell indices were reduced and periph-eral blood films demonstrated moderate morphological changes and noinclusion bodies. On electrophoresis with alkaline pH, an abnormal fraction

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594 V. Douna et al.

was detected running close to the Hb S position, whereas on electrophoresiswith acid pH, the abnormal fraction was detected running close to the Hb Cposition. The elution pattern following HPLC analysis showed Hb A, Hb F, HbA2 and the abnormal Hb fraction (30%) eluting soon after Hb S. Instabilitytests were not done. The proband’s brother had similar hematological find-ings, including a comparable percentage (29.3%) of the abnormal Hb fraction.

The mother showed no anemia but slightly reduced red cell indices,consistent with her heterozygous Hb Agrinio status (6,7,10). No inclusionbodies were detected and she had no abnormal Hb fractions. Hematologi-cal parameters of the father were in the normal range, as shown in Table 1.No inclusion bodies were observed on peripheral blood film, but he hadan abnormal Hb fraction on electrophoresis and chromatography com-prising 16.5% of the total Hb, comparable to previously reported Hb Setifheterozygotes (1–5). The proband and his brother, both compoundheterozygotes for Hb Setif and Hb Agrinio, had similar hematological andclinical findings. As could be expected, they had about double percentageof the abnormal Hb Setif fraction compared to their father, since thehyperunstable Hb Agrinio acted as an α-thal mutation, reducing the rela-tive synthesis of normal α-globin, thus increasing the relative amount ofHb Setif. The relatively low levels of Hb A2 detected in both brothers andtheir father may be attributed to the fact that a percentage of the δ-globinchains likely associate with the Hb Setif α chains. Reevaluation of the chro-matogram of the HPLC analysis in fact revealed a small fraction (up to 1%)assigned as ‘unknown’ and eluting after the Hb A2 peak. Owing to thefrequency and population distribution of Hb Setif and Hb Agrinio, thecases represent a very rare interaction.

TABLE 1 Laboratory Data at First Clinical Evaluation of the Proband and his Family

Parameters Proband Mother Father Brother

Genotype αSetifα/αAgrinioα αα/αAgrinioα αSetifα/αα αSetifα/αAgrinioαSex-Age (years) M-6 F-37 M-45 M-10Hb (g/dL) 10.9 13.9 13.3 10.9PCV (L/L) 0.35 0.44 0.40 0.34MCV (fL) 65.4 76.2 83.7 67.7MCH (pg) 20.3 24.1 27.7 21.5RDW (%) 14.1 13.1 12.1 14.6Red cell morphologya [++] [+] [–] [++]Reticulocytes (%) 1.1 1.2 1.0 1.6Hb A2 (%) 1.2 2.0 1.6 1.4Hb F (%) 0.2 0.2 0.2 0.2Abnormal fraction (%) 30.0 – 16.5 29.3Ferritin (ng/μg) 30.0 93.0 N.D. N.D.

ND = no data.aHypochromia, microcytosis, anisocytosis, target cells, basophilic stippling.

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Interaction of Hb Setif and Hb Agrinio 595

ACKNOWLEDGMENTS

The authors thank Ms. Stavroula Papadopoulos (Medical Genetics,Athens University Medical School, Athens, Greece) for technical assistance.This study was partially supported by a European Commission CoordinationAction Contract grant (no. 026539), titled “Infrastructure for ThalassaemiaResearch Network.”

Declaration of Interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of this article.

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