Hemoglobin, 32 (4):361–369, (2008)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260802173890

361

LHEM0363-02691532-432XHemoglobin, Vol. 32, No. 4, May 2008: pp. 1–17Hemoglobin

ORIGINAL ARTICLE

A RARE THALASSEMIC SYNDROME CAUSED BY INTERACTION

OF Hb ADANA [a59(E8)Gly®Asp] WITH AN a+-THALASSEMIA

DELETION: CLINICAL ASPECTS IN TWO CASES

Hb Adana and Thalassemia Intermedia in GreeceV. Douna et al.

Varvara Douna,1 Ioannis Papassotiriou,2 Anastasia Garoufi,3

Eleni Georgouli,3 Vassilis Ladis,4 Alexandra Stamoulakatou,5

Anna Metaxotou-Mavrommati,6 Emmanuel Kanavakis,6

and Joanne Traeger-Synodinos6

1Hematology Laboratory, “P. and A. Kyriakou” Children’s Hospital, Athens, Greece2Department of Clinical Biochemistry, “Aghia Sophia” Children’s Hospital, Athens, Greece3Second Department of Pediatrics, University of Athens, “P. and A. Kyriakou” Children’s Hospital, Athens, Greece4Thalassemia Unit, First Department of Pediatrics, University of Athens, “Aghia Sophia” Children’s Hospital, Athens, Greece5Department of Hematology, “Aghia Sophia” Children’s Hospital, Athens, Greece6Laboratory of Medical Genetics, University of Athens, “Aghia Sophia” Children’s Hospital, Athens, Greece

� Hb Adana is a highly unstable and rare a-globin hemoglobin (Hb) variant, to date describedin only three families, in interaction with other a-thalassemia (a-thal) deletions. We describe theclinical and hematological findings in two cases from independent families of Albanian origin,who have an interaction of the codon 59 (Gly®Asp) a2-globin gene variant in trans to a 3.7 kba+-thal deletion (acodon 59a/-a). We report their presenting symptoms and laboratory findings aswell as complications and differences in their clinical management. Both cases can be characterizedas thalassemia intermedia and illustrate the problems associated with selecting the most appropriateoptions for patient management, especially in cases with rare underlying genotypes.

Keywords Hb Adana, Unstable hemoglobin (Hb), α-Thalassemia (α-thal), Thalassemiaintermedia

Received 15 October 2007; accepted 21 December 2007.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.:+30-210-746-7461; Fax: +30-210-779-5553; E-mail: jtraeger@med.uoa.gr

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INTRODUCTION

Globin gene mutations can either result in deficient synthesis of thepolypeptide globin chains (thalassemia) or in abnormal hemoglobin(Hb) variants. In thalassemia, the excess of the normally synthesizedglobin chain leads to ineffective erythropoiesis, phenotypically mostapparent in individuals who inherit two β-thalassemia (β-thal) mutations.On the other hand, unstable abnormal Hbs are associated with variousdegrees of peripheral hemolysis, although highly unstable Hb variants areoften associated with both ineffective erythropoiesis and peripheralhemolysis and have thus been termed thalassemic hemoglobinopathies(1). In comparison to highly unstable β-globin chain variants, due to agene dosage effect, highly unstable α-globin variants are often identifiedin individuals only when they interact with other α-thal mutations, typi-cally causing either Hb H (β4) disease, or occasionally, a state with clinicaland hematological findings similar to β-thal intermedia (2,3). The pheno-typic expression of such interactions is potentially influenced by whetherthe mutation for the unstable variant is carried on the α1-globin gene, orthe more dominantly expressed α2-globin gene, by the mechanism anddegree of protein instability, and also by the type of interacting α-thalmutation (2–6).

In this paper we describe the clinical and hematological findings in twocases with the rare interaction of the Hb Adana [α59(E8)Gly→Asp (α2)]variant in trans to a 3.7 kb α+-thal deletion (αcodon 59α/−α). The α-globinchain variant with a Gly→Asp substitution at residue 59 has also been previ-ously described in three families, resulting from an underlying mutation atcodon 59 (GGC>GAC) in either the α1-globin gene (7) or the α2-globingene (8,9). The codon 59 α chain mutation interacted with either an α0-thal mutation in trans (7,8) or an α+-thal deletion (9). Here we report afourth family with the Hb Adana mutation, again on the α2-globin gene,with an α+-thal deletion in trans, along with a follow-up evaluation of thepreviously described patient with the identical genotype (3,9), illustratingthe dilemma associated with choice of clinical management in such rarecases.

MATERIALS AND METHODS

Clinical, Hematological and Hemoglobin Studies

This study included two children from two unrelated families of Alba-nian origin. For hematological and Hb studies, blood samples (10–20 mL)were collected in EDTA. Red blood cell indices were measured using aSiemens Advia 120 (Siemens Medical Solutions, Erlangen, Germany) whole

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Hb Adana and Thalassemia Intermedia in Greece 363

blood analyzer. The reticulocyte count was evaluated by an experiencedhematologist after methylene blue staining.

Hemolysates were analyzed by cellulose acetate electrophoresis(pH 9.0) and by isoelectric focusing (IEF) on agarose gels (pH 6.0–8.0).Weak cation exchange high performance liquid chromatography (HPLC)was performed using a Bio-Rad Variant Hemoglobin Testing system withthe β-Thalassemia Short Program (Bio-Rad Laboratories, Hercules, CA,USA), also modified to quantitate Hb Bart’s (γ4) and Hb H levels (10).Heinz body formation, thermal and isopropanol Hb stability tests wereperformed by standard methods (11). Serum ferritin levels were measuredusing the IMXsystems Ferritin kit (Abbott Laboratories, Abbott Park, IL,USA). Bilirubin levels were measured with the Siemens Advia 1650 ClinicalChemistry System (Siemens Medical Solutions).

Genomic DNA was isolated from white blood cells (WBC) by thesalting-out method or using the Mini Blood DNA kit (Qiagen GmbH,Hilden, Germany) as previously described (12,13). Common Mediterra-nean deletional and nondeletional α-thal mutations were investigatedusing Southern blot analysis and polymerase chain reaction (PCR)-basedprotocols as previously described (13,14). The codon 59 mutation on theα2-globin gene was characterized by direct sequencing of the selectivelyamplified α2 gene and analysis on an automatic DNA sequencer (VisibleGenetics, Toronto, Ontario, Canada) (13). Pathological mutations in theβ-globin gene were excluded by denaturing gradient gel electrophoresis(DGGE) analysis (15).

RESULTS

The probands in both families (case 1 and case 2) were characterizedwith the 3.7 kb α+-thal deletion on one allele in trans to a single mutation atcodon 59 of the α2-globin gene (GGG>GAC), predicted to cause the unsta-ble Hb Adana variant [α59(E8)Gly→Asp]. The fathers of both probandscarried the Hb Adana mutation, while both of the mothers carried the 3.7kb α+-thal deletion.

Case 1 and her parents originated from Albania. This patient was origi-nally described almost 8 years ago, when aged 12 (9). At 10 years of age, shewas admitted to the Athens University Pediatric Clinic at the “Aghia Sophia”Children’s Hospital (Athens, Greece) with a history of persistent moderateanemia, initially observed at 3 months of age, at which time she had beengiven a single blood transfusion. Her laboratory findings are shown inTable 1.

At the age of 12, she showed normal growth and development for her age(weight 39 kg, 50th percentile; height 145 cm, 20th percentile), althoughshe was pale and slightly jaundiced, with mild thalassemic facies. X-rays

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364 V. Douna et al.

showed mild osteoporosis, and moderate splenomegaly was evident by a pal-pable spleen, 7 cm below the costal margin.

Between the ages of 10 to 16 years, case 1 maintained Hb levels ofaround 8.0–9.0 g/dL without blood transfusions. Red cell indices werealways slightly reduced and peripheral blood films consistently demon-strated Hb H-like inclusion in about 20% of red cells, moderate reticulocytosis(6%) and occasional erythroblasts (2/100 WBC). The elution pattern fol-lowing cation exchange HPLC analysis was identical on all follow-up analysesand showed Hb A, Hb F (1.3%), Hb A2 (1.3%), traces of Hb Bart’s; noabnormal Hb fractions have ever been observed with starch gel or celluloseacetate electrophoresis, nor abnormal globin chains with perfusion chro-matography. Heinz bodies were observed after incubation of red cells in thepresence of oxidative dyes, while both heat and isopropanol instability weremarginally positive.

At the age of 16 she was splenectomized and transfused with 2 units ofpacked red cells. Post splenectomy, her clinical and laboratory findingswere no different compared to those observed prior to splenectomy andshe has maintained the same Hb level (8.0–9.0 g/dL).

The patient is now 20 years old and recently suffered an episode ofautoimmune hemolytic anemia (Hb 6.5 g/dL; PCV 0.255 vol/vol), whichwas diagnosed and treated in another hospital and was apparently resolved

TABLE 1 Clinical, Hematological, Biochemical and Molecular Data atDiagnosis

Parameters Case 1 Case 2

Genotype −α3.7/αcodon 59α −α3.7/αcodon 59αAge at diagnosis 10 years 36 daysPresent age 20 years 20 monthsHb (g/dL) 8.4 6.4PCV (vol/vol) 0.27 0.21MCV (fL) 79.0 74.7MCH (pg) 24.6 23.2MCHC (g/dL) 31.1 31.0Reticulocytes (%) 5.6 4.5Normoblasts 2/100 WBC 1/100 WBCInclusion bodies positive negativeRDW (%) 21.1 18.6Hb F (%) 2.0 45.0Hb A2 (%) 1.3 1.3Hb Bart’s (%) trace presentHb H (%) absent presentFerritin (μg/L) 138.0 –Bilirubin (mg/dL) 2.6 8.9Transfusions two every 4–6 weeksHepatosplenomegaly splenectomized [++]

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Hb Adana and Thalassemia Intermedia in Greece 365

without complications following standard cortisone treatment. She showsnormal appearance and growth. She has been prescribed a folate supple-ment of 2.5 mg daily.

Case 2 and her parents also originated from Albania. Following anormal pregnancy, birth and postnatal period, the infant was first hospital-ized at the age of 36 days with jaundice (which had been initially observedon the third day post delivery) and moderate anemia. Her weight, heightand head circumference were in the 90th, 50th and 90th percentiles,respectively. She was afebrile and the clinical evaluation was unremarkable,with the exception of a palpable spleen (2 cm below the costal margin).

Hematological parameters on admission (36 days old) are shown inTable 1. Direct and indirect Coombs tests were negative. The peripheralblood film showed hypochromasia, micro- and anisopoikilocytosis, poly-chromatophilia and basophilic stippling. Electrophoretic and chromato-graphic analyses of Hb fractions revealed two fast eluting fractions: HbBart’s, Hb Facetylated + Hb B (probably traces of Hb H) with 1.3% Hb A2 and45% Hb F (Table 1). Stability tests and in vitro chain synthesis were notdone. She was discharged after 5 days with a Hb level of 11.0 g/dL followinga single transfusion. Since the time of discharge, the proband has beentransfused every 4 weeks to maintain a Hb level of around 12.0 g/dL;between transfusions, her Hb level drops to 9.0–10.0 g/dL. She is now 20months old, and during the last year she has been transfused every 3 weeks.She shows retardation in growth and development for her age (weight 11 kg,25th percentile; height 85 cm, 25th percentile), moderate splenomegaly andhepatomegaly (2–3 cm and 2 cm, respectively, below the costal margins).She is not jaundiced. She has also been prescribed a folate supplement of2.5 mg twice per week.

Hematological analyses in both mothers showed red cell indices withinthe normal range. Occasional red cells from the mother of case 1 had typicalHb H inclusions. Hematological parameters of both the fathers were at thelower end of the normal range, with a mild degree of hypochromasia. Themain laboratory findings for the father of case 1 were as follows: Hb 12.8 g/dL,PCV 0.410 vol/vol, MCV 83.0 fL, MCH 25.9 pg, RDW 13.9%, reticulocytes1.0%, and for the father of case 2: Hb 12.9 g/dL, PCV 0.409 vol/vol, MCV79.7 fL, MCH 21.5 pg, RDW 13.0%, reticulocytes 1.1%. No Hb H inclusionsor Heinz bodies were observed on peripheral blood films and neither fatherhad detectable abnormal Hb fractions. Their Hb A2 and Hb F values werewithin normal limits (Hb A2 2.5 and 3.1%, Hb F 0.6 and 0.3%, respectively).

DISCUSSION

The two cases described in this report are consistent with the state ofthalassemia intermedia, which is generally characterized by Hb levels

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around 7–10 g/dL without the need of regular blood transfusions alongwith varying degrees of spleen enlargement and skeletal changes. However,there are no clear guidelines for managing thalassemia intermedia patients(16,17), particularly with regards as to whether the patients should be trans-fused and how frequently, and also the management of complications, espe-cially the issue of splenectomy. It is especially difficult to evaluate how tomanage patients with rare underlying genotypes for whom a limited num-ber of similar cases preclude accumulated experience and more definedmanagement strategies (16,17). Although, even for thalassemia patientswith common globin gene mutation interactions, there are not always clearcorrelations between genotype and phenotype due to other modifyingfactors (18). These two cases demonstrate quite different choices in man-agement, most likely due to the prevailing opinion at the time in the clinicsin which they were hospitalized and subsequently treated.

Case 1, who is now 20 years old, has been transfused only twice, has anormal appearance and growth, and has never suffered any of the typicalcomplications of thalassemia intermedia, with the exception of splenome-galy and subsequent splenectomy. We have no information about the deci-sion to splenectomize, other than the apparent size of the spleen, since theproband has maintained steady and adequate Hb levels and there was noevidence of leukopenia or thrombocytopenia.

Case 2 has been transfused regularly since she was initially diagnosed.With the observation that her Hb levels had a tendency to drop below 9.5–10.0 g/dL (which is considered the borderline level required to adequatelysuppress the bone marrow activity, promote better growth and minimizeiron absorption), the decision was taken to initiate a regular transfusionregimen. There is evidence that there are age-related changes in adaptationto severe anemia in childhood, whereby transfusion is indicated in order tosuppress erythroid over-expression which is responsible for many of thecomplications seen in young children with hemoglobinopathies (19).Although case 2 still shows some signs of growth retardation, the cliniciansare intending to increase the intervals between transfusions. When transfu-sions start early, it also helps to reduce the risk for alloimmunization andautoimmunization, and complications are better controlled (16,17,20,21).On the other hand, the disadvantage of a regular transfusion regimenis that the patient requires safe blood supplies and effective chelationtherapy.

In both these cases with an identical and rare genotype, it is noteworthythat anemia was the presenting symptom and apparently very early in life.We have inadequate data regarding the jaundice reported in case 1, andsince the steady state levels of bilirubin in both patients is generally below3.5 mg/dL, we did not consider it necessary to investigate the patients forthe polymorphism in the UGT1A1 gene promoter.

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Hb Adana and Thalassemia Intermedia in Greece 367

The substitution of glycine for aspartic acid at residue 59 of the α-globinchain has been independently described to be caused by a GGG>GACsubstitution at codon 59 of the α1- or α2-globin genes (7–9). With respectto the protein, this substitution affects the internal glycine residue at posi-tion 59 of the α-globin chain, and is predicted to affect the contacts with21Ala, 22Gly, 25Gly, 26Ala, 29Leu and 64Asp within the α-globin chain,overall disrupting its folding (3,7). Thus, the consequences of this on theprecipitation of globin and/or Hb in the red cells and the constitution ofred cell inclusions observed is only speculative.

The interaction of an α0-thal deletion and the Hb Adana (α1) mutationhas been described to underlie a Hb H-like disease (7), with detectable levelsof Hb Bart’s rather than Hb H, and with clinical manifestations that were notsevere, since two such patients survived into adulthood. The case with the α0-thal and Hb Adana (α2) mutation had the severe condition of Hb H hydropsfetalis in the perinatal period, subsequently developing into a transfusion-dependent disease with very low levels of α-globin chain synthesis (8). Theimplication is that the more severe phenotype in the latter case wasaccounted for by involvement of the more dominantly expressed α2-globingene with respect to the Hb Adana variant (2,8). Our patients, with anα+-thal deletion and a Hb Adana (α2) mutation have a phenotype of mildthalassemia intermedia, which, when compared to the Hb H hydrops fetaliscase with an α2 mutation (8), is likely attributed to the co-inheritance of amilder α+-thal deletion rather than an α0-thal deletion in trans.

However, despite the fact that these patients are left with the expression oftwo genes producing normal α chains, their clinical expression is more severethan would be expected for an α+-thal compound heterozygous condition. Itwould appear that the intermediate phenotype is likely due to the fact that thehighly unstable Hb variant and/or globin chains exacerbate the abnormal redcell physiology already caused by the excess β-globin chains, analogous to thehighly unstable “dominant” β-thal hemoglobinopathies (22).

To date, more than 20 unstable α chain variants have been described(3,23–28). Patients co-inheriting α-thal mutations along with unstable αchain variants are rare, and there is minimal experience reported on theirclinical outcome and management. Thus, since heterozygotes for hyperun-stable α chain Hb variants often have no abnormal hematological findings,as in the heterozygous Hb Adana fathers, prevention programs cannoteasily facilitate their detection since definitive diagnosis is often onlyachieved through DNA analyses.

ACKNOWLEDGMENTS

The authors wish to thank Ms. Stavroula Papadopoulos (Medical Genetics,Athens University Medical School, Athens, Greece) for technical assistance.

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This study was partially supported by a European Commission CoordinationAction, Contract grant (no. 026539), titled “Infrastructure for ThalassaemiaResearch Network.”

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