Hemoglobin, 30 (4):409–418, (2006)Copyright © Informa HealthcareISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260600867842

409

LHEM0363-02691532-432XHemoglobin, Vol. 30, No. 4, July 2006: pp. 1–19Hemoglobin

ORIGINAL ARTICLE

OBSERVATION OF A RARE HEMOGLOBIN VARIANT [Hb LULU

ISLAND, b107(G9)Gly®Asp, GGC®GAC] CO-INHERITED WITH

A b+-THALASSEMIA MUTATION [IVS-I-110 (G®A)] OR IN THE

HETEROZYGOUS STATE IN A GREEK-ALBANIAN FAMILY

Hb Lulu Island [β107(G9)Gly→Asp] and ThalassemiaI. Papassotiriou et al.

Ioannis Papassotiriou,1 Alexandra Stamoulakatou,2 Henri Wajcman,3 Jean

Kister,4 Gerasimos Dimisianos,5 Christina Lazaropoulou,1 Ino Kanavaki,1

Efstathios Vavourakis,2 Antonios Kattamis,6 Emmanuel Kanavakis,5 and

Joanne Traeger-Synodinos5

1Department of Clinical Biochemistry, “Aghia Sophia” Children’s Hospital, Athens, Greece2Hematology Laboratory, “Aghia Sophia” Children’s Hospital, Athens, Greece3INSERM U468, Hôpital Henri Mondor, Créteil, France4INSERM U473, CHU de Bicêtre, Le Kremlin Bicêtre, France5Medical Genetics, Athens University Medical School, Athens, Greece6First Department of Pediatrics, Athens University Medical School, Athens, Greece

� We report clinical, hematological, biochemical, functional and molecular studies carried out ontwo first cousins from a Greek-Albanian family who have clinical and hematological findingsconsistent with the diagnosis of thalassemia intermedia. DNA studies determined that they hadco-inherited a common Mediterranean b-thalassemia (thal) mutation, IVS-I-110 (G®A), in transto a b-globin gene mutation at codon 107 (GGC®GAC), predicted to give rise to a rare unstable bchain variant Hb Lulu Island or b107(G9)Gly®Asp.

Keywords Unstable hemoglobins (Hbs), Thalassemia (thal), Erythropoiesis

INTRODUCTION

Almost 690 hemoglobin (Hb) variants affecting the β-globin chainshave been described to date (http://globin.cse.psu.edu/hbvar/menu.html),the majority of which are due to single amino acid replacements and have

Received 20 February 2006, accepted 15 March 2006.Address correspondence to Ioannis Papassotiriou, Ph.D., Department of Clinical Biochemistry,

“Aghia Sophia” Children's Hospital, 115 27 Athens, Greece; Fax: +30-210-746-7171; E-mail: biochem@paidon-agiasofia.gr; jpapasotiriou@ath.forthnet.gr

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been observed in sporadic cases only. The altered globin polypeptide mayhave consequences on the stability and/or functional properties of the variantHb molecule. The variants with pronounced alteration in structure and/orfunction usually cause an observable phenotypic expression in the individualswho carry them. On the other hand, amino acid substitutions causing moresubtle structural and/or functional changes in the variant Hb only becomephenotypically apparent when co-inherited with a β-thalassemia (thal)mutation in trans (1–3).

We report clinical, hematological, biochemical, functional and molecularstudies carried out on two first cousins from a Greek-Albanian family whohave clinical and hematological findings consistent with the diagnosis ofthalassemia intermedia. DNA studies determined that they had co-inheriteda common Mediterranean β-thal mutation, IVS-I-110 (G→A), in trans to a β-globin gene mutation at codon 107 (GGC→GAC), predicted to give rise toa rare unstable β chain variant Hb Lulu Island [β107(G9)Gly→Asp]. Thefamily also provided us with the opportunity of studying the phenotypic fea-tures in Hb Lulu Island heterozygotes, as this variant has only beenreported once before in a female thalassemia intermedia patient of EastIndian origin, who was a compound heterozygote for Hb Lulu Island and aβ0-thal nonsense mutation [codon 15 (TGG→TAG)] (4).

MATERIALS AND METHODS

Hematology and Blood Chemistry

Blood samples (10–20 mL) were collected in EDTA. Blood films werestudied using standard techniques. Red blood cell (RBC) indices for acomplete blood count (CBC) were measured using a Bayer-ADVIA 120whole blood auto analyzer (Bayer, Tarrytown, NY, USA). Hemoglobinswere quantitated using weak cation exchange high performance liquidchromatography (HPLC) with the VARIANT™ Hemoglobin Testing systemand the β-Thalassemia Short Program (Bio-Rad Laboratories, Hercules, CA,USA). Heinz body formation, thermal and isopropanol instability testswere performed by standard techniques.

Erythropoietin, soluble transferrin receptor (sTfR) and ferritin levels inthe sera of the probands and their parents were measured in duplicate usinga two-site chemiluminescence immunoassay (Nichols Institute Diagnostics,San Clemente, CA, USA). Erythroid marrow activity was calculated as the ratioof sTfR levels in the case to the mean levels in normal controls as measuredin our laboratory. Oxygen equilibrium curves (OEC) were performed inwhole blood and in purified hemolysates using an automatic continuousmethod (Hemox-Analyzer; TCS, Medical Products Division, Southampton,PA, USA) as previously described (5). The P50 values were calculated from

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Hb Lulu Island [b107(G9)Gly®Asp] and Thalassemia 411

the OECs. Red cell 2,3-bisphosphoglycerate (2,3-BPG) levels were measuredenzymatically (Roche Diagnostics, Mannheim, Germany).

Blood chemistry parameters were measured using the Bayer Advia 1650Clinical Chemistry System (Bayer). Specific antioxidants and catabolicproducts were measured as follows: red cell oxidized/reduced glutathione(GSSG and GSH, respectively) by reversed phase HPLC with fluorimetricdetection (excitation 385 nm and emission 515 nm); plasma malonyldialde-hyde (MDA) levels by reversed phase HPLC with fluorimetric detection(excitation 532 nm and emission 550 nm), and vitamins A and E levels byreversed phase HPLC with UV detection at 295 nm. All HPLC procedureswere performed using the HP 1100 Series HPLC system (Hewlett Packard,Palo Alto, CA, USA).

DNA and Protein Studies

Genomic DNA was isolated from white blood cells using the QIAampDNA Blood Mini Kit (Qiagen, Hilden, Germany). The β-globin gene wasanalyzed using denaturing gradient gel electrophoresis analysis (DGGE) tolocalize nucleotide changes that were subsequently characterized by directsequencing of β gene-specific amplicons on an automatic DNA sequenceras previously described (5). The α-globin genes were analyzed for deletionsusing gap-polymerase chain reaction (gap-PCR), and for point mutationsusing PCR-based methods as previously described (6). Hemoglobinpolypeptide chains were analyzed by reversed phase HPLC using a Lichro-spher 100 RP8 phase (Merck, Darmstadt, Germany) (7), and the abnormalglobin chain was analyzed with electrospray mass spectrometry (ES-MS)andpeptide mapping (8).

RESULTS

Clinical Evaluation

The study included a male proband (proband I), his female first cousin(proband II) and their parents, all of Greek-Albanian origin. Proband I wasfirst admitted to hospital with gastroenteritis at the age of 2.5 years. Theresults of CBC and Hb electrophoresis were consistent with the presence ofa hemoglobinopathy, which remained undefined at that time (Table 1). Atthe age of 4 years, he was hospitalized with a chicken-pox infection, andfollowing the observation that his Hb level had fallen to 5.5 g/dL hereceived his first blood transfusion. Clinical examination at this time foundthe child pale, with a palpable spleen (5 cm below the costal margin) and amediocre general condition. Over the 3-year period during which the childhas been followed at the “Aghia Sophia” Children’s Hospital, Athens,

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412 I. Papassotiriou et al.

Greece, he has maintained a steady-state Hb level of around 9.0 g/dL,which had been observed to fall to 5.5–6.0 g/dL during infections andfebrile episodes. A cranial X-ray at the age of 5 years demonstrated a smalldegree of osteoporosis, and physical examination had consistently found apalpable spleen, about 2 cm below the costal margin.

Proband II has only been examined once at the “Aghia Sophia” Children’sHospital, Athens, Greece. At 3 years of age, clinical examination excludedremarkable clinical features. Hematological analysis demonstrated a mildanemia with Hb levels around 10.0 g/dL, mild microcytosis, hypochromasiaand reticulocytosis (Table 1).

Hematology and Blood Chemistry

Hematological findings in the mothers of both probands were consistentwith the diagnosis of a β-thal carrier state. The fathers of both probands(who were brothers) had slightly reduced red cell indices, borderline raisedHb A2 levels and mild reticulocytosis (Table 1). Positive Hb instability testswere observed in both probands and both fathers. The estimated percentageof the variant Hb was approximately 20% in the heterozygote fathers, asassessed by perfusion chromatography of globin polypeptide chains. Ofnote was the observation of red cell inclusions that were ellipsoid-like, acharacteristic finding associated with variant Hbs with a tendency to dimerformation.

Whole blood oxygen affinities in the two probands and their fathers werenormal with P50 values of 28.5, 31.0, 26.5 and 27.0 mm Hg, respectively,with normal 2,3-BPG concentrations for the degree of anemia. The oxygen

TABLE 1 Hematological Finding in the Probands and Their Parents

Parameters Father Mother Proband I Father Mother Proband II

Sex-Age M-32 F-28 M-2.5 M-5 M-29 F-26 F-3Hb (g/dL) 14.3 11.5 8.9 8.9 15.7 11.7 9.9PCV (L/L) 0.458 0.388 0.309 0.330 0.504 0.392 0.335MCV (fL) 86.3 65.5 78.6 79.1 92.4 72.1 74.2MCH (pg) 27.1 19.5 22.7 21.8 28.8 21.6 21.8MCHC

(g/dL)31.5 29.7 28.8 27.5 31.2 29.9 27.4

RDW (%) 14.7 15.1 18.6 18.4 14.5 14.4 18.4Hb F (%) 2.4 1.6 21.7 10.6 0.8 0.9 2.8Hb A2 (%) 3.7 5.4 4.9 4.8 3.8 4.7 5.0Reticulocytes

(%)2.8 1.2 15.4 13.0 2.5 1.2 8.6

Red cell inclusions

[+] [−] [+++] [+++] [++/+++] [−] [+++]

Genotype β107/βA IVS-I-110/βA IVS-I-110/β107 β107/βA IVS-I-110/βA IVS-I-110/β107

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Hb Lulu Island [b107(G9)Gly®Asp] and Thalassemia 413

binding properties of the purified hemolysate from proband I, are summarizedin Table 2a and 2b, and demonstrate a mild increase in oxygen affinity anda moderately raised Bohr effect.

The parameters indicating iron status, hemolytic and erythropoietic statusin both probands, are summarized in Table 3, and demonstrate a normaliron status. However, both probands had increased erythropoietic activity,reflected by sTfR levels more than 8-fold normal along with increasederythropoietin levels within the range expected for their degree anemia.Furthermore, the fathers of both probands, who were heterozygous for theHb Lulu Island variant, had a 1.9- and 2.9- fold increase in erythroid marrowactivity (data not shown).

TABLE 2a Oxygen Binding Properties of a Purified β+-Thalassemia/Hb Lulu Island Hemolysate

Hb AHemolysate

β+-Thal/Hb LIa P50 Hb A/P50 Hb LIa

Experimental conditions P50 (torr) (n50) P50 (torr) (n50)pH 7.2 4.8 (2.7) 4.1 (2.7) 1.17pH 7.2 + 2,3-BPG (1 mM) 14.5 (2.8) 12.4 (2.5) 1.17pH 6.5 11.0 (2.6) 12.0 (2.3) 0.92

Other conditions: NaCl 0.1 M, Bis-Tris 0.05 M, EDTA 50 μM, catalase 20 μg/mL, [heme] = 70–80 μM,25°C.

aHb LI: Hb Lulu Island.

TABLE 2b Heterotropic Effects of a Purified β+-Thalassemia/Hb Lulu Island Hemolysate

Hb A β+-Thal/Hb Lulu Island

Bohr effect: (Δlog P50/ΔpH) −0.51 −0.672,3-BPG effect: (Δlog P50 ± 1 mM 2,3-BPG) +0.48 +0.48

TABLE 3 Blood Chemistry Parameters Associated with Iron Status,Hemolysis and Erythropoiesis

Parameters Proband I Proband II

Bilirubin (μmol/L) 35.9 18.8LDH (U/L) 645.0 412.0Fe (μmol/L) 24.0 15.2TIBC (μmol/L) 92.4 75.8Transferrin saturation (%) 26.0 20.0Ferritin (μg/L) 18.0 34.2sTfR (mg/L) 12.1 (×10) 9.9 (×8)Epo (IU/L) 61.1 55.1

LDH: lactate dehydrogenase; Fe: iron; TIBC: total iron bindingcapacity; sTfR: soluble transferrin receptor; Epo: erythropoietin.

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Indicators of oxidant and antioxidant status are shown in Table 4, dem-onstrating a mild impairment of the glutathione system associated withlipid peroxidation in both probands. In addition, the antioxidant vitaminlevels were low in proband I, although in proband II they were within thenormal range.

DNA analysis in both probands demonstrated a common β+-thal mutation[IVS-I-110 (G→A)] in trans to a single base substitution in exon 3 of theβ-globin gene [codon 107 (GGC→GAC)] (Figure 1). The mothers of bothprobands were heterozygous for the β+-thal mutation [IVS-I-110 (G→A)],

TABLE 4 Blood Chemistry Parameters Associated with Oxidative Stress

Parameters Proband I Proband II Reference Values

GSHtotal (μmol/L) 653.0 1014.0 872.0 ± 157.0GSH (μmol/L) 469.0 724.0 814.0 ± 171.0GSSG (μmol/L) 92.0 145.0 <100.0GSSG/GSHtotal (%) 14.0 14.0 <10.0MDA (μmol/L) 0.77 0.76 0.68 ± 0.17Vitamin A (μmol/L) 3.5 1.2 1.0–2.8Vitamin C (μmol/L) 10.8 78.4 23.0–85.0Vitamin E (μmol/L) 8.5 15.8 12.0–42.0

GSH: reduced glutathione; GSSG: red cell oxidized; MDA: malonyldialdehyde.

FIGURE 1 Direct sequence analysis of exon 3 of the β-globin gene in proband I demonstrated theposition of the mutation at codon 107.

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Hb Lulu Island [b107(G9)Gly®Asp] and Thalassemia 415

and both fathers carried the codon 107 (GGC→GAC) mutation. α-Globingene variants were not observed in any of the family members.

The reversed phase HPLC elution pattern of the globin chains (Figure 2)demonstrates that the β-Lulu Island chain constitutes around 80% of totalβ-globin chains. The abnormal β chain was much more hydrophilic thanthe normal β chain, with a retention time of 5.5 (on an arbitrary scalewhere normal β-globin chains elute at 10.0 and normal α-globin chains at20.0). Electrospray mass spectrometry of the abnormal globin chaindemonstrated a difference in mass of +58 AMU compared to normal, whilepeptide mapping confirmed the predicted substitution of Gly→Asp in theβ-globin chain, consistent with the rare variant Hb Lulu Island.

DISCUSSION

To date, only one other patient has been reported with Hb Lulu Island.The patient was an adult female with thalassemia intermedia patient origi-nating from East India, who also had a β0-thal nonsense mutation [codon15 (TGG→TAG) in trans (4). The observation of Hb Lulu Island in a familyof Greek-Albanian origin probably represents an independent occurrence of

FIGURE 2 Reversed phase HPLC elution pattern of the globin chains.

Heme

Lulu Island

A

A

A 280nm

RT(arbitrary units)10 205.5

αβ

β

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416 I. Papassotiriou et al.

the DNA mutation, although haplotype studies to confirm this were notpossible.

In Hb Lulu Island, the amino acid substitution occurs in the α1β1 inter-face inducing instability of the Hb molecule, while oxygen affinity in wholeblood in the probands of this study (and in the original case) were almostnormal. However, the functional studies of purified hemolysate in probandI (performed for the first time), demonstrated an increased oxygen affinityand a mildly increased Bohr effect, indicating an increased dissociation rateof the tetramers into dimers.

The family in this study represents the first observation of Hb LuluIsland in the heterozygous state, as characterized in the fathers of bothprobands. Their hematological features were normal, with the exception ofborderline raised Hb A2 levels and erythroid marrow activity comparablewith those of β-thal heterozygotes probably due to the instability of thisvariant, a finding that seems to be consistent in carriers of unstable β chainvariants (9,10).

The interaction of rare Hb variants with β+- and β0-thal has beenreported in several cases before, including the single previous reported caseof Hb Lulu Island (2–4). In such cases, the major Hb component in theerythrocytes is that of the variant, as found to be the case in the twoprobands in the study who had more than 85% Hb Lulu Island. In the twoprobands of Greek-Albanian origin, the trans allele was a β+-thal mutation.Both cases had clinical and hematological features of thalassemia intermedia,including a chronic moderate anemia, although proband I demonstratedsevere anemia during episodes of infection and inflammation. The intermedi-ate forms of β-thal refer to patients without a requirement for regular bloodtransfusions and with clinical and hematological manifestations that aremilder than those associated with β-thal major. There are many underlyinggenetic causes of β-thal intermedia (11–14), and one such category includesthe co-inheritance of unstable β chain variants and a β-thal mutation (15).

Both probands manifested increased erythroid marrow activity withdyserythropoiesis. However, despite the identical genotypes of the twoprobands, the phenotypic expression of their disease varied significantly.The more pronounced erythroid marrow activity of proband I may possiblybe attributed to his higher levels of Hb F, which could be due to geneticfactors and/or stress erythropoiesis (16,17). Impairment of the glutathionesystem in both probands and low antioxidant vitamin levels in proband Iindicate that environmental factors, including diet, also play an importantrole in the clinical expression of patients with hemoglobinopathies (18).

There is minimal experience for the management of such rare andatypical cases. This case further illustrates that it is probably insufficient tomonitor clinical status in patients with hemoglobinopathies based on Hblevels alone, and evaluation should also include biochemical parameters (19).

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Hb Lulu Island [b107(G9)Gly®Asp] and Thalassemia 417

The indication that this unusual hemoglobinopathy may lead to a state ofmoderate oxidative stress, implies that such patients should be carefullymonitored, and possibly, that vitamin supplementation could be beneficial.

ACKNOWLEDGMENTS

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

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