Hemoglobin, 32 (4):379–385, (2008)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260802174021

379

LHEM0363-02691532-432XHemoglobin, Vol. 32, No. 4, May 2008: pp. 1–14Hemoglobin

ORIGINAL ARTICLE

FURTHER IDENTIFICATION OF THE HYPERUNSTABLE a-GLOBIN

CHAIN VARIANT Hb HERAKLION [codons 36/37 (–CCC); Pro®0

(a1)] IN GREEK CASES WITH CO-INHERITED a+-THALASSEMIA

MUTATIONS

Four Cases of Hb Heraklion in GreeceV. Douna et al.

Varvara Douna,1 Ioannis Papassotiriou,2 Anna Metaxotou-Mavrommati,3

Alexandra Stamoulakatou,4 Dimitra Liapi,5 Dimitrios Kampourakis,6

Amalia Tsilimigaki,7 Emmanuel Kanavakis,3 and Joanne Traeger-Synodinos3

1Hematology Laboratory, “P. and A. Kyriakou” Children’s Hospital, Athens, Greece2Department of Clinical Biochemistry, “Aghia Sophia” Children’s Hospital, Athens, Greece3Department of Medical Genetics, University of Athens, “Aghia Sophia” Children’s Hospital, Athens, Greece4Hematology Laboratory, “Aghia Sophia” Children’s Hospital, Athens, Greece5Hematology Laboratory, Venizelio-Pananio General Hospital, Heraklion, Greece6Pediatric Clinic, Agios Nikolaos General Hospital, Agios Nikolaos, Greece7Second Pediatric Clinic, Venizelio-Pananio General Hospital, Heraklion, Greece

� We report four Greek cases (from three unrelated families), who all had a similar atypicalthalassemia intermedia phenotype, characterized by chronic moderate anemia, mild hemolysis andsplenomegaly in the absence of abnormal hemoglobin (Hb) fractions. In all four cases (two unre-lated children and two siblings), DNA analysis identified common a+-thalassemia (a+-thal) muta-tions in trans to the in frame 3 bp deletion (-CCC) on the a1-globin gene between codons 36 and37, which has previously been reported as Hb Heraklion in a single Greek case. Clinical, hematologicaland biochemical findings in all cases, including a follow-up evaluation of the original case, aredescribed. All the cases originated from the Greek island of Crete.

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

Received 24 January 2008; accepted 1 February, 2008.Address correspondence to Joanne Traeger-Synodinos, D.Phil., Laboratory of 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

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

380 V. Douna et al.

INTRODUCTION

The synthesis of α-globin chains is directed by the duplicated α-globingenes located at the tip of the short arm of chromosome 16 (16p13.3). Muta-tions that reduce the synthesis of α-globin chains cause α-thalassemia(α-thal), and over 80 have been described worldwide (http://globin.cse.psu.edu/hbvar/menu.html ). The majority of the most common α-thaldeterminants are deletions that remove some, or all of, the α-globin genecluster (1,2). Less common are single nucleotide (nt) substitutions or microdeletions within either the α1- or α2-globin genes, also known as nondele-tional mutations. An apparent reduction in α-globin chain synthesis can alsobe caused by substitutions that give rise to hyperunstable α-globin chains. Inmost cases, these variants are so unstable that they cannot be detected at theprotein level, and can only be deduced from the DNA sequence. Althoughthese variants are generally considered rare, through the wider applicationof improved DNA analytical technologies, the number identified continueto grow (3–13). Several have even been identified in the heterozygous statedespite usually normal or borderline hematology. However, the hyperunsta-ble α-globin variants become phenotypically apparent in the homozygousstate or when they interact with other α-thal determinants (3–13).

In this report we describe four patients who presented with hematologicaland clinical findings consistent with a relatively severe dyserythropoieticanemia comparable to thalassemia intermedia. All were found to haveco-inherited the rare, highly unstable α-globin variant Hb Heraklion, an inframe 3 bp deletion (−CCC) on the α1-globin gene, between codons 36 and37 in trans to a common α+-thal mutation (14). All originated from theGreek island of Crete.

MATERIALS AND METHODS

For hematological, hemoglobin (Hb) and DNA studies, blood samples(10–20 mL) were collected in EDTA. All hematological and Hb analyseswere performed by standard procedures as previously described, as wellas blood chemistry measurements, such as serum ferritin and bilirubinlevels (15,16).

Genomic DNA was isolated from white blood cells using the QIAGENMini Blood DNA kit (Qiagen GmbH, Hilden, Germany) as previouslydescribed (17). Common Mediterranean deletional and nondeletionalα-thal mutations were investigated using polymerase chain reaction (PCR)-based protocols, and subsequently, direct sequencing of selectivelyamplified α1 or α2 genes, as previously described (17,18). Pathologicalmutations in the β-globin genes were excluded by denaturing gradient gelelectrophoresis (DGGE) analysis (19).

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

Four Cases of Hb Heraklion in Greece 381

RESULTS

The patients in this study were from three unrelated families andincluded two unrelated children and two adult siblings. Based on analysisof the α-globin genotypes, all four cases had a deletion of 3 bp (−CCC)between codons 36 and 37, previously described as Hb Heraklion. Threeof these cases had the common Mediterranean nondeletional IVS-I donorsplice site mutation (αHphα) in trans, and the fourth had the commonα+-thal 3.7 kb deletion (see Table 1).

TABLE 1 Clinical, Hematological and Biochemical Data at First Clinical Evaluation of the HbHeraklion Compound Heterozygotes

Parameters Case 1 Case 2 Case 3–1 Case 3-II

Sex-Age F-10 M-19 F-33 F-32 (approximately)

First evaluation September 2005 September 1989 November 1982 February 2002Symptoms Fever, jaundice Anemia Anemia, splenomegaly,

thalassemia faciesAnemia,

splenomegalyHb (g/dL)

at first evaluation

8.3 8.5 7.46 9.5

PCV (L/L) 0.253 0.290 0.235 0.290MCV (fL) 73.5 73.0 77.6 88.8MCH (pg) 24.1 22.9 24.4 23.9RDW (%) 17.7 32.8 21.7 23.3Morphological

changesYes [+] Yes [+++] Yes [++] Yes [++]

Normoblasts 1/100 WBC N.D. 78/100 WBC NoneReticulocytes

(%)4.2 10.0 9.0 N.D.

Hb A2 (%) 1.6 3.5 1.7 2.2Hb F (%) 2.5 3.8 3.6 1.2Inclusion

bodiesN.D. Yes Yes Yes

Bilirubin (mg/dL)

7.1 3.2 4.3 (0.6) N.D.

LDH N.D. N.D. 540.0 N.D.Ferritin

(ng/mL164.0 N.D. 71.0 13.0

sTfR (mg/dL) N.D. 16.8 71.1 185.0Epo (IU/L) N.D. 72.0 78.4 56.5Spleen 8 cm 16.5 × 6 cm

(ultrasound 1999)

3–4 cm N.D.

Liver 2 cm N.D. 1.5–2 cm N.D.Genotype ααcodons 36/37a/

–α3.7ααcodons 36/37a/

αHphαααcodon 36/37a/

αHphαααcodons 36/37a/

αHphα

N.D. = No data.aHb Heraklion [codons 36/37 (−CCC) Pro→0 (α1)].

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

382 V. Douna et al.

All patients were diagnosed in childhood. In cases 2, 3-I and 3-II,anemia was the main presenting symptom, which, although well tolerated,was persistent and led the patients to seek medical attention. Case 1 wasadmitted to hospital at the age of 8 years following an episode of fever andjaundice. In all four cases, the Hb levels were consistent with a moderateanemia, and abnormal red cell morphology was present in all cases. Thevalues of the red cell indices as well as all laboratory findings are shown inTable 1. Reticulocytes were elevated, particularly in cases 2 and 3-I, and incases 1 and 3-I, erythroblasts were additionally observed in the peripheralblood. Inclusion bodies were observed in cases 2, 3-I and 3-II, althoughnone had abnormal Hb fractions with either electrophoresis or high perfor-mance liquid chromatography (HPLC), and Hb A2 and Hb F values werewithin normal limits. With the exception of case 3-II (for whom there wasinadequate data), splenomegaly, as well as hyperbilirubinemia, were themain clinical findings.

Case 2, the first case of Hb Heraklion described (14), was diagnosed ininfancy (14 months of age) and has received three blood transfusions todate, initiated in childhood. At the age of 9 years, he suffered an episodeof allergic purpura, at which time he had mild anemia (Hb 9.0 g/dL,PCV 0.28 L/L) which required no transfusions, and his clinical conditionwas evaluated as being generally good. He is now 19 years old but has beenlost to follow-up. Case 3-I was transfused twice, the first time at the age of8 years after a hemolytic episode induced by a fever and again at the ageof 28 when she was given nine units of packed red cells after a stomachhemorrhage.

DISCUSSION

Hb Heraklion is an unstable α chain variant caused by a deletion of aproline residue at position 37 in the α-globin chain (14). The α37 residue isin the central cavity of the α chain and is normally involved in the α1β2contacts of the Hb molecule through interaction with the histidine residueat β147. In addition, α37 is in close proximity to residues arginine 31 andphenylalanine 36 of the α chain in the α1β1 interface. Proline α37 alsoplays an important role in stabilization of the T-conformation of the Hbmolecule. The mutation causes instability and is expected to alter oxygenaffinity. Two other Hb variants have been described with a substitution atα37(C2)Pro: Hb Boumerdes (→Arg) (20) and Hb Manawatu (→Leu) (21).In Hb Manawatu, the molecular instability was confirmed by a positiveisopropanol test, although the altered oxygen affinity was not confirmed byoxygen affinity measurements. Hb Boumerdes was detected electrophoreti-cally, but no stability or oxygen affinity data were reported. The HbHeraklion carriers (the parents of our patients) were hematologically and

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

Four Cases of Hb Heraklion in Greece 383

clinically indistinguishable from normal (data not shown), although itcould be expected that the deletion of the proline residue at α37 in HbHeraklion is more detrimental than an amino acid substitution, likelycausing severe instability of the α-globin chain and Hb molecule, elicitingα-globin chain deficiency analogous to an α-thal mutation. This is analo-gous to the mutation in the hyperunstable Hb Taybe which is caused by a3 bp deletion at neighboring codons on the α1-globin gene [codons 38/39(−ACC); Thr→0] (22).

To date, Hb Heraklion has been observed in a single Greek patient whois included in this report (14). Like many other highly unstable α-globinchain variants described, it was detected in an individual when it interactedwith another α-thal mutation, probably due to a gene dosage effect (13,23–25). Hb Heraklion homozygotes have not yet been observed.

The Greek patients whom we describe have all inherited Hb Heraklionin trans either to a Mediterranean nondeletional IVS-I donor splice sitemutation (αHphα) or to the common α+-thal 3.7 kb deletion. They all havemoderate anemia but without blood transfusion requirements; mild-to-moderate splenomegaly is the main clinical finding, present in three of thefour patients. It is interesting to note that their phenotypic presentationis comparable to that of Hb Taybe compound heterozygotes, as hasbeen described by Oron-Karni et al. (23) and recently in four Greek casesco-inheriting Hb Taybe with a common α-thal mutation (24). In all cases,the absence of characteristic clinical and hematological findings preclude adefinitive diagnosis based on these factors alone, and a definitive diagnosismust ultimately be achieved by DNA analysis.

Apparently, the incidence of Hb Heraklion in Greece is low, since up tonow it has only been reported in these four cases, and has never been iden-tified following DNA analysis in over 3,000 samples with normal/borderlinehematology, which have been referred to our laboratory in the last fewyears. This, coupled with the evidence that the clinical course of compoundheterozygotes can be considered clinically mild, indicates that screening forHb Heraklion in the general population is not indicated. However, DNAanalysis to detect the anomaly in spouses of α-thal carriers should be consid-ered to optimize genetic counseling.

ACKNOWLEDGMENTS

The authors wish to thank the members of the Laboratory of MedicalGenetics, Athens University Medical School, Athens, Greece, Irene Fylaktou,B.Sc., for sequencing analyses and Stavroula Papadopoulou for technicalassistance. This study was partially supported by a European CommissionCoordination Action Contract grant (no. 026539) titled “Infrastructure forThalassaemia Research Network.”

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

384 V. Douna et al.

REFERENCES

1. Waye JS, Chui DHK. The α-globin gene cluster: genetics and disorders. Clin Invest Med 2001;24(2):103–109.

2. Higgs DR. Molecular mechanisms of α thalassemia. In: Steinberg MH, Forget BG, Higgs DR, NagelRL, Eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management.Cambridge: Cambridge University Press. 2001; 405–430.

3. Goossens M, Lee KY, Liehaber SA, Kan YW. Globin structural mutant α125Leu→Pro is a novelcause of α-thalassaemia. Nature 1982; 296(5860):864–865.

4. Honig GR, Shamsuddin M, Vida LN, Mompoint M, Vacourt E, Bowie LJ, Jones EC, Powers PA,Spritz RA, Guis M, Embury SH, Conboy J, Kan YW, Mentzer WC, Weil SC, Hirata RK, Waloch J,O’Riordan JF, Goldstick TK. Hemoglobin Evanston (α14 Trp→Arg). An unstable α-chain variαntexpressed as α-thalassemia. J Clin Invest 1984; 73(6):1740–1749.

5. Weiss I, Cash FE, Coleman MB, Pressley A, Adams JG, Sanguansermsri T, Liebhaber SA, SteinbergMH. Molecular basis for α-thalassemia associated with the structural mutant Hemoglobin Suan-Dok (α2 109Leu→Arg). Blood 1990; 76(12):2630–2636.

6. Darbellay R, Mach-Pascual S, Rose K, Graf J, Beris P. Haemaoglobin Tunis-Bizerte: a new α1globin 129 Leu→Pro unstable variant with thalassaemic phenotype. Br J Haematol 1995;90(1):71–76.

7. Ho PJ, Rochette J, Rees DC, Fisher CA, Huehns ER, Will AM, Thein SL. Hb Sun Prairie: diagnosticpitfalls in thalassemic hemoglobinopathies. Hemoglobin 1996; 20(2):103–112.

8. Préhu C, Mazurier E, Riou J, Kister J, Promé D, Richelme-David S, Al Jassem L, Angellier E,Wajcman H. A new unstable α2-globin gene variant: Hb Chartres [α33(B14)Phe→Ser]. Hemoglobin2003; 27(2):111–115.

9. Lacerra G, Testa R, De Angioletti M, Schilirò G, Carestia C. Hb Bronte or α93(FG5)Val→Gly: anew unstable variant of the α2-globin gene, assocaited with a mild α+-thalassemia phenotype. Hemo-globin 2003 27(3):149–159.

10. Dutley F, Fehr J, Goede JS, Morf M, Troxler H, Frischknecht H. A new highly unstable α chain vari-ant causing α+-thalassemia: Hb Zurich Albisrieden [α58(E8)Gly→Arg (α2)]. Hemoglobin 2004;28(4):347–351.

11. Harteveld CL, Steen G, Vlasveld LT, van Delft P, Giordano PC. Hb Bronovo, a new globin genemutation at α2 103 (His→Leu) associated with an α thalassemia phenotype. Haematologica 2006;91(4)570–571.

12. Giordano PC, Zweegman S, Akkermans N, Arkesteijn SG, van Delft P, Versteegh FGA, Wajcman H,Harteveld CL. The first case of Hb Groene Hart [α119(H2)Pro→Ser, CCT→TCT (α1)] homozygos-ity confirms that a thalassemia phenotype is associated with this abnormal hemoglobin variant.Hemoglobin 2007; 31(2):179–182.

13. Douna V, Papassotiriou I, Garoufi A, Georgouli E, Ladis V, Stamoulakatou A, Metaxotou-MavrommatiA, Kanavakis E, Traeger-Synodinos J. A rare thalassemic syndrome caused by interaction of HbAdana [α59(E8)Gly→Asp] with an α+-thalassemia deletion: clinical aspects in two cases. Hemoglo-bin 2008; 32(4): (this issue).

14. Traeger-Synodinos J, Papassotiriou I, Metaxotou-Mavrommati A, Vrettou C, Stamoulakatou A,Kanavakis E. Distinct phenotypic expression associated with a new hyperunstable α globin variant(Hb Heraklion, α1cd37(C2)Pro→0): comparison to other α-thalassemic hemoglobinopathies.Blood Cells Molec Dis 2000;26(4):276–284.

15. Papassotiriou I, Traeger-Synodinos J, Vlachou C, Karagiorga M, Metaxotou A, Kanavakis E,Stamoulakatou A. Rapid and accurate quantitation of Hb Bart’s and Hb H using weak cationexchange high performance liquid chromatography: correlation with the α-thalassemia genotype.Hemoglobin 1999; 23(3):203–211.

16. Carrell RW. Methods of determining hemoglobin instability (unstable hemoglobins). In: HuismanTHJ, Ed. The Hemoglobinopathies. Methods in Hematology, Vol.15. Edinburgh: Churchill Living-stone. 1986; 109–123.

17. Dimisianos G, Traeger-Synodinos J, Vrettou C, Papassotiriou I, Kanavakis E. A rare 33 bp in-framedeletion (α63–74 or α64–74 or α65–75) in the α1-globin gene causing α+-thalassemia: a secondobservation. Hemoglobin 2004; 28(20):137–143.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

Four Cases of Hb Heraklion in Greece 385

18. Kanavakis E, Papassotiriou I, Karagiorga M, Vrettou C, Metaxotou-Mavromati A, Stamoulakatou A,Kattamis C, Traeger-Synodinos J. Phenotypic and molecular diversity of Haemoglobin H disease.A Greek experience. Br J Haematol 2000; 111(3):915–923.

19. Kanavakis E, Traeger-Synodinos J, Vrettou C, Maragoudaki E, Tzetis M, Kattamis C. Prenataldiagnosis of thalassemia syndromes by rapid DNA analytical methods. Mol Hum Reprod 1997;3(6):523–528.

20. Dahmane-Arbane M, Blouquit Y, Arous N, Bardakdjian J, Benamani M, Riou J, Benabadji M, RosaJ, Galacteros F. Hemoglobin Boumerdès α237(C2)Pro→Argβ2: a new variant of the α chain associ-ated with Hemoglobin S in an Algerian family. Nouv Rev Fr Hematol 1987; 29(5):317–320.

21. Brennan SO, Sheen C, Johnson S. Hb Manawatu [α37(C2)Pro→Leu]: a new mildly unstablemutation at an invariant proline residue. Hemoglobin 2002; 26(4):389–392.

22. Galacteros F, Girodon E, M’Rad A, Martin J, Goossens M, Jaber L, Cohen IJ, Tamary H, Goshen Y,Zaizov R, Wajcman H. Hb Taybe (α38 or 39 THR deleted): an α-globin defect, silent in the het-erozygous state and producing severe hemolytic anemia in the homozygous. C R Acad Sci 1994;317(5):437–444.

23. Oron-Karni V, Filon D, Shifrin Y, Fried E, Pogrebijsky G, Oppenheim A, Rund D. Diversity ofα-globin mutations and clinical presentation of α-thalassemia in Israel. Am J Hematol 2000;65(3):198–202.

24. Douna V, Liapi D, Kampourakis D, Repapinou Z, Papassotiriou I, Stamoulakatou A, PoziopoulosH, Kanavakis E, Traeger-Synodinos J. First observation of Hb Taybe [codons 38/39 (–ACC) Thr→0(α1)] in Greece: clinical and hematological findings in patients with co-inherited α+-thalassemiamutations. Hemoglobin 2008; 32(4):371–378.

25. Pobedimskaya DD, Molchanova TP, Streichman S, Huisman THJ. Compound heterozygosity fortwo α-globin gene defects, Hb Taybe (α1; 38 or 39 minus Thr) and a poly A mutation (α2;AATAAA→AATAAG), results in a severe hemolytic anemia. Am J Hematol 1994; 47(3):198–202.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UB

Mai

nz o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.