Hemoglobin, 32 (4):371–378, (2008)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260802173973

371

--LHEM0363-02691532-432XHemoglobin, Vol. 32, No. 4, May 2008: pp. 1–15Hemoglobin

ORIGINAL ARTICLE

FIRST OBSERVATION OF Hb TAYBE [codons 38/39 (-ACC) Thr®0

(a1)] IN GREECE: CLINICAL AND HEMATOLOGICAL FINDINGS IN

PATIENTS WITH CO-INHERITED a+-THALASSEMIA MUTATIONS

Hb Taybe [codons 38/39 (−ACC) Thr→0 (α1)] in GreeceV. Douna et al.

Varvara Douna,1 Dimitra Liapi,2 Dimitrios Kampourakis,3 Zoe Repapinou,4

Ioannis Papassotiriou,5 Alexandra Stamoulakatou,6 Christos Poziopoulos,7

Emmanuel Kanavakis,8 and Joanne Traeger-Synodinos8

1Hematology Laboratory, “P. and A. Kyriakou” Children’s Hospital, Athens, Greece2Hematology Department, Venizelio-Pananio General Hospital, Heraklion, Greece3Pediatric Clinic, Agios Nikolaos General Hospital, Agios Nikolaos, Greece4Hematology Laboratory, Heraklion University Hospital, Heraklion, Greece5Department of Clinical Biochemistry, “Aghia Sophia” Children’s Hospital, Athens, Greece6Hematology Laboratory, “Aghia Sophia” Children’s Hospital, Athens, Greece7Hematology Department, 401 Military Hospital, Athens, Greece8Laboratory of Medical Genetics, “Aghia Sophia” Children’s Hospital, Athens, Greece

� This report describes four unrelated Greek patients (one child and three adults) who all had anatypical thalassemia intermedia phenotype, characterized by chronic moderate anemia with mildhemolysis in some cases, and the absence of abnormal hemoglobin (Hb) fractions. DNA analysisidentified the inheritance of common a+-thalassemia (a+-thal) mutations in trans to an in-frame3 bp deletion at codons 38/39 (-ACC) on the a1-globin gene, previously described as Hb Taybe.Hematological findings in the parents of three of the Hb Taybe carrier cases, together with a fourthunrelated carrier, are also presented. These cases represent the first observation of Hb Taybe in theGreek population, as to date, it has only been observed in Israeli-Arab families. With the exceptionof one patient and his mother who both originate from Corfu, all our cases come from the Greekisland of Crete.

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

Received 15 January 2008; accepted 25 January 2008.Address correspondence to Joanne Traeger-Synodinos, D.Phil., Laboratory of Medical Genetics,

University of Athens, “Aghia Sophia” Children’s Hospital, Thivon and Levadias St., 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

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.

372 V. Douna et al.

INTRODUCTION

α-Thalassemia (α-thal) mutations are among the most common causeof monogenic disorders in humans. Of the more than 80 different α-thalmutations that have been described, the most frequent are deletions whichpartially or completely remove the α-globin genes from the α-globin genecluster at the tip of chromosome 16 (16p13.3, ζ2–ψζ1–ψα2–ψα1–α2–α1–θ).Point mutations within either of the duplicated α-globin genes, also knownas nondeletional mutations, tend to be less commonly observed in casescharacterized with α-thal phenotypes. Nondeletional mutations usuallyreduce α-globin synthesis by impeding RNA processing or translation(haplo-insufficient), but some cause posttranslational instability of theabnormal polypeptide, and when the variant α-globin chain synthesis ishyperunstable, it mimics an α-thal allele since the overall synthesis ofα-globin chains is reduced. Interaction of nondeletional α-thal determi-nants with other α-thal mutations usually causes Hb H disease, a chronicmoderate anemia in which excess α-globin chains form Hb H (β4) (1–3).However, some unstable α-globin chain variants, either in the homozygousstate or in interaction with other α-thal determinants, may present ashemolytic anemia or with an atypical thalassemia phenotype that has manyfeatures of β-thal intermedia but without the raised levels of Hb F or Hb A2.In addition, electrophoretically or chromatographically detectable fractionsof Hb H are absent (4,5). In rare cases, unstable α-globin variants havebeen described to cause Hb H hydrops fetalis in the homozygous state orwhen co-inherited with severe haplo-insufficient α-thal determinants (6,7).

This report describes four unrelated Greek patients (one child andthree adults) who all had a similar atypical thalassemia intermedia pheno-type, characterized by chronic moderate anemia with mild hemolysis, in theabsence of abnormal hemoglobin (Hb) fractions. In all cases, DNA analysesidentified the inheritance of common α+-thal mutations in trans to anin-frame 3 bp deletion at codons 38/39 (−ACC) on the α1-globin gene,previously described as Hb Taybe. Hematological findings in the parents ofthree of the Hb Taybe carrier cases, together with a fourth unrelatedcarrier, are also presented. These cases represent the first observation of HbTaybe in the Greek population, as to date, it has only been observed inIsraeli-Arab families. With the exception of one patient and his mother whoboth originate from Corfu, all our cases come from the Greek island ofCrete.

MATERIALS AND METHODS

Blood samples (10–20 mL) were collected in EDTA and subjected tohematological and Hb studies by standard methods as previously described,

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.

Hb Taybe [codons 38/39 (-ACC) Thr®0 (a1)] in Greece 373

including measurement of red blood cell (RBC) indices, cellulose acetateelectrophoresis (pH 9.0), isoelectric focusing (IEF) on agarose gels(ph 6.0–8.0), weak cation exchange high performance liquid chromatogra-phy (HPLC) (Bio-Rad Laboratories, Hercules, CA, USA) with the β-Thalassemia Short Program and the program for quantitation of Hb Bart’s(γ4) and Hb H (8).

Heinz body formation and thermal and isopropanol Hb stability testswere performed by standard methods (9), and evaluation of red cell mor-phology was done by an experienced hematologist. Serum ferritin levelswere measured using the IMXsystems Ferritin Kit (Abbott Laboratories,Chicago, IL, USA) and bilirubin levels were measured by standard methods.

Genomic DNA was isolated from white blood cells using the Qiagen MiniBlood DNA Kit (Qiagen GmbH, Hilden, Germany) (10). Common Mediter-ranean deletional and nondeletional α-thal mutations were investigatedusing polymerase chain reaction (PCR)-based protocols and subsequently,direct sequencing of selectively amplified α1 or α2 genes as previouslydescribed (3,10,11). Pathological mutations in the β-globin genes wereexcluded by denaturing gradient gel electrophoresis (DGGE) (12).

RESULTS

Based on analysis of the α-globin genotypes, all four patients had a 3 bpdeletion (−ACC) between codons 38 and 39, previously described as HbTaybe. Three of them had the common α+-thal 3.7 kb deletion in trans,while the fourth had the common Mediterranean nondeletional IVS-Idonor splice site mutation (αHphα) (see Table 1). Anemia was the mostcommon presenting symptom, observed from early life in two patients (case1 and case 2), although cases 3 and 4 were young adults when diagnosed.The hematology presented in Table 1 represents “steady state” values in allcases except case 1 in whom the hematology data was obtained duringhospitalization following an infection. Hemoglobin levels in all four caseswere consistent with mild-to-moderate anemia, and abnormal red cellmorphology was present in all patients. Reticulocyte counts were mildlyelevated in cases 2, 3 and 4, and more marked in case 1 due to his febrilestate. Additionally, erythroblasts were observed on peripheral blood smearsin cases 1 and 2. Inclusion bodies were observed in cases 2, 3 and 4 (therewas inadequate data for case 1). Hb A2 and Hb F levels were within thenormal range and no abnormal Hb fractions were observed with eitherelectrophoresis or HPLC. Cases 2 and 3 had mild jaundice, and mild-to-moderate splenomegaly was observed in all patients but to date, nonehave been splenectomized.

Case 2 received a single blood transfusion at 37 years of age followingreported dizziness, despite the observation that her hematocrit at that time

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.

374 V. Douna et al.

(0.23 L/L) was no lower that observed throughout her life (0.23–0.24 L/L),even during two uneventful pregnancies, which notably did not requiresupport with blood transfusions. All the patients, three of whom are pres-ently adults, demonstrate normal growth and appearance and live normalhealthy lives.

The parents of three of the Hb Taybe carriers were available for studytogether with another Hb Taybe heterozygote who was characterized coin-cidentally during routine carrier screening. All four Hb Taybe carriers hadnormal hematological findings with the exception of minor morphologicalred cell changes (Table 2). None of them had any abnormal clinical findings,such as a history of anemia, jaundice or splenomegaly.

TABLE 1 Hematological and Biochemical Data at First Clinical Evaluation of the Hb Taybe CompoundHeterozygotes

Parameters Case 1 Case 2 Case 3 Case 4

Sex-Age M-6 F-42 M-27 M-25Age at first

evaluationChildhood Childhood Young adulthood Young adulthood

Reason for evaluation

Febrile episode with anemia and splenomegaly

Anemia and splenomegaly (2 cm)

Mild anemia, splenomegaly and jaundice

Anemia and splenomegaly

Genotype −α3.7/ααcodons38/39a −α3.7/ααcodons38/39a αHphα/ααcodons38/39a −α3.7/ααcodons38/39a

Hb (g/dL) 8.8 9.2 12.6 10.9PCV (L/L) 0.28 0.31 0.40 0.34MCV (fL) 77.1 87.2 78.4 77.0MCH (pg) 24.2 N.D. 23.6 24.8RDW (%) 27.7 21.6 18.7 18.3Red cell

morphology[++/+++] [+] [+] [++]

Normoblasts 9/100 WBC 3/100 WBC None N.D.Reticulocytes

(%)8.8 4.0 4.0 4.5

Hb A2 (%) 2.5 2.2 2.2 2.3Hb F (%) 3.9 2.4 1.0 1.4Inclusion

bodiesN.D. Fine inclusions in

occasional red cells

Fine inclusions in occasional red cells

[+]

Bilirubin (mg/dL)

N.D. 1.5 3.41 N.D.

Ferritin (ng/mL

N.D. 54.60 25.47 147.00

Splenomegaly [+] [+] (2 cm) [+] [+]Present status Good general

condition; Hb 9.5–10.0 g/dL; transfusion free

Good general condition; Hb 9.0 g/dL;transfused once at age 37

Good general condition; transfusion free

Good general condition; transfusion free

N.D. = No data.aHb Taybe [codons 38/39 (−ACC) Thr→0 (α1)].

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.

Hb Taybe [codons 38/39 (-ACC) Thr®0 (a1)] in Greece 375

DISCUSSION

Hb Taybe is an unstable α chain variant caused by a deletion of a thre-onine residue at codon 38 or 39 of the α1-globin chain. The structuralabnormality of Hb Taybe is located in helix C which participates in theα1β2 contact and is close to the α1β1 interface. The consequences of themutation at the protein level have been well described (14,15). Fractionsof the abnormal Hb Taybe have been observed in red cells only in somehomozygotes, especially following splenectomy. It appears that the physi-cochemical properties of the variant globin chains and Hb preclude theformation of Hb H. Although it could be expected that when Hb Taybeco-exists with another α-thalassemic gene, the reduced production of theα chains may support the formation of trace amounts of Hb Taybetogether with some Hb H, we did not observe any abnormal fractions onelectrophoresis or with HPLC analyses. However, it cannot be excludedthat detection of Hb Taybe may require fresh blood samples, and theapplication of other methods such as reversed phase HPLC or mass spec-trometry. In three of the cases, fine inclusions were observed in occasionalred cells.

Previous reports describe carriers as asymptomatic, without hematologi-cal abnormalities, although some cases have mild hypochromic microcyticanemia (13,14). The Greek Hb Taybe carriers we report here had Hb levelswithin the normal range, normal red cell indices and Hb fractions (Table 2).

Homozygosity for Hb Taybe has been reported to cause severe hemolyticanemia in adults, and in rare cases, has been described in association with

TABLE 2 Clinical, Hematological and Molecular Data of the Hb Taybe Heterozygotes (αα/ααcodons38/39)a

Parameters Father of Case 1 Father of Case 3 Mother of Case 4 Unrelated Carrier

Hb (g/dL) 15.3 16.0 13.5 15.6

PCV (L/L) 0.46 0.48 0.45 0.45MCV (fL) 81.9 85.3 88.0 82.0MCH (pg) 27.1 28.6 26.3 27.4RDW (%) 13.7 12.6 15.1 13.3Red cell morphology [+] [+] [+] [++]Erythroblasts None None None NoneReticulocytes (%) N.D. 4.0 2.0 1.7Hb A2 (%) 2.4 2.7 2.8 2.8Hb F (%) 0.30 0.60 0.39 1.70Inclusion bodies N.D. Yes No N.D.Ferritin (ng/mL) None None None NoneSplenomegaly No No No NoGenotype αα/ααcodons38/39a αα/ααcodons38/39a αα/ααcodons38/39a αα/ααcodons38/39a

N.D. = No data.aHb Taybe [codons 38/39 (−ACC) Thr→0 (α1)].

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.

376 V. Douna et al.

hydrops fetalis (6,13,14). On the other hand, compound heterozygotes withHb Taybe and interacting α+-thal mutations usually have a clinical presenta-tion analogous to mild thalassemia intermedia (16). A single case has beenreported with a severe hemolytic anemia unresponsive to splenectomy withthe need for occasional blood transfusions, in whom Hb Taybe was co-inherited with the severe nondeletional polyadenylation signal (poly A)mutation (AATAAA>AATAAG) (15), implying that the phenotypic severityin compound heterozygotes is influenced by the severity of the interactingα-thal mutation.

The Greek patients that we describe have all inherited Hb Taybe intrans, either to the common α+-thal 3.7 kb deletion or to the commonMediterranean nondeletional IVS-I donor splice site mutation (αHphα).They all have relatively mild anemia without transfusion requirements;mild-to-moderate splenomegaly was the only clinical finding, present inall four patients. These clinical characteristics were comparable to thecases previously reported by Oron-Karni et al. (16), in whom analogousgenotypes are also described. It is interesting to note that the pheno-typic presentation of Hb Taybe compound heterozygotes is also compa-rable to that which has been observed in cases co-inheriting theα2-globin gene αHphα mutation with Hb Heraklion [(4) and manuscriptin preparation]. Hb Heraklion is also a rare α-globin chain variant thatis caused by a 3 bp deletion on the α1-globin gene at the neighboringcodon [α37(C2)Pro→0] to that underlying Hb Taybe, and is likely tohave a comparable consequence at the protein level. Overall, theabsence of characteristic clinical and hematological findings precludeda definitive diagnosis, which was ultimately achieved in all patients byDNA analysis.

To date, Hb Taybe has only been observed in Israeli-Arabs, in whom itis the most frequent α-thal point mutation (13). It is of interest that allpatients and carriers in this study originated from the island of Crete inGreece with the exception of case 4 and his carrier mother who origi-nated from the island of Corfu. No data is available as to whether theobservation of this variant in Israeli-Arabs and Greeks is due to a foundermutation or represents two independent mutation events. However, HbTaybe carriers in Greece are very rare, implying that the risk of findingHb Taybe homozygotes is low, as is the likelihood of interaction withsevere α-thal determinants, which are also quite rare in the Greek popula-tion (3). This, coupled with the observation that the clinical course ofcompound heterozygotes with most other α-thal is mild, means thatscreening for Hb Taybe in the general population is not indicated inGreece, although DNA analysis could be useful in spouses of α-thal carri-ers in order to support comprehensive genetic counseling in the contextof prevention programs.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.

Hb Taybe [codons 38/39 (-ACC) Thr®0 (a1)] in Greece 377

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.”

REFERENCES

1. Adams JG, Coleman MB. Structural hemoglobin variants that produce the phenotype of thalas-semia. Semin Hematol 1990; 27:229–238.

2. Chui DHK, Fucharoen S, Chan V. Hemoglobin H disease: not necessarily a benign disorder. Blood2003; 101(3):791–800.

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

4. 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.

5. Waye JS, Eng B, Patterson M, Carcao MD, Chang L, Olivieri NF, Chui DHK. Identification oftwo new α-thalassemia mutations in exon 2 of the α1-globin gene. Hemoglobin 2001;25(4):391–396.

6. Arnon S, Tamary H, Dgany O, Litmanovitz I, Regev R, Bauer S, Dolfin T, Yacobovich J, Wolach B,Jaber L. Hydrops fetalis associated with homozygosity for Hemoglobin Taybe (α 38/39 THR dele-tion) in newborn triplets. Am J Hematol 2004; 76(3):263–266.

7. Lorey F, Charoenkwan P, Witkowska HE, Lafferty J, Patterson M, Eng B, Waye JS, Finklestein JZ,Chui DHK. Hb H hydrops foetalis syndrome: a case report and review of literature. Br J Haematol2001; 115(1):72–78.

8. 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.

9. Carrell RW. Methods of determining hemoglobin instability (unstable hemoglobins). In: HuismanTHJ, Ed. The Hemoglobinopathies. Methods in Hematology, Vol. 15. Edinburgh: ChurchillLivingstone. 1986; 109–124.

10. 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(2):137–143.

11. Traeger-Synodinos J, Kanavakis E, Tzetis M, Kattamis A, Kattamis C. Characterization ofnondeletion α-thalassemia mutation in the Greek population. Am J Hematol 1993; 44(3):162–167.

12. Kanavakis E, Traeger-Synodinos J, Vrettou C, Maragoudaki E, Tzetis M, Kattamis C. Prenatal diag-nosis of the thalassemia syndromes by rapid DNA analytical methods. Molec Hum Reprod 1997;3(6):523–528.

13. Galacteros F, Girodon E, M’Rad A, Martin J, Goossens M, Jaber L, Cohen IJ, Tamary H, Goshen Y,Zaikov R, Wajcman H. Hb Taybe (α 38 or 39 THR deleted): an α-globin defect, silent in the het-erozygous state and producing severe hemolytic anemia in the homozygous. CR Acad Sci Paris, Scide la Vie/Life Sci 1994; 317(5):437–444.

14. Bassat B, Simjanovska L, Jaber L, Efremov GD. Hb Taybe: description of genetics and laboratoryfindings in an Israeli Arab family. Hemoglobin 1998; 22(2):161–166.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.

378 V. Douna et al.

15. Pobedimskaya DD, Molchanova TP, Streichman S, Huisman THJ. Compound heterozygosityfor two 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.

16. 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.

Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ath

on 1

1/02

/14

For

pers

onal

use

onl

y.