Distribution and frequency of b-thalassemiamutations in northwestern and centralGreece

b-Thalassemia is a common autosomal recessivedisorder resulting from over 200 different muta-tions of the b-globin gene that result either in theabsence of the b-globin chains (b0 thal) or areduction in their output (b+ thal). The inabilityto synthesize b-globin chains results in an excess ofa-globin chains that precipitate and damage the redcell precursors and their progeny (1, 2).

b-Thalassemia is one of the most common singlegene disorders and a major public health concernin Greece, although control programs consisting ofscreening and prenatal antenatal diagnosis havesucceeded in minimizing the frequency of neonateseffected by b-thalassemia in 80–100% (3). Thespectrum of b-thalassemia mutations in Greece has

been previously described in the population of thecapital city of Athens, or in b-thalassemia patientshaving transfusion therapy (4–6). Molecular ana-lysis of b-thalassemia mutations has been per-formed by PCR-based protocols mostly forprenatal diagnostic needs (7). The most commonmutation found in those studies was IVS-I-110 andthis was followed, in order of frequency, by themutations Cd-39, IVS-I-1, IVS-I-6, IVS-II-745,Cd-6, IVS-II-1, Cd-5, IVS-I-6 and 44 bp del.Mutation frequencies in these studies were calcu-lated from the mutations found in patients whilein this study most samples (more than 1000) wereanalyzed on carriers identified in carrier screeningprograms.

Georgiou I, Makis A, Chaidos A, Bouba I, Hatzi E, Kranas V, Zilidis C,Bourantas KL. Distribution and frequency of b-thalassemia mutationsin northwestern and central Greece.Eur J Haematol 2003: 70: 75–78. � Blackwell Munksgaard 2003.

Abstract: Objectives: b-Thalassemia is a common autosomal recessivedisorder resulting from over 200 different mutations of the b-globingenes. The spectrum of b-thalassemia mutations in Greece has beenpreviously described in the population of the capital city of Athens, or inb-thalassemia patients having transfusion therapy. The aim of the pre-sent study was to identify the distribution of the most commonb-thalassemia mutations in the population of northwestern and centralGreece. Methods: The data for this study were derived from a total of1130 unrelated subjects including 46 b-thalassemia major, threeb -thalassemia intermedia and 1081 carriers identified in our antenatalscreening program. b-Thalassemia mutations were identified by ARMS,DGGE and Reverse Dot Blot. Results: The most common mutation,IVS-I-110, is followed, in order of frequency, by the mutations Cd-39,IVS-I-1, IVS-II-1, Cd-6, IVS-I-6, IVS-I-5, IVS-II-745, Cd-5 and 44 bpdel. IVS-I-110 and Cd-39 frequencies are similar with those found inother Balkan countries. Significant differences in regional distributionwere observed. The results showed a clear drift of the distribution of themost frequent IVS-I-110 mutation in the south–north (29.4, 40.0, 44.6and 61.7%) and the east–west axis (31.8 and 44.6%). Conclusions:Population screening and prenatal diagnosis are significantly facilitatedby these data. Furthermore, the detailed distribution tables ofb-thalassemia mutations are essential for counseling and extraction ofgenetic diversity estimates for population genetic studies in otherinherited disorders.

I. Georgiou1, A. Makis2, A. Chaidos2,I. Bouba1, E. Hatzi1, V. Kranas1,C. Zilidis2, K. L. Bourantas21Genetics Unit, Department of Obstetrics andGynecology, 2Hematology Unit, Department of InternalMedicine, Medical School, University of Ioannina,Ioannina, Greece

Key words: thalassemia; b-globin mutations; Greece

Correspondence: K. L. Bourantas, MD, Department ofInternal Medicine-Haematology, P.O. Box 37,GR-45 002 Ioannina, GreeceE-mail: kbouran@cc.uoi.gr

Accepted for publication 13 December 2002

Eur J Haematol 2003: 70: 75–78Printed in UK. All rights reserved

Copyright � Blackwell Munksgaard 2003

EUROPEANJOURNAL OF HAEMATOLOGY

ISSN 0902-4441

75

The aims of the present study were to: identifythe detailed distribution of the most commonb-thalassemia mutations in the population ofnorthwestern and central Greece and SouthernAlbania (Fig. 1); and construct detailed frequencymaps in order to perform effective prenatal diag-nosis in the region and extract accurate riskestimates.

Patients and methods

Patients

The data for this study were derived from a total of1130 unrelated subjects and of those 1081 individ-uals were identified as carriers by red cell indices,Hb electrophoresis and HbA2 levels. One hundredand twenty-four mutation analyses were donein couples who requested prenatal diagnosis forb-thalassemia and hemoglobinopathies. Our studyalso included 46 b-thalassemia major patients andthree b-thalassemia intermedia.Each region was represented in the population

study origin as follows: Ioannina 32.9%, Arta21.0%, Preveza 15.7%, Thesprotia 9.8%, Corfu8.2%, Thessaly 7.5%, Albanian patients 4.9%(Fig. 1). This representation is approximatelyanalogous to the actual population size in the four

regions of Epirus while the population of Corfuand Thessaly was sampled in the major publichospitals of the capital cities.

Methods

Blood samples were collected in ethylenediamine-tetraacetic acid-containing tubes. For prenataldiagnosis, chorionic villus sampling at 10–13 wkof gestation was used for the identification ofmutations (8). In all instances, genomic DNA wasisolated from white blood cells by the standard saltextraction method. The samples were tested for the10 most common b-thalassemia mutations des-cribed in other Greek populations (4–6) by thefollowing methods: amplification refractory muta-tion system (ARMS) (9), denaturating gradient gelelectrophoresis (DGGE) (10), and reverse dot blot(Strip A� VeinnaLab, Vienna, Austria). The resultsof prenatal diagnosis were verified in the cord bloodat birth. Although the study was designed to lookfor the 10 most common mutations in Greece, atotal of 22 mutations was included in Strip A andall the abnormal DGGE patterns were sequenced.This approach did not uncover any unknownmutations and only the rare bd-Sicilian, bd-Corfuand Hb Lepore were found in less than 5% ofcarriers from Epirus and Corfu.

Ioannina 32.9%Thesprotia 9.8%Arta 21%Preveza 15.7%Corfu 8.2%Thessaly 7.5%Albanian patients 4.9%

Fig. 1. Map of Greece: regional distribution of the population study.

Georgiou et al.

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Results

The prevalence of b-thalassemia mutations amongdifferent regions in northwestern and centralGreece is shown in Table 1.The most common mutation is IVS-I-110 and isfollowed, in order of frequency, by the mutationsCd-39, IVS-I-1, IVS-II-1, Cd-6, IVS-I-6, IVS-I-5,IVS-II-745, Cd-5 and 44 bp del. Five of thesemutations account for 91.1% of the chromosomes.A significantly higher frequency (9.1 vs. 2.0%) ofthe IVS-II-1 mutation was found in the generalpopulation of central Greece. In particularly, theneighboring regions of Arta and Preveza had higherpercentages of 15.7 and 13.0%, respectively.The distribution of mutations was differentamong the four regions of Epirus (Ioannina, Arta,Preveza and Thesprotia). Significant differences inthe frequency and distribution were also observedin the neighboring regions of Corfu, Thesally andAlbania. The results show a clear drift of thedistribution of the most common mutation, IVS-I-110, in the south–north axis (29.4, 40.0, 44.6and 61.7%) and the east–west axis (31.8 and44.6%).

Discussion

In the past two decades, the molecular pathologyand the mutation–phenotype correlation ofb-thalassemia have been extendedly elucidated.The b-globin-like genes are arranged in a clusteron the short arm of chromosome 11 over approxi-mately 60 000 nucleotide bases (11). Most of themutations affecting the b-globin gene are pointmutations or oligonucleotide additions or deletions(12). To date, nearly 200 different mutations of theb-globin gene have been described (3).In spite of this marked heterogeneity, everypopulation in the world with high-frequencyb-thalassemia seems to carry a few common muta-tions that are unique to a particular region,together with a number of rare ones. This know-ledge has been applied to molecular diagnosis,carrier screening, prenatal diagnosis, genetic coun-seling, and gene therapy. In our study, we con-firmed the universal b-thalassemia mutationscharacteristic in northwestern and central Greece.IVS-I-110 is the most common mutation and isfollowed, in order of frequency, by Cd-39, IVS-I-1,IVS-II-1, Cd-6, IVS-I-6, IVS-I-5, IVS-II-745, Cd-5and 44 bp del. Five of these mutations account for91.1% of the chromosomes. These data are inaccordance with previous studies in Greek patients(4). The frequencies of the most common mutationsIVS-I-110 and Cd39 are similar with those foundin the Balkan peninsula (13). Differences are

Table1.

Frequencyandregionaldistributionof

b-thalassemiamutationsinnorthwesternandcentralGreece

Mutation

NWIoannina

SWArta

SWPreveza

NWThesprotia

NEThessaly

NWCorfu

NWSouthern

Albania

Total

Overall

distribution

inGreece1

(%)

n%

n%

n%

n%

n%

n%

n%

n%

IVS-I-110

173

44.6

7329.4

7440.0

7161.7

2831.8

4445.4

3458.6

497

42.2

42.5

Cd-39

6115.7

6325.4

4122.2

4034.8

2123.9

3233.0

1627.6

274

23.2

16.9

IVS-I-1

5213.4

3112.5

105.4

10.9

1011.4

99.3

113

9.6

13.2

IVS-II-1

307.7

3915.7

2413.0

89.1

33.1

104

8.8

2Cd-6

297.5

2510.1

105.4

33.4

675.7

2.9

IVS-I-6

102.6

72.8

147.6

21.7

33.4

22.1

46.9

423.6

7.2

IVS-I-5

20.5

20.8

10.5

10.9

33.4

11.0

100.8

0.9

IVS-II-745

123.1

20.8

21.1

89.1

55.2

292.5

6.9

Cd-5

51.3

20.8

94.9

161.4

1.12

44bpdel

20.5

11.1

35.2

60.5

0.6

HbLepore

10.3

20.8

11.1

40.3

db-Sicilian

112.8

20.8

22.3

11.7

161.4

db-Corfu

11.0

10.1

Total

388

248

185

115

8897

581179

n,numberofchormosomes.

1From

ref.(4).

b-Thalassemia mutations in Greece

77

significant in the frequency of Cd39, which is highin Italy (more than 40%) and low in Turkey (lessthan 5%) (14). In Greece, and especially in ourregion, the frequency is between the Italian and theTurkish values (approximately 20%). Likewise, thepercentage of IVS-I-110 in Italy is low (approxi-mately 11%) while in Greece and other Balkancountries, Cyprus and Turkey, it is approximately40% and higher (14). IVS-I-1 also seems to have ahigher overall distribution in Greece (13%) and incentral Greece (10% in this study), while in Italyand Turkey it is approximately 5% or less. In theother Balkan countries, the percentages seem tovary at 10% with the exception of Albania (lessthan 3%) (13). Difference in the frequencies of theless common mutations is difficult to extrapolatebecause of the low mean percentages.When the percentage of the 10 most common

b-thalassemia mutations in the population ofnorthwestern and central Greece is compared withthe overall frequency of mutations in the country(4), some interesting differences are revealed.Increased percentage of the mutations Cd-39,Cd-6, IVS-II-1 and decreased percentage of IVS-II-745 and IVS-I-6 were observed compared to thegeneral population. The highest frequencies ofCd-39 were found in Thesprotia and Corfu, ofCd-6 in Arta and Ioannina, and IVS-II-1 in Artaand Preveza. The IVS-II-745 was absent in thesamples from Thesprotia and Albania and IVS-I-6was very low in Ioannina and Corfu.In this study, IVS-I-110 was the most frequent

mutation in all the regions of northwestern andcentral Greece. However, notable differences inregional distribution of this mutation are observed,showing a clear drift in the south–north and east–west axes.The identification of the spectrum of b-thalasse-

mia mutations in our region is necessary forpopulation carrier screening and for accurate pre-natal diagnosis. Furthermore, the detailed distribu-tion tables of b-thalassemia mutations are essentialfor counseling and extraction of genetic diversityestimates for similar studies in other inheriteddisorders.

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