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The molecular heterogeneity of β-thalassemia in Greece
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Blood Cells, Molecules, and Diseases 40 (2008) 317 – 319 www.elsevier.com/locate/ybcmd
The molecular heterogeneity of β-thalassemia in Greece Marina Boussiou ⁎, Photini Karababa, Klio Sinopoulou, Panagiotis Tsaftaridis, Eleni Plata, Aphrodite Loutradi-Anagnostou National Centre for Thalassemia, WHO Collaborating Centre for the Community Control of Hereditary Diseases, “Laikon” General Hospital, KCS, GRE-21, Greece Submitted 4 September 2007; revised 28 October 2007 Available online 21 December 2007 (Communicated by G. Stamatoyannopoulos, M.D., Dr. Sci., 9 November 2007)
Abstract β-thalassemia is the most predominant genetic defect in Greece. In this study, we investigated the heterogeneity and the frequency of β-thalassemia mutations among 3796 heterozygotes detected in the course of DNA based diagnoses. The diagnostic strategy included Denaturing Gradient Gel Electrophoresis (DGGE), Allele Specific Oligonucleotide Hybridization (ASO), GAP PCR, Restriction Enzyme (RE) analysis and direct sequencing and led to 100% identification of the underlying molecular lesion. Six out of 33 different β-globin defects identified accounted for more than 91.4% of the total β-thalassemia chromosomes in Greece. The β-globin gene mutations cd29 C→T, IVS-I-2 T→C, IVS-I-5 G→T, cd37 G→A and poly A Kurdish AATAAA→AATAAG are for the first time reported in Greece, whereas cd7 GAG→TAG is a new β0-thalassemia mutation detected in an adult man from Albania residing in Greece. Three DNA single nucleotide polymorphisms (IVS-I-85 T→C, IVS-I-91 C→T and IVS-I-108 T→C) were also revealed; among these, IVS-I-85 T→C and IVS-I-91 C→T are new and described for the first time worldwide. © 2007 Elsevier Inc. All rights reserved. Keywords: β-thalassemia; β-globin gene mutations
Introduction The hemoglobinopathies are genetic disorders of hemoglobin that result either from structural hemoglobin variants or by defects in the synthesis of one or more globin chains, so called thalassemias [1]. Greece is a hot spot of thalassemia with a mean prevalence of thalassemia heterozygotes of approximately 8% [2]. The thalassemias are heterogeneous and several types of mutations such as β-, δβ- and α-thalassemia have been detected. Their distribution in Greece is heterogeneous. Some thalas-
Abbreviations: Hb, hemoglobin; SNP, single nucleotide polymorphism; DGGE, denaturing gradient gel electrophoresis; RE, restriction enzyme; ASO, allele specific oligonucleotides; CVS, chorionic villi sampling. ⁎ Corresponding author. National Thalassemia Centre, Prenatal diagnosis, “Laikon” General Hospital of Athens, Greece, 16 Sevastoupoleos str. 11526 Athens, Greece. Fax: +30 210 7789476. E-mail address: [email protected] (M. Boussiou). 1079-9796/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2007.11.003
semic mutants have been introduced in Greece recently by the influx of immigrants mainly from the North West (Albania), but also by East (Kurds, Iraq, Iran, Pakistan and Philippines) and South (Egypt). The high frequencies of thalassemia in Greece have a major impact on public health. A National Program for Prevention of Thalassemia introduced in 1973 aimed at preventing the accumulation of new cases through genetic counseling and prenatal diagnosis and in addition at providing optimal care to the patients under the auspices of this program. Services are provided by a Central Unit in Athens, by 21 Units attached to hospitals of Athens and other towns in areas with high frequencies and a Unit for Prenatal Diagnosis. Identification of β-thalassemia mutations in Greece has been reported before by Kattamis et al. [3] on 348 chromosomes of patients and by our Centre [4] on 744 chromosomes from both carriers and patients. Data in the present study were collected by molecular analysis of 3796 adult heterozygotes that came for prenatal diagnosis at our Centre. The results showed that along
318
M. Boussiou et al. / Blood Cells, Molecules, and Diseases 40 (2008) 317–319
with a few prevalent defects, we have also identified a large number of rare alleles. Materials and methods We studied the β-globin genotype from DNA samples of couples “at risk” who were referred to our Centre from all over Greece for genetic counseling and/or prenatal diagnosis. In total, we have studied 4489 chromosomes. Hematological and biochemical data were determined by standard techniques. DNA was extracted from the buffy coat using salt precipitation [5]. The β-globin gene was initially scanned by Denaturing Gradient Gel Electrophoresis (DGGE) [6,7] to localize the nucleotide changes. These were subsequently identified by Allele Specific Oligonucleotide hybridization (ASO) normal [8] and reverse [9] with the BioRad mDx Betha commercial kit, GAP PCR and restriction enzyme analysis or by direct sequencing [10] in the case of new mutations. Results The diagnostic strategy that followed led to the identification of the underlying molecular defect in all cases studied. The most common molecular defect among β-thalassemia carriers are single point mutations (3796 chromosomes, 84.6%). Thirtythree different β-globin gene mutations were identified, leading to both β0 and β+ phenotypes. The distribution of the 11 most prevalent β-globin gene mutations (frequency N0.5%) is listed in Table 1. According to these results, IVS-1-110 G→A is present in 42.1% of carriers and together with CD39 C→T, IVS-I-1 G→A, IVS-I-6 T→C, IVS-II-745 C→G and IVS-II-1 G→A, they account for 91.4% of the thalassemia genes. Twenty-two other single point thalassemia mutations or small deletions are very rare (Table 2). Large deletions are responsible for δβ-thalassemia of both Sicilian and Macedonian or Turkish type (frequency 1.4% among the heterozygote parents). Three DNA single nucleotide substitutions were also revealed in the first intervening sequence (IVS-I) of the β-globin Table 1 Distribution and relative frequencies of the common and rare β-thalassemia mutations in Greece Mutation
Phenotype
Case number
Frequency %
IVS-I-110 G→A CD39 C→T IVS-I-1 G→A IVS-I-6 T→C IVS-II-745 C→G IVS-II-1 G→A CD6 − A − 101 C→T − 87 C→G CD5 − CT CD8 − AA Rare mutations Total
β+ β0 β0 β+ β+ β0 β0 β+ β+ β0 β0
1599 714 487 307 239 125 63 60 40 31 30 101 3796
42.1 18.8 12.8 8.1 6.3 3.3 1.7 1.6 1.0 0.8 0.8 2.7 100
Table 2 Rare β-thalassemia mutations in Greece Mutation
Phenotype
No. of cases
−90 C→T −30 T→A −28 A→C CD7 GAG→TAG CD8/9 +G CD15 TGG→TAG CD29 C→T CD30 AGG→ACG IVS-I-2 T→C IVS-I-5 G→A IVS-I-5 G→T IVS-I-116 T→G IVS-I-130 G→C DEL IVSI − 25 bp DEL − 44 bp CD37 TGG→TGA CD44 − C IVS-II-848 C→A +1480 C→G +1570 T→C PolyA AATAAA→AATGAA polyA Kurdish AATAAA→AATAAG Total
β+ β+ β+ β0 β0 β0 β+ β0 β0 β+ β+ β0 β0 β0 β0 β0 β0 β+ β+ β+ β+ β+
1 1 1 1 4 1 2 10 2 14 1 10 3 1 10 1 8 10 4 7 8 1 101
gene further indicating heterogeneity; these include IVS-I-85 T to C, IVS-I-91 C to T and IVS-I-108 T to C. Discussion β-thalassemia is the most common genetic defect in Greece as in many Mediterranean and Balkan countries. Previous reports [3,4] have investigated the prevalence and distribution of the different β- thalassemia mutations in the Greek population based on a fairly small number of heterozygotes and patients; these reports showed that Greece shared the same set of thalassemia mutations as the other Mediterranean countries and also revealed the molecular heterogeneity as reflected by the presence of a few frequent mutations and a large number of rare defects. In the present study, we described a wider spectrum of β-globin gene defects by molecular analysis of DNA from 3796 thalassemic heterozygotes. The majority were Greeks whose origin represented the whole country. Results reflect the current changes in the population synthesis in which 10% is comprised by immigrants. Most of them are Albanian (∼ 58%) who has settled down in Greece the last decade [11]. The diagnostic methodology that followed included initial scanning of the β-globin gene by DGGE analysis in order to localize the nucleotide change. Known electrophoretic patterns were subsequently identified directly by ASO hybridization, RE digestion or GAP PCR. Unknown patterns were further investigated by direct sequencing and in every case the mutation was confirmed by ASO hybridization. The above strategy proved a rapid and reliable approach to the characterization and identification of the underlying molecular defects leading to 100% molecular diagnosis.
M. Boussiou et al. / Blood Cells, Molecules, and Diseases 40 (2008) 317–319
Thirty-three different β-thalassemia mutations were identified. The order and the frequency of the most prevalent are the same as in the previous reports [3,4]. Among the 22 rare mutations detected in the present study, six had never been described before in Greece and one of them is a new mutation; these mutations were CD7 (G to T), cd29 C→T (two samples), IVS-I-2 T→C (two samples), IVS-I-5 G→T, cd37 G→A and poly A Kurdish AATAAA→AATAAG (Table 2). It should be noted that IVS-I-5 (G to T) and CD29 (C to T) were found in carriers of Greek origin, the polyA Kurdish type (AATAAA to AATAAG) in a Jewish individual from N. Greece, whereas IVS-I2 (T to C), CD37 (G to A) and CD7 (G to T) in immigrants from Albania. The substitution CD7 G to T is a new mutation that causes β0-thalassemia by changing codon 7 GAG (Glu) to a termination codon TAG. It was found in an adult male from Albania. The new allele presented an unknown pattern at DGGE analysis of the first exon of the β-globin gene. Identification involved direct sequencing and ASO hybridization for final confirmation. Genetic heterogeneity is further confirmed by the presence of three single nucleotide polymorphisms which were identified in the first intervening sequence (IVS-I) of the β-globin gene at nucleotides 85, 91 and 108, respectively. The SNPs lied on the non-thalassemic chromosome as revealed by family studies. Carriers of these polymorphisms were all of Greek origin. IVSI-108 (T to C) was first reported in a person of Iranian origin [12]. The current study is the first report worldwide for IVS-I-85 T→C and IVS-I-91 C→T. Acknowledgments The present study is a long collaborative effort that involved many colleagues and technical staff who are not cited by name. The authors acknowledge with thanks the valuable contribution of the Staff of the National Thalassemia Centre; the Units of
319
Prevention all over Greece; and the collaborating obstetricians and other colleagues. References [1] D.J. Weatherall, J.B. Clegg, The Thalassemia Syndromes, 4th ed., Blackwell Science, Oxford, 2001. [2] C. Tegos, A. Voutsadakis, N. Paleologou, et al., The incidence and distribution of thalassemias in Greece (a study on 64.814 recruits), Hell. Armed Forces Med. Rev. 21 (1987) 27–36. [3] C. Kattamis, H. Hu, G. Cheng, et al., Molecular characterization of β-thalassemia in 174 Greek patients with thalassemia major, Br. J. Haematol. 74 (1990) 342–346. [4] P. Kollia, Ph. Karababa, K. Sinopoulou, et al., b-Thalassemia mutations and the underlying b gene cluster haplotypes in the Greek population, Gene Geogr. 6 (1992) 59–70. [5] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic Acids Res. 16 (3) (1988) 1215. [6] N. Ghanem, E. Girodon, M. Vidaud, et al., A comprehensive scanning method for rapid detection of β-globin gene mutations and polymorphisms, Human Mutat. 1 (1992) 229–239. [7] R. Fodde, M. Losekoot, Mutation detection by denaturing gradient gel electrophoresis (DGGE), Human Mutat. 3 (1994) 83–94. [8] R.K. Saiki, D.H. Gelfand, S. Stoffel, et al., Primer directed enzymatic amplification of DNA with a thermo stable DNA polymerase, Science 239 (1988) 487–491. [9] L.A. Ugozzoli, J.D. Lowery, A.A. Reyes, et al., Evaluation of the Be Tha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations, Am. J. Hematol. 59 (3) (1998) 214–222. [10] D.R. Engelke, P.A. Hoener, F.S. Collins, Direct sequencing of enzymatically amplified human genomic DNA, Proc. Natl. Acad. Sci. 85 (1988) 544–548. [11] Results of the Population Census 2001. General Secretariat of National Statistical Services in Greece. [12] C. Badens, N. Jassim, N. Martini, et al., Characterization of a new polymorphism, IVS-I-108 (T→C), and a new beta-thalassemia mutation, −27 (A→T), discovered in the course of a prenatal diagnosis, Hemoglobin 23 (4) (1999) 339–344.
Blood Cells, Molecules, and Diseases 40 (2008) 317 – 319 www.elsevier.com/locate/ybcmd
The molecular heterogeneity of β-thalassemia in Greece Marina Boussiou ⁎, Photini Karababa, Klio Sinopoulou, Panagiotis Tsaftaridis, Eleni Plata, Aphrodite Loutradi-Anagnostou National Centre for Thalassemia, WHO Collaborating Centre for the Community Control of Hereditary Diseases, “Laikon” General Hospital, KCS, GRE-21, Greece Submitted 4 September 2007; revised 28 October 2007 Available online 21 December 2007 (Communicated by G. Stamatoyannopoulos, M.D., Dr. Sci., 9 November 2007)
Abstract β-thalassemia is the most predominant genetic defect in Greece. In this study, we investigated the heterogeneity and the frequency of β-thalassemia mutations among 3796 heterozygotes detected in the course of DNA based diagnoses. The diagnostic strategy included Denaturing Gradient Gel Electrophoresis (DGGE), Allele Specific Oligonucleotide Hybridization (ASO), GAP PCR, Restriction Enzyme (RE) analysis and direct sequencing and led to 100% identification of the underlying molecular lesion. Six out of 33 different β-globin defects identified accounted for more than 91.4% of the total β-thalassemia chromosomes in Greece. The β-globin gene mutations cd29 C→T, IVS-I-2 T→C, IVS-I-5 G→T, cd37 G→A and poly A Kurdish AATAAA→AATAAG are for the first time reported in Greece, whereas cd7 GAG→TAG is a new β0-thalassemia mutation detected in an adult man from Albania residing in Greece. Three DNA single nucleotide polymorphisms (IVS-I-85 T→C, IVS-I-91 C→T and IVS-I-108 T→C) were also revealed; among these, IVS-I-85 T→C and IVS-I-91 C→T are new and described for the first time worldwide. © 2007 Elsevier Inc. All rights reserved. Keywords: β-thalassemia; β-globin gene mutations
Introduction The hemoglobinopathies are genetic disorders of hemoglobin that result either from structural hemoglobin variants or by defects in the synthesis of one or more globin chains, so called thalassemias [1]. Greece is a hot spot of thalassemia with a mean prevalence of thalassemia heterozygotes of approximately 8% [2]. The thalassemias are heterogeneous and several types of mutations such as β-, δβ- and α-thalassemia have been detected. Their distribution in Greece is heterogeneous. Some thalas-
Abbreviations: Hb, hemoglobin; SNP, single nucleotide polymorphism; DGGE, denaturing gradient gel electrophoresis; RE, restriction enzyme; ASO, allele specific oligonucleotides; CVS, chorionic villi sampling. ⁎ Corresponding author. National Thalassemia Centre, Prenatal diagnosis, “Laikon” General Hospital of Athens, Greece, 16 Sevastoupoleos str. 11526 Athens, Greece. Fax: +30 210 7789476. E-mail address: [email protected] (M. Boussiou). 1079-9796/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2007.11.003
semic mutants have been introduced in Greece recently by the influx of immigrants mainly from the North West (Albania), but also by East (Kurds, Iraq, Iran, Pakistan and Philippines) and South (Egypt). The high frequencies of thalassemia in Greece have a major impact on public health. A National Program for Prevention of Thalassemia introduced in 1973 aimed at preventing the accumulation of new cases through genetic counseling and prenatal diagnosis and in addition at providing optimal care to the patients under the auspices of this program. Services are provided by a Central Unit in Athens, by 21 Units attached to hospitals of Athens and other towns in areas with high frequencies and a Unit for Prenatal Diagnosis. Identification of β-thalassemia mutations in Greece has been reported before by Kattamis et al. [3] on 348 chromosomes of patients and by our Centre [4] on 744 chromosomes from both carriers and patients. Data in the present study were collected by molecular analysis of 3796 adult heterozygotes that came for prenatal diagnosis at our Centre. The results showed that along
318
M. Boussiou et al. / Blood Cells, Molecules, and Diseases 40 (2008) 317–319
with a few prevalent defects, we have also identified a large number of rare alleles. Materials and methods We studied the β-globin genotype from DNA samples of couples “at risk” who were referred to our Centre from all over Greece for genetic counseling and/or prenatal diagnosis. In total, we have studied 4489 chromosomes. Hematological and biochemical data were determined by standard techniques. DNA was extracted from the buffy coat using salt precipitation [5]. The β-globin gene was initially scanned by Denaturing Gradient Gel Electrophoresis (DGGE) [6,7] to localize the nucleotide changes. These were subsequently identified by Allele Specific Oligonucleotide hybridization (ASO) normal [8] and reverse [9] with the BioRad mDx Betha commercial kit, GAP PCR and restriction enzyme analysis or by direct sequencing [10] in the case of new mutations. Results The diagnostic strategy that followed led to the identification of the underlying molecular defect in all cases studied. The most common molecular defect among β-thalassemia carriers are single point mutations (3796 chromosomes, 84.6%). Thirtythree different β-globin gene mutations were identified, leading to both β0 and β+ phenotypes. The distribution of the 11 most prevalent β-globin gene mutations (frequency N0.5%) is listed in Table 1. According to these results, IVS-1-110 G→A is present in 42.1% of carriers and together with CD39 C→T, IVS-I-1 G→A, IVS-I-6 T→C, IVS-II-745 C→G and IVS-II-1 G→A, they account for 91.4% of the thalassemia genes. Twenty-two other single point thalassemia mutations or small deletions are very rare (Table 2). Large deletions are responsible for δβ-thalassemia of both Sicilian and Macedonian or Turkish type (frequency 1.4% among the heterozygote parents). Three DNA single nucleotide substitutions were also revealed in the first intervening sequence (IVS-I) of the β-globin Table 1 Distribution and relative frequencies of the common and rare β-thalassemia mutations in Greece Mutation
Phenotype
Case number
Frequency %
IVS-I-110 G→A CD39 C→T IVS-I-1 G→A IVS-I-6 T→C IVS-II-745 C→G IVS-II-1 G→A CD6 − A − 101 C→T − 87 C→G CD5 − CT CD8 − AA Rare mutations Total
β+ β0 β0 β+ β+ β0 β0 β+ β+ β0 β0
1599 714 487 307 239 125 63 60 40 31 30 101 3796
42.1 18.8 12.8 8.1 6.3 3.3 1.7 1.6 1.0 0.8 0.8 2.7 100
Table 2 Rare β-thalassemia mutations in Greece Mutation
Phenotype
No. of cases
−90 C→T −30 T→A −28 A→C CD7 GAG→TAG CD8/9 +G CD15 TGG→TAG CD29 C→T CD30 AGG→ACG IVS-I-2 T→C IVS-I-5 G→A IVS-I-5 G→T IVS-I-116 T→G IVS-I-130 G→C DEL IVSI − 25 bp DEL − 44 bp CD37 TGG→TGA CD44 − C IVS-II-848 C→A +1480 C→G +1570 T→C PolyA AATAAA→AATGAA polyA Kurdish AATAAA→AATAAG Total
β+ β+ β+ β0 β0 β0 β+ β0 β0 β+ β+ β0 β0 β0 β0 β0 β0 β+ β+ β+ β+ β+
1 1 1 1 4 1 2 10 2 14 1 10 3 1 10 1 8 10 4 7 8 1 101
gene further indicating heterogeneity; these include IVS-I-85 T to C, IVS-I-91 C to T and IVS-I-108 T to C. Discussion β-thalassemia is the most common genetic defect in Greece as in many Mediterranean and Balkan countries. Previous reports [3,4] have investigated the prevalence and distribution of the different β- thalassemia mutations in the Greek population based on a fairly small number of heterozygotes and patients; these reports showed that Greece shared the same set of thalassemia mutations as the other Mediterranean countries and also revealed the molecular heterogeneity as reflected by the presence of a few frequent mutations and a large number of rare defects. In the present study, we described a wider spectrum of β-globin gene defects by molecular analysis of DNA from 3796 thalassemic heterozygotes. The majority were Greeks whose origin represented the whole country. Results reflect the current changes in the population synthesis in which 10% is comprised by immigrants. Most of them are Albanian (∼ 58%) who has settled down in Greece the last decade [11]. The diagnostic methodology that followed included initial scanning of the β-globin gene by DGGE analysis in order to localize the nucleotide change. Known electrophoretic patterns were subsequently identified directly by ASO hybridization, RE digestion or GAP PCR. Unknown patterns were further investigated by direct sequencing and in every case the mutation was confirmed by ASO hybridization. The above strategy proved a rapid and reliable approach to the characterization and identification of the underlying molecular defects leading to 100% molecular diagnosis.
M. Boussiou et al. / Blood Cells, Molecules, and Diseases 40 (2008) 317–319
Thirty-three different β-thalassemia mutations were identified. The order and the frequency of the most prevalent are the same as in the previous reports [3,4]. Among the 22 rare mutations detected in the present study, six had never been described before in Greece and one of them is a new mutation; these mutations were CD7 (G to T), cd29 C→T (two samples), IVS-I-2 T→C (two samples), IVS-I-5 G→T, cd37 G→A and poly A Kurdish AATAAA→AATAAG (Table 2). It should be noted that IVS-I-5 (G to T) and CD29 (C to T) were found in carriers of Greek origin, the polyA Kurdish type (AATAAA to AATAAG) in a Jewish individual from N. Greece, whereas IVS-I2 (T to C), CD37 (G to A) and CD7 (G to T) in immigrants from Albania. The substitution CD7 G to T is a new mutation that causes β0-thalassemia by changing codon 7 GAG (Glu) to a termination codon TAG. It was found in an adult male from Albania. The new allele presented an unknown pattern at DGGE analysis of the first exon of the β-globin gene. Identification involved direct sequencing and ASO hybridization for final confirmation. Genetic heterogeneity is further confirmed by the presence of three single nucleotide polymorphisms which were identified in the first intervening sequence (IVS-I) of the β-globin gene at nucleotides 85, 91 and 108, respectively. The SNPs lied on the non-thalassemic chromosome as revealed by family studies. Carriers of these polymorphisms were all of Greek origin. IVSI-108 (T to C) was first reported in a person of Iranian origin [12]. The current study is the first report worldwide for IVS-I-85 T→C and IVS-I-91 C→T. Acknowledgments The present study is a long collaborative effort that involved many colleagues and technical staff who are not cited by name. The authors acknowledge with thanks the valuable contribution of the Staff of the National Thalassemia Centre; the Units of
319
Prevention all over Greece; and the collaborating obstetricians and other colleagues. References [1] D.J. Weatherall, J.B. Clegg, The Thalassemia Syndromes, 4th ed., Blackwell Science, Oxford, 2001. [2] C. Tegos, A. Voutsadakis, N. Paleologou, et al., The incidence and distribution of thalassemias in Greece (a study on 64.814 recruits), Hell. Armed Forces Med. Rev. 21 (1987) 27–36. [3] C. Kattamis, H. Hu, G. Cheng, et al., Molecular characterization of β-thalassemia in 174 Greek patients with thalassemia major, Br. J. Haematol. 74 (1990) 342–346. [4] P. Kollia, Ph. Karababa, K. Sinopoulou, et al., b-Thalassemia mutations and the underlying b gene cluster haplotypes in the Greek population, Gene Geogr. 6 (1992) 59–70. [5] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic Acids Res. 16 (3) (1988) 1215. [6] N. Ghanem, E. Girodon, M. Vidaud, et al., A comprehensive scanning method for rapid detection of β-globin gene mutations and polymorphisms, Human Mutat. 1 (1992) 229–239. [7] R. Fodde, M. Losekoot, Mutation detection by denaturing gradient gel electrophoresis (DGGE), Human Mutat. 3 (1994) 83–94. [8] R.K. Saiki, D.H. Gelfand, S. Stoffel, et al., Primer directed enzymatic amplification of DNA with a thermo stable DNA polymerase, Science 239 (1988) 487–491. [9] L.A. Ugozzoli, J.D. Lowery, A.A. Reyes, et al., Evaluation of the Be Tha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations, Am. J. Hematol. 59 (3) (1998) 214–222. [10] D.R. Engelke, P.A. Hoener, F.S. Collins, Direct sequencing of enzymatically amplified human genomic DNA, Proc. Natl. Acad. Sci. 85 (1988) 544–548. [11] Results of the Population Census 2001. General Secretariat of National Statistical Services in Greece. [12] C. Badens, N. Jassim, N. Martini, et al., Characterization of a new polymorphism, IVS-I-108 (T→C), and a new beta-thalassemia mutation, −27 (A→T), discovered in the course of a prenatal diagnosis, Hemoglobin 23 (4) (1999) 339–344.