doi: 10.1046/j.1529-8817.2004.00138.x Genetic Heterogeneity of Beta Thalassemia in Reflects Historic and Recent Population Migration

N. J. Makhoul1,R.S.Wells2,H.Kaspar1,3,H.Shbaklo3,A.Taher1,4,N.Chakar1 and P. A. Zalloua1,5∗ 1Department of Obstetrics and Gynecology, American University of , Beirut, Lebanon 2Oxford University, London, UK 3Genetics Research Laboratory, Chronic Care Center, Beirut, Lebanon 4Department of Internal Medicine, American University of Beirut, Beirut, Lebanon 5Program for Population Genetics, Harvard School of Public Health, Boston, USA

Summary

Beta thalassemia is an autosomal recessive disorder characterized by reduced (β+)orabsent (β0) beta-globin chain synthesis. In Lebanon it is the most predominant genetic defect. In this study we investigated the religious and geographic distribution of the β-thalassemia mutations identified in Lebanon, and traced their precise origins. A total of 520 β-globin chromosomes from patients of different religious and regional backgrounds was studied. Beta thalassemia mutations were identified using Amplification Refractory Mutation System (ARMS) PCR or direct gene sequencing. Six (IVS-I-110, IVS-I-1, IVS-I-6, IVS-II-1, cd 5 and the C>T substitution at cd 29) out of 20 β-globin defects identified accounted for more than 86% of the total β-thalassemia chromosomes. Sunni had the highest β-thalassemia carrier rate and presented the greatest heterogeneity, with 16 different mutations. Shiite Muslims followed closely with 13 mutations, whereas represented 11.9% of all β-thalassemic subjects and carried 7 different mutations. RFLP haplotype analysis showed that the observed genetic diversity originated from both new mutational events and gene flow from population migration. This study provides information about the types and distribution of β-thalassemia mutations within each religious group and geographic region, which is essential for the implementation of screening and prevention programs.

Introduction are genetically protected against malaria and therefore selectively advantaged (Haldane, 1949). Beta thalassemia is an autosomal recessive disorder char- Although over 300 different mutations in the β- acterized by reduced (β+)orabsent (β0) beta-globin globin gene have been described (Huisman et al. 1997), chain synthesis (Weatherall & Clegg, 1981). The disor- each population or ethnic group has been found to have der is manifested by severe hemolytic anemia, requiring its own characteristic set of mutations. Identification aregular blood transfusion and iron chelation therapy. of both common and rare β-thalassemia mutations has Beta thalassemia occurs with high frequency in areas proved essential for the implementation of screening and endemic for malaria such as Mediterranean countries, prenatal diagnosis programs. In addition, these muta- Africa and Southeast Asia, where carriers of the disease tions can be used as genetic markers to study the origin and spread of β-thalassemia genes, revealing historical relationships between populations. ∗ Corresponding author: Pierre A. Zalloua, Ph.D. Department In Lebanon, β-thalassemia is the most predominant of Obstetrics and Gynecology, American University of Beirut, genetic defect (Hamamy & Alwan, 1994). Being located PO Box: 113-6044, Beirut, Lebanon. Phone: 961-1-350000 ext in the Eastern Mediterranean Basin, Lebanon is a 4805. Fax: 961-1-370829. E-mail: [email protected] crossroads between Europe, Asia and Africa,

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allowing settlement and intermingling of succes- 30 (G>C), IVS-I-1 (G>A), IVS-I-6 (T>C), IVS-I- sive different racial groups throughout history. 110 (G>A), cd 44 (−C), IVS-II-1 (G>A), IVS-II-745 Consequently, a melting-pot of seventeen religious (C>G)] were identified using the Amplification Re- groups resides in Lebanon, with over three million fractory Mutation System (ARMS) (Bravo et al. 1999; inhabitants, contributing to its particular heterogeneity. Newton et al. 1989), a PCR technique based on allele- There are two major communities: the Muslim specific priming. The 25 bp deletion and 290 bp dele- community, mainly consisting of Shiites, Sunnis and tion were detected by gap PCR (Faa et al. 1992). The , and the Christian community, mainly consisting βS mutation was studied by PCR amplification followed of Maronites, Greek Orthodox, Greek Catholics by digestion with the restriction enzyme Bsu36I. The and Armenians. Intercommunity marriages are rare, remaining mutations were characterized by direct se- whereas consanguineous marriages exceed 25% in quencing of the amplified DNA using an ABI Prism certain groups (Klat & Khudr, 1986), increasing the 310 genetic analyzer. incidence of affected offspring. Beta thalassemia carrier frequency is estimated to be RFLP Haplotype Analysis between 2% and 3% in Lebanon (Cabannes et al. 1965). β-globin gene RFLP haplotypes were determined us- This frequency is highly variable according to religious ing PCR amplification of fragments containing seven groups, the highest being previously reported for the polymorphic restriction sites around and within the ε- Sunnis, followed closely by the Shiites and then by the Gγ -Aγ -β-δ-β globin gene complex. Following di- Maronites (Zahed et al. 1997). gestion with the specific restriction enzymes (HincII 5 In the present report a total of 520 β-globin chromo- to ε, HindIII within Gγ and Aγ , HincII within and 3 somes from patients with different religious and regional to β, AvaII within β and HinfI 3 to β), samples were backgrounds were studied, reflecting a wide spectrum visualized by ethidium bromide staining after agarose of the population. The aims of this study were to inves- or polyacrylamide gel electrophoresis. RFLP haplotypes tigate the religious and geographic distribution of the were named according to Orkin et al. (1982). β-thalassemia mutations identified in Lebanon, and to trace their precise origins, in the hope of revealing some of the historical relationships that exist between the main Phylogenetic Analysis Lebanese communities and their neighbours. The frequencies of β-thalassemia mutations in Lebanese Materials and Methods samples were used to derive a tree of population relation- ships. The programs GENDIST, SEQBOOT, NEIGH- Subjects BOR and CONSENSE of PHYLIP (Felsenstein, 1993) were used, with 100 bootstraps. Some popula- β β We studied 255 -thalassemia and 5 Sickle Cell/ - tions were dropped from the analysis because of the small thalassemia patients receiving treatment at the Chronic number of chromosomes. Care Center in Lebanon, the country’s only professional β unit for the prevention and treatment of -thalassemia. Results The patients included in the study were unrelated and information about their religious and geographic origin Mutational Analysis was noted. Of the total 520 chromosomes investigated for β-globin gene mutations, only one remained unknown, even af- DNA Preparation and Mutation Screening ter β-globin gene sequence analysis. Twenty different DNA was extracted from peripheral blood leucocytes β-globin mutations were identified, leading to both using salt precipitation (Miller et al. 1988). The most β0 (thalassemia major) and β+ (thalassemia intermedia) frequent β-thalassemia mutations [−88 (C>T), −87 phenotypes. The relative frequencies of mutant chro- (C>G), cd 5 (−CT), cd 8 (−AA), cd 29 (C>T), cd mosomes are listed in Table 1. All mutations have been

56 Annals of Human Genetics (2005) 69,55–66 C University College London 2004 Genetic Heterogeneity of β-thalassemia

Table 1 Types and Frequencies of β-Thalassemia Mutations and β-Globin variants in Lebanon

Mutation Phenotype Number of Chromosomes Number of homozygotes/heterozygotes Frequency (%) IVS-I-110 (G>A) β+ 178 65/48 34.2 IVS-I-1 (G>A) βo 78 29/20 15.0 IVS-I-6 (T>C) β+ 75 28/19 14.4 cd 29 (C>T) β+ 50 22/6 9.6 IVS-II-1 (G>A) βo 45 17/11 8.6 cd5(−CT) βo 26 9/8 5.0 cd 30 (G>C) βo 14 6/2 2.7 cd8(−AA) βo 13 5/3 2.5 cd 44 (−C) βo 8 3/2 1.5 IVS-II-745 (C>G) β+ 6 3/0 1.1 βs βs 5 0/5 1.0 −87 (C>G) β+ 4 1/2 0.8 IVS-I-5 (G>C) β+ 4 2/0 0.8 −88 (C>T) β+ 3 1/1 0.6 290 bp deletion βo 3 1/1 0.6 25 bp deletion βo 2 1/0 0.4 δβ-thalassemia (Sicilian type) δβo 2 1/0 0.4 cd 8/9 (+G) βo 1 0/1 0.2 cd 36/37 (−T) βo 1 0/1 0.2 cd 39 (C>T) βo 1 0/1 0.2 Unknown 1 0/1 0.2 Total 520 194/132 100 previously reported in Lebanon (Chehab et al. 1987; Most heterogeneous was the Sunni group with 16 Zahed et al. 1997), except for the frameshift at cd 36/37 different mutations. The Mediterranean IVS-I-110 mu- (−T). Six mutations accounted for 87% of the Lebanese tation was the most common, followed by IVS-I-6 and β-thalassemia chromosomes; these were, in decreasing IVS-II-1. Two mutations seemed to be exclusive to order of frequency, IVS-I-110 (G>A), IVS-I-1 (G>A), this group, the Asian Indian IVS-I-5 (G>C) and the IVS-I-6 (T>C), cd 29 (C>T) and cd 5 (−CT). Mediterranean cd 8 (-AA), and these were not found in any other religious group in our study. Religious Distribution of β-Thalassemia Thirteen different β-globin mutations were present Mutations in Shiite subjects. The cd 30 (G>C) mutation was ex- The frequency and distribution of β-thalassemia mu- clusive to the Shiite group and all but two of the cd 29 tations by the various religious groups in Lebanon are mutations were found in this group. presented in Figures 1 and 2. Four hundred and eighteen For the Druze, a closed religious group, six muta- (80%) of the mutant chromosomes were found in Mus- tions were found in 12 β-thalassemia chromosomes, of lim subjects: Shiites and Sunnis were predominant and which one was apparently restricted: the rare β+ pro- represented 42% and 36% of all thalassemic patients re- moter mutation C>Tatposition −88. spectively, whereas the Druze accounted only for 2.3%. No major particularities were found among the While the Shiites were proportional in number to their Christian groups except for the IVS-I-6 mutation, representation in the Lebanese population, the Sunni which represented the most common mutation in the thalassemics were over-represented as they account for Greek Orthodox patients, but was detected in only 27% of the total population (census taken in 1985), and 5 chromosomes in the Maronites. In addition, Christians thus were considered to have the highest frequency of were found to be relatively more homogeneous than β-thalassemia in Lebanon. The Christians, with a total Muslims in their β-globin mutation distribution; the of 102 mutant chromosomes (20%), were represented Maronites, being the most heterogeneous group among mostly by the Maronites followed by Greek Orthodox. Christians, had 7 different β-globin mutations.

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Figure 1 Distribution of β-thalassemia patients by different religious groups. β-Thalassemia was studied in 260 patients. Most of these patients belong to the Shiite (41.9%) and Sunni (36.2%) religious groups, followed by the Maronite (11.9%), the Orthodox (5%), the Catholic and Druze with equal proportions (2,3%), and finally the Latin (0.4%).

Figure 2 Distribution of the most common β-thalassemia mutations within different religious groups. The diagram represents the most common mutations found in the 520 chromosomes studied. Some mutations are common to several groups, whereas others are specific to only one group.

β-Globin RFLP Haplotype Analysis graphic locations from each group. The association of β-globin RFLP haplotypes with the different muta- To determine the origin of mutant chromosomes within tions identified is presented in Table 2. IVS-I-110 and each religious group, RFLP haplotype analysis was per- IVS-I-1, found in all religious groups, occurred on the formed on a number of patients from different geo- same RFLP haplotype background: I and V respectively.

58 Annals of Human Genetics (2005) 69,55–66 C University College London 2004 Genetic Heterogeneity of β-thalassemia

Table 2 Association of β-Thalassemia Mutations with β-Globin Haplotypes within the Different Religious Groups

Haplotype Muslims Christians Mutation Sunnis Shiites Druze Maronites Orthodox Catholics

IVS-I-110 I I I I I I IVS-I-6 VI - VII - I VI - VII VI VI VI VI - I IVS-I-1 V V V V V V cd 29 I II - I IVS-II-1 III III III cd 5 V V V V cd 8 IV -VII cd 30 IX cd 44 I - IX IX IVS-II-745 VII VII −87 V V V −88 V IVS-I-5 VII 290bp del VI 25bp del IX cd 8/9 I cd 36/37 I cd 39 II

IVS-I-6, also found in all religious groups, occurred on within these 5 main geographic regions (Bekaa, South three different RFLP haplotype backgrounds: VI, VII Lebanon, Beirut, North Lebanon and ). and I, with frequencies of 88.2%, 7.8% and 3.9% respec- The frequency of each mutation was remarkably altered tively. IVS-I-6 haplotype VII was only found in Sunni within each geographic area. The IVS-I-110 mutation, and Shiite subjects, whereas IVS-I-6 haplotype I was which alone accounted for more than half of the mutant only found in Sunnis and Greek Catholics. Three addi- alleles in the South, was also the most frequent in the tional mutations were found to be linked to more than North and Mount Lebanon. In Beirut, IVS-II-1 was one RFLP haplotype: cd 29 was associated with RFLP the most prevalent, and in the Bekaa area the predomi- haplotypes I (77.3%) and II (22.7%), cd 8 with RFLP nant mutation was cd 29, with significantly higher fre- haplotypes IV (88.9%) and VII (11.1%), and finally cd quencies than in any other region in the country. The 44 with RFLP haplotypes IX (75%) and I (25%). South displayed the most heterogeneity but presented aregion-specific highly prevalent mutation, the cd Geographic Distribution of β-Thalassemia 30 mutation (14 chromosomes). Three rare mutations Mutations also seemed to be regionally restricted; these were the 290 bp deletion and the IVS-I-5 mutation within Although all religious groups reside in all parts of the Beirut, and the C>T substitution at position −88 country, each tends to cluster in a specific geographic re- within Mount Lebanon. gion where they may have initially settled centuries ago: Shiites aggregate in the Bekaa and the South; Sunnis in Phylogenetic Analysis Beirut and the North; Druze and Maronites in Mount Lebanon; and Greek Orthodox individuals are mainly A population tree (Figure 4) shows the clustering of concentrated in the North. Figure 3 displays the relative populations based on their mutation frequencies. The frequencies and distribution of β-thalassemia mutations two populations of Greek Christians, Catholic and

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Figure 3 Distribution of the most common β-globin mutations within different lebanese geographic regions. N represents the total number of chromosomes studied from each geographic region.

Orthodox, cluster together, as do the Sunni and Shi- ties and geographic regions; however, this report showed ite Muslims. This reflects their similar β-thalassemia no notable religious and/or regional specificities, and mutation frequencies. The Maronite and Druze pop- was based on the analysis of a fairly small number of ulations are not significantly associated with either of subjects. these two clusters, or with each other. In the case of the In the present study we described a wider spectrum of Druze, with only 12 sampled chromosomes, this could β-thalassemia mutations in Lebanon by analyzing 520 be due to sampling error. The Maronite sample, with β-globin chromosomes, and defined a scale of these mu- amuch larger number of chromosomes, is more likely tations according to religion and geography. Twenty dif- to represent the actual frequency of mutations in this ferent β-thalassemia mutations were identified, of which population. the frameshift at cd 36/37 (−T) had never been re- ported before in Lebanon. In addition, four previously described (Chehab et al. 1987; Waye et al. 1994; Zahed Discussion et al. 1997) mutations have not been identified in our Beta thalassemia is the most common genetic disorder in subjects; these mutations were the IVS-II-1 (G>T) Lebanon, as in many Mediterranean countries. A previ- substitution, the C>G substitution at position −86, ous report (Zahed et al. 1997) investigated the distribu- the T>A transversion at position –30, and finally the tion of β-thalassemia mutations by religious communi- G>A change at cd 30. If these mutations are included,

60 Annals of Human Genetics (2005) 69,55–66 C University College London 2004 Genetic Heterogeneity of β-thalassemia

have arisen in this religious group; the remaining two cd 29 chromosomes found in Sunni patients may be the consequence of intermarriages occurring between these two Muslim communities. As for the cd 29 mutations encountered in Azerbaijan and former Yugoslavia, these may have been introduced by the recent and frequent migration of Lebanese Shiites to these countries. A striking feature of this study was the high preva- lence of patients homozygous for single β-thalassemia mutations, including rare mutations. Of the 260 sub- jects studied, 194 were true homozygotes (Table 1), in spite of the high heterogeneity of β-thalassemia alle- les described above. This considerable degree of ho- mozygosity reflects the high consanguinity rate among the Lebanese population, reaching 40% in certain com- munities (Klat & Khudr, 1986). This may be the re- sult of religious and socio-cultural ethnic endogamy in the Lebanese population. Modern Lebanon is mainly composed of Arabs belonging to several religious en- dogamous societies, largely grouped into Muslims and Christians, and subsequently into Armenians, Kurds, Figure 4 Neighbor-joining tree of Lebanese populations based Assyrians and other ethnicities. However, migration oc- on β-Thalassemia mutation frequencies. Nei genetic distances were used, and internal numbers show the confidence (number curs between communities inside each main religion, of times out of 100 in a bootstrap analysis) in the internal nodes. but rarely between communities of different religions. Scale of genetic distance is shown at bottom left. The distribution of β-thalassemia according to the different religious groups in this study correlated with the number of β-thalassemia mutations found in the the distribution of hemoglobinopathies in Lebanon. Lebanese population is 24, reflecting an even more het- Sunni Muslims had the highest β-thalassemia carrier erogeneous population than previously described. rates and presented the greatest heterogeneity with 16 Six of the 20 β-globin defects identified in our study different mutations. The Shiite Muslims followed closely accounted for more than 86% of all the β-thalassemia with 13 mutations, whereas the Maronites, the predom- chromosomes. These included five of the common inant community of Christians, represented 11.9% of Eastern Mediterranean mutations (IVS-I-110, IVS-I-1, total β-thalassemic subjects and carried only 7 different IVS-I-6, IVS-II-1 and cd 5) and the C>T substitution mutations, of which six were common Mediterranean at cd 29. The latter mutation, first reported in two ho- mutations. mozygous Lebanese patients (Chehab et al. 1987), has This high mutational heterogeneity of Lebanese Mus- been also identified in three chromosomes from Azer- lims may partly be explained by their interaction with baijan (Curuk et al. 1992) and two from former Yu- neighbouring Muslim countries for centuries, resulting goslavia (Dimovski et al. 1990). In the current study, in genetic admixture. Lebanese Sunni Muslims origi- 50 chromosomes were found carrying the cd 29 mu- nate from Arab tribesmen who entered South Lebanon tation, representing 9.6% of the total Lebanese β- in the 7th century AD after the Muslim conquest of thalassemic chromosomes, a frequency never observed . In the 11th century many were converted to the anywhere else worldwide. This suggests that this mu- Druze faith, evolved in southeast Lebanon, and later tation may have originated in Lebanon. Furthermore, filtered north into the southern Lebanese mountains. since 48 of the 50 cd 29 chromosomes were found in At the end of the 11th century Shiite Muslim groups Shiite patients, we could assume that this mutation may from several areas migrated into Lebanon’s northern

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Bekaa and later to the South. After expelling the Cru- 3.8 saders in 1187, Muslim Mamluks and then Ottoman 4.3 Turks governed Lebanon and neighbouring countries until World War I. This Muslim dominion that lasted seven centuries in Lebanon, in addition to Muslim sur- 1992) 8- (Kattamis roundings within and around Lebanon, isolated the Christian endogamous societies and consequently lim- et al. ited their genetic permeability. This is evidenced by 1

the fact that only 3 mutations (IVS-I-110, IVS-I-1 and < IVS-I-6) accounted for more than 90% of all Christian β-thalassemia chromosomes. Since the prevalence of 5.5 1.3 1.1

these mutations (Table 3) is relatively similar to that ob- 1992b) 7- (Efremov served in Macedonia and surrounding countries, which et al.

were under ancient Macedonian dominion, this may 1 < confirm the presumed Macedonian origin of certain Lebanese Christians. Christianity became deeply rooted in Lebanon during Byzantine sovereignty, which came

after the Macedonian Greek and Roman periods, and 2.1 1.1 2.2 4.3 1994) 6- (Rosatelli lasted until their defeat in 630 AD. Maronites, the 1 1 1 et al. < 5.0 7.0 5.0 5.8 < biggest Christian community in Lebanon, fled Syria in < the 7th century and migrated into the northern moun- tains of Lebanon absorbing the indigenous peasants. 1

The Druze are a relatively small Middle Eastern es- < oteric group, derived from Islam in the 11th century; they evolved mostly in the Lebanese mountains, with 1991) 5- (Bennani 1995) 12- (el-Kalla and Mathews 1993). 2.3

smaller communities in northern Israel and southern et al. Syria. They permit no conversion, either away from et al. 1 < or into their religion, and no intermarriage. Thus they 1.3 6.8 have lived as a closed religious group for centuries. Tak-

ing into consideration their long genetic isolation and 1 2.3 <

the small number of patients, we anticipated a relatively 2001) 4- (Fattoum -Thalassemia Mutations in Lebanon and in Arab and Mediterranean Countries β 1

homogeneous population with a limited number of mu- 1992) 11- (el-Hazmi et al. 6.0 10.5 4.6 40.8 27.7 40.1 3.8 17.0 3.8 1.9 12.9 5.4 < tations. Surprisingly, six different mutations were de- 123 4 5 67 8 9 101112 et al. tected in a total of only twelve chromosomes from Druze

extraction. One of these mutations, the −88 substitu- 1 < tion, seemed to be unique to this religious group. This unexpected diversity in the was how- 1994) 3- (Sadiq 1 et al.

ever not observed among Israeli Jews (Filon et al. 1994). 1998) 10- (Baysal < The latter were found to carry only one mutation, the et al. IVS-II-1 allele, which was not found in our Druze subjects.

β Lebanon As for the distribution of -thalassemia by geo- 2001) 2- (Filon

graphic regions, we found that the majority of our tha- ∗ et al. lassemic patients originated from South Lebanon, the Frequency Distribution of the Most Common Bekaa plains and North Lebanon. Since malaria was re- 1990) 9- (Tadmouri ble 3 Mutation Christians Muslims Overall Syria Israel Jordan Tunisia Algeria Italy Macedonia Greece Turkey Cyprus Saudi Arabia UAE 1- (Old cd44IVSII-745cd39 cd37OthersReferences 3 1.4 1.9 1.2 1.2 5.6 1.0 5.3 2.3 8.5 14.2 32.8 25.2 1.4 11.1 5.0 16 9.1 6.3 31.7 6.6 21.4 10 23.1 1.6 47.3 72.5 cd8 3.1 2.5 IVSI-110IVSI-1IVSI-6 35.3cd29 34.3IVSII-1 20.6cd5 34.0cd30 3.9 10.3 34.2 12.9 2.9 15.0 24.0 9.8 14.4 12 21.5 17.0 5.5 25.0 4.0 5.0 8.7 3.3 9.6 11.0 20.5 10.0 5 4.1 8.3 24.7 2.7 7.0 9.0 23.0 15.0 39.3 11.7 1.5 3.3 3.8 10.2 42.0 10.5 9.9 4.5 39.3 21.7 13.0 77.0 3.9 5.0 7.2 1.2 26.9 6.3 10.1 2.0 6.7 1.6 4.7 3.8 12.9 3.2 ∗ Ta ported to have spread mostly in these marshy regions et al.

62 Annals of Human Genetics (2005) 69,55–66 C University College London 2004 Genetic Heterogeneity of β-thalassemia

(Rafie & Rafie, 1984; Taleb et al. 1964), sparing Beirut at their current levels following a long period of en- and the central parts of the country and the mountains, dogamy, in agreement with the historical record. these results might also explain the high prevalence of When compared to other countries (Table 3), the thalassemia among Muslims who reside in these areas, distribution and types of β-thalassemia mutations in corroborating the fact that malaria-infested regions ex- Lebanon were found to be generally similar; a small hibit the highest frequencies of β-thalassemia. number of mutations predominated and the most com- Combining the geographic findings with the reli- mon ones were geographically the most widespread and gious distribution of β-thalassemia mutations we found theoretically the oldest. This was the case for the IVS-I- that the cd 30 mutation followed an interesting pat- 110 mutation, which represented 34.2% of all Lebanese tern among the Lebanese population. All 14 chromo- thalassemic chromosomes. This mutation, possibly hav- somes carrying this mutation belonged to subjects from ing an ancient Greek origin (Cao et al. 1989), reached the Shiite community, originating from the same dis- its highest frequency in Cyprus (77%) (Baysal et al. trict (Bint Jbeil) in South Lebanon. In addition, the cd 1992), and decreased along the North-East to South- 30 mutation was found to be associated with haplo- West axis, and towards Eastern Asia. In contrast, the type IX in all 14 chromosomes. This marked example cd 39 mutation that has been suggested to have a Ro- of clustering is probably due to a founder effect in a man origin (Boletini et al. 1994) followed an opposite highly consanguineous community. As a result, the cd trend, reaching a frequency of 40% in Tunisia (Fattoum 30 mutation was found to be the second most common et al. 1991) and Italy (Rosatelli et al. 1992b), and 96% mutation in the South, and accounted for 9.7% of all in Sardinia (Rosatelli et al. 1992a). Although Romans β-thalassemia mutations in that region. Another exam- ruled Phoenicia (ancient Lebanon) from 64 BC until ple of a region-religion-specific mutation was the −88 395 AD, only one chromosome was found to carry the mutation, detected only within the Druze of Mount cd39 mutation. This could be explained by the lim- Lebanon. ited impact of the Romans on the Phoenician popu- The population tree shown in Figure 4 suggests that lation. In Lebanon the IVS-I-110 mutation may have Greek Christian populations share similar distributions been brought by the migratory Phoenicians, who col- th of β-thalassemia mutations as the Muslim groups. This onized a large part of Cyprus by the 9 century BC, or could be due to a common geographic origin or sub- later after the conquest of Phoenicia by Alexander the stantial admixture. The latter explanation is unlikely, Great in 333 BC and subsequent Macedonian dynasties since all four groups are highly endogamous. Thus, it (323-83 BC). However, the possibility of a more recent is more probable that the geographic origin of the pop- introduction of the IVS-I-110 mutation by the Turks ulations (Greece in the case of the Christians, the Levant during the Ottoman occupation of Lebanon (16th to in the case of the Muslims) is reflected in their muta- early 20th century) was invoked by Zahed et al. (2002), tion frequencies. Interestingly, the Maronites, an ancient and also argued by Bennani et al. (1994) and Perrin Levantine Christian population, have a mutation pattern et al. (1998) in their studies of β-thalassemia distribu- more typical of the Sunni and Shiite Muslim popula- tion in Algeria. Although the Turkish settlement lasted tions. For instance, Maronites have detectable frequen- for about 400 years in Lebanon, we believe it did not cies of the typically Levantine/Muslim –290, cd5 and result in a substantial exchange of population because of cd7 mutations (none of which are found in the Greek the nature of the occupation, during which ethnic clus- Christians), as well as a very high frequency of IVS-I- tering became more stressed. The IVS-I-110 mutation 110. They also, however, have a very high frequency of may have been reintroduced in Lebanon by the Turks, the IVS-I-1 mutation, which is more common in the butitismore likely to have a more ancient origin, since Greek Christians. This mutation is found in Muslim it is by far the most predominant mutation in Lebanon. populations as well, although at much lower frequen- Perrin et al. (1998) suggested that a possible spread of cies. Overall, the Maronite pattern is consistent with a the IVS-I-110 mutation could have occurred from the model in which this population is composed of typi- Greeks or the Phoenicians after the colonization of sev- cally Levantine markers whose frequencies have arrived eral coasts and islands, but attributed the origin of this

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mutation to Anatolia, mainly because of the high degree tation may have been introduced by the Turksduring the of heterogeneity found in the genetic background of the Ottoman rule (1516-1920 AD), since the same RFLP IVS-I-110 mutation in the Turkish population. Indeed haplotype has been described in Turkey and Northern 4 IVS-I-110 RFLP haplotypes were found in Turkey Cyprus (Diaz-Chico et al. 1988). The cd 5 mutation is an (93.1% to haplotype I), 2 in Algeria (92% to haplotype I) eastern Mediterranean mutation, relatively frequent in (Zahed et al. 2002), and only one RFLP haplotype (hap- Lebanon and immediate surroundings. It was found to lotype I) was found to be associated with the IVS-I-110 be associated with RFLP haplotype V in the Lebanese, mutation in the current study. However, in two previous and with RFLP haplotype III in Palestinians (El-Latif studies, 2 additional RFLP haplotypes (II and 12) were et al. 2002), suggesting a different origin. The cd 37 mu- described associated with 1 and 3 IVS-I-110 Lebanese tation, also present in neighbouring countries, was not chromosomes respectively (Chehab et al. 1987; Zahed detected within the Lebanese population. This mutation et al. 2002), and 93% of the IVS-I-110 mutations were wasfirst observed in a Saudi Arabian patient (Boehm associated with haplotype I. Based on these results, and et al. 1986) and is most common in Israel and Jordan the fact that 6 sequence haplotypes were found to be where it may have originated. The IVS-I-5 mutation associated with this mutation in the Turkish population is rare in the Mediterranean and found mainly in the (Tadmouri et al. 2001), a more extensive study of the Asian subcontinent on RFLP haplotype VII (Varawalla genetic background of IVS-1-110 mutations, including et al. 1992). In a previous study (Chehab et al. 1987) this sequence haplotypes, must be carried out in Lebanon mutation was found to be linked to RFLP haplotype IX in order to explain the origin as well as the spread of in a Lebanese homozygote, and thus was suggested to this mutation. In addition to IVS-I-110 and cd 39, two have a different origin. However, in our patients RFLP other common Mediterranean mutations were frequent haplotype VII was found to be associated with this mu- in Lebanon: IVS-I-1 and IVS-I-6. These two mutations tation, which may have been introduced, along with were present in all Eastern Mediterranean populations, the cd 8/9 mutation, by recent migration from South- with no unusual frequencies except for IVS-I-6, which east Asia or the Arabian Peninsula. As for the cd 8, cd accounted for almost half of the mutant chromosomes 44 and cd 30 mutations, they probably originated from found in the West Bank (El-Latif et al. 2002). However, the Turks, Kurdish Jews and Tunisians, respectively. this high frequency was explained by genetic drift oc- The heterogeneity observed in the Lebanese tha- curring in an isolated and highly consanguineous popu- lassemic patients reflects the ethnic diversity of this pop- lation. In Lebanon, the IVS-I-6 mutation was found to ulation. Throughout history, Lebanon has been a land be linked to RFLP haplotypes VI and VII, as in Israeli of migration for several civilizations that contributed and Palestinian subjects, and also to RFLP haplotype to its genetic admixture. Traces of human existence in I, which argues more in favour of an ancient Eastern Lebanon date back to the Paleolithic period. Around Mediterranean origin. As for the IVS-I-1 mutation, it 3000 BC, the Phoenicians arrived and settled in the was found to be predominant in Hungary (Ringelhann land that became Phoenicia. Known as the merchants et al. 1993) and Czechoslovakia (Indrak et al. 1992), of the Levant, they extended their influence from the reaching a frequency of 29.4% and 45.2% respectively; 2nd millennium BC until the 8th century BC by a se- thus it is possible that it may have originated in East- ries of settlements, from Anatolia to North Africa and ern Europe. Interestingly, the IVS-II-1 mutation ob- South Spain, passing through several Mediterranean is- served among Palestinians and Kurdish Jews in Israel was land, and therefore allowing racial admixture with local linked to different RFLP haplotypes than those found populations. Phoenicia was first penetrated and ruled among the Lebanese population. In Lebanon this muta- by the Amorites and the Egyptians, then by the As- tion was associated with RFLP haplotype III, whereas it , Babylonians, Persians and Macedonian Greeks. was linked to RFLP haplotype I in Palestinians (El-Latif Afterwards, Phoenicia was incorporated into the Ro- et al. 2002) and RFLP haplotype V in the Kurdish Jews man and then the Byzantine empires, until its conquest (Filon et al. 1994), suggesting three different paths of by Muslim Arabs in the 7th century AD. Four centuries gene flow by migration. In Lebanon, the IVS-II-1 mu- later Lebanon became a part of the crusaders’ states, and

64 Annals of Human Genetics (2005) 69,55–66 C University College London 2004 Genetic Heterogeneity of β-thalassemia was then re-conquered by the Egyptian Muslim Mam- Cao, A., Gossens, M. & Pirastu, M. (1989) Beta thalassaemia luks, followed by the Ottoman Turks until the French mutations in Mediterranean populations. Br J Haematol 71, mandate after World War I. In addition, several immi- 309–312. Chehab, F. F., Der Kaloustian, V., Khouri, F. P., Deeb, S. S. & grant groups displaced from their homelands, including Kan, Y. W. (1987) The molecular basis of beta-thalassemia the Palestinians, the Kurds and the Armenians, found in Lebanon: application to prenatal diagnosis. Blood 69, refuge in Lebanon throughout the last century. These 1141–1145. successive waves of people throughout history have, no Curuk, M. A., Yuregir, G. T., Asadov, C. D., Dadasova, T., doubt, contributed to the genetic diversity of the cur- Gu, L. H., Baysal, E., Gu, Y. C., Ribeiro, M. L. & Huis- rent Lebanese population. man, T. H. (1992) Molecular characterization of beta- thalassemia in Azerbaijan. Hum Genet 90, 417–419. Conclusion Diaz-Chico, J. C., Yang, K. G., Stoming, T. A., Efremov, D. G.,Kutlar, A., Kutlar, F., Aksoy, M., Altay, C., Gurgey, Considering the high mutational heterogeneity ob- A., Kilinc, Y. et al. (1988) Mild and severe beta-thalassemia served in Lebanon, this study has provided useful infor- among homozygotes from Turkey: identification of the mation about the types and distribution of β-thalassemia types by hybridization of amplified DNA with synthetic mutations within each religious group and geographic probes. Blood 71, 248–251. region, thus allowing rapid detection of mutations in Dimovski, A., Efremov, D. G., Jankovic, L., Juricic, D., Zisovski, N., Stojanovski, N., Nikolov, N., Petkov, G. T., couples at risk, within the frames of prevention pro- Reese, A. L., Stoming, T. A. et al. (1990) Beta-thalassemia grams. 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