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Abstract: HLA alleles have been determined in individuals from the Republic
of Macedonia by DNA typing and sequencing. HLA-A, -B, -DR, -DQ
allele frequencies and extended haplotypes have been for the first time
determined and the results compared to those of other Mediterraneans, particularly
with their neighbouring Greeks. Genetic distances, neighbor-joining
dendrograms and correspondence analysis have been performed. The
following conclusions have been reached: 1) Macedonians belong to the
‘‘older’’ Mediterranean substratum, like Iberians (including Basques), North
Africans, Italians, French, Cretans, Jews, Lebanese, Turks (Anatolians), Armenians
and Iranians, 2) Macedonians are not related with geographically
close Greeks, who do not belong to the ‘‘older’’ Mediterranenan substratum,
3) Greeks are found to have a substantial relatedness to sub-Saharan (Ethiopian)
people, which separate them from other Mediterranean groups. Both
Greeks and Ethiopians share quasi-specific DRB1 alleles, such as *0305,
*0307, *0411, *0413, *0416, *0417, *0420, *1110, *1112, *1304 and *1310.
Genetic distances are closer between Greeks and Ethiopian/sub-Saharan
groups than to any other Mediterranean group and finally Greeks cluster
with Ethiopians/sub-Saharans in both neighbour joining dendrograms and
correspondence analyses. The time period when these relationships might
have occurred was ancient but uncertain and might be related to the displacement
of Egyptian-Ethiopian people living in pharaonic Egypt.
TUKA
| "Our results show that Macedonians are related to other Mediterraneans and do not show a close relationship with Greeks; however they do with Cretans (Tables 3, 4, Figs 1–3). This supports the theory that Macedonians are one of the most ancient peoples existing in the Balkan peninsula, probably long before arrival of the Mycaenian Greeks (10) about 2000 B.C." |
slicna studija
HLA genes in Southern Tunisians (Ghannouch area) and their Relationship with other Mediterraneans
A. Hajjej a, S. Hmida a,*, H. Kaabi a,A. Dridi a,A. Jridi a, A. El Gaa1ed b, K. Boukef a
a National Blood Transfusion Centre, Tunis, Tunisia
b Laboratory of Immunogenetics, Department of Biology, University of Tunis, El Manar
Received 23 December 2004
Available online 10 February 2005
Abstract
South Tunisian HLA gene profile has studied for the first time. HLA-A, -B, -DRB1 and
-DQB1 allele frequencies of Ghannouch have been compared with those of neighboring populations,
other Mediterraneans and Sub-Saharans. Their relatedness has been tested by genetic distances,
Neighbor-Joining dendrograms and correspondence analyses. Our HLA data show that both southern
from Ghannouch and northern Tunisians are of a Berber substratum in spite of the successive incursions
(particularly, the 7th–8th century A.D.Arab invasion) occurred in Tunisia. It is also the case of
other North Africans and Iberians. This present study confirms the relatedness of Greeks to Sub-
Saharan populations. This suggests that there was an admixture between the Greeks and Sub-
Saharans probably during Pharaonic period or after natural catastrophes (dryness) occurred in Sahara.
© 2005 Elsevier SAS. All rights reserved.
Keywords: HLA polymorphism; Tunisians; Berbers; Mediterranean; Basques
1. Introduction
The HLA system is one of the most polymorphic genetic systems in humans. The investigation
of HLA polymorphisms has provided valuable information for understanding the
genetic relationships between populations from different ethnics. Alleles and haplotypes
frequencies as well as the strong linkage disequilibrium between HLA neighboring loci
differences are observed among several populations from different geographic areas. The
comparisons between populations using genetic distances calculated from HLA allele frequencies
have led to draw their history. The HLA markers are useful tools for determining
the origins of populations.
In the North African region, there were many population incursions, such as Phoenicians,
Roman, Arab, Turkish and European, in addition to the original ethnic settlers. In
pre-Neolithic times, The Western region (Maghreb) was first peopled by hunter-gatherers
of a late Palaeolithic culture (Iberomarusian) extending from Spain to Morocco, Algeria
and Tunisia and then by populations belonging to the Capsians [1,2].
Berbers may be the descendants both of the Mesolithic Capsian populations, and of the
later Neolithic people who possibly introduced the Afro-Asiatic languages. The Neolithic
people spread from the Middle East to Egypt between 9,500 and 7 000 B.C. Since then, the
first important invasion was that of the Phoenicians coming from the East Mediterranean
sea-coast around 1,000 B.C, who represented almost one-tenth of the North African population
at the time of the Roman Conquest, in 146 B.C. Berber kingdom declined under the
impact of the Greek invasions (457–404 B.C), Roman Punic wars (246–266 B.C), and
Roman Settlements in the area. Arabs coming from the Middle East invaded the area during
7–8th centuries.A massive Bedouin immigration also occurred in the 11th century [3].
Other immigrants came from the North (Andalusians, Romanians) and from the South
(black slaves from Sudan). All these populations probably contributed to the present Tunisian
genetic pool. The present Tunisian population is constituted by the Berbers and Arabicspeaking
groups. Also, in the South, Tunisian Blacks are clearly more frequent than in the
North and their contribution to the southern Tunisian genetic pool is probable.
The aim of this paper is to study the HLA class I and class II polymorphisms in the
southern population from Ghannouch and to compare it with northern population, other
North Africans, Europeans from several Mediterranean countries and Sub-Saharan populations.
The polymorphism revealed by DNA typing is higher than that defined by serological
methods. This allows us to discriminate the most of HLA class I alleles described in international
nomenclature [4] and to provide a molecular basic for the future HLA studies.
2. Material and Methods
Eighty-two unrelated healthy individuals from Ghannouch (southern town belonging to
Gabes, situated at 400 km from Tunis) were recruited. The origin of other populations used
for calculation comparisons are detailed in Table 1.
DNA was extracted from blood collected in EDTA using a salting out method after
digestion with proteinase K [5].
Generic HLA class I (A and B) and high-resolution class II (DRB1, DQB1) genotyping
were done by Reverse dot blot technique (Innogenetics N.V., Zwijndrecht, Belgium) [6,7].
In this technique, DNA was amplified using A, B, DRB1 and DQB1 primers labelled by
a biotin at their 5’end. All the primers exist in second exon. The amplified product is hybridized
with specific probes immobilized on nylon membrane. Then, the visualization of posi-
tive signals is done by adding streptatvidin labelled with alkaline phosphatase to biotinylated
hybrid. This enzyme metabolises BCIP/NBT chromogen in coloured precipitate. The
genotypes were interpreted using INNOLIPA program.
The HLA-A and -B allele and haplotype frequencies were estimated by maximumlikelihood
(ML) using the computer package Arlequin v1.1 [8]. The Standard deviations
(SD) were calculated from 500 bootstrap iterations [9]. The departure from Hardy–
Weinberg equilibrium was tested at each locus by applying a Markov chain approach with
100.000 steps [10]. The linkage disequilibrium between two alleles at two different loci,
the level of significance (p) for 2 × 2 comparisons and also the relative linkage disequilibrium
(RLD or D’), was calculated [11] by Arlequin. The latter was also used to estimate
maximum-likelihood multi-locus haplotype frequencies from genotypic data through an
expectation-maximization (EM) algorithm [12,13]. Phylogenetic trees (dendrograms) were
constructed with the allele frequencies by applying the Neighbor-Joining (NJ) method [14]
with the standard genetic distances (SGD) [15], by using DISPAN software containing the
programs GNKDST and TREEVIEW [16,17].A three-dimensional correspondence analy-
sis and its bidimensional representation were carried out using the VISTAV5.02 computer
program ([18], http:/forrest.psych.unc.edu). Correspondence analysis comprises a geometric
technique that may be used for showing a global view of the relationship among populations.
According to HLA (or other) allele frequencies, this methodology is based on the allele
frequency variance among populations (similarly to the classical principal components methodology)
and on the display of a statistical projection of the differences.
3. Results
The hypothesis of Hardy–Weinberg equilibrium is accepted for all the HLA A, B,
DRB1 and DQB1 loci. Fifteen alleles were examined for HLA-A locus in Ghannouch
population (Table 2). The most frequent allele is A*24 (15.24%). This value is among the
highest in Mediterranean region. High frequencies of this allele have been reported in Italians
(14.1%) [19], Basques (10.4%) [20] and Sardinians (10.5%) [21], while it is rare in
Sub-Sahara Africans [19,22]. The second most frequent allele is A*02 (14.63%). The latter
is frequent in the most of populations; however, significant differences between ethnic
groups were reported [19]. The A*29, which is frequent in Ghannouch (9.14%), is present
at high frequencies inWestern Europe and, in particular, in the Iberian peninsula (13% in
Spanish Basques) [20].A high frequency of A*6601 (4.26%) was observed in Ghannouch.
This allele is very rare (even absent) in all populations tested [19]. A total of 18 HLA-B
alleles were detected from which the B*50 (13. 41%) is the most frequent allele in Ghannouch
as well as in other Arab populations [23–25], while it is infrequent in Sub-Sahara
Africans and in Europeans [19,22]. The B*07, B*08, B*35,B*44 and B*51, which are
common in Mediterranean populations [19,21,24,26], were also frequent in Ghannouch.
The B*41 (8.5%), B*39 (6.1%), B*45 (6.5%) and B*52 (5.4%) are relatively frequent in
Ghannouch. These frequencies are higher compared to other Mediterranean populations
[19] [22]. Noteworthy is the presence of rare allele B*82 (1.83%) in this population. For
HLA-DRB1, 13 alleles were detected, the most relevant result is a very high frequency for
DRB1*0701 (28.65%). This frequency is one of the highest observed among all populations
tested [19]. The closest frequencies for this allele were observed in Spanish (21.3%),
Basques (19.4%) [21], Tunisians (20%) [24] and Moroccans (20.5%) [27]. A high frequency
of DRB1*1001 (7.31%) allele was recorded in Ghannouch. This allele is very rare
in northern Tunisian population (1.5%) [7]. The closest frequency for this allelewas observed
in Sardinians (6.2%) and in certain Sub-Saharan tribes (Remaibe (8.9%), Nubians (6.6%))
[19]. Nineteen alleles were observed at the DQB1*locus, among which DQB1*0302 is the
most frequent (20.73%). The most relevant result is the low frequency of DQB1*0301
(3.04%) in Ghannouch comparing to other Tunisians and Mediterranean populations
[7,19,24].
Two types of analysis were carried out to compare Ghannouch HLA frequencies with
other Mediterranean population frequencies: (1) with HLA-A, B, DR and DQ data (Fig. 1)
(2) with only HLA-DRB1 data (high resolution), which is probably a more informative and
discriminating methodology (Figs. 2 and 3).
These two types of analysis were both performed because some of the populations used
for comparisons lacked HLA-A and B data [Lebanese (NS, kZ, andY), Moroccans-Agadir,
Jews (Ashkenazi, Moroccans), French-Rennes, Tunisians], genericDQ[Greeks and Greeks-
Cyprus], or high-resolution HLA-DRB1 data [Portuguese, Turks]. These partially HLAtyped
populations should have been ignored, but they could be analysed conjointly taking
in account only either DRB1 or generic A, B, DR and DQ frequencies (Table 2, Figs. 2
and 3).
Fig. 1. Neighbor-Joining dendrogram showing relatedness between Ghannouch and other populations. Standard genetic distances (SGD) between populations were calculated by using generic HLA-A, -B, -DRB1 and DQB1 genotyping. Data from other populations were taken from references detailed in Table 1. Bootstrap values
from 1.000 replicates are shown.
Results obtained by using HLA-A, B, DR andDQdata differ slightly from those obtained
by only DRB1data [23,28,29]. In fact, the Neighbor-Joining tree constructed with HLA-A,
B, DR and DQ allele frequencies (Fig. 1) shows lower bootstrap values. This is probably
due to that one HLA antigen obtained by generic DNA typing may contain several HLA
alleles, while this is not the case for most DRB1 alleles. This phenomenon can homogenize
the comparisons.
The Fig. 2 depicts an HLA-DRB1 Neighbor-Joining tree. The latter shows that there is a
smooth gradient of relatedness betweenWestern to Eastern Mediterranean populations; on
each side the groups tend to cluster together. The Neighbor-Joining present three main
branches with high bootstrap values: the first one groups Western Mediterraneans (both
Europeans and North Africans, including Ghannouch). The second branch is formed by
Eastern Mediterraneans (and Moroccan Jews). The third one includes the Greeks and Sub-
Saharan populations.
It is clear that the Ghannouch are closer toWestern Mediterraneans (both Europeans and
North Africans) than to Eastern Mediterraneans. Ghannouch population shows the closest
genetic distance with Moroccans-Agadir (Berbers) followed by Basques-Arratia (9.12 10–2),
Spaniards, Moroccans Eljadida, Algiers, Basques and Tunisians. Thus, Ghannouch were
relatively distant to Tunisians (20.73 10–2). The Ghannouch were separate from Eastern
Mediterraneans including Arabs as Lebanese.
The Greeks have been shown a substantial Sub-Saharan admixture [30,31].
Bushmen and Japanese form an outgroup. On the other hand, the HLA-DRB1 correspondence
analysis (Fig. 3) grouped together Western Europe-
ans and North Africans, placing together Eastern Mediterranean populations except for the
Greeks, who were putted together with Sub-Saharan population, and locating Bushmen
and Japanese as an outgroup. These results correlate with those obtained by genetic distances
and Neighbor-Joining trees.
Fig. 2. Neighbor-Joining dendrogram showing relatedness between Ghannouch and other populations. Standard
genetic distances (SGD) between populations were calculated by using high resolution HLA-DRB1 genotyping.
Data from other populations were taken from references detailed in Table 1. Bootstrap values from 1.000 replicates are shown. Only Individuals with defined DRB1 subtypes are considered.
4. HLA-A, -B, -DRB1 and -DQB1 linkage disequilibria in Ghannouch
The HLA Ghannouch haplotypes (Tables 4 and 5) have been defined for the first time
and allow us to compare them with those previously reported in other populations. The
HLA-A-B, DRB1-B and DRB1-DQB1 two-loci linkage disequilibrium data show that the
most frequent.
Combinations are characteristic of Mediterraneans, particularlyWestern Europeans and
North Africans. However, no high frequency haplotypes are found in Ghannouch. This
may reflect the existence of a higher admixture of Mediterranean populations in Ghannouch.
The most frequent extended haplotypes (Table 5)A3201-B41-DRB1*1001-DQB1*0501,
A01-B51-DRB1*0402/03-DQB1*0302,A23-B50-DRB1*0701-DQB1*0303 andA29-B39-
DRB1*0701-DQB1*0202 may characterize the southern Tunisian population and they are
absent even in north Tunisian population (our own unpublished results). However, The
other HLA-A-B-DRB1-DQB1 extended haplotypes found in southern Tunisian population
reflect a basic Mediterranean background. Indeed, the A2-B50-DRB1*0701-DQB1*02 is
present in Spaniards (1.2%), Italians (0.5%), Turks (1.3%) and Moroccan Jews (2%)
[22,29,32]. A24-B8-DRB1*0301-DQB1*0201 is found in Europeans and frequently associated
with A1 as in Spaniards (3.4%), Basques (5%), Macedonians (4.9%), in British
(2.9%), Germans (4.8%), Yugoslavians (7.7%) [22,30,33]. The A2-B44-DRB1*0701-
DQB1*02was observed in Basques (1.3%) and Pasiegos (1.6%) [33].A1-B52-DRB1*1502-
DQB1*0601 is found in Macedonians (1.7%) [28] and partially (B52-DRB1*1502-
DQB1*0601) found in Moroccans (1.5%) [23], Cretans (2.5%), Spaniards (1.1%) and
Italians (0.8%) [19,22]. The A02-B44-DRB1*0301-DQB1*02 is also present in Cornish
(3.2%) [19]. These haplotype results are concordant with those obtained by allele frequency
analysis (genetic distance, Neighbor-Joining trees and correspondence analysis).
5. Discussion
5.1. Tunisians, North Africans, Berbers and Eastern Arabs
Our study shows that Ghannouch population (Fig. 4) is related toWestern Mediterraneans,
particularly, to North Africans and Spaniards. This result was confirmed by all analyses
carried out in this study: generic and high-resolution Neighbor-Joining trees (Figs. 1
and 2), high-resolution correspondence analysis (Fig. 3), high-resolution DRB1 genetic
distances (Table 3) and haplotype studies (Tables 4 and 5). Western Mediterraneans are
separate from Eastern Mediterraneans.
The relatedness of Tunisians (Southerns and Northerns) to other North Africans is evident
because all the North Africans share, with slight differences, the same history (as
previously described).
The southern Tunisians from Ghannouch, Northerns from Tunis, Algerians and Moroccans
(Eljadida) are all related to Moroccan Berbers from Souss area (Table 3) and Tunisians
Berbers (our own unpublished results). Therefore, the present-day North African populations
are not genetically different from Berbers. In addition, Lebanese (Table 3) and Arabs
from the Arabian Peninsula [23] show bigger distances to North Africans. This suggests
that the 7th century A.D. Arab invasion (and even the 11th century A.D. massive Bedouin
immigration) of North Africa does not deeply modify the North African genetic pool, in
contrary, they relatively kept their Berber genetic profile. It may be due to that the number
of newcomers from the Middle East was probably very low in comparison with the number
of established Berbers. Therefore, southern Tunisians have kept their Berber substratum in
spite of the successive invasions of the area by: Phoenicians (814–146 B.C.), Romans
(146 B.C.–439 A.D.), Vandals (439–534 A.D.), Byzantines (534–647 A.D.), Arabs (since
644 A.D.), Turks (1574–1957 A.D.) and French settlement (1881–1956) [34]. Also, the
contribution of Negroid to the southern Tunisian genetic pool was low in spite of their high
number in the South of Tunisia. This may be due to cultural barriers. Indeed, The most part
of the Negroid live in small communities as those in Gabes (Mdou,Arram),Kebili, Tataouine
and Mednine; however, Arab invasions (in 7th and 11th centuries A.D.) had clearly strong
social and cultural effects. Indeed, Arabic and Islam are respectively the official language
and religion of the southern Mediterranean countries. EvenTamazight, NorthAfrican Berberlanguage,
was highly influenced by the Arab language [35]. Indeed, Tunisian Berbers speak
Shluh (Chleuch), which is a mixture of Arab language and ancient Berber-language. So,
Shluh has changed rapidly locally owing to the strong Arabic influence. Under the impact
of the Arab invasions (particularly in the 11th century A.D.), a part of Berbers migrated to
the southern mountains of Tunisia (it is the case of other North African Berbers) [36]. Now,
they live in small isolates (some thousands) in the mountains of Tunisian South. The main
ones are in Matmata region (25 km from Gabes), Tataouine and Djerba. All these Berber
isolates are Muslim and speak Arabic in addition to Shluh. Thus, North Africans are genetically
Berbers but culturally Arabs.
5.2. North Africans and Iberian
Many genetic studies (23, 33 and present study) show that Basques are related to Berbers
(Agadir) (Table 3, Figs. 2 and 3). The admixture of these two populations (isolates)
with the 8th century A.D. Arab invaders was very low even absent. Also, Spaniards are not
genetically distinguishable from both Basques and Berbers, in contrary, they show close
genetic distances to them. Moreover, many linguistic studies demonstrate that Berber languages
have several similarities with present-day Basque and ancient Iberian languages
and all have been included in the Na-dene Caucasian group languages [37]. These languages
were widely spoken in Eurasia and North Africa. All these observations confirm
that the genetic contribution of the 8th century A.D. Arab invasion in Iberians was low and
the main part of their genetic pool came from North Africa (Berbers), but before the 8th
century A.D. It was probably at after 10,000 B.C., when the Berbers were forced to emigrate
to the northern Mediterranean coast during the hyperarid conditions established in the
Sahara [28]. In summary, the present-day North Africans, Iberians and Basques are not
genetically distinguishable from Berbers, and all these populations show big distances to
Eastern Mediterraneans (Figs. 2 and 3) and Arabs from Middle East [23]. This suggests
that they have a common Saharan origin and the main part of the Iberian-North African
genetic pool came during the Northward Saharan migration. So, the 7th–8th century A.D.
Arab invasion of the area had low gene flow, but strong social and cultural effects in both
Iberia and North Africa.
5.3. Greeks and Sub-Saharans
Our study shows that the Greeks are separate from other Mediterranean populations and
tend to cluster with Sub-Saharans (Figs. 2 and 3).
This result confirms the Sub-Saharan origin of Greeks.
The Greek/Sub-Saharan relatedness is probably due to that several quasispecific
Greek alleles were also found in some tribes ofWest Africa (Rimaibe, Fulani and
Mossi) and others from East Africa (Oromo, Amhara, Nubians).
This observation suggests that there was an admixture between the Greeks and Sub-Saharans at an
ancient time. The admixture has probably occurred during Egyptian pharaonic times.
Indeed, ancient Greeks thought that their culture and religion came from Egypt.
In this period, there was an influx of people to Greece represented by the migration
of Negroid Egyptian dynasties with their followers (who were expelled) towards Greece [38].
Other emigrations to Greece have probably occurred [31].
Why Greeks cluster with Sub-Saharans as Rimaibe (Figs. 2 and 3).
The latter is one from related tribes of West Africa (Fulani, Mossi and Rimaibe, Burkina
Faso). The Rimaibe Blacks were slaves belonging to the Fulani and frequently mixed with
them. The facials and skin colour parameters of Fulani suggest a Caucasian admixture.
Moreover, many linguistic and cultural studies confirm that Fulani have several characteristics
in common with ancient Egyptians, which suggest that they may come from pharaonic
Egypt [31]. Also, Many genetic studies show that the West African and the East Afri-
can tribes (Nubians, Oromo...) are inter-related. This is supported by linguistic and cultural
evidences. Two types of ancient Nubians were described: reds and blacks, which may reflect
a Caucasian admixture. The present-day Nubians are the descendants of the ancient Nubians
that ruled Egypt between 8th–7th centuries B.C. [39]. All these observations confirm
the relationship between Western African tribes and ancient Egyptians and thus between
the Western Africans and Greeks. An other possible influx of Negroid people into Greece
was occurred, when the weather of desert became hyperarid (5000 B.C.) [31].
European Journal of Medical Genetics 49 (2006) 43–56
http://www.elsevier.com/locate/ejmg |
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Тема: iGENEA - тест за гените
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