Republic of Macedonia

HLA alleles have been determined in individuals from the Re¬public 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, par¬ticularly with their neighbouring Greeks. Genetic distances, neighbor-join¬ing 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), Ar¬menians 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 (Ethiop¬ian) 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 displace¬ment of Egyptian-Ethiopian people living in pharaonic Egypt.

The highly polymorphic HLA system has been validated as useful for distinguishing and/or relating populations (and individuals) in many research studies since the first International HLA Anthropology Workshop (Evian, 1973) and in all the subsequent seven International Workshops. HLA gene frequencies correlate with geographically re¬lated populations.
The existence or absence of gene flow among neighbouring ethnic groups may be assessed with the study of HLA frequencies and the corresponding genetic distances (1,2).
Ancient Macedonians were among the peoples that lived be¬tween northern Greece (Thessaly) and Thrace in the Balkans and were considered by the classical Greeks as ‘‘non-Greek barbarians’’ that could not participate in the Greek Olympic Games (3).
Hero dotus wrote that "Macedonians" were "Dorians" and were never admitted to the Greek community (4).
They did not speak Greek but another language presently unknown and of which only proper names remain; nowadays, they speak a Slavic language (5).
Mace¬donians fought against the Greeks between 357-336 B.C. under King Philip II. They defeated the Greeks at the Battle of Chaironea (338 B.C.). The Macedonian empire extended from the Balkan Penin¬sula to the Himalayas and to North Africa during the reign of Phil¬ip's son, Alexander the Great (6).
Thereafter, Macedonia was conqu¬ered by the Romans and has been disputed in more recent times by Serbs and/or Bulgars. Ottoman Turks controlled Macedonia be¬tween 1380-1912 A.D., and it was integrated into Yugoslavia in 1946. In 1991, after the partition of Yugoslavia, a referendum gave Macedonia its independence.
The present ethnic groups within the country are:
1) Macedonians: 1,279,000;
2) Albanians: 377,000;
3) Turks: 87,000;
4) Serbs: 44,000; and
5) others: 40,000.
The northern¬most region of Greece is also known as Macedonia and this is why Greece has opposed the independence of the country while it bears the same name (7).
Furthermore, we have found that the Greeks did not cluster to¬gether with other Mediterranean populations, including both west¬ern (Iberians, Algerians, Berbers) and eastern (Cretans, Jews, Leb¬anese, Egyptian, Turks-Anatolians) Mediterraneans (8-10).
The aim of the present work is to determine the relative contri¬butions of Macedonians and Greeks to the present-day genetic pool of Mediterranean peoples.
For these purpose, both HLA class I and class IIDNA typings have been studied in Macedonians for the first time.
The genetic relationship of Macedonians and Greeks to other Mediterraneans, including North Africans (Berbers from Agadir and El Jadida areas and Algerians from Algiers), Iberians (Spani¬ards, Basques and Portuguese) and Greeks (from Attica, Aegean and Cyprus) were calculated.
In addition, sub-Saharan and other Africans were compared with all available Mediterranean groups in order to solve the question of the unique Greek HLA profile.

Material and methods

Population samples

Samples from one hundred and seventy-two unrelated Macedonians in Skopje (Institute of Blood Transfusion, Tissue Typing Labora¬tory), the Republic of Macedonia capital, were used for HLA geno-typing and phylogenetic calculations. All were Macedonian lan¬guage speakers and their ancestors did not belong to a country minority group (detailed above). The origin of all other populations used for comparisons is given in Table 1.

Populations used for the present work

Table 1
HLA genotyping, DNA sequencing and statistics

Generic HLA class I (A and B) and high-resolution HLA class II (DRB1 and DQB1) genotyping was performed using a reverse dot-blot technique with the Automated Innolipa system (Innogenetics N.V., Zwijndrecht, Belgium).
HLA-A, -B, -DRB1, and -DQB1 allele DNA sequencing was only done when indirect DNA typing (reverse dot-blot) yielded ambiguous results (11).
Statistical analysis was performed with Arlequin v1.1 software kindly provided by Ex-coffier and Slatkin (12).
In summary, this program calculated HLA-A, -B, -DRB1 and -DQB1 allele frequencies, Hardy-Weinberg equilib¬rium and the linkage disequilibrium between two alleles at two dif¬ferent loci.
Linkage disequilibrium (Dø; also named LD, see ref. 13) and its level of significance (P) for 2x2 comparisons were deter¬mined using the formulae of Mattiuz and co-workers (14) and the 11th International Histocompatibility Workshop methodology (13).
In addition, the most frequent complete haplotypes were deduced following a methodology used in the 11th International Histocom¬patibility Workshop:
1) the 2, 3, and 4 HLA loci haplotype frequen¬cies (2,15,16);
2) the haplotypes previously described in other popu¬lations (2, 16); and
3) haplotypes which were assigned if they ap¬peared in two or more individuals and the alternative haplotype was well defined.
In order to compare allelic and haplotype HLA frequencies with other populations, the reference tables used were those of the 11th and 12th International HLA Workshops (2, 16; see also Table 1).
Phylogenetic trees (dendrograms) were constructed with the allelic frequencies by applying the Neighbor-Joining (NJ) method (17) with the genetic distances between populations (DA, 18) and using DISPAN software containing the programs GNKDST and TREEVIEW (19, 20).
A three-dimensional correspondence analysis and its bidimensional representation was carried out using the VISTA v5.02 computer program (21, http:/forrest.psych.unc. edu).
Correspondence analysis comprises a geometric technique that may be used for displaying a global view of the relationships among populations according to HLA (or other) allele frequencies.
This methodology is based on the allelic frequency variance among populations (similarly to the classical principal components method¬ology) and on the display of a statistical projection of the differ¬ences.

Table 2

Genetic distances between populations (DA) between Macedonians and other populations (xlO2) obtained by using HLA-DRB1 allele frequencies (see Table 1 for populations identification)


Table 3

Characteristic HLA allele frequencies of the Macedonian population compared to other Mediterraneans

The expected and observed allele frequencies for HLA-A, -B, -DRB1 and -DQB1 loci do not significantly differ and the population sample is in Hardy-Weinberg equilibrium. Table 2 shows the HLA allele frequencies found in the Macedonian population. Fourteen different HLA-A and twenty-eight different HLA-B alleles were observed in the Macedonian population. Six HLA-A alleles and seven HLA-B alleles had frequencies higher than 5% (A*01, A*02, A*03, A* 11, A*24, A*26, B*07, B*08, B*18, B*35, B*38, B*44 and B*51) and these are characteristic of Mediterranean populations (8-10, 22).

With regard to the HLA class II alleles, thirty-one different DRB1 alleles were found and only six had frequencies higher than 5%; DQ allele frequencies reflect the DRB1 locus allele distribution due to the strong linkage disequilibrium between these two loci.
Two types of analyses were carried out to compare Macedonian HLA frequencies with other Mediterranean population frequencies: 1) with DRB1 data, which is probably a more informative and discrimi¬nating methodology; and 2) with generic (low-resolution) DR-DQ data. These two types of analysis were both performed because some of the populations used for comparison lacked HLA-A and -B data [Berbers (from Souss, Agadir area, Morocco), Jews (Ashkenazi), Jews (Morocco), Jews (non-Ashkenazi), Lebanese (NS and KZ), see Table 1], or high resolution HLA-DQ data [(Greeks (Attica), Greeks (Cyprus), Greeks (Attica-Aegean), see Table 1]], or only generic HLA-DR and DQ data were available [Portuguese, Turks, Iranians, Armenians and Egyptians, see Table 1]. These partially HLA-typed populations should have been ignored, but they could be analyzed conjointly tak¬ing into account only either DRB1 or generic DR and DQ frequencies (Tables 3, 6, Figs 1-3). Analyses using DRB1and DQB1 conjointly were made but are not shown because only a few populations could be used and the results are concordant with the DRB1 analysis. Finally, it should be pointed out that class I generic typing tends to homogenize the comparisons based on DRB1 high-resolution typing (see ref. 22); one class I allele obtained by generic DNA typing may contain several class I alleles, while this is not the case for most DRB1 alleles.
Fig. 1 depicts an HLA class II (DRB1) neighbor-joining tree. Populations are grouped into three main branches with high boots¬trap values: the first one groups both eastern (including Macedoni¬ans, Cretans, Jews, Lebanese) and western Mediterraneans (Euro¬peans and North Africans; Sardinians are also included in the first group). The second branch is formed by African Negroid popula tions and the third one includes Greek and sub-Saharan popula¬tions. This distribution is also confirmed in the correspondence analysis (Fig. 2): the three groups are clearly delimited and a west to east Mediterranean gradient is shown. The Macedonian population shows the closest genetic distance with Cretans (Table 3) and no discontinuity is observed with eastern and western Mediterraneans reflecting the genetic similarity among these populations. It is evi¬denced that Cretans-Greeks distance is high. These results are con¬firmed using DR and DQ generic typings (see Fig. 3 and data not shown) which were used in order to include other Mediterranean populations (Iranians, Armenians, Egyptians and Turks, see Table 1). A DR-DQ neighbour-joining tree (data not shown) maintains the West to East Mediterranean gradient and also the group formed by Greeks and sub-Saharan populations. Turks (old Anatolians), Kurds, Iranians and Armenians have been shown specifically to cluster with the eastern Mediterranean groups (Arnaiz-Villena et al., submitted). On the other hand, genetic distances obtained by using DR-DQ generic typing allele frequencies (data not shown) show that Iranians (LlOxlCr2) and Cretans (1.54X1CT2) are the two populations closest to the Macedonians followed by the other Mediterranean populations. A discontinuity is found between Berbers (Souss) and Greeks (Attica) (9.59X1CT2 vs. 12.42X1CT2) showing that the latter have a distant relationship with Mediterran ean populations as previously described (10, 22) and cluster together with the sub-Saharan populations.

 Fig. 2. Correspondence analysis showing a global view of the re¬lationship between Mediterraneans and sub-Saharan and Black African populations according to HLA allele frequencies in three dimensions (bidimensional representation). HLA-DRB1 allele fre¬quencies data.

Fig. 3. Correspondence analysis showing a global view of the re¬lationship among West Mediterraneans (green),
East Mediterran¬eans (orange),
Greeks and sub-Saharan populations (red) and
Blacks (grey)
according to HLA allele frequencies in three dimen¬sions (bidimensional representation).
HLA-DR and DQ (low-resolution) allele frequencies data.

Common HLA-DRB1 alleles between Greeks and sub-Saharan Africans


Fig. 1. Neighbor-Joining dendrogram showing relatedness be¬tween Macedonians and other populations. Genetic distances between populations (DA) were calculated by using HLA-DRB1 (high-resolution). Data from other populations were from references detailed in Table 1. Boots¬trap values from 1000 replicates are shown.

Most frequent HLA-A, -B, -DRB1, and -DQB1 extended haplotypes in the Mace¬donian population and their possible origin

HF: Haplotype frequency. aAlso found in Basques (2.4%), Spaniards (3.4%), Britons (2.9%), Danes (3.4%), Cretans (1.1%), Germans (4.8%), Austrians (5.3%) and Yugoslavs (7.7%) (2, 9, 10, 15, 16). bThis haplotype has been found in Albanians (3.9%), Italians (2.1%), Yugoslavs (3.5%), Turks (1.1%), Spaniards (1.1%) and Greeks (4.0%) (2, 16 and our own unpublished results). candd Pres¬ent only in Macedonians. ePartially (B52-DRB1*1502-DQB1*0601) found in Moroccans (1.5%), Cretans (2.5%), Spaniards (1.1%) and Italians (0.8%) (2, 16, 22). fHaplotype found in Armenians (2.1%) and Italians (0.7%) (2, 16). gOnly found in Italians (0.8%) (2, 16). hHaplotype found only in Iberians, Portuguese (1.5%) and Spaniards (0.3%) (15). iPresent in Turks (0.9%) and in Jews (our own unpublished results and 33). Other low frequency haplotypes present in Macedonians are also shared with central Europeans (A*03-B*07-DRB1*1501-DQB1*0602, HF: 0.8; A*02-B*13-DRB1*0701-DQB1*02, HF: 0.8; A*02-B*44-DRB1*0701-DQB1*02, HF: 0.6), western Europeans (A*02-B*07-DRB1*1501-DQB1*0602, HF: 0.6), north Africans (A*02-B*07-DRB1*1001-DQB1*0501, HF: 0.6) and Mediterranean-Europeans (A*23-B*44-DRB1*0701-DQB1*02, HF: 0.6) (2, 8-10, 16 and our own unpublished results)
Table 4
AF: Allele frequency. Some of these HLA-DRB1 alleles are also present in other populations: aNot found in other populations. bPresent in Hungarians (0.4%). cFound in Amerindians and some Pacific peoples. dNot found in other populations. eFound in Hungarians (1.2%). fPresent in Hva Island (Croatia, 0.3%) and Amerindian Yukpa (2.3%). gFound in Lebanese 0.1%. hFound in Hva Island (Croatia, 0.9%) and Hungarians (2.6%). iAlso found in Lebanese (2.3%) and Hungarians (2.6%). jNot found in other populations. kAlso present in Hvar Island (Croatia, 1.0%) (2, 23)
Table 5
HLA-A, -B, -DRB1, and -DQB1 linkage disequilibria in Macedonians

Extended HLA haplotypes were determined in Macedonians and compared with those previously reported in other populations (Table 4). HLA-A-B and DRB1*-DQB1* two-loci linkage disequilib¬rium data (not shown) show that the most frequent combinations are characteristic of European and Mediterranean (western and eastern) populations (B*18-DRB1*1104, Haplotype Frequency (HF): 9.0; A*02-B*18, HF: 8.1; A*01-B*08, HF: 5.5; B*08-DRB1*0301, HF: 5.2; A*24-B*35, HF: 4.9 and B*07-DRB1*1501, HF: 4.1). The HLA-A-B-DR-DQ extended haplotypes found in the Macedonian population (Table 4) reflect common characteristics with the other "older" Medi¬terranean background (see footnote to Table 4). These haplotype results are concordant with those obtained by the allele frequency analyses (genetic distances, neighbor-joining trees and correspon¬dence, see above).

Common alleles of Greeks with sub-Saharan Africans

In order to study the possible origin of the Greeks who remain outliers among Mediterraneans (10, 22), specific DRB1 alleles pres¬ent in Greeks and not present in the other Mediterranean popula¬tions were searched in other geographically not very distant popu lations. Our own data, the 11th and 12th International Histocom-patibility Workshops reference panels (2, 16, 23) and other previously described data were used (see Table 1). Table 5 shows the presence of these Greek alleles mainly in sub-Saharan popula¬tions from Ethiopia (Amhara, Oromo), Sudan (Nuba) and West Africa (Rimaibe, Fulani, Mossi). Some of these alleles are sporadi¬cally present in other populations without any relationships among them (see footnote to Table 5). It may be deduced from these data that sub-Saharans and Greeks share quasi-specific HLA-DRB1 alleles. The neighbor-joining tree (Fig. 1) and the cor¬respondence analyses (Figs 2 and 3) confirm this Greek/sub-Sahar-an relatedness. The HLA-DRB1 genetic distances between Greeks and other Mediterraneans are shown in Table 6 and also support a sub-Saharan/Greek relatedness; genetic distances with HLA-DR and -DQ generic typings (not shown) give essentially the same re¬sults. No relationship of Greeks is seen with the Senegalese and South African Blacks (Bantu and people coming from the Guinea Gulf after the Bantu expansion, respectively (24)), nor with the present day Bushmen (24).
Two different types of problem regarding the obtained data are discarded: 1) mistakes in the HLA typings and 2) mistakes in the assignation of these specific alleles (DRB1*0417, *1112, etc, see Table 5). These problems are not likely to exist in the present work because; 1) HLA typings have been made by genetic technologies in three different Greek populations (2, 23) and 2) similar results are obtained when generic typing is used (DR-DQ analysis in Fig. 3; see also ref. 22).

Genetic distances (DA) between the different groups of Greeks and other populations (xlO2)
obtained by using HLA-DRB1 allele frequencies
(see Table 1 for identification of populations)

Table 6



Our results show that Macedonians are related to other Mediterran¬eans 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. Other possible explanation is that they might have shared a genetic background with the Greeks before an hypothetical admixture between Greeks and sub-Saharans might have occurred. The cultural, historical and genetic identity of Macedonians is established according to our results. However, 19th century historians focused all the culture in Greece ignoring all the other Mediterranean cultures present in the area long before the classical Greek one (25).

Greeks are genetically related to sub-Saharans

Much to our surprise, the reason why Greeks did not show a close relatedness with all the other Mediterraneans analyzed (Tables 5, 6 and Figs 1-3) was their genetic relationship with sub-Saharan eth¬nic groups now residing in Ethiopia, Sudan and West Africa (Burki-na-Fasso). Although some Greek DRB1 alleles are not completely specific of the Greek/sub-Saharan sharing, the list of alleles (Table 5) is self-explanatory. The conclusion is that part of the Greek gen¬etic pool may be sub-Saharan and that the admixture has occurred at an uncertain but ancient time.
The origin of the West African Black ethnic groups (Fulani, Mossi and Rimaibe sampled in Burkina-Fasso) is probably Ethiopian (26, 27) (Fig. 4). The Fulani are semi-nomadic hunters and gatherers and one of the few people in the area to use cows' milk and its by¬products to feed themselves and to trade; their facial parameters show a Caucasian admixture. The Rimaibe Blacks have been slaves belonging to the Fulani and have frequently mixed with them (27). The Nuba people are now widespread all over Sudan, but are de¬scendants of the ancient Nubians that ruled Egypt between 8th-7th centuries B.C. (28) and later established their kingdom at Meroe, North Khartoum. Two kinds of Nubians were described in ancient times: Reds and Blacks, probably reflecting the degree of Caucasian admixture. Both the Oromo and Amharic peoples live in the Ethiop¬ian mountains (27). They obviously have in common a genetic back¬ground with the west-African groups mentioned above. Linguistic, social, traditional and historical evidence supports an east-to-west migration of peoples through the Sahel (southern Sahara strip), al¬though this is still debated (26, 27).
Thus, it is hypothesized that there could have been a migration from southern Sahara which mixed with ancient Greeks to give rise to a part of the present day Greek genetic background. The admix¬ture must have occurred in the Aegean Islands and Athens area at least (Figs 1 and 2). The reason why this admixture is not seen in Crete is unclear but may be related to the influential and strong Minoan empire which hindered foreigners establishment (10). Also, the time when admixture occurred could be after the overthrown of some of the Negroid Egyptian dynasties (Nubian or from other periods) or after undetermined natural catastrophes (i.e.: dryness). Indeed, ancient Greeks believed that their religion and culture came from Egypt (4, 25).

Fig. 4.
Map showing the location of the populations tested in the present work..

The complete scientific report on the Macedonian Genes done by the Spanish University of Complutense, now is back online for free (at the original website you must pay in order to view the complete report).
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