Genetic studies in 5 Greek population samples using 12 highly polymorphic DNA loci

Kondopoulou, Helena

Abstract Two minisatellite (D1S80, D17S5) and 10 microsatellite (D2S1328, TPO, D3S1358, D9S926, D11S2010, THO1, VWF, FES, D16S310, and D18S848) polymorphic loci were analyzed in 5 Greek population groups (eastern Macedonia, central Macedonia, Thessaly, Epirus, and Greeks from Asia Minor) using the polymerase chain reaction. The genotypes at these loci conformed to Hardy-Weinberg equilibrium, and pairwise comparisons between them were in agreement with the expectation of independence between loci. This along with the low values of the coefficient of gene differentiation (G^sub ST^) and the high heterozygosity levels of all loci allows the use of allele frequency data from the 12 hypervariable DNA markers for medicolegal casework in the Greek population groups studied. The small genetic distances indicate a genetic affinity among the 5 population samples. However, a few markers seem to allow some discrimination among the groups. No significant differences with other European populations were found for the loci studied.


To date, a large number of hypervariable region (HVR) loci have been described and characterized in the human genome. This polymorphism arises from variation in the number of tandem repeats of a DNA sequence that is generated by mutations that lead to new length alleles. Mutation processes include replication slippage, unequal sister chromatid exchange, and gene conversion (Schlotterer and Tautz 1992; Jeffreys et al.1994). Because of their multiallelic variation and, consequently, high level of informativeness, these HVR loci can be used for human gene mapping (Nakamura et al. 1987), genetic analysis of inherited diseases (Peake et al. 1990; Hearne et al. 1992), study of human evolutionary history (Bowcock et al. 1994), and personal identification in the medical and forensic sciences (Jeffreys et al. 1991; Gill et al. 1995).

The HVR loci include minisatellites and microsatellites. Minisatellite loci are composed of 6-100-bp repeats, and they are preferentially located in the proterminal regions of human autosomes (Royle et al. 1988). They present stable inheritance patterns and are characterized by a large number of alleles per locus and by high heterozygosities (Nakamura et al. 1987; Wong et al. 1987). Thus they are extremely useful in evolutionary studies of genetically close populations. Microsatellite loci consists of several repeats of 1-5 bp, and they are widely dispersed throughout the genome at an estimated frequency of 1 microsatellite per Cr-10 kbp (Oldroyd et al. 1995). Because of their small size (100-400 bp), they can be easily amplified from minimal amounts of even old and/or highly degraded material.

Several genetic studies using multiallelic systems have been conducted for worldwide human populations representing the major geographic groups (Balazs et al. 1992; Devlin and Risch 1992; Deka et al. 1995; Perez-Lezaun et al. 1997). Furthermore, during the last few years, a number of specific local populations have been investigated (Furedi et al. 1995; Hochmeister et al. 1995; Martin et al. 1995; Sjerps et al. 1995; Rose et al. 1996).

In Greece allele frequency data are available for only a few HVR loci (mostly minisatellites) and refer to either the whole population (Hatzaki et al. 1995; Lambropoulos et al. 1995) or a certain geographic area of Greece (De Benedictis et al. 1993; Falcone et al. 1995).

In our ongoing survey 12 highly polymorphic DNA loci were tested in Greek population samples residing in 4 geographic areas of Greece (eastern Macedonia, central Macedonia, Thessaly, Epirus) and 1 population of Greeks from Asia Minor living in eastern and central Macedonia since 1923 (Figure 1). These population groups were chosen to provide material that is relevant to the cultural, historical, and genetic questions that present themselves when Europe is considered within the wider global context.

Eastern Macedonia is a region north of the Aegean where Neolithic settlements appeared. In central Macedonia there are many excavation sites, such as Dion, Aiges, Pella, Edessa, and Sindos, and the oldest Neolithic settlement was found in Nicomedia, between the Aliakmonas and Axios rivers, which were important for the transmission of farming to Europe from the Axios valley. In Thessaly the fertile plains are of central importance for the first farmers of Europe, where the Neolithic (5000-4000 B.C.) settlement of Sesklo was found. In the mountain region of Epirus there are possible preNeolithic elements (Tsaktsitas 1995). Finally, the Greeks from Asia Minor were chosen because presumably the first farmers of Europe emanated from the Near East, more specifically, the Fertile Crescent, in the southeastern and south-central part of Asia Minor. From there they moved through Thrace and Macedonia (northern Greece) to Europe (Cavalli-Sforza et al. 1994).

For the analysis of the 5 groups we chose 2 minisatellites and 10 microsatellites. For the microsatellites only tetranucleotide repeat loci were used, because analysis of dinucleotide loci has revealed polymerase slippage during amplification, resulting in artifactual stutter or shadow bands (Hauge and Litt 1993) and making unambiguous designation of alleles difficult. In contrast, tetranucleotide loci are less prone to these truncated polymerase chain reaction (PCR) products.

This study is part of a project that aims to investigate the genetic composition of the Greek population for the following reasons: first, to establish a genetic data bank for native individuals from each geographic area and for some particular population groups; and, second, to correlate the results from genetic data with archeological and historical data.

Materials and Methods

Population Samples and Genetic Loci. Two minisatellite and 10 microsatellite loci were tested in the 5 population groups (Figure 1). Approximately 100 unrelated native individuals (their ancestries have been traced for 3 generations) were analyzed for each population sample.

The 2 minisatellite loci were DIS80 (Budowle et al. 1991) and D17S5 (Batanian et al. 1990), and the 10 microsatellites were D2S1328, D9S926, DII S2010, and D18S848 (Perez-Lezaun et al. 1997), TPO (Anker et al. 1992), D3S1358 (Li et al. 1993), THO1 (Edwards et al. 1992), VWF (Kimpton et al. 1992), FES (Polymeropoulos et al. 1991), and D16S310 (Hudson et al. 1992). The microsatellites have a 4-bp repeat unit and represent the microsatellites chosen as part of a greater pan-European study. Chromosomal localization and primer sequences are given in Table 1.

DNA Analysis. Genomic DNA was extracted from whole blood using standard procedures (Yancopoulos and Alt 1990). PCR amplification of all loci was carried out for 30 cycles (D17S5 for 32 cycles) in a 25-,ul reaction volume containing approximately 100 ng of DNA.

The amplification conditions used for minisatellites were as follows: for D1S80, 95 deg C for 1 min, 72 deg C for 4 min; for D17S5, 95 deg C for 1 min, 55 deg C for 1 min, 72 deg C for 3 min. For microsatellites the denaturation (95 deg C for 45 s) and extension (72C for 1 min) conditions were constant, whereas the annealing temperatures varied: 55 deg C for D18S848, 56oC for D3S1358, 58oC for D11S2010, 59 deg C for FES/FPS, 60 deg C for D9S926 and D16S310, and 61C for D2S1326, VWF, TPO, and THO]. The annealing time was 30 s. The products from loci DI S80 and D17S5 were resolved by agarose gel electrophoresis. Allele separation of microsatellites was performed using an 8% polyacrylamide gel. Visualization of bands was achieved by silver staining. The 100-bp ladder was used as a standard size for D17S5, whereas phi X174/HaeIII and PBR322/MspI were used for D1S80 and for the microsatellites. Also, allelespecific ladders composed of amplified DNA from individuals with known alleles were constructed for each microsatellite.

Statistical Analysis. Allele frequencies were calculated using the gene counting method. The observed and expected heterozygosities and standard errors were computed according to the method of Nei (1978). Hardy– Weinberg equilibrium was tested for each locus and population sample using the chi-square test (BIOSYS-1) (Swofford and Selander 1989) and the exact test (Guo and Thompson 1992) (GENEPOP, version 3.1; Raymond and Rousset 1995b). GENEPOP uses the Markov chain algorithm, and this approach was set to 50,000 steps and 1,000 steps of dememoration. To test for linkage disequilibrium (i.e., allelic independence across loci), we used GENEPOP (Raymond and Rousset 1995a).


Allele frequencies for the 12 HVR loci are given in Table 2. Their patterns of distribution showed differences among loci. Some are unimodal and others are bimodal, and still others demonstrate a more complex pattern. One hundred twenty-one alleles were revealed for the 12 HVR loci. The mean number of alleles per locus was 10.08, with a range of 6 to 25. Table 3 shows the mean observed and expected heterozygosities for all tested loci for each population sample. The mean observed heterozygosity values were similar among the samples and ranged from 0.703 +/- 0.019 for Epirus to 0.744 0.020 for Thessaly. Similarly, the mean expected heterozygosity values did not vary among samples.

In 5 of 60 loci per population sample the chi-square and exact tests showed a deviation from Hardy-Weinberg equilibrium that is slightly more than what was expected. It was in only 2 of these 5 cases, for the D17S5 locus, that both tests gave a significant result at the p

Linkage disequilibrium showed that from the 330 pairwise comparisons between loci in all the population samples studied, 17 deviated from expectation of independence; this corresponds approximately to the expected proportion of 5%.

Table 4 shows average GST values for each locus. Values vary from 0.003 for D16S310 and FES to 0.010 for D2S1328. The mean G^sub ST^ value for all the tested loci was 0.006. The exact test for population differentiation showed significant differences at the D2S1328 locus between the group of Greeks from Asia Minor and each of the other 4 population groups (p

Two neighbor-joining trees were constructed using a matrix of D^sub 8^ and D^sub A^ genetic distances among the 5 population groups. The D^sub S^ genetic distances are given in Table 5. D^sub S^ and D^sub A^ distance trees are supported by bootstrap values of over 62% and 42%, respectively. It seems that Ds performs better than the D^sub A^ distance, and consequently only the tree based on Ds distances is shown here (Figure 2). However, it is clear from both trees that the population samples of eastern Macedonia, central Macedonia, Thessaly, and Epirus form a cluster that is close to the group of Greeks from Asia Minor.

Figure 3 illustrates a 2-dimensional principal components plot where the first principal component accounts for 92% of the total genetic variation and the second principal component accounts for 2.4%. The plot further illustrates the genetic similarities of the 5 Greek population groups for the loci studied.


The present study is a survey of polymorphism in 5 Greek population groups using 2 minisatellite and 10 tetranucleotide microsatellite loci. The general pattern of the allele frequency distribution (Table 2) appears to be similar to what has been reported for other European populations (Buscemi et al. 1994; Rose et al. 1996; Perez-Lezaun et al. 1997). The number of alleles detected, 121, provides a higher level of genetic information compared with classical markers.

Because there is little evidence of departure from Hardy-Weinberg and linkage equilibrium, we believe that the 5 population groups are randomly mating. Therefore this allows the use of the allele frequency data from the 12 HVR loci for paternity and identification casework.

Despite the low G^sub ST^ values (Table 4), which indicate the relative lack of substructuring among the population groups analyzed, the exact test for population differentiation showed that the D2S1328 locus is able to distinguish the group of Greeks from Asia Minor from each 1 of the other population groups. Also, the DI 7S5 locus is able to distinguish the group of Greeks from Asia Minor from the groups of central Macedonia, Thessaly, and Epirus. Moreover, the D9S926 locus can discriminate the population sample of Epirus from the samples of central and eastern Macedonia. These results suggest that, although the 4 native Greek population groups are close geographically and interpopulation gene flow is not restricted, 1 marker seems to be able to distinguish these groups. Furthermore, the Greeks from Asia Minor who lived for centuries in Anatolia can be distinguished from other Greeks with only 2 of the 12 markers used, indicating gene flow between these populations.

The small genetic distances (Table 5) indicate great genetic affinity among the native individuals of the 4 population groups residing in neighboring geographic areas (Figure 1) of Greece (eastern Macedonia, central Macedonia, Thessaly, and Epirus). Furthermore, the small genetic distance between these 4 populations and the Greeks from Asia Minor could be explained by expansions of the populations in the prehistorical and historical periods. More specifically, in the Neolithic Period the first farmers of Europe moved first from Asia Minor to the fertile plains of Macedonia and later to Thessaly (Cavalli-Sforza et al. 1994; Tsaktsitas 1995). From 1100 to 1000 B.c. and from 700 to 650 B.c., 2 colonizing waves were developed from continental Greece to coastal areas of Asia Minor (Tsaktsitas 1995). In the Hellenistic Period (323-146 B.C.), under Alexander the Great and his successors, migration took place from Greece to Asia Minor (Tsaktsitas 1995). Since that time, the flow of populations has continued to the present (Barmaze 1995; Koliopoulos 1995). The close genetic relationships between the 5 Greek population groups are further supported by the phylogenetic tree and the principal components map.

No statistically significant heterogeneity (p > 0.30) was observed in the gene frequencies between the pooled Greek data and other European populations (Schnee-Griese et al. 1993; Buscemi et al. 1994; Klintschar and Kubat 1995; Martin et al. 1995; Pestoni et al. 1996; Rose et al. 1996; PerezLezaun et al. 1997). At the DI S80 and DI 7S5 loci the allele frequencies were not significantly different between our pooled data and data from other studies on the Greek population (p = 0.48 and p = 0.61, respectively) (Hatzaki et al. 1995).

These results support the notion of Alonso et al. (1995) that the allele frequencies of microsatellite loci are well preserved in Europeans. The study of more populations and/or other micro- and minisatellite loci might support or reject Alonso’s hypothesis.

In conclusion, this survey provides the allele frequencies for the construction of a Greek genetic database, indicates the peculiarities of some markers that may be able to discriminate between population samples from Greece, points toward the similarity of the Greek population sample to other European populations, and tries to interpret the genetic data in light of the known history of the studied populations.


This research was supported by the European Commission within the framework of the ERBCHRX-CT 940676 project. R. Loftus was supported by the project. We thank D. MacHugh for his help with the analyses. Also, we warmly thank the people who helped us to collect blood samples.

Received 16 December 1997; revision received 6 July 1998.

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1 Department of Genetics, Development, and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 54006 Thessaloniki, Macedonia, Greece.

2 Genetics Department, Trinity College, Dublin 2, Ireland.

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