Materials and Methods
ResultsMaterials. We have studied 343 DNA male DNA samples from the DNA bank at our institution, of which 211 were Macedonians, 111 Albanians and 21 Roma, Serbs or Turks. The study was approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia (R. Macedonia).
Methods. The set of 28 Y-SNP markers was grouped hierarchically into five multiplex polymerase chain reaction (mPCR)/primer extension reactions, so as to determine the most frequent haplogroups using one or two multiplexes (Figure 1). Most of the PCR and minisequencing primers have already been described , the remainder were designed by us.
DiscussionMultiplex 1 consists of eight SNPs and allows detection of the more frequent major clades in Europe. It also includes the 12f2 deletion, which determines haplogroup J and was typed by presence/absence in the PCR amplification (Figure 2). Multiplex 1 determines haplogroups R1b1-P25, R1a1-SRY1532 and I2a-P37b. We performed multiplex 1 in all samples, and depending on the results, multiplex 2, 3, 4 or 5 was performed in order to define the haplogroup more precisely.
Multiplex 2 comprises four SNPs, multiplex 3 and 4 comprise six SNPs each, while multiplex 5 is a combination of five SNPs. Multiplex 2 subdivides haplogroup I, multiplex 3 haplogroup E, multiplex 4 haplogroup J, while haplogroup 5 determines haplogroups G-M201, H-M69, L-M22, N1c-Tat and T-M70.
Among the DNA samples we studied, we detected 20 different Y haplogroups, of which five haplogroups (E1b1b1a-M78, I2a-P37b, J2b2-M241, R1a1-SRY1532 and R1b1-P25) comprised 72.6% of the studied Y chromosomes (Table 3). The distribution of the Y haplogroups in Macedonians, Albanians and males of other ethnic origin (Roma, Serbs and Turks) is given in Table 3. The most common Y haplogroup in Macedonians was I2a-P37b (27.5%), which was followed by E1b1b1a-M78 (15.6%), R1a1-SRY1532 (14.2%) and R1b1-P25 (11.4%). In the Albanians, E1b1b1a-M78 was found in 28.8%, R1b1-P25 in 18.0%, J2b2-M241 in 13.5% and R1a1-SRY1532 in 12.6%. In the small group of Roma, Serbs or Turks, R1b1-P25 was found in 23.8% and E1b1b1a-M78 and H-M69 in 14,3% each. All three males with haplogroup H-M69 were of Roma ethnic origin.
SourceThe R. Macedonia has a multiethnic population consisting of Macedonians (64.2%), Albanians (25.2%) and Roma, Serbs, Turks, Aromuns and others (10.6%) (2002 census). The most common Y haplogroups in this population are E1b1b1a-M78, I2a-P37b, J2b2-M241, R1a1-SRY1532 and R1b1-P25. Y haplogroup composition of the population conforms with previous findings in the Southeast European (SEE) population .
The Y haplogroup E1b1b1a-M78 was the most frequent haplogroup in Albanians and the Roma, Serb or Turk group, but was second in frequency in Macedonians. It is the most common haplogroup E lineage in Europe with a frequency peak centered in the Balkans [11,12]. It is also found in the Middle East and in eastern and northern Africa. Its frequency in Kosovar Albanians (46%) and Macedonian Roma (30%) is most likely a result of genetic drift .
The most frequent haplogroup in Macedonian males is I2a-P37b (27.5%), which has maximum frequency in Herzegovinians (64%) and Bosnians (52%) and substantial frequencies in all SEE populations except for two reproductively isolated and non slavic speaking populations, Kosovar Albanians and Macedonian Romani . In the Albanians in Macedonia, I2a-P37b had a very low frequency (1.8%) (Table 3), similar to Albanians from Kosovo (2.7%) and different from Albanians in Albania (17%) .
The R1b1-P25 haplogroup showed an overall frequency of 14.3%, being 18.0% in Albanian and 11.4% in Macedonian subjects. This lineage shows a frequency peak (40-80%) in western Europe and decreases in eastern (with the exception of 43% in the Ossetians) and southern Europe [13,14]. However, it shows two intermediate local peaks in mainland Croatians and Serbians, and among Kosovar Albanians, Albanians and Greeks .
The R1a1-SRY1532 haplogroup showed similar frequencies in Macedonians and Albanians and was less frequent in the Roma, Serb or Turk groups. The frequency in Macedonia agrees with that of 16% determined in SEE . Its distribution increases from west to east with peaks in Finno-Ugric and Slavic populations. R1a1-SRY1532 frequency decreases slowly to the south of Europe.
Haplogroup J is defined by a 12f2 polymorphism and has two major subclades, J1-M267 and J2-M172, of which the latter is more prevalent in Europe . The J2b-M102 lineages are more frequent in SEE comprising 5% of all chromosomes, with a peak in Kosovar Albanians . The J2a4b-M67 cluster is predominant in Greeks and Italians . The J2b2-M241 was the fifth most frequent Y haplogroup in the populations of R. Macedonia due to its high prevalence in Albanians (13.5%).
The most significant difference (p <0.0001) was found in the case of Hgr I2a-P37b, which was prevalent (27.5%) in Macedonians and infrequent in Albanians (1.8%). Although present at a relatively low frequency in both groups, I1-M253 was significantly more frequent in Albanians (6.3 vs. 1.9% in Macedonians, p = 0.0383). The E1b1b1a-M78 haplogroup was statistically more frequent in Albanians (28.8 vs. 15.6% in Macedonians, p = 0.0050). The same applies to J2b2-M241 (13.5 vs. 5.2%), being more common in Albanians (p = 0.0093). The R1a1 and R1b1 haplogroups were present with similar frequencies in both populations.
In conclusion, we have developed a simple, robust and efficient Y-SNP typing assay that can find application in evolutionary and forensic studies in the major ethnic groups of R. Macedonia. The hierarchical strategy using the SNaPshot multiplex kit (Applied Biosystems) made Y chromosome SNP typing rapid and inexpensive.
What is interesting is the absence of I2a among Macedonian Albanians just as the Kosovo Albanians.
Macedonian Albanians have slightly lower R1a percentage(12.6%) than Macedonians but they have 6.3% I1-M253 a similar percentage to Kosovo Albanians.