Genetic analysis of a Sicilian population using 15 short tandem repeats

Human Biology, Apr 2003 by Calo, C M, Garofano, L, Mameli, A, Pizzamiglio, M, Vona, G

Abstract The genetic structure of the population of Alia (Sicily, Italy) was analyzed using 15 short tandem repeats: TPOX, D2S1338, D3S1358, FIBRA, D5S818, CSF1PO, D7S820, D8S1179, TH01, VWA, D13S317, D16S539, D18S51, D19S433, and D21S11. Two of these markers, D2S1338 and D19S433, have never before been used in research on population genetics and only recently have they been put to use in forensic medicine. Results of the analysis underline the genetic isolation of the Alia population and show it to be a recent bottleneck as a consequence of a cholera epidemic in 1837. While comparing the Alia population with other populations from Sicily, a genetic heterogeneity within Sicily was uncovered, thus confirming previous results obtained from the analysis of classical markers. This heterogeneity underlines the existence of genetic boundaries within the island. Comparisons with other Italian, Mediterranean, and European populations highlight the differentiation of the Sicilian population, reflecting the presence of a genetic boundary that separates Sicily from northern and central Italy and from the western Mediterranean basin.

KEY WORDS: SICILY, ALIA, STRS, BOTTLENECK

Located 800 meters above sea level and about 80 km from Palermo (Sicily, Italy), Alia has a population of around 4000. In 1837 Alia, as well as all of Sicily, suffered a cholera epidemic that resulted in a dramatic reduction in its population. In 1995 an excavation uncovered the remains of about 300 individuals who had died from cholera. Interest in this population was highlighted by other studies based on analyses of mitochondrial DNA (mtDNA) (Vona et al. 2001) and classical genetic markers (Ghiani et al. 2002) that stressed the uniqueness of allele frequencies in the Alia population. The genetic peculiarity of this population may be due to its isolation and very low density. These two factors combined could have amplified the consequences of genetic drift. The aim of this study is to increase our knowledge about the genetic features of the population of Alia. Using some of the markers that we have analyzed in the present study, further information may be obtained from the comparison between the present population of Alia and the ongoing research into the subfossil remains resulting from the 1837 cholera epidemic.

Alia has undergone all of the historical incursions that have affected Sicily. Following the arrival of the first settlers on the island (Sycanians, Siculi, Elymians), Sicily, because of its position in the middle of the Mediterranean, has been invaded by Phoenicians, Greeks, Romans, Vandals, Goths, Arabs, Normans, and Spaniards. The contribution of all these different groups to the complex genetic constitution of the Sicilian population has yet to be accurately studied (Piazza et al. 1988; Rickards et al. 1992, 1998; Vona et al. 2000).

Materials and Methods

Blood samples were obtained from 50 unrelated, apparently healthy adults of both sexes, whose families were born and have lived in the same village of Alia for at least three generations. One of our researchers in Alia gathered the samples after obtaining informed consent from the donors. These samples were then taken to the Department of Experimental Biology of Cagliari University as well as the Raggruppamento Investigazioni Scientifiche of Carabinieri (RIS) in Parma for analysis. DNA was extracted from anonymous, whole blood samples using the traditional phenol-chloroform technique. Amplification by polymerase chain reaction (PCR) technique was performed using commercially available kits according to the manufacturers' recommendations (PE-ABD, Promega, USA). PCR was performed in a PE-ABD 2400 Thermal Cycler.

Electrophoresis was carried out on 5% polyacrylamide denaturing sequencing gels on a 377 automated system (PE-ABD). Data were analyzed by both Gene Scan v.3.1 and Genotyper v.2.0 software. The 15 short tandem repeats (STRs) studied were: TPOX, D2S1338, D3S1358, FIBRA, D5S818, CSF1PO, D7S820, D8S1179, TH01, VWA, D13S317, D16S539, D18S51, D19S433, and D21S11. Their characteristics are shown in Table 1.

Statistical Analysis. Allele frequencies were calculated from the genotypes using the gene counting method. Possible divergence from Hardy-Weinberg equilibrium was determined by the exact test method based on Markov chain analysis according to Guo and Thompson (1992) through the Genepop computer program (v.1.2) (Raymond and Russel 1995). Through the same program linkage disequilibrium for loci located on the same chromosome was tested. In addition, polymorphic information content (PIC) values were calculated by the use of the formula suggested by Hearne at al. (1992). The coefficient of gene diversity (G^sub ST^) (Nei 1973) and the genetic distance evaluations were obtained by employing the methods of Nei (Nei 1972) through the Phylip computer program, v.3.5c (Felsenstein 1989). Dendrogram construction of the genetic distances was performed with the neighbor-joining method (Saitou and Nei 1978). Furthermore, the existence of genetic boundaries within the island was tested through the Delauney network (Brassel and Reif 1979) and Monmonier's algorithm (1973). Genetic evidence for recent bottlenecks was evaluated for subpopulations with the Bottleneck computer program (Cornuet and Luikart 1996). This program tests whether H^sub obs^ significantly differs from H^sub exp^ under the mutation-drift equilibrium.