Population genetic studies on the tetrameric short tandem repeat loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 in Egypt

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1 Forensic Science International 104 (1999) 31 locate/ forsciint Population genetic studies on the tetrameric short tandem repeat loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 in Egypt Michael Klintschar a *, Nabil Al-Hammadi, Barbara Reichenpfader a, b a Department of Legal Medicine, University of Graz, Universitatsplatz 4, A-8010 Graz, Austria b Department of Forensic Medicine, College of Medicine, Sanaa University, Sanaa, Yemen Received 24 March 1999; received in revised form 28 June 1999; accepted 12 July 1999 Abstract The short tandem repeat loci (STRs) D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 and a locus allowing for sex-discrimination (amelogenin) can be co-amplified by the polymerase chain reaction using a commercially available kit (AmpFlSTR Profiler plus, Perkin-Elmer Biosystems, San Jose, CA) and subsequently typed using capillary electrophoresis (ABI Prism 310 Genetic analyzer, Perkin Elmer Applied Biosystems, San Jose CA). To establish databases for these loci for an Arab population sample from Egypt, 140 unrelated persons were typed. Analysis of these data revealed that all loci except for VWA were in Hardy-Weinberg equilibrium, that the combined mean paternity exclusion chance (MEC) was and that the combined discriminating power (DP) was The allelic distributions found in the Egyptian sample were significantly different at four loci from those found for an Austrian Caucasian population, at all nine loci from an African-American sample and at six of six loci from a Chinese sample. No evidence of linkage equilibrium between any of the co-amplified loci was found. Our results support that the combination of multiplex PCR and capillary electrophoresis can both save time and yield excellent results for paternity testing and stain analysis Elsevier Science Ireland Ltd. All rights reserved. Keywords: Forensics; DNA profiling; capillary electrophoresis; short tandem repeat; Egypt *Corresponding author. Tel.: ; fax: address: michael.klintschar@kfunigraz.ac.at (M. Klintschar) / 99/ $ see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S (99)

2 24 M. Klintschar et al. / Forensic Science International 104 (1999) Introduction Short tandem repeat loci (STRs, microsatellites) are highly variable DNA polymorphisms consisting of a variable number of repetitive elements of 2 to 6 bp length which can easily be amplified using the polymerase chain reaction and have thus become powerful tools for forensic stain analysis and paternity testing [1 3]. For these purposes tetrameric or pentameric STRs with amplified fragment lengths below 300 bp are optimal because of their relatively low occurence of stutter bands and their high sensitivity even when using highly degraded DNA as template [4]. The STRs D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 are among those selected for the US Combined DNA Index System (CODIS) and typed in crime labs throughout the world. It would be therefore of benefit for the forensic community to make data on the genetic variation of these loci available in as many populations as possible. However, up to now, no allelic frequencies on Arab populations like the Egyptian one have been published. Although the Egyptian population is Caucasian by descend and should therefore be closely related to other Caucasian populations, older studies have observed significant differences between the Egyptian and European population samples for most STRs tested [5 7]. The goal of this paper is therefore to study the allelic distribution of these 9 STRs which can be coamplified with a locus allowing for sex-discrimination (amelogenin) by PCR using a commercially available kit (AmpFlSTR Profiler plus, Perkin-Elmer Biosystems, San Jose, CA), and subsequently be typed using capillary electrophoresis, in a population sample from Egypt. 2. Materials and methods 2.1. Sample preparation Whole EDTA blood was obtained by venipuncture from 140 unrelated Egyptian Caucasians from the Cairo area. Bloodstains were prepared on sterilized cotton cloth and subsequently air dried. The DNA was extracted using a modified alkaline lysis protocol as described [8] PCR amplification and typing Aliquots of 1.25 ml of the extracts with a DNA content of approximately 2 ng/ml were used for amplification without prior quantification of the DNA content. Amplification was performed using the AmpFlSTR Profiler plus kit (Perkin-Elmer Biosystems, San Jose, CA) according to the manufacturers recommendations except that a total assay volume of ml instead of 50 ml was used and that thermocycling was performed in a TRIO-Thermoblock (Biometra, Gottingen, FRG). Capillary electrophoresis was carried out using an ABI Prism 310 Genetic analyzer (Perkin-Elmer Applied Biosystems) according to the manufacturers recommendations.

3 M. Klintschar et al. / Forensic Science International 104 (1999) Table 1 Allelic frequencies and forensic efficiency parameters of 6 STR loci in 140 unrelated Egyptians. (H. obs.: observed heterozygosity; H. exp.: expected heterozygosity, MEC: Mean exclusion chance; DP: Discriminating power) D5S H. obs.: 0.72 H. exp.: 0.76 DP: MEC: D13S H. obs.: 0.77 H. exp.: 0.79 DP: MEC: D7S H. obs.: 0.74 H. exp.: 0.78 DP: MEC: D3S H. obs.: 0.77 H. exp.: 0.77 DP: MEC: 0.555

4 26 M. Klintschar et al. / Forensic Science International 104 (1999) 31 Table 1. Continued VWA H. obs.: 0.74 H. exp.: 0.80 DP: MEC: D8S H. obs.: 0.82 H. exp.: 0.79 DP: MEC: Statistical analysis The frequency of each allele for each locus was calculated from the numbers of each genotype obtained in the sample set (i.e. the gene count method). The mean exclusion chance (MEC) was calculated according to Kruger et al. [9], and the discriminating power (DP) was calculated according to Fisher [10]. Exact tests for checking the Hardy Weinberg expectations were performed using the GENEPOP software version 1.2 (M. Raymond and F. Rousset, Montpellier). Comparisons of the allele frequencies between different populations were performed using two-way contingency tables. The computer program was kindly provided by G. Carmody, Ottawa. 3. Results and discussion The distributions of the genotypes for the nine STR loci found in Egypt and the respective forensic efficiency parameters are shown in Tables 1 and 2. The observed allelic frequencies are shown in Table 3. The most common allele frequencies ranged from (FGA, allele 24) to (D5S818, allele 12). The observed heterozygosity

5 M. Klintschar et al. / Forensic Science International 104 (1999) Table 2 Allelic frequencies and forensic efficiency parameters of the STR loci FGA, D21S11 and D18S51 in 140 unrelated Egyptians. (H. obs.: observed heterozygosity; H. exp.: expected heterozygosity, MEC: Mean exclusion chance; DP: Discriminating power) FGA H. obs.: 0.75 H. exp.: 0.81 DP: MEC: D21S H. obs.: 0.81 H. exp.: 0.82 DP: MEC: D18S H. obs.: 0.84 H. exp.: 0.86 DP: MEC: 0.711

6 28 M. Klintschar et al. / Forensic Science International 104 (1999) 31 Table 3 Allelic frequencies for nine STR loci in 140 unrelated Egyptians Allele D5S818 D13S317 D7S820 D3S1358 VWA FGA D8S1179 D21S11 D18S (H. obs.) ranged between 0.72 (D5S818) and 0.84 (D18S51), the Mean Exclusion Chance (MEC) between (D5S818) and (FGA), and the Discriminating power (DP) between (D5S818) and (FGA). The combination of these nine 211 STRs proved to be extremely discriminating; the combined DP was , and the combined MEC was The maximum frequency of a combination of nine genotypes (i.e. a combination of nine genotypes consisting of the two most common alleles at each locus) was calculated to be one in 142 million.

7 M. Klintschar et al. / Forensic Science International 104 (1999) Table 4 Check for Hardy Weinberg expectations using an exact test Locus P SE D5S D13S D7S D3S VWA FGA D8S D21S D18S All loci except for VWA were in Hardy-Weinberg equilibrium (HWE) (P.0.05) (Table 4). For this locus an excess of homozygotes (H. obs: 0.80, H. exp: 0.74) could be demonstrated. Most remarkable was the excess of individuals typed for 14,14 (three observed, 0.9 expected), 16,16 (14 vs. 9.5) and 18,18 (7 vs. 4.3). Nevertheless also two heterozygote genotypes (17,19 (13 vs. 6.2); 17,20 (6 vs. 1.2) were substantially more often observed than expected. However, as the P-value was close to 0.05 (0.031), the departure at the VWA locus is (unlike e.g. for a British Caucasian sample [11]) not highly significant. One explanation could be that this observation is a statistical artifact due to multiple testing of the same population sample. As after having tested 140 DNA samples for nine STRs no shared combined genotypes were observed, this hypothesis can be refused. Other reasons for the deviation observed at the VWA locus include inbreeding, population substructure, selection, or silent alleles [12]. Given the structure of the Egyptian population with contributions from both Caucasian and African ancestry to the genepool, population substructure might be a likely explanation. The Egyptian population allele frequency data for these 9 PCR-based loci differ substantially from a European population sample from Austria [13] at four loci (D7S820, D8S1179, D21S11, D18S51) and from an Afro-American reference sample [14] at all nine loci (Table 5). Moreover, when comparing the Egyptian data for the loci Table 5 Comparison between Egyptians (this study), African-Americans [14], Chinese [15] and Austrians [13] using two-way contingency tables (probability values; bold print: statistically significant differences). The loci D8S1179, D21S11 and D18S51 were not typed in the Chinese reference sample African-Americans Chinese Austrians D5S818,10, D13S317,10, D7S , D3S1358,10, VWA,10, FGA,10, D8S D21S11,10,10 D18S51,

8 30 M. Klintschar et al. / Forensic Science International 104 (1999) 31 D5S818, D13S317, D7S820, D3S1358, VWA and FGA with Chinese data [15] evidence of population heterogeneity was found for all these six loci. In conclusion, the PCR multiplex kit and the automated CE system were an efficient combination, reducing expediture of time and handling of toxic substances to a minimum. The nine STR loci described here proved furthermore to be sufficiently polymorphic for most forensic purposes. Nevertheless, extensive mutation studies as already performed for other loci [16] should be performed, before using these polymorphisms for paternity testing. Acknowledgements The authors are indebted to to S. Schweitzer, I. Wolfbeis and R. Frizberg for valuable support and to M. Abdull Fatah, Cairo, for providing a part of the blood samples. This study was supported by grant 65/1 from the Jubilee Fund of the Austrian National Bank to M. Klintschar References [1] A. Edwards, A. Civitello, H.A. Hammond, C.T. Caskey, DNA typing and genetic mapping with trimeric and tetrameric tandem repeats, Am. J. Hum. Genet. 49 (1991) [2] M. Benecke, DNA typing in forensic medicine and in criminal investigations: a current service, Naturwissenschaften 84 (1997) [3] R.L. Alford, H.A. Hammond, I. Coto, C.T. Caskey, Rapid and efficient resolution of parentage by amplification of short tandem repeats, Am. J. Hum. Genet. 55 (1994) [4] B. Brinkmann, The use of STRs in stain analysis, in: Procedings From the Third International Symposion On Human Identification, Promega, Madison, USA, 1992, pp [5] M. Klintschar, N. Al-Hammadi, T. Lux, B. Reichenpfader, Genetic variation at the short tandem repeat loci HumVWA, HumFXIIIB, and HumFES/ FPS in the Egyptian and Yemenian populations, J. Forensic Sci. 43 (1998) [6] M. Klintschar, Z. Kozma, N. Al Hammadi, M. Abdull Fatah, C. Nohammer, A study on the short tandem repeat systems HumCD4, HumTHO1, and HumFIBRA in a Yemenian and an Egyptian population sample, Int. J. Legal Med. 111 (1998) [7] M. Klintschar, U. Ricci, N. Al-Hammadi, B. Reichenpfader, A. Ebner, M.L. Giovannucci Uzielli, Genetic variation at the STR loci D12S391 and CSF1PO in four populations from Austria, Italy, Egypt and Yemen, Forensic Sci. Int. 97 (1998) [8] M. Klintschar, F. Neuhuber, Evaluation of an alkaline lysis method for the extraction of DNA from whole blood and forensic stains for STR analysis. J. Forensic Sci (in press) [9] J. Kruger, W. Fuhrmann, K.H. Lichte, C. Steffens, Zur Verwendung des Polymorphismus der sauren Erythrocytenphosphatase bei der Vaterschaftsbegutachtung, Dtsch. Z. Gerichtl. Med. 64 (1968) [10] R. Fisher, Standard calculations for evaluating a blood group system, Heredity 5 (1951) [11] M.A. Drozd, L. Archard, P.J. Lincoln, N. Morling, L.J. Nellemann, C. Phillips, B. Soteriou, D. Syndercombe Court, An investigation of the HUMVWA31A locus in British Caucasians, Forensic Sci. Int. 69 (1994) [12] National Research Council, The Evaluation of Forensic Dna Evidence, National Academy Press, Washington, DC, [13] M. Klintschar, A. Ebner, B. Reichenpfader, Population genetic studies on 9 tetrameric short tandem repeat loci using fluorescence dye labeled primers and capillary electrophoresis in the Austrian population. Electrophoresis (in press)

9 M. Klintschar et al. / Forensic Science International 104 (1999) [14] AmpFlSTR Profiler Plus Users Manual, PE Applied Biosystems, San Jose 1998, [15] C.-E. Pu, F.-C. Wu, C.-L. Cheng, K.-C. Wu, C.-H. Chao, J.-M. Li, DNA short tandem repeat profiling of Chinese population in Taiwan determined by using an automated sequencer, Forensic Sci. Int. 97 (1998) [16] B. Brinkmann, M. Klintschar, F. Neuhuber, J. Huhne, B. Rolf, Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat, Am. J. Hum. Genet. 62 (1998)