Genetic variation at the STR loci D12S391 and CSF1PO in four populations from Austria, Italy, Egypt and Yemen

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1 Forensic Science International 97 (1998) Genetic variation at the STR loci D1S391 and CSF1PO in four populations from Austria, Italy, Egypt and Yemen a, c b Michael Klintschar *, Ugo Ricci, Nabil Al Hammadi, a a Barbara Reichenpfader, Alexander Ebner, Maria Luisa Giovannucci c Uzielli a Department of Legal Medicine, University Graz, Universitatsplatz 4, A-8010 Graz, Austria b Department of Forensic Medicine, College of Medicine, Sanaa University, Sanaa, Yemen c Azienda Meyer University of Florence Human Genetics Service, Via Masaccio 09, I-5013 Florence, Italy Received 7 May 1998; received in revised form 3 August 1998; accepted 6 August 1998 Abstract The short tandem repeat systems (STRs) D1S391 and CSF1P0 were amplified by the polymerase chain reaction (PCR) on blood samples from 100 to 158 unrelated Austrians, Italians, Yemenians and Egyptians. The samples were analyzed by both native and denaturing electrophoresis and two primer pairs were tested for the CSF1PO locus. Except for the CSF1PO data on the Egyptians, no deviations from the Hardy Weinberg equilibrium were detected. For D1S391, no significant differences were found between the two Arab populations and between the two European populations, but the differences between both Arab populations and the Italians were significant. For CSF1PO, differences were only observed between the Yemenians and all three other populations. No evidence of linkage disequilibrium between the two STRs was found. The observation of a D1S391 allele consisting of only 14 repeats was confirmed by sequencing Elsevier Science B.V. All rights reserved. Keywords: D1S391; CSF1PO; Austria; Italy; Egypt; Yemen; Population study * Corresponding author. Tel: ; fax: ; michael.klintschar@kfunigraz.ac.at / 98/ $ see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S (98)

2 38 M. Klintschar et al. / Forensic Science International 97 (1998) Introduction Tetrameric short tandem repeat (STR) polymorphisms have become powerful tools for forensic stain analysis and paternity testing [1 3]. For those purposes it is necessary to obtain data on the genetic variation of the loci applied in various populations. The goal of this paper was to study a recently introduced STR, D1S391 [4] and one of the most frequently used STRs, CSF1PO [5], in four populations from Europe, Northern Africa and the Arabian peninsula, since especially the two latter populations have not been widely studied up to now. Moreover, the applicability of two electrophoretic setups for both loci and two different primer pairs for the CSF1PO locus were compared and a possible linkage disequilibrium between both loci was investigated.. Materials and methods.1. Sample preparation Whole EDTA blood was obtained by venipuncture from 100 unrelated Austrians from Styria, 158 unrelated Italians from Tuscany, 104 unrelated Yemenians from the Sanaa area and 100 unrelated Egyptians 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 [6]... PCR Amplification and typing Aliquots of.5 ml of the extracts with a DNA content of approximately ng/ml were used for amplification without prior quantification of the DNA content. The D1S391 locus was amplified as described [1]. For the CSF1PO locus, the Italian samples and 50 Austrian samples were amplified as described by Hammond et al. [5], whereas all Austrian samples and all Arab samples were amplified using a different primer pair [7]. Two electrophoretic setups were tested; native horizontal polyacrylamide gels [8] and vertical denaturing polyacrylamide gels [5] followed by silver staining. A sequenced allelic ladder for the D1S391 locus was kindly provided by A. Carracedo, Santiago de la Compostela. The allelic ladder for the CSF1PO locus amplified using the primers described by Hammond et al. [5] was purchased from Promega (Madison, WI). The CSF1PO ladder for the samples amplified using the primers by Yoshida et al. [7] was constructed from samples previously typed using the Promega ladder..3. Sequencing To confirm the sequence of the rare allele, the sample was run in an agarose gel and the DNA was eluted using the QIAquick-Gel Extraction kit (QIAGEN, Germany). The amplicon was then cloned into the plasmid vector pcrtmii, supplied by Invitrogen TA cloning kit (Invitrogen, USA), according to the protocol provided by the supplier. The

3 M. Klintschar et al. / Forensic Science International 97 (1998) nucleotide sequence of the cloned fragment was determined using the standard technique described by Sanger et al. [9]..4. Statistical analysis The mean exclusion chance (ME) was calculated according to Kruger et al. [10] and the discriminating power (DP) was calculated according to Fisher [11]. Exact tests were performed using the GENEPOP software version 1. (M. Raymond and F. Rousset, Montpellier) for checking the Hardy Weinberg expectations. For linkage analysis the data of all four populations were pooled and an exact test was performed using the same software. Comparisons of the allele frequencies between different populations were performed by using two-way contingency tables. The computer programme was kindly provided by G. Carmody, Ottawa. 3. Results Allele frequencies of the two STRs obtained by denaturing PAGE for D1S391 and for both denaturing PAGE (Italians: Lareu primers [4]) and native PAGE (Austrians, Egyptians and Yemenians: Yoshida primers [7]) for CSF1PO are shown in Table 1. Observed and expected genotype frequencies are given in Tables and 3. Parameters of forensic efficiency for each locus and both loci combined are shown in Table 4. At the CSF1PO locus (Table 3), significant differences between the Yemenians and each of the three other populations were found (Table 5). At the D1S391 locus (Table ), a population heterogeneity between both Arab samples and the Italians was observed. No other significant differences in interpopulation comparisons were found (Table 5). Deviations from Hardy Weinberg expectations were found for the CSF1PO data on the Egyptian sample (Table 4). Furthermore, no evidence of linkage disequilibrium between both STRs was found (P50.63). Fifty Austrian samples which were typed for CSF1PO using both Lareu primers followed by denaturing PAGE and Yoshida primers followed by native PAGE gave consistent results. Sequencing of the rare D1S391 allele confirmed that it consisted of 14 repeats with an overall fragment length of 05 bp, as suspected from its electrophoretic mobility. The structure of the polymorphic region was 59-AGAT AGAT AGAT AGAT AGAT AGAT AGAT AGAC AGAC AGAC AGAC AGAC AGAC AGAT-39 and therefore one AGAT repeat shorter than the smallest allele in the ladder (15). 4. Discussion We have analysed the STR loci D1S391 and CSF1PO in samples from two European and two Arab countries to obtain allele and genotype frequencies and to investigate the usefulness of these loci for forensic stain analysis and paternity testing in these

4 40 M. Klintschar et al. / Forensic Science International 97 (1998) Table 1 Allelic frequencies for the STRs CSF1PO and D1S391 in two Arab and two European population samples Allele Egyptians Yemenians Italians Austrians D1S n CSF1PO n The most common alleles are in bold print (n, number of chromosomes analysed). populations. The degree of polymorphism of both loci was similar in all four populations. For the CSF1PO locus departures from Hardy Weinberg proportions were observed in the Egyptian population. Possible reasons include inbreeding, population substructure and selection [1]. Given the structure of the Egyptian population with contributions from both Caucasian and African ancestry to the genepool, population substructure appears to be the most likely explanation. Both loci were tested using both native and denaturing polyacrylamide gels. The application of native gels to these polymorphisms proved to be problematic. Especially for the D1S391 locus, no reliable typing was possible as already predicted by Lareu et al. [4]. Moreover, the alleles of the CSF1PO locus amplified using the primers published by Hammond et al. [5] proved to be too long for sufficient resolution on native gels. The resolution of the CSF1PO alleles amplified with the Yoshida primers [7], was satisfactory. On the other hand, both loci could be reliably typed on denaturing gels. Interestingly, significant differences were found for the CSF1PO locus between both Arab populations, while no differences were observed between the geographically considerably more remote populations of Austria and Egypt. Nevertheless, no differ-

5 M. Klintschar et al. / Forensic Science International 97 (1998) Table Observed and expected genotypes at the CSF1PO locus in four populations Genotype Egyptians Yemenians Italians Austrians Exp. Obs. Exp. Obs. Exp. Obs. Exp. Obs. 8, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Total ences between the Yemenian and the Egyptian population were found for D1S391, and for six other STRs [6,13] differences between the same samples were observed at one locus (HumCD4) only. In conclusion, the two STRs CSF1PO and D1S391 proved to be valuable for differentiation of individuals of both Arabic and European descent. Both loci could be easily typed using denaturing PAGE, while native PAGE was only suitable for the locus CSF1PO amplified with shorter, recently introduced primers [7].

6 4 M. Klintschar et al. / Forensic Science International 97 (1998) Table 3 Observed and expected genotypes at the D1S391locus in four populations Genotype Egyptians Yemenians Italians Austrians Exp. Obs. Exp. Obs. Exp. Obs. Exp. Obs. 14, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

7 M. Klintschar et al. / Forensic Science International 97 (1998) Table 3. Continued Genotype Egyptians Yemenians Italians Austrians Exp. Obs. Exp. Obs. Exp. Obs. Exp. Obs. 0, , , , , , , , , , , , , , , , , , Table 4 Forensical efficiency parameters and P values for the exact test for Hardy Weinberg equilibrium (HWE) for the two STRs in the four populations studied CSF1PO D1S391 Combined H. obs. Egyptians Yemenians Italians Austrians DP Egyptians Yemenians Italians Austrians MEC Egyptians Yemenians Italians Austrians HWE: P values (exact test) Egyptians Yemenians Italians Austrians H. obs., observed heterozygosity; DP, discriminating power; MEC, mean exclusion chance.

8 44 M. Klintschar et al. / Forensic Science International 97 (1998) Table 5 x - and G-statistic comparisons of the Egyptian, Yeminian, Italian and Austrian populations Populations CSF1PO P D1S39 P Egyptians Yemenians x x * G G* Egyptians Italians x x * G G * Egyptians Austrians x x G G Yemenians Italians x x * G G* * Yemenians Austrians x x * G G Italians Austrians x x G G *Statisticaly significant differences. Acknowledgements The authors are indebted to M. Hochmeister and Ch. Gehrig, Berne, and to S. Schweitzer, I. Wolfbeis and R. Frizberg for valuable support. This study was supported by grant 653/1 from the Jubilee Fund of the Austrian National Bank to M. Klintschar. References [1] A. Edwards, A. Civitello, H.A. Hammond, T.C. Caskey, DNA typing and genetic mapping with trimeric and tetrameric tandem repeats, Am. J. Hum. Genet. 49 (1991) [] 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] M.V. Lareu, C. Pestoni, M. Schurenkamp, S. Rand, B. Brinkmann, A. Carracedo, A highly variable STR at the D1S391 locus, Int. J. Legal Med. 109, [5] H.A. Hammond, L. Jin, Y. Zhong, C.T. Caskey, Evaluation of 13 short tandem repeat loci for use in personal identification applications, Am. J. Hum. Genet. 55 (1994) [6] 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) (in press). [7] K. Yoshida, K. Sekiguchi, K. Kasai, H. Sato, S. Seta, G.F. Sensabaugh, Evaluation of new primers for CSF1PO, Int. J. Legal Med. 110 (1997) [8] P. Wiegand, B. Budowle, S. Rand, B. Brinkmann, Forensic validation of the STR systems SE33 and TC11, Int. J. Legal Med. 105 (1993) [9] F. Sanger, S. Nicklen, A.R. Coulson, DNA sequencing with chain terminating inhibitors, Proc. Natl. Acad. Sci. USA 74 (1977) [10] 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) [11] R. Fisher, Standard calculations for evaluating a blood group system, Heredity 5 (1951)

9 M. Klintschar et al. / Forensic Science International 97 (1998) [1] National Research Council, The Evaluation of Forensic DNA Evidence, National Academy Press, Washington, DC, [13] 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)