Forensic Science International105 (1999) 131–136

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Italian population data on two new short tandemrepeat loci: D2S1338 and Penta E

a , a a*Luciano Garofano , Marco Pizzamiglio , Francesco Donato ,a aFulvio Biondi , Matteo Rossetti , Bruce Budowle (Senior Scientist for

bBiology)aReparto Carabinieri Investigazioni Scientifiche, Parco Ducale 3, 43100 Parma, Italy

bLaboratory Division, Forensic Science Research and Training Center, FBI Academy, Quantico, VA, USA

Received 14 June 1999; received in revised form 28 August 1999; accepted 3 September 1999

Abstract

A population study on two new short tandem repeat (STR) loci D2S1338 (a tetranucleotiderepeat) and Penta E (a pentanucleotide repeat) was performed on 208 unrelated Italian Caucasians.The DNA was amplified by polymerase chain reaction (PCR) and separation and detection of theamplified STR fragments were carried out by use of a PE/ABD PRISM 377 DNA Sequencer 377automated system (Applied Biosystems Division /Perkin-Elmer). Both loci meet Hardy–Weinbergexpectations. There is no evidence for departures from expectations between the two loci. Thecombined Probability of Discrimination and Probability of Exclusion for the two STR loci are0.999155 and 0.944925, respectively. The results demonstrate that these two regions can be usefulfor differentiating among individuals, particularly in concert with other STR loci. 1999Elsevier Science Ireland Ltd. All rights reserved.

Keywords: DNA; STR; Population studies; Forensic science

1. Introduction

This paper presents allele /genotype frequency data on the two short tandem repeat(STR) loci D2S1338 (a tetranucleotide repeat) and Penta E (a pentanucleotide) in apopulation sample of 208 unrelated Italian individuals.

*Corresponding author. Tel. / fax: 139-521-231-507.E-mail address: scienarma@provincia.parma.it (L. Garofano)

0379-0738/99/$ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved.PI I : S0379-0738( 99 )00132-2

132 L. Garofano et al. / Forensic Science International 105 (1999) 131 –136

2. Materials and methods

2.1. Sample preparation

Whole blood samples from unrelated Italians were drawn in EDTA vacutainer tubes.Our survey comprises 208 blood donors sampled from many different regions in Italywith respect to the population distribution. Approximately 150–200 ml blood sampleswere placed onto cotton cloth and allowed to air dry. A 2 mm32 mm portion of thestain was used for analysis. The DNA was extracted organically and quantified using theslot-blot procedure described by Waye et al. [1] and Budowle et al. [2]. The samepopulation sample was typed at 15 other STR loci, which are the core CODIS loci plustwo additional STR loci, D6S433 and D19S477, Garofano and co-workers [3,4].

2.2. Polymerase chain reaction (PCR) amplification

Amplification by PCR of the STR loci was performed according to the manufacturer’srecommendations, using the final version of the SGM Plus STR system kit (Perkin-Elmer, Foster City, CA, USA) and the PowerPlex 2.2 System Prototype kit, kindlyprovided by Perkin-Elmer and Promega (Madison, WI, USA), respectively. The PCRwas performed in a Perkin-Elmer 2400 thermal cycler.

2.3. STR typing

Electrophoresis was carried out on 5% polyacrylamide denaturing sequencing gels (36cm well-to-read) on a PE/ABD PRISM 377 DNA Sequencer 377 automated system(Applied Biosystems Division /Perkin-Elmer). The length of the amplified DNAfragments was determined based on the internal lane standard ROX-400 HD (Perkin-Elmer) and CXR 60-500 (Promega) for the first and the second kit, respectively. Alleledesignations were made using Genescan PCR analysis software (Genescan Analysis2.1.1) by comparison with allelic ladders that were on each gel, followed by a secondsize allele calling performed with the Genotyper DNA fragment analysis software(Genotyper Analysis 2.0). Statistical analyses were performed as previously described[3,4].

3. Results and discussion

To the best of our knowledge, this is the first published report on the STR lociD2S1338 and Penta E (located on 15q) in an Italian sample population. The degree ofstutter bands (i.e., number observed and peak height /area percentage compared with theallelic product) observed during typing of the D2S1338 locus was below 5% of the trueallele, and most samples never showed stutter bands for alleles at the Penta E locus.Fourteen and 16 alleles were observed at each locus, respectively. For either locus,consecutive alleles differ in size by a tetranucleotide or a pentanucleotide repeat unit.

L. Garofano et al. / Forensic Science International 105 (1999) 131 –136 133

Table 1Distribution of allele counts and frequencies for D2S1338 and Penta E STR loci in 208 unrelated Italians

D2S1338 Penta E

Allele n Frequency Allele n Frequency

15 1 0.002 5 25 0.0616 28 0.067 7 57 0.13717 96 0.23 8 14 0.03318 35 0.084 9 9 0.02119 45 0.108 10 36 0.08620 53 0.127 11 60 0.14421 11 0.026 12 71 017022 12 0.028 13 62 0.14923 43 0.103 14 13 0.03124 44 0.105 15 15 0.03625 39 0.093 16 19 0.04526 7 0.016 17 12 0.02827 1 0.002 18 10 0.02428 1 0.002 19 6 0.014

20 6 0.01421 1 0.002

The distribution of observed allelic frequencies for the two loci are shown in Table 1.There were 56 and 72 different genotypes observed for the D2S1338 and Penta E loci,respectively. These numbers of genotypes are noticeably higher than previously reportedfor the D21S11, D18S51, FGA, D6S477 and D19S433 loci [3,4]. These range from 45to 53 observed genotypes in similarly sized databases. With so many genotypes in only208 individuals, both loci demonstrate a high degree of heterozygosity (84.13% and89.90% for D2S1338 and Penta E, respectively). In fact, only the five genotypesD2S1338 17-17, D2S1338 17-20, Penta E 7-13, Penta E 11-13 and Penta E 12-13exceed an observed frequency of 0.05 (Table ). Both loci meet HWE (Table 3). ThePower of Discrimination and the Probability of Exclusion for our Italian CaucasianDatabase are listed in Table 4.

A pairwise interclass correlation test for the two loci did not detect any departure fromexpectations. Garofano and co-workers [3,4] previously typed these same Italian samplesfor 13 other STR loci, which are the core CODIS loci, and for two additional STR loci,D6S433 and D19S477. Therefore, the pairwise interclass correlation test was performedwith those loci also. There were only six departures out of a total of 136 tests, which isclose to expectations of 5% (6/13654.41%). The only newly observed pairwisedeparture for the 17 STR loci was between the loci D5S818 and D2S1338 (P50.002).Furthermore, after Bonferroni correction, none of the six departures were significant.Thus, the use of the product rule to estimate the rarity of a multiple locus STR profile isvalid.

In conclusion, the use of the two loci D2S1338 and Penta E (and certainly in concertwith other STR loci) in multiplex systems enables forensic scientists to obtain a highdegree of discrimination for forensic analyses, paternity testing and linkage studies.

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Table 2Distribution of genotype counts and frequencies for D2S1338 and Penta E STR loci in 208 unrelated Italians

D2S1338 Penta E

Genotype n Frequency Genotype n Frequency

15-17 1 0.0048 5-5 1 0.004816-16 2 0.0096 5-7 2 0.009616-17 4 0.0192 5-8 1 0.004816-18 2 0.0096 5-10 3 0.014416-19 6 0.0288 5-11 5 0.024016-20 2 0.0096 5-12 4 0.019216-21 1 0.0048 5-13 3 0.014416-23 3 0.0144 5-15 2 0.009616-24 1 0.0048 5-17 1 0.004816-25 5 0.0240 5-18 1 0.004817-17 15 0.0721 5-20 1 0.004817-18 7 0.0336 7-7 4 0.019217-19 7 0.0336 7-9 1 0.004817-20 16 0.0769 7-10 4 0.014417-21 1 0.0048 7-11 6 0.028817-22 8 0.0384 7-12 10 0.048017-23 6 0.0288 7-13 14 0.067317-24 10 0.0480 7-14 3 0.014417-25 5 0.0240 7-15 1 0.004817-26 1 0.0048 7-16 4 0.019218-18 2 0.0096 7-18 3 0.014418-19 6 0.0288 7-20 1 0.004818-20 3 0.0144 8-8 1 0.004818-21 2 0.0096 8-10 1 0.004818-22 1 0.0048 8-11 2 0.009618-23 4 0.0192 8-14 1 0.004818-24 3 0.0144 8-15 2 0.009618-25 2 0.0096 8-16 3 0.014418-26 1 0.0048 8-17 1 0.004819-19 3 0.0144 8-21 1 0.004819-20 6 0.0288 9-10 1 0.004819-22 1 0.0048 9-11 1 0.004819-23 4 0.0192 9-12 3 0.014419-24 4 0.0192 9-16 1 0.004819-25 5 0.0240 9-18 1 0.004820-20 3 0.0144 9-19 1 0.004820-21 1 0.0048 10-10 3 0.014420-23 8 0.0384 10-11 4 0.019220-24 5 0.0240 10-12 6 0.028820-25 5 0.0240 10-13 5 0.024020-28 1 0.0048 10-14 1 0.004821-23 3 0.0144 10-16 1 0.004821-24 1 0.0048 10-17 2 0.009621-25 1 0.0048 10-18 1 0.004821-26 1 0.0048 10-19 1 0.0048

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Table 2. Continued

D2S1338 Penta E

Genotype n Frequency Genotype n Frequency

22-24 1 0.0048 11-11 4 0.019222-25 1 0.0048 11-12 9 0.043223-23 1 0.0048 11-13 11 0.052823-24 4 0.0192 11-14 3 0.014423-25 6 0.0288 11-15 5 0.024023-26 3 0.0144 11-16 2 0.009624-24 5 0.0240 11-17 1 0.004824-25 4 0.0192 11-18 1 0.004824-26 1 0.0048 11-20 2 0.009625-25 2 0.0096 12-12 6 0.028825-27 1 0.0048 12-13 14 0.0673

12-15 1 0.004812-16 4 0.019212-17 4 0.019212-19 3 0.014412-20 1 0.004813-13 2 0.009613-14 4 0.019213-15 2 0.009613-16 2 0.009613-17 2 0.009613-19 1 0.004814-15 1 0.004815-16 1 0.004816-18 1 0.004817-18 1 0.004818-20 1 0.0048

Table 3Summary of Hardy–Weinberg tests

D2S1338 Penta E

Observed homozygosity 15.9% 10.1%Expected homozygosity (unbiased) 12.3% 10.8%Homozygosity test (probability values) 0.120 0.755Likelihood test (probability values) 0.540 0.273Exact test (probability values) 0.405 0.245

Table 4Power of discrimination and probability of exclusion of the two loci

D2S1338 Penta E

Probability of discrimination (observed data) 0.968 0.972Probability of discrimination (expected data) 0.972 0.978Probability of discrimination combined (observed data) 0.999Probability of exclusion 0.750 0.779Probability of exclusion (combined) 0.944

136 L. Garofano et al. / Forensic Science International 105 (1999) 131 –136

References

[1] J.S. Waye, L. Presley, B. Budowle, G.G. Shuttle, R.M. Fourney, A simple method for quantifying humangenomic DNA in forensic specimen extracts, Biotechniques 7 (1989) 852–855.

[2] B. Budowle, F.S. Baechtel, C.T. Comey, A.M. Giusti, L. Klevan, Simple protocols for typing forensicbiological evidence: chemiluminescent detection for human DNA quantitation and RFLP analyses andmanual typing of PCR amplified polymorphisms, Electrophoresis 16 (1995) 1559–1567.

[3] L. Garofano, M. Pizzamiglio, C. Vecchio, G. Lago, T. Floris, G. D’Errico, G. Brembilla, A. Romano, B.Budowle, Italian population data on thirteen short tandem repeat loci: HUMTHO1, D21S11, D18S51,HUMVWFA31, HUMFIBRA, D8S1179, HUMTPOX, HUMCSF1PO, D16S539, D7S820, D13S317D5S818, D3S1358, Forensic Sci. Int. 97 (1998) 53–60.

[4] L. Garofano, M. Pizzamiglio, G.P. Bizzaro, F. Donato, M. Rossetti, B. Budowle, Italian population dataon two new short tandem repeat loci: D6S477 and D19S433, Forensic Sci. Int. 101 (1999) 203–208.