Forensic Science International97 (1998) 53–60

Italian population data on thirteen short tandemrepeat loci: HUMTH01, D21S11, D18S51,

HUMVWFA31, HUMFIBRA, D8S1179, HUMTPOX,HUMCSF1PO, D16S539, D7S820, D13S317,

D5S818, D3S1358

a , a a*Luciano Garofano , Marco Pizzamiglio , Cesare Vecchio ,a a aGianpietro Lago , Tiziano Floris , Giancarlo D’Errico ,a a bGiorgio Brembilla , Alessandro Romano , Bruce Budowle

aServizio Carabinieri Investigazioni Scientifiche, Via Porco Ducale 3, 53100 Parma, ItalybProgram Manager for DNA Research, Forensic Science Research and Training Center, FBI Academy,

Quantico, VA, USA

Received 24 February 1998; received in revised form 13 August 1998; accepted 17 August 1998

Abstract

A population study on thirteen short tandem repeat (STR) loci was performed on 223 unrelatedItalian Caucasians. The DNA was amplified by PCR. Separation and detection of the amplifiedSTR fragments was carried out by use of 377 automated system (Applied Biosystems Division /Perkin Elmer). All loci meet Hardy–Wienberg expectations, and the data show only fivedepartures out of seventy-eight pairwise locus tests which is close to expectations of 5%(5/7856.4%). When correcting for multiple tests, there is little evidence for departures fromexpectations between loci. The combined Power of Exclusion for the thirteen STR loci is0.99999270. The results demonstrate that these loci will be very useful for human identification inforensic cases in Italy. 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: DNA; Short tandem repeat; Population studies; Forensic science

*Corresponding author.

0379-0738/98/$ – see front matter 1998 Elsevier Science Ireland Ltd. All rights reserved.PI I : S0379-0738( 98 )00132-7

54 L. Garofano et al. / Forensic Science International 97 (1998) 53 –60

1. Introduction

Many forensic laboratories worldwide are evaluating and implementing highlypolymorphic DNA loci, whose polymorphism derive from short tandem repeated (STR)core sequences. Simultaneous amplification and subsequent typing of a number of theseSTR loci have significantly enhanced the capabilities of forensic scientists to analyzeDNA derived from biological specimens.

The current study investigated Italian population data for 13 STR loci: HUMTH01,D21S11, D18S51, HUMVWFA31, HUMFIBRA, D8S1179, HUMTPOX,HUMCSF1PO, D16S539, D7S820, D13S317, D5S818, D3S1358 [1–9]. These loci alsoform the core PCR-based genetics markers for the US Combined DNA Index System(CODIS). The data obtained demonstrate that STR loci are very useful for humanidentification in forensic case-work in Italy.

2. Materials and methods

2.1. Sample preparation

Whole blood samples from unrelated Italians were drawn into EDTA vacutainer tubes.Our survey comprises 223 donors sampled from 20 different regions in Italy. Approxi-mately 150–200 ml blood samples were placed onto a cotton cloth and allowed to airdry. A 232 mm portion of the stain was used for analysis. The DNA was extractedorganically and quantified using the slot-blot procedure described by Waye et al. [10].

2.2. PCR amplification

Amplification by PCR of the STR loci was performed using the Profiler STR systemskit (Perkin Elmer), Powerplex STR systems kit (Promega) and Second GenerationMultiplex (Forensic Science Service), according to the manufacturers’ recommendations.The PCR was 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 377 automated system (Applied Biosystems Division /PerkinElmer). The length of the amplified DNA fragments was determined based on internallane standard Genescan-500 ROX (Perkin Elmer). Allele designations were made usingGenescan PCR Analysis software (GENESCAN ANALYSIS 2.0.1) and by comparison withallelic ladders that were on each gel.

2.4. Statistical analysis

The frequency of each allele for each locus was calculated from the numbers of eachgenotype obtained in the sample set (i.e., the gene count method). Unbiased estimates of

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Table 1Distribution of allele frequencies counts of eleven STR loci in 223 unrelated Italians

STR HUMTH01 HUMTPOX HUMCSF1PO D16S539 D7S820 D13S317 D5S818 D18S51 HUMVWFA3-

1 D3S1358 D8S1179

Allele n Freq. n Freq. n Freq. n Freq. n Freq. n Freq. n Freq. n Freq. n Freq. n Freq. n Freq.

6 124 0.278 2 0.004

7 59 0.132 2 0.004 1 0.002 11 0.025

8 49 0.11 237 0.531 4 0.009 13 0.029 86 0.193 57 0.128 2 0.004 12 0.027

8.3 1 0.002

9 90 0.202 56 0.126 14 0.031 53 0.119 39 0.087 37 0.083 14 0.031 5 0.011

9.2 1 0.002

9.3 120 0.269

10 2 0.004 25 0.056 114 0.256 24 0.054 122 0.274 24 0.054 31 0.07 3 0.007 38 0.085

11 11 0.002 113 0.253 136 0.305 128 0.287 111 0.249 130 0.291 159 0.356 12 0.027 25 0.056

12 11 0.025 145 0.325 133 0.298 63 0.141 142 0.318 152 0.341 84 0.188 37 0.083

13 25 0.056 83 0.186 11 0.025 40 0.09 78 0.175 82 0.184 1 0.002 2 0.004 134 0.3

14 7 0.016 12 0.027 3 0.007 14 0.031 6 0.013 64 0.143 37 0.083 36 0.081 109 0.244

15 2 0.004 3 0.007 51 0.114 49 0.11 128 0.287 59 0.132

16 53 0.119 104 0.233 104 0.233 23 0.052

17 42 0.094 111 0.249 109 0.244 4 0.009

18 30 0.067 95 0.213 62 0.139

19 9 0.02 42 0.094 5 0.011

20 12 0.027 7 0.016

21 2 0.004

22 1 0.002

23 1 0.002

56 L. Garofano et al. / Forensic Science International 97 (1998) 53 –60

Table 2Distribution of allele frequencies counts of D21S11 locus in 223 unrelated Italians

Allele Nunber Frequency

24.2 1 0.00226 1 0.00227 12 0.02728 65 0.14628.2 1 0.00229 108 0.24230 111 0.24930.2 8 0.01831 21 0.04731.2 48 0.10832 6 0.01332.2 49 0.1133.2 14 0.03134 1 0.002

expected heterozygosity were computed as described by Edwards et al. [11]. Possibledivergence from Hardy–Weinberg expectations (HWE) was tested by calculating theunbiased estimate of the expected homozygote /heterozygote frequencies [12–15], thelikelihood ratio test [11,13,16] and the exact test [17]. An interclass correlation criterion(Karlin test) [18] for two locus associations was used for detecting disequilibriumbetween STR loci. The Power of Exclusion (PE) was calculated according to Garber andMorris [19]. The Power of Discrimination (PD) was calculated according to Fisher [20].Analyses were facilitated using a computer program kindly provided by Dr. RanajitChakraborty (Houston, TX, USA).

Table 3Distribution of allele frequencies counts of HUMFIBRA locus in 223 unrelated Italians

Allele Number Frequency

18 3 0.00719 27 0.06120 57 0.12821 85 0.19121.2 2 0.00422 68 0.015222.2 3 0.00723 66 0.14823.2 1 0.00224 60 0.13524.2 3 0.00725 50 0.11226 16 0.03627 3 0.00728 2 0.004

L. Garofano et al. / Forensic Science International 97 (1998) 53 –60 57

Table 4Summary of Hardy–Weimberg tests

1 THO1Observed homozygosity 26.5%Expected homozygosity (unbiased) 21.8%

aHomozygosity test 0.094aLikelihood test 0.588

aExact test 0.514

2 D21S11Observed homozygosity 22.4%Expected homozygosity (unbiased) 16.8%

aHomozygosity test 0.025aLikelihood test 0.282

aExact test 0.059

3 D18S51Observed homozygosity 12.1%Expected homozygosity (unbiased) 13.0%

aHomozygosity test 0.678aLikelihood test 0.926

aExact test 0.888

4 vWAObserved homozygosity 18.4%Expected homozygosity (unbiased) 18.8%

aHomozygosity test 0.876aLikelihood test 0.518

aExact test 0.498

5 FGAObserved homozygosity 13.5%Expected homozygosity (unbiased) 13.2%

aHomozygosity test 0.900aLikelihood test 0.959

aExact test 0.970

6 D8S1179Observed homozygosity 17.9%Expected homozygosity (unbiased) 18.7%

aHomozygosity test 0.783aLikelihood test 0.668

aExact test 0.496

7 TPOXObserved homozygosity 35.9%Expected homozygosity (unbiased) 36.5%

aHomozygosity test 0.853aLikelihood test 0.412

aExact test 0.697

8 CSF1POObserved homozygosity 28.3%Expected homozygosity (unbiased) 26.7%

aHomozygosity test 0.597aLikelihood test 0.447

aExact test 0.568

58 L. Garofano et al. / Forensic Science International 97 (1998) 53 –60

Table 4. Continued

9 D16S539Observed homozygosity 25.6%Expected homozygosity (unbiased) 22.3%

aHomozygosity test 0.239aLikelihood test 0.618

aExact test 0.640

10 D7S820Observed homozygosity 22.4%Expected homozygosity (unbiased) 20.1%

aHomozygosity test 0.387aLikelihood test 0.763

aExact test 0.65311 D13S317Observed homozygosity 23.3%Expected homozygosity (unbiased) 22.0%

aHomozygosity test 0.628aLikelihood test 0.789

aExact test 0.764

12 D5S818Observed homozygosity 30.5%Expected homozygosity (unbiased) 27.8%

aHomozygosity test 0.374aLikelihood test 0.271

aExact test 0.24513 D3S1358Observed homozygosity 24.2%Expected homozygosity (unbiased) 22.1%

aHomozygosity test 0.440aLikelihood test 0.814

aExact test 0.755a These values are probability values.

3. Results and discussion

The distribution of observed allelic frequencies in our Italian population sample forthe loci HUMTH01, D21S11, D18S51, HUMVWFA31, HUMFIBRA, D8S1179,HUMTPOX, HUMCSF1PO, D16S539, D7S820, D13S317, D5S818, D3S1358 areshown in Tables 1–3. All thirteen loci meet HWE (Table 4).

Pairwise interclass correlation tests were performed for all possible two-locuscombinations. Only five departures out of 78 pairwise comparisons were detected, whichis close to expectations of 5% (5/7856.4%). There was no detectable departure fromindependence for the loci CSF1PO and D5S818, which both reside on chromosome 5 at5q33.3-34 and 5q23.3- 32, respectively. When correcting for multiple tests, there is littleevidence for departures from expectations.

The PD and the PE for our Italian Caucasian database are listed in Table 5. Thecombination of the thirteen STR loci is extremely discriminating; the maximum

211frequency for a combined 13-locus genotype frequency is 1.52?10 .

L. Garofano et al. / Forensic Science International 97 (1998) 53 –60 59

Table 5Power of discrimination and probability of exclusion of the 13 loci

a bLocus PD PD PE

TH01 0.91978524 0.91651493 0.56712407D21S11 0.94640954 0.95026632 0.66630064D18S51 0.96406523 0.96824276 0.73332154VWA 0.93414306 0.93710698 0.62393151FGA 0.96603591 0.96738496 0.72958774D8S1179 0.93772246 0.94117262 0.63776403TPOX 0.81924028 0.81602129 0.39109541CSF1PO 0.88206077 0.87993972 0.48877024D16S539 0.91580366 0.91528505 0.56656438D7S820 0.92666251 0.92901216 0.60113161D13S317 0.91982545 0.91976619 0.57928376D5S818 0.87240845 0.87391869 0.47986244D3S1358 0.91230469 0.91475118 0.56312631a PD calculated using observed data.b PD calculated using expected data.

The Italian population allele frequency data for these thirteen PCR-based loci do notdiffer substantially from other Caucasian data for the same loci (data not shown).

In conclusion, the use of multiplex systems enables forensic scientists the ability toanalyze a large number of genetic markers relatively easily and to obtain a high degreeof discrimination for forensic analyses, paternity testing, and linkage studies.

With regard to the future possibility of setting up an Italian National DNA Databaseto provide investigative leads in a variety of violent crimes a selection of core STR lociwill be needed. These 13 STR loci reported in this study are the same set of loci used fordatabasing in the US CODIS National Felon Database. The same 13 loci, or a subset,should be considered.

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