Forensic Science International101 (1999) 203–208

Italian population data on two new short tandemrepeat loci: D6S477 and D19S433

a , a a*Luciano Garofano , Marco Pizzamiglio , Giovanni Paolo Bizzaro ,a a bFrancesco Donato , Matteo Rossetti , Bruce Budowle

aReparto Carabinieri Investigazioni Scientifiche, Parco Ducale 3, 43100 Parma, ItalybLaboratory Division, Forensic Science Research and Training Center, FBI Academy, Quantico, VA, USA

Received 21 January 1999; received in revised form 15 March 1999; accepted 16 March 1999

Abstract

A population study on two new short tandem repeat (STR) loci D6S477 and D19S433 wasperformed on 214 unrelated Italian Caucasians. The DNA was amplified by PCR and separation

Eand detection of the amplified STR fragments were carried out by use of a PE/ABD PRISM 377DNA sequencer 377 automated system (Applied Biosystems Division /Perkin Elmer). Both locimeet Hardy–Weinberg expectations. There is no evidence for departures from expectationsbetween the two loci. The combined probability of discrimination and probability of exclusion forthe two STR loci are 0.997161 and 0.883183, respectively. The results demonstrate that these locican be useful for human identification in forensic cases in Italy. 1999 Elsevier Science IrelandLtd. 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 D6S477 and D19S433 in a population sample of 214 unrelated Italianindividuals.

*Corresponding author. Tel.: 139-521-231-507; 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 )00030-4

204 L. Garofano et al. / Forensic Science International 101 (1999) 203 –208

2. Materials and methods

2.1. Sample preparation

Whole blood samples from unrelated Italians were drawn in EDTA vacutainer tubes.Our survey comprises 214 donors sampled from 20 different regions in Italy. Approxi-mately 150–200-ml blood samples were placed onto cotton cloth and allowed to air dry.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. [1] andBudowle et al. [2].

2.2. PCR amplification

Amplification by PCR of the STR loci was performed using the SGM Plus STRsystem kit (Perkin Elmer, Foster City, CA), according to the manufacturer’s recom-mendations. 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 (36Ecm well-to-read) on a PE/ABD PRISM 377 DNA sequencer 377 automated system

(Applied Biosystems Division /Perkin Elmer, Foster City, CA). The length of theamplified DNA fragments was determined based on the internal lane standard ROX-400HD (Perkin Elmer, Foster City, CA). Allele designations were made using GenescanPCR Analysis software (Genescan Analysis 2.1.1) and by comparison with allelicladders 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 ofexpected heterozygosity were computed as described by Edwards et al. [3]. Possibledivergence from Hardy–Weinberg expectations (HWE) was tested by calculating theunbiased estimate of the expected homozygote /heterozygote frequencies [4–7], thelikelihood ratio test [3,5,8] and the exact test [9]. An interclass correlation criterion(Karlin test) [10] for two locus associations was used for detecting disequilibriumbetween STR loci. The power of exclusion (PE) was calculated according to Garber andMorris [11]. The power of discrimination (PD) was calculated according to Fisher [12].Analyses were facilitated using computer programs kindly provided by Giovanni PaoloBizzaro (Reparto Carabinieri Investigazioni Scientifiche, Parma, Italy) and Dr RanajitChakraborty (University of Texas School of Biomedical Sciences, Houston, TX, USA)

L. Garofano et al. / Forensic Science International 101 (1999) 203 –208 205

Table 1Distribution of genotype counts and frequencies for D6S477 and D19S433 STR loci in 214 unrelated Italians

D6S477 D19S433

Genotype n Freq. Genotype n Freq.

12.2–15 1 0.005 11–13 1 0.00512.2–16 1 0.005 11–14 1 0.00512.2–18 4 0.019 11–14.2 1 0.00513–16 1 0.005 11–15.2 2 0.00913.2–13.2 2 0.009 12–12 3 0.01413.2–14.2 1 0.005 12–13 14 0.06513.2–15 3 0.014 12–14 14 0.06513.2–15.2 3 0.014 12–14.2 1 0.00513.2–16 11 0.051 12–15 8 0.03713.2–17 3 0.014 12–15.2 4 0.01913.2–18 11 0.051 12–16.2 1 0.00513.2–19 4 0.019 12–17.2 1 0.00514–16 1 0.005 12.2–14 1 0.00514–17 2 0.009 13–13 13 0.06114–18 4 0.019 13–13.2 1 0.00514–19 3 0.014 13–14 29 0.13614.2–16 1 0.005 13–14.2 4 0.01914.2–17 2 0.009 13–15 11 0.05114.2–18 3 0.014 13–15.2 9 0.04214.2–19 1 0.005 13–16 6 0.02814.2–20 1 0.005 13–16.2 1 0.00515–15.2 1 0.005 13–17 1 0.00515–16 4 0.019 13.2–14 1 0.00515–16.2 1 0.005 13.2–15.2 1 0.00515–17 2 0.009 14–14 23 0.10715–18 9 0.042 14–14.2 5 0.02315–19 6 0.028 14–15 17 0.07915–20 1 0.005 14–15.2 9 0.04215.2–16 1 0.005 14–16 5 0.02315.2–17 2 0.009 14–16.2 1 0.00515.2–18 4 0.019 14–17 1 0.00515.2–19 1 0.005 14–17.2 1 0.00516–16 11 0.051 14.2–14.2 1 0.00516–16.2 1 0.005 14.2–15 4 0.01916–17 4 0.019 14.2–15.2 2 0.00916–18 23 0.107 14.2–17 1 0.00516–19 13 0.061 15–15 4 0.01917–17 7 0.033 15–15.2 2 0.00917–18 10 0.047 15–16.2 2 0.00917–19 6 0.028 15–17.2 1 0.00517–20 2 0.009 15.2–15.2 2 0.00918–18 11 0.051 15.2–16 2 0.00918–19 20 0.093 15.2–16.2 2 0.00918–20 2 0.00919–19 9 0.042

206 L. Garofano et al. / Forensic Science International 101 (1999) 203 –208

Table 2Distribution of allele counts and frequencies for D6S477 and D19S433 STR loci in 214 unrelated Italians

D6S477 D19S433

Allele n Freq. Allele n Freq.

12.2 6 0.014 11 5 0.01213 1 0.002 12 49 0.11413.2 40 0.094 12.2 1 0.00214 10 0.023 13 103 0.24114.2 9 0.021 13.2 3 0.00715 28 0.065 14 131 0.30615.2 12 0.028 14.2 20 0.04716 83 0.194 15 53 0.12416.2 2 0.005 15.2 37 0.08717 47 0.110 16 13 0.03018 112 0.262 16.2 7 0.01619 72 0.168 17 3 0.00720 6 0.014 17.2 3 0.007

3. Result and discussion

To the best of our knowledge, this is the first published report on the STR lociD6S477 and D19S433 in an Italian sample population. Both loci demonstrate a highdegree of heterozygosity (82.3 and 78.5% for D6S477 and D19S433, respectively).There were 45 and 43 different genotypes observed for the D6S477 and D19S433 loci,respectively. Only the genotypes D6S477 16–18 and D19S433 13–14 and D19S43314–14 exceed an observed frequency of 0.10 (Table 1). Thirteen alleles were observedat each locus. For both loci, consecutive alleles differ in size by either a tetranucleotiderepeat unit or by only two bases. The distribution of observed allelic frequencies for thetwo loci are shown in Table 2. Both loci meet HWE (Table 3). The power ofdiscrimination and the probability of exclusion for our Italian caucasian database arelisted in Table 4.

A pairwise interclass correlation test for the two loci did not detect any departure fromexpectations. Garofano et al. [13] previously typed these same Italian samples for 13other STR loci, which are the core CODIS loci. Therefore, the pairwise interclasscorrelation test was performed with those loci also. There were only six departures out

Table 3Summary of Hardy–Weinberg tests

D6S477 D19S433

Observed homozygosity 18.7% 21.5%Expected homozygosity (unbiased) 16.0% 18.9%Homozygosity test (probability values) 0.277 0.338Likelihood test (probability values) 0.459 0.931Exact test (probability values) 0.473 0.850

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

D6S477 D19S433

Probability of discrimination (observed data) 0.953 0.939Probability of discrimination (expected data) 0.954 0.939Probability of discrimination combined (observed data) 0.997Probability of exclusion 0.681 0.632Probability of exclusion (combined) 0.883

of a total of 105 tests, which is close to expectations of 5% (6/10555.7%). The onlynewly observed pairwise departure for the 15 STR loci was between the loci vWA andD19S433 (P50.007). After Bonferroni correction, none of the six departures weresignificant. Thus, the use of the product rule to estimate the rarity of a multiple locusSTR profile is valid.

In conclusion, the use of the two loci D6S477 and D19S433 in multiplex systemsenables forensic scientists to obtain a high degree of discrimination for forensic analyses,paternity testing, and linkage studies. In addition, the size of the amplicons for these lociranges from 100–150 base pairs. Thus, typing success is extremely good even withsubstantially degraded DNA samples.

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