P-073

Investigation of single nucleotide polymorphisms associated with ethnicity

Daniel R, Walsh SJ, Piper A

Centre for Forensic Science, University of Technology Sydney, NSW, Australia

Single nucleotide polymorphisms (SNPs) have been widely investigated as markers in human genetic studies ranging from comparative population variation to disease linkage studies. As a result of the low mutation rate of SNPs, they are also considered to be useful markers of biogeographical ancestry.  

Recently, in forensic genetics, attention has returned to SNPs, particularly due to their association with ethnicity and physical appearance.  Such developments are potentially of great benefit to forensic investigators who are unable to match crime scene samples to database profiles. Unfortunately, physical characteristics are usually polygenic traits that are influenced by a number of genes and, in some cases, by environmental factors. However, many genes contain SNPs that are highly polymorphic in different ethnic groups. 

As the first component of an investigation into the utility of SNPs as markers of ethnicity or appearance, we have developed the initial stage of an ethnicity multiplex. From an extensive literature survey, six autosomal SNPs were selected on the basis of strong associations with particular ethnic groups.  The SNPs were specifically chosen for their potential to distinguish the major ethnic groups in the Australian population.

The ABI Prism® SNaPshot^TM Multiplex kit (Applied Biosystems) based on single base extension of an unlabelled oligonucleotide (extension) primer was utilised for the development of the multiplex. Primer concentration optimisation experiments were conducted prior to genotyping over 200 hundred buccal swab samples collected from participants representing a cross-section of the Sydney community.  Allele and genotype frequency data has been used to assess the usefulness of the multiplex as a predictor of ethnicity. Results have been cross-compared to genealogical information, self-declared by the participant over three generations.

The results from this preliminary research are promising in that distinct genotype distributions are evident among the predominant populations under study. Statistical analysis has been applied to empirically evaluate the observed trends.

DNA phenotyping is as yet in its infancy.  The development of rapid and robust tests suitable for identification of phenotypes specific to the Australian population will provide a valuable intelligence tool for forensic investigators.  

Contact: Runa.Daniel@student.uts.edu.au 

P-074

Artificial blood chimerism due to graft-versus-host-disease after liver transplantation

Dauber EM¹, Müller CJ², Schöniger-Hekele M.², Dorner G¹, Wenda S¹, F.Mühlbacher³, Mayr WR¹

¹Division of Blood Group Serology, Medical University of Vienna, Austria

²Division of Gastroenterology and Hepatology, Medical University of Vienna, Austria

³Division of Surgery, Medical University of Vienna, Austria

After transplantation of solid organs a small amount of the donor’s cells can be detected in the recipient’s blood which usually indicates a good prognosis for organ survival in liver transplantation. But in case of graft-versus-host disease (GVHD), however, donor’s cells proliferate and produce an immune response against the recipient. In this case a higher degree of chimerism is observed.

Two months after liver transplantation a recipient developed diarrhea and leucopenia, which were interpreted to be  side effects of therapy with ganciclovir for cytomegalovirus infection. After cessation of ganciclovir, however, no improvement was observed and a blood sample was sent to our laboratory to exclude graft-versus-host disease as a possible reason for his condition.

Multiplex-STR-typing has been carried out applying the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, Foster City, USA). A blood chimerism with a higher percentage of donor’s than recipient’s cells was observed. Two buccal swab samples taken inside from each cheek also showed a mixture of the DNA profiles of donor and recipient. Only the eye brows showed the recipient’s DNA profile itself. Another blood sample was taken 2 weeks later, three days before the patient deceased. This DNA profile was identical with the donor’s profile, which was identified in a sample stored after tissue typing prior to transplantation.

To find out, whether the chimerism could already have been observed in tissue sections of a bone marrow puncture taken on the first onset of clinical symptoms, a singleplex PCR of the ACTBP2 (SE33) locus was carried out: 15% of the nucleated cells derived from the donor. Histopathology had just described hypocellular bone marrow without giving any clues to GVHD. Additionally, material from 21 different biopsies taken during autopsy was investigated. A chimerism was detectable in all samples except the transplanted liver, which only showed the donor’s alleles.

In course of progression of clinical symptoms, the recipient increasingly showed the donor’s DNA profile and his blood sample was found to be identical with the donor at the zenith of graft-versus-host disease. Just his hairs were found to be free of the donor’s DNA genotype and exhibited only his own alleles. Therefore, STR-typing of bone marrow samples should be performed whenever an early stage of graft-versus-host disease is suspected. Hair samples of the recipient and material of the donor, if available, have to be investigated, in order to identify the two cell lines, as the major component does not necessarily represent the recipient’s cell line.

contact: eva-maria.dauber@meduniwien.ac.at

P-075

Two apparent mother/child mismatches due to mispriming at the D3S1358 and the SE33 locus

Dauber EM¹, Parson W², Glock B¹, Mayr WR¹

¹Division of Blood Group Serology, Medical University of Vienna, Austria

²Institute of Legal Medicine, Innsbruck Medical University, Austria

We report two cases of apparent mother/child mismatch due to opposite homozygosity. They were observed at the D3S1358 locus after amplification with the AmpFlSTR IDfiler PCR Amplification Kit (Applied Biosystems, Foster City, USA) amongst 825 meioses and at the SE33 locus after PCR with the original primers published by Polymeropoulos et al. [1] amongst 1219 meioses.

The D3S1358 results were identical with the Geneprint Powerplex 16 System (Promega, Madison, USA). After lowering the annealing temperature in a singleplex PCR at the D3S1358 [2] and the SE33 locus the mendelian inheritance between mother and child was restored in both cases. Therefore a point mutation in the primer binding region had to be supposed.

A PCR with alternative primers lying outside of the primer binding sites of the original oligonucleotides confirmed these results. The alternative amplicons were sequenced and proved a point mutation in the binding site of the original primers. In case of the mother/child mismatch at the SE33 locus the failure of PCR was due a base substitution in the reverse primer region, which was already reported by other authors [3]. A point mutation near the 3’ end of the reverse primer was found to be the reason for non-amplification of the D3S1358 allele, which has not been reported so far.

To overcome the problems of isolated parent/child mismatches due to opposite homozygosity a singleplex PCR with lower annealing temperatures can easily be performed to reestablish mendelian inheritance in case of base exchanges in the primer binding site.

[1] Polymeropoulos et al. 1992 Nucl Acids Res 20(6):1432

[2] Li et al. 1993 Hum Mol Genet 2(8):1327

[3] Hering et al. 2002 Int J Legal Med 116:365-367

contact: eva-maria.dauber@meduniwien.ac.at

P-076

PCR-based diagnosis of cytomegaloviruses in paraffin-embedded heart tissue

Dettmeyer R, Müller J, Poster S, Madea B

Institute of Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany

Introduction. Immunohistochemical and molecular-pathological techniques have improved the diagnosis, but the incidence of virus-induced lethal myocarditis still remains unclear. Studies of myocarditis in adults demonstrated that numerous cases of acute myocarditis can not be diagnosed, according to the Dallas criteria, by traditional hematoxylin-eosin staining of endomyocardial biopsies. Previously, we reported on detection of enteroviruses (EV) including coxsackieviruses B3 (CVB3), parvovirus B19 (PVB19), adenoviruses (AV) and Epstein-Barr virus (EBV). We analysed cytomegalovirus DNA from paraffin-embedded heart tissue with PCR. Therefore, we established a reliable method to isolate DNA from formadehyde-fixed and paraffin-embedded material.

Materials and methods. Postmortem samples were obtained from 70 cases with suspected sudden infant death syndrome (SIDS). Eight myocardial samples were taken from each heart at standardized locations. Viral DNA was extracted from paraffin-embedded myocardial, liver and spleen samples with the Genial First-DNA-Kit (Genial, Troisdorf, Germany). The prerequisite for virus PCR was the amplification of cyclophilin (cyc). To avoid false-positive results due to contamination, negative controls were performed in all experiments. PCR products were sequenced on a ABI 310 sequencer. Sequence comparison was performed by a BLAST search of NCBI Gen-Bank. PCR products were also analysed on polyacrylamide gels.

Results. Cytomegalovirus-DNA was detected in 2 out of 70 cases of suspected SIDS. In all SIDS cases, the myocardial samples revealed no signs of myocarditis according to the Dallas criteria using conventional histologic stainings.

Discussion. Acute myocarditis can be diagnosed by PCR as a rapid method. Given the fact that in endomyocardial biopsies, the detection of cytomegaloviruses would be regarded as a pathological finding, this can be regarded as the cause of death, especially in SIDS cases presenting immunohistological signs of myocarditis in addition

contact: rdettmey@uni-bonn.de

P-077

Y Chromosome Polymorphisms in Argentine Population

Di Lonardo AM, Santapá O, Valente S, Filippini S

Banco Nacional de Datos Genéticos, Unidad Inmunología, Hospital Carlos G. Durand, J. B. Ambrosetti 743 (1405), Buenos Aires, Argentina

Short tandem repeats (STRs) loci are the most informative PCR based genetic markers available to date for attempting to individualize biological material. The full use of DNA typing technology in forensic science has grown up by the development of National DNA databases. That is the reason why today, many efforts are made to build up Y STRs databases for forensic purposes. Knowledge about mutation rates and mutational process of short tandem repeats (STRs), microsatellite loci used in paternity testing and forensic analysis, is crucial for the correct interpretation of genetic profiles. In our study, we analyzed Y Chromosome Polymorphisms for the loci: DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS439, DYS438, in unrelated Argentine individuals, most of them from Buenos Aires. Statistical interpretation of the results let us create a database of our own population, and we also studied paternity cases to discover genetic inconsistencies in father-son biological relationship testing.

Materials and methods: Blood specimens were collected from 301 unrelated males, most of them from Buenos Aires city, and 63 father-son pairs. DNA was extracted by the salting out method (Miller et. al.). Multiplex PCR amplification of six loci: DYS19, DYS385, DYS389II, DYS390, DYS391 and DYS393 was performed using Y-Plex™ 6 (Reliagene Technologies, Inc.) kit, and the PCR amplification of five loci: DYS389I, DYS389II, DYS392, DYS438 and DYS439 was performed using Y-Plex™ 5 (Reliagene Technologies, Inc) kit, according to the user’s manual provided by the manufacturer. The amplified products were detected using an ABI PRISM^® 3100 Genetic Analyzer (PE Applied Biosystems).The results were analyzed using GeneScan Analysis v 3.7 software (PE Applied Biosystems) and the alleles were typed using Genotyper v3.7 software (PE Applied Biosystems). The recommendations of the International Society for Forensics Genetics (ISFG) were followed for typing and interpretation.

Results: A total of 301 male unrelated individuals were analyzed for all 10 Y-STR loci and produced 274 haplotypes, of wich 258 haplotypes were unique, 11 were found in two individuals , 3 were found in three individuals, 1 was found in four individuals and the most common haplotype. DYS19 14, DYS389I 13, DYS389II 29, DYS390 24, DYS391 11, DYS392 13, DYS393 13, DYS385 11/14, DYS438 12, DYS439 12, was found in five individuals. The haplotype diversity calculated from the 10 Y-STR loci was 99.92% and the Genetic Identity:  0.0041. Most frequent haplotypes in our population sample have been compared with the Y-STR Haplotype Reference Database (www.yhrd.org, Institute of Legal Medicine, Medical Faculty, Charité, Humboldt University, Berlin-Germany) considering eight loci for the minimal haplotype and ten loci for the extended haplotype. The study of 63 alleged father-child non-exclusion cases with 15 autosomal STRs performed with Amp FlSTR^® Identifiler™, showed three alleles 14/16/17 at DYS385 locus, in one case. We observed one double mutation displaying two genetic inconsistencies at two different loci: DYS389I and DYS389II, between father (DYS389I: 12, DYS389II: 28) and son (DYS389I:13, DYS389II: 29) with W= 99,9999991 % (15 autosomal STRs). We also found mutational events in two unrelated individuals, three alleles at DYS385 locus: 12/13/14, and a biallelic pattern at DYS19 locus: 15/16.

E-mail: bndg@infovia.com.ar.

P-078

FOUR HIGHLY POLYMORPHIC STR-LOCI AS A “SCREENING TEST” IN PATERNITY CASES

Dorner G, Dauber EM, Wenda S, Glock B, Mayr WR

Division of Blood Group Serology, Medical University of Vienna, Austria

The aim was to design a “screening method” for paternity cases by investigation of 4 loci in a single run. We chose 4 highly polymorphic markers with a high chance of paternity exclusion: SE33 (0.905), D12S391 (0.791), D8S1132 (0.708) and D6S389 (0.845). The expected cumulative CPE (chance of paternity exclusion) for these 4 loci is 99.9%, the calculated probability to find 3 or more exclusions is 81%.

A triplex PCR (SE33, D12S391 and D8S1132), which can be detected simultaneously with a singleplex PCR (D6S389) in the same electrophoresis run, has been established.

74 paternity cases (48 non-exclusions, 26 exclusions), already investigated with conventional markers (red cell antigens, red cell enzymes, protein polymorphisms), 4 VNTR- (D1S80, YNZ, COL2A1, APO-B) and 11 STR- (SE33, TH01, vWA, FGA, D12S391, D8S1132, FES/FPS, F13B, CD4, LPL) loci were included in the study.

All non-fathers were detected in this paternity screening approach with at least 2 exclusions, in 21 out of 26 cases (81%) three or more exclusions were found. A single exclusion at the D6S389 locus, which was probably due to a mutation in the paternal germline, was found in a non-exclusion case.

In 66% of the non-exclusion cases the CPE was between 99% and 99.9%, in 34% the CPE was higher than 99.9%; in 36 of these 47 cases (77%) the probability of paternity was >99.75%, which corresponds to the attribute “paternity practically proven”.

contact: gudrun.dorner@meduniwien.ac.at

P-079

A TRIPLEX-PCR FOR SE33, D12S391, D8S1132 AND A SINGLEPLEX-PCR FOR D6S389 IN A SINGLE RUN

Dorner G, Dauber EM, Wenda S, Glock B, Mayr WR

Division of Blood Group Serology, Medical University of Vienna, Austria

A triplex-PCR was developed for the highly polymorphic STR loci SE33, D12S391 and D8S1132. The primers were labelled with different dyes as the amplicons have partially overlapping size ranges. The highly informative STR locus D6S389 had to be amplified separately, as a multiplex-PCR without changing the primer sequences of the other loci was not possible. The D6S389 singleplex-PCR products were labelled with a fourth dye and could therefore be analysed in a single run together with the triplex-PCR products.

In this study and we investigated the triplex-PCR loci and D6S389 in a sample of 342 unrelated Austrian Caucasoid individuals. These data were in concordance with former results obtained after singleplex PCR and native polyacrylamide gel electrophoresis (SE33, D8S1132) or denaturing fragment analysis on an A.L.F. DNA Sequencer (D12S391). Some rare and new SE33 alleles have been detected and sequenced. Population data and statistic parameters were calculated for all loci. No deviation from Hardy-Weinberg equilibrium was observed.

Parameter	SE33	D12S391	D8S1132	D6S389
Observed heterozygosity	0.953	0.898	0.857	0.924
χ2	86.86	25.91	39.20	48.28
df	78	28	28	36
p	0.234	0.573	0.065	0.078
Polymorphism information content	0.940	0.870	0.840	0.890
Matching probability	0.009	0.026	0.039	0.023
Power of discrimination	0.991	0.974	0.961	0.977
Power of exclusion	0.905	0.791	0.708	0.845
Typical paternity index	10.69	4.89	3.49	6.58

These 4 markers have been used to establish a “screening test” for paternity cases (see presentation of Dorner et al., Four Highly Polymorphic STR-Loci as a “Screening Test” in Paternity Cases)

contact: gudrun.dorner@meduniwien.ac.at

P-080

A new primer set in a SRY gene for sex identification: its implication in forensic applications and prenatal diagnosis

Drobnič K

Forensic Science Centre, Ministry of the Interior, Ljubljana, SLOVENIA

Sex determination can be an important piece of information in various forensic investigations, especially in sexual assault cases, but can be also useful in prenatal diagnosis of foetus with a known family history of genetic disorder affecting only male child. Because of that a gender determination has nowadays become a part of a human identification PCR kits. Although different PCR-based methods are known to identify a sample as originating from a male or a female, the only sex test included in commercially available human identification PCR kits for gender determination is based on the amelogenin sex test described by Sullivan et al., with primers spanning a part of the first intron, which results in two PCR products that vary from each other by 6 bp. The test is quick and effective but new studies showed that is not always reliable. The frequency of a deletion of Y copy of the amelogenin gene occurs between 1.85 % and 0.02 % depending on a population and “deleted-amelogenin males” (designed as DAMs) would have been identified as women. In the Slovene national DNA database the number of phenotypic male individuals reached 3713 and one individual showed a sex test failure. The observed frequency of the amelogenin sex failure in our Caucasian sample is thus 0,027 %. Considering the consequences of the result obtained only using an amelogenin marker, we have tried to design a new primer set to facilitate the integration of the SRY sex test into multiplex STR human identification kits. This poster presents strategies and results for solving problems of a sex test reliability. As forensic samples are usually low in quantity and mostly very degraded, we decided to design a primers set which after amplification gave a small amplicon only 96 bp in lengths. Another benefit of the small amplicon is that SRY fragment will not overlap with alleles in multiplex STR kits. At first, different amplification conditions and primers concentrations were tested using DNA isolated from 9947A and 9948 cell-lines. In the end, the amplification resulted in only one band peak for a male sample and no reproducible peaks were observed over minimum threshold in a female sample even with high quantity of female DNA. To evaluate the sensitivity of the primers we tested the minimum required input of male DNA. We obtained peak even with 0.25 ng of template. After optimalization of concentration we tested the amplification under PCR condition of commercially kits AmpSTR SGM Plus (Applied Biosystems) and PowerPlex 16 (Promega) not only using control DNAs from the kit but also DNA isolated from reference samples taken from a man and a woman. We succeeded to coamplify the SRY fragment with STR loci under both condition without any artefact using male DNAs, but it was absent from females. Finally, we tested new primers with a phenotypically normal male, who was genotyped as female, using either the AmpFlSTR® SGM Plus kit or the PowerPlex® 16 kit by lacking the amelogenin Y-specific PCR product. Identical results were obtained by using three different primer sets for amplification of this region of the amelogenin gene. The presence of Y chromosome was determined by using six Y-STR markers. The male genotype of the individual was also confirmed by the amplification of a 96 bp long fragment of the SRY gene. Because it is very important that gender detemination tests give correct prediction in some forensic cases and prenatal diagnosis, we propose that this kind of amplicon from SRY locus is included as an additional safety measure of the sex status, especially in suspicious samples.

Contact: katja.drobnic@mnz.si

P-081

Forensic validation of the X-chromosomal STR-markers GATA165B12, GATA164A09, DXS9908 and DXS7127 in German population

Edelmann J¹, Lessig R¹, Willenberg A¹, Wildgrube R¹, Hering S², Szibor R³

¹Institute of Legal Medicine, University of Leipzig, Germany

²Institute of Legal Medicine, Technical University of Dresden, Germany

³Institute of Legal Medicine, Otto-von-Guericke University of Magdeburg, Germany

The Chromosome X-STRs (ChrX STRs) were recently recognised as useful tools in forensic kinship testing, mainly in solving of complex cases. The highly effective strategy of ChrX microsatellite haplotyping requires the description of numerous STR markers. The aim of this presentation is to add four STRs to the known panel of ChrX markers and to describe their forensic suitability. GATA165B12 and GATA164A09 were characterised recently and population data were published for Korean population samples (Shin SH et al. 2005 and Son JY et al. 2002). DXS9908 and DXS7127 are not forensically evaluated yet to our knowledge.

We report here primer sequences, PCR protocols, allele structures and population data for a German population sample. We investigated for the four STRs up to 766 unrelated individuals and up to 333 meioses. The markers described here revealed a moderate degree of variability (Het = 0.69, 0.67, 0.83, 0.76 and PIC = 0.65, 0.68, 0.72, 0.78, respectively) low mutation rates and no problems in handling when the automated fragment analysis was performed on the ABI PRISM™ 310 Analyzers. Information regarding location on the ChrX are drawn from NCBI and by performing an own recombination study. Performing the exact test for genotype distribution of the STRs we found no significant deviation from Hardy-Weinberg equilibrium. All markers are suitable for forensic purpose.

contact: jeanett.edelmann@medizin.uni-leipzig.de

P-082

Relevant aspects for forensic STR analysis of canine DNA:

Repeat based nomenclature, allelic ladders and PCR multiplexes

Eichmann C, Berger B, Parson W

Institute of Legal Medicine, Innsbruck Medical University, Austria

As the dog is deemed to be our closest companion and most popular pet, it can also be considered as the most interesting animal species from a forensic point of view. Canine saliva as well as dog hairs can remain everywhere where contact between dogs and humans have taken place. Canine-specific short tandem repeat (STR) analysis discloses a new approach for investigating dog attacks and other forensically important incidents involving dogs. As forensic identity testing of canine DNA using STRs is becoming commonplace in resolving criminal cases it has become increasingly important to have a set of minimum guidelines, such as common used STR markers and a reliable nomenclature, which enables exchange of data and international collaborations. The majority of canine STR markers described in the literature are not yet characterized with respect to their sequence structure and earlier studies have not been using a uniform repeat-based nomenclature for the STR alleles. Mostly the alleles were reported by the estimated fragment size as determined by electrophoresis of the PCR-products. The lack of a uniform harmonized nomenclature makes the application of these markers difficult. Here we present a nomenclature for a set of forensically useful STR markers that is adopted from the recommendations of the International Society of Forensic Genetics (ISFG) for the nomenclature of human STRs. We describe two newly designed PCR multiplexes sensitive to degraded DNA for 8 polymorphic canine STR markers (FH2087Us, FH2611, PEZ15, FH2054, PEZ2, PEZ6, WILMS-TFs, FH2328l). The sequence structure of selected alleles of these markers was the basis for the implementation of a repeat based nomenclature. Additionally, allelic ladders containing the common alleles for all markers used in both multiplexes are shown, which allow an unequivocal allele designation of unknown samples.

Contact: burkhard.berger@uibk.ac.at 

P-083

Molecular analysis of in vitro damaged DNA samples

Fattorini P¹, Tomasella F¹, Grignani P², Sanchez P³, Ricci U⁴, Carracedo A³, Previderè C ²

¹ UCO di Medicina Legale, Dipartimento di Scienze di Medicina Pubblica, Università di Trieste, Italy

² Dipartimento di Medicina Legale e Sanità Pubblica, Università di Pavia, Italy

³ Institute of Legal MedicineUniversity of Santiago de Compostela, Santiago de Compostela, Spain

⁴ Azienda Ospedaliera-Universitaria “A. Meyer”, U.O. Genetica Medica, Firenze, Italy

A large extraction of DNA was performed from 500 ml of a male donor blood by phenol/chloroform procedure. After spectrophotometric quantification at O.D.₂₆₀/O.D.₂₈₀, about 26 micrograms of the sample were aliquoted in 72 different eppendorf tubes. These samples then underwent different treatments with several physical and chemical agents (UV radiation at 254 nm, formic aldeyde, HCl, H₂0₂, FeCl₃, CuSO₄, FeCl₃ plus H₂0₂, CuSO₄ plus H₂0₂ and NaOH) for a comparable time (from 1 to 10min). All the treatments were performed in duplicate. After ethanol precipitation, the samples were redissolved in H₂O and analyzed by the following methods: EtBr staining, Alu probing, Real Time PCR, STR typing and SNPs analysis. In addition, to evaluate the degree of chemical damage of the DNA bases, Capillary Electrophoresis (CE) was also performed.

Our data show that most of the above treatments caused a chemical damage of the DNA template. In addition, we observed that PCR fidelity was strongly influenced by the integrity of the template.

(contact: fattorin@univ.trieste.it)

P-084

Analysis of Y chromosome and mtDNA variability in the Madeira Archipelago population

Fernandes AT, Gonçalves R, Rosa A, Brehm A

Laboratório de Genética Humana, Universidade da Madeira

The Atlantic archipelago of Madeira is composed of two islands (Madeira and Porto Santo) with 250.000 inhabitants.These islands were discovered and settled by the Portuguese in the 15th century and played an important role in the complex Atlantic trade network in the following centuries.

The history of local settlements and constrains on the populations mobility especially due to orography is a possible explanation for the differences found between different regions of the Madeira Island. The genetic composition of the Madeira Islands’ population was investigated by analyzing Y chromosomal bi-allelic and STR markers in three different regions of the Main Island plus Porto Santo Island. We compared the results with mtDNA data and used the Y chromosome STRs to determine the variability within each haplogroup. A sample of 143 unrelated males divided into four groups (Funchal n=35, West Madeira n=39, North and East Madeira n=46 and Porto Santo n=23) were analyzed.

Significant genetic differences between these regions and the population of Funchal were found. The population of Funchal had a lower gene diversity than expected.

Contact: atgf@uma.pt

P-085

MtDNA analysis of ancient samples from Castellón (Spain): diachronical variation and genetic relationships.

Fernández E.^1,2, Oliver A.³, Turbón D.², Arroyo-Pardo E.¹

¹⁾ Depto. Toxicología y Legislación Sanitaria, Facultad de Medicina, Universidad Complutense, Madrid, Spain.

²⁾ Unidad de Antropologia. Depto Biología Animal, Facultad de Biología, Universidad de Barcelona, Spain.

³⁾ Sección de Arqueología. Museo de Bellas Artes de Castellón, Castellón, Spain.

Thirty seven bone and teeth samples from Calcolithic and Iberian ages from several archaeological sites located in Castellón (Spain) were analyzed for mitochondrial DNA HVRI polymorphism. Despite of the presence of high amounts of PCR inhibitors in the ancient extracts it was possible to recover 150bp fragments in 9 cases. Recovered lineages suggest a close relationship among individuals from the same archaeological site, this suggesting a possible familiar relationship or the presence of an homogenous ethnic group. Moreover, Calcolithic haploypes differed so much from those recovered from Iberian samples. This points out a possible genetic replacement between both periods in the Spanish Levant.

Contact: earroyop@med.ucm.es 

P-086

The distribution of Y-chromosomal haplotypes and haplogroups in two population samples from the Romagna region (North Italy): differences between urban (Rimini) and rural area (Valmarecchia)

G. Ferri, S. Ceccardi, F. Lugaresi, F. Ingravallo, C. Bini, A. Cicognani, S. Pelotti

Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, Bologna, Italy

We have studied the distribution of Y chromosomal haplotypes and haplogroups in two population samples from the Romagna region (North Italy) by analyzing male-specific markers, that reflect past and recent history, like SNPs and STRs. On the basis of previous studies on human Y-chromosomal single-nucleotide polymorphisms (Y-SNPs), the non-recombining part of Y chromosome has been shown useful for the investigation of population movements. These slowly evolving markers permit the detection of differences and similarities among populations without problems due to recurrent mutations in STR-based haplotypes. By contrast, Y-STRs are capable to detect more recent historical events and to resolve population stratification, but they are significantly dependent on an accurate sample collection, especially for neighbouring populations.

Our population samples were collected in the urban area of Rimini, an ancient port in Roman age and in the near rural area of Valmarecchia, that is more isolated and geographically out of ancient trading ways. 100 autochthonous unrelated males from Rimini and 70 from Valmarecchia were selected.

We analyzed 11 Y STRs (DYS391, DYS389I, DYS389II, DYS439, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390 and DYS385) by a commercial kit and 20 binary polymorphisms, grouped in three multiplexes for determining the most frequent haplogroups, by minisequencing analysis.  Statistical parameters were calculated using Arlequin 2.0 package.

The aim of this study is to analyse the microgeographic heterogeneity of Y chromosome in a Northern Italian region and to link it to geographical and historical perspectives.

Contact: susi.pelotti@unibo.it

P-087

BPA analysis as a useful tool to reconstruct crime dynamics. Part III

Fratini P¹, Pizzamiglio M¹ , Floris T¹ , Ceneroni G² , Talamelli L¹, Sampò G  and Garofano L¹

¹ Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Parma, Italy

² Raggruppamento Carabinieri Investigazioni Scientifiche, Roma, Italy

On 18 June 2001, a 42 year-old man was killed with a single gunshot aimed at his head. The weapon used was a shotgun “Beretta” cal.20 mod.A300. The victim was shot while sitting on a sofa placed in the dining room of his former wife’s friend’s home.

The lady claimed that her friend, who was a hunter, had come into the room handling his rifle at the end of a long discussion which had taken place beforehand. She also showed her friend’s position, added that he just wanted to scare the victim with no intention of shooting him, but that an accidental shot had been fired.

The Prosecutor asked our lab to reconstruct the dynamics of the events, in order to establish what had really happened, especially as regards the exact positions of the shooter and the victim when the shot was fired.

We first examined the gun and all its components in order to exclude mechanical failures or any other fault which could justify the accidental shot. The weapon was in perfect order.

In order to reconstruct the trajectory followed by the bullet and the probable position of the shooter,  we examined the report written by the forensic pathologist, analyzed data acquired at the crime scene (i.e. evidence on bullet impact, measurements, etc.), and applied the BPA technique to bloodstains.

In this regard, particularly interesting were the bloodstains which had projected around the victim’s head as they allowed to establish the position of the body and both the orientation and height of the victim’s head, when it was hit by the bullet. By elaborating the evidence on the bullet impact, it was then possible to trace the second part of the trajectory (from head to wall) and hence estimate the first part (from shotgun barrel to target) on the basis of the conclusions reached by the forensic pathologist.

At the end of our study, by using 3D graphic software (AutoCAD 2000^â), we were able to show that at the moment of the shot :

-       the shotgun was working perfectly excluding any accidental shot ;

-       the victim was sitting on the sofa with his head turned to the right and his legs apart;

-       the shooting distance ranged between 2 and 3 meters;

^-            the position of the shooter was different from the one stated by the lady, indicating the possibility of a voluntary murder. Contact: lugaro@tin.it

P-088

BPA analysis as a useful tool to reconstruct crime dynamics. Part I

Fratini P¹, Pizzamiglio M¹ ,Floris T¹ ,Pierni M¹ and Garofano L¹

¹ Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Parma, Italy

This paper concerns a case of two bodies which were found dead in their bedroom, shot several times with a semiautomatic pistol.

It was essential to establish if we were dealing with a double homicide or rather the shooting of the first victim, followed by suicide of the second.

We refer to technical activities we conducted at the crime scene and the analytical approach we adopted, based on DNA as well as on BPA analyses of the bloodstains we recovered, studied and collected during CSI.

Following this method, also supported by ballistic exams, it was possible to establish the exact position of the first victim, as well as that of the shooter and reconstruct the dynamic of the event.

This shows, once more, that to obtain affordable and useful results for investigation we need to look to an integrated analytical approach which uses contributions from all aspects of forensics, especially when DNA and BPA analyses are available. 

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