Kamis, 17 Juli 2014





P-027
Evaluation of seven autosomal STR loci in a German population

Becker D, Vogelsang D, Brabetz W

Biotype AG, Moritzburger Weg 67, 01109 Dresden, Germany


Population data for the seven autosomal STR loci D4S2366, D6S474, D14S608, D19S246, D20S480, D21S226 and D22S689 were analysed in a German population of unrelated individuals (n =189) by capillary gel electrophoresis. For this purpose two screening multiplex polymerase chain reactions (triplex and quadruplex) were developed with fluorescently labelled primers.
Different alleles for all loci were sequenced and an allelic nomenclature consistent with the ISFG recommendations was defined. Simple, compound and complex repeats could be distinguished. Allele frequencies and further population statistical data for all loci were described and will be discussed with regard to forensic applications.


P-028
The Comparison of mtDNA and Y chromosome Diversity in Malay Populations
Bekaert B, Hadi S, Goodwin W

Department of Forensic& Investigative Sciences, University of Central Lancashire, Preston, UK

Analysis of the mtDNA hypervariable region I of 106 modern Malay samples and 59 Orang Asli samples had previously found that the mtDNA diversity in the modern Malay was comparable to other Caucasian and Asian populations while, unsurprisingly the diversity in the isolated Orang Asli was very low, with only 14 different haplotypes being observed. 
A follow up study was carried out to assess the effect of isolation on the levels of diversity of the Y chromosome in the Orang Asli polulation. Thrity three samples from the Orang Asli and thirty eight from the modern Malay population were analysed using the Promega Y-Plex kit. The Orang Asli population consisted of two subpopulations: the Jahai population and the Kensiu population. Fifteen samples were profiled from the Jahai population with the most common haplotype occuring in 3 individuals, over all the gene diversity value was 0.9536. Eighteen Kensiu samples were profiled and the most common haplotype again occurred 3 times, the gene diversity was 0.961. In 38 modern Malay samples no common haplotype was found and the gene diversity value was calculated as 0.9999. In both the Orang Asli and Malay population the diversity of the Y chromosome was higher than had been detected in the mtDNA genome.
The different frequency of haplotypes in isolated populations is an important consideration when applying lineage markers to casework.


P-029
A Multiplex SNP Typing Approach for the DNA Pyrosequencing Technology

Bender K1, Nehlich C1, Harrison C2, Musgrave-Brown E2,  Syndercombe-Court D2, Schneider PM1
and the SNPforID Consortium

1 Institute of Legal Medicine, Johannes Gutenberg University Mainz, Germany
2 Centre for Haematology, ICMS, Barts and The London, Queen Mary’s School of Medicine and Dentistry, UK
www.snpforid.org

Multiplex Pyrosequencing enables simultaneous analyses of multiple target DNA. Single and multiplex PCR was employed to amplify target DNA templates each containing one of 23 single nucleotide polymorphisms (SNPs) from genomic DNA selected by the SNPforID Consortium. In our investigations we have looked for the multiplex capacity of the PSQ™ 96MA instrument (Biotage AB). To test the reliability of the SNP typing by Pyrosequencing we have analysed each of the SNPs by using the SNaPshot minisequencing technique in parallel as reference method.
For developing a multiplex assay, in the first step 23 PCR products were divided into 8 aliquots of equal volume and each aliquot was typed in parallel with a set of three different SNPs. In the next step the same set of SNPs was typed by using one duplex and seven 3plex PCR reactions side by side. Because the amount of DNA is limited in the majority of casework samples it is necessary to amplify all relevant SNPs in one or only a few PCR reactions. Therefore we have addressed the questions, whether it is possible to perform a successive typing of two 3plex SNP typing reactions out of a 6plex PCR reaction and how often this SNP typing reaction can be repeated. Finally we could show that the typing of 23 SNPs out of a 23plex PCR reaction seems to be possible under optimized conditions. Due to the lack of an adequate instrument software for our strategy the dispensation orders for the nucleotides used in the pyrosequencing had to be designed manually in a time-consuming step. To improve the method different purification steps and the use of single strand binding protein (SSB) were tested.



P-030

Y Chromosome variation in Gabon


Gemma Berniell-Lee, Elena Bosch, Jaume Bertranpetit, David Comas

Unitat de Biologia Evolutiva, Departament de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, Spain

Of the 6,900 known living languages worldwide, one third are spoken in Africa. African languages are divided into four main language families or phyla, the largest of these being the Niger – Congo family (both in terms of geographical area, the number of speakers, and the number of different languages). In turn, one third of the Niger-Congo phyla are Bantu languages. Bantu languages are spoken throughout Sub-Saharan Africa (i.e. the Congo Basin, Angola, Mozambique etc...) and are thought to have obtained this distribution through one of the major cultural expansions in Africa; the Bantu Expansion. This expansion is thought to have taken place around 5,000 years ago, and to have originated in southern Nigeria and/or northwestern Cameroon. Although its linguistic side has been widely studied, little is known about the demographic processes associated to it. Our aim is to provide insights into the origin and diffusion of Bantu and Bantu-speaking populations by means of genetic data. Since the human Y chromosome is uniparentally inherited, and its phylogeny has been exhaustively described, it is possible to reconstruct a phylogeography of the human male lineages in sub-Saharan Africa. We have typed 18 STRs in over 1,100 samples from multiple ethnic groups (i.e. Galoa, Benga, Nzebi etc...) from the area of Gabon, located in the Guinea Gulf, which together with SNP data, should enable us to identify admixture, possible migration routes, and to study correlations between languages, cultures and genes.


P-031
Diversity of maternal and paternal lineages in the geographic extremes of the Azores (Santa Maria and Flores Islands): insights from mtDNA, Y-Chromosome and Surname data

Bettencourt C1, Montiel R1, Santos C2,3, Prata MJ4, Aluja MP2, Lima M1

1Center of Research in Natural Resources (CIRN), University of the Azores, Ponta Delgada, Portugal
2Unity of Anthropology, Department BABVE, Autonomous University of Barcelona, Barcelona, Spain
3Department of Anthropology, University of Coimbra, Coimbra, Portugal
4Institute of Pathology and Molecular Immunology of the University of Porto (IPATIMUP), Porto, Portugal

The Azores Islands were discovered, uninhabited, by Portuguese navigators in the early 15th century. The 9 islands that form this Archipelago are clustered in three geographic groups (Eastern, Central and Western). The peopling process was initiated in 1439 by the Eastern group (S. Miguel and Santa Maria, proceeding slowly to the remaining islands. Santa Maria (population of 5 490 inhabitants; area of 96.9 km2) and Flores (population of 3 949 inhabitants; area of 141 km2) occupy respectively the eastern and western limits of the Archipelago. These two small islands represent not only geographic extremes but also are chronologically distant in terms of settlement history, since Santa Maria was the first to be peopled whereas Flores was the last to be occupied. With the purpose of analysing the impact that the effective population size, geographic distance and chronology of settlement had on the genetic structure of these islands, we characterized the maternal and paternal lineages of both populations by:
a) determining the sequence of HVRI region and specific polymorphic positions of the non-coding region of mitochondrial DNA (mtDNA);
b) analysing 20 binary polymorphisms located in the non-recombining portion of the Y-chromosome (NRY); and c) studying patterns of surname composition.

P-032
Validation of a single expert system to automate the interpretation of STR data, including mixtures

Bill M1, Gill P1, Young R1, Maguire C1, Healy M1, Thornton L1, Curran J2

1Forensic Science Service, Trident Court, 2960 Solihull Parkway, Solihull B620LS UK
2Department of Statistics, University of Waikato, New Zealand.

The Forensic Science Service® (FSS) has utilised computer software to automate the interpretation of STR data since 1998. The introduction of these systems has resulted in dramatic improvements in the quality, speed and efficiency of the analytical stage of the DNA profiling process. The FSS have developed an expert system suite called FSS-i³ (FSS i-cubed) which brings together the technical knowledge and experience acquired .The suite uses complex heuristic rule-sets developed with the Forensic Science
Service's most experienced reporting officers (ROs) and analysts, and is designed for use with any STR multiplex and any PCR cycle number.  The software is used to completely automate the designation of alleles so that genotypes are now down-loaded to the UK national DNA database without the need of an operator interface. In addition to the 'core' interpretative processes, the software has alternate algorithmic solutions using least squared approach and geometric means to interpret mixtures. Apart from completely de-skilling the interpretative process, the net outcome is a significant reduction in unit's costs and an increase in the success rate of crime-stain data by circa 20%.  The FSS-i³ software has proven to be so robust in its ability to correctly interpret data that its usage as a single expert system has been approved.

P-033
CYP2C9 Polymorphism in Iranian population with three different ethnicity

Biramijamal F2 ,Tanhaei s1,Sanati M.H2,Sheidai M1

1Shahid Beheshti University,Tehran-Iran
2National Institute for Genetic Engineering and Biotechnology(NIGEB),Tehran-Iran


The Cytochrome P450 2C9 gene has a function in detoxification of carcinogenic compounds. Recently, described the polymorphism at codon 144 of the CYP2C9gene (Cys/Arg) and susceptibility of several types of cancer. Also it is reported that CYP2C9 polymorphism is involved in drug resistance.
To investigate the CYP2C9 codon 144 polymorphism among different ethnicity, we collected samples from healthy population from three different ethnicity groups. The CYP2C9 Cys144Arg genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing analysis in 120 healthy controls.
Among the healthy subject with Mazandarani, Turkaman and Kord ethnicity, the allelic frequency of CYP2C9 Cys144Arg were 16%,9% and 14% for Cys allele,84%,91% and 86% for Arg allele.
No significant difference in CYP2C9 allele distribution was observed between Mazandarani, Turkaman and Kord healthy individuals.In each group distribution of genotypes fits the Hardy-Weinberg equilibrium. Our initial study of 120 Iranian healthy individuals calls for future work in Iranian population genetics, also finding the CYP2C9 genotypes between Iranian cancer patient and comparing it with healthy controls. Indeed pharmacogenetics studies can be done according our data.
This work was supported by NIGEB project number 197.


P-034
Analysis of Y chromosome lineages in native South American population

Blanco-Verea A, Brion M, Sanchez-Diz P, Jaime JC, Lareu MV, Carracedo A

Institute of Legal Medicine. University of Santiago de Compostela. Spain


The object of this work is to try and identify both the evolutionary footprints and the origin of native populations of Argentina, and to compare them with those of other populations from South America.   We analyzed 32 SNPs and 11 STRs of the Y chromosome in 126 samples from three different native populations from Argentina (Kollas, Mapuches and Diaguitas). The STR markers were amplified by means of the commercial kit PowerPlex Y system (Promega Corporation), the SNPs were amplified by means of four multiplex reactions and genotyped using the SNaPshot minisequencing Multiplex Kit (Applied Biosystems), and the products were analyzed with an ABI Prism 3100 Genetic Analyzer. Our results reveal that haplogroups R1b, Q3, G, I are the main haplogroups present in these populations, indicating the introduction of European Y chromosome lineages during the colonization of the American continent.

Contact: brioniml@usc.es

P-035
Rapid Microarray-based Typing of Forensic SNPs.

Bogus M,1, Sobrino B2, Bender K1, Carracedo A2, Schneider PM1
and the SNPforID Consortium

1 Institute of Legal Medicine, Johannes Gutenberg University Mainz, Germany
2 Institute of Legal Medicine, CEGEN, University of Santiago de Compostela, Spain
www.snpforid.org

The Single Base Extention-Tag Array (SBE-Tag Array) method is carried out on glass slides and combines the specificity of minisequencing for SNP typing with the high troughput capacity of microarrays. Following multiplex PCR, a single tube SBE reaction is carried out, and the labelled extension products are hybridized to an array for locus-specific analysis. The aim is to prove and optimise the conventional microarray reaction on accuracy and efficiency for forensic applications.
From a list of non-cross-reacting sequences, 29 tag sequences were chosen and the complementary sequences were spotted as capture probes in duplicates on glass slides. Each slide contains four to ten arrays (MWG/CodeLink), which can provide results for the same number of individuals, using a design called “array of arrays” (Pastinen et al., Genome Res. 2000, 10:1031). In a minisequencing reaction containing fluorophore (Cy5, Cy3, Rox) labelled ddNTPs and 5´-tagged SBE primers, the extention reaction is performed and finally demultiplexed by hybridization to the arrays. Genotyping is carried out using an Affymetrix 428 scanner. Detection is carried out at three wavelengths, therefore the assays have been designed to avoid A/C polymorphisms, as these bases had to be labelled with the same dye. Alternatively, if a two-wavelength scanner is used, minisequencing can be performed using only a single dye label in four separate reactions. Then the reaction products have to be hybridized to four separate arrays on the same slide, and analyzed individually for each base. At present, 23 SNPs are combined into a single reaction.
The SBE-Tag array on glass slides is a promising and cost-efficient genotyping technology, which can be further extended in respect of the number of simultaneously anlysed individuals and the size of the multiplex PCR reaction.


P-036
Internal Validation of AmpFlSTR Identifiler PCR Amplification Kit with detection on ABI Prism 3100 Genetic Analyzer for Use in Forensic Casework at the Department of Chemistry, Malaysia.
 
Lim Kong Boon
 
Department of Chemistry Malaysia
 
According to the guidelines of quality assurance standards for forensic DNA testing laboratories, prior to implementing a new DNA analysis procedure or an existing DNA analysis procedure developed by another laboratory, the forensic laboratory must first demonstrate reliability of the procedure by carrying out internal validation. Seven elements were design by the forensic laboratory at the Department of Chemistry, Malaysia to validate the use of the AmpFlSTR(r) Identifiler PCRTM Amplification Kit with detection on ABI Prism(r) 3100 Genetic Analyzer  using POP-4TM polymer. The presentation summarizes the results obtained for each of the seven elements of the validation studies, which include the following evaluation: sensitivity, precision, reproducibility, non-probative casework, stutter, heterozygous ratio and mixtures. With these data, guidelines for the interpretation of STR DNA profiles based on the AmpFlSTR(r) Identifiler genetic loci were documented for use by the DNA laboratory.
 
Contact: kblim@kimia.gov.my 

P-037
Comparison of calculated paternity indices based on the typing of 15 STRs, 7 VNTRs, and 52 SNPs in 50 Danish mother-child-father trios

Børsting C, Sanchez JJ, Birk AH, Bruun HQ, Hallenberg C, Hansen AJ, Hansen HE, Simonsen BT, Morling N

Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Denmark

Fifty Danish paternity cases from the year 2004 were selected based on the results obtained with the AmpFlSTRÒ IdentifilerÒ PCR amplification kit (Applied Biosystems). In all cases, the calculated paternity index (PI) was higher than 10,000, and there was not observed any genetic inconsistensies between mother and child, or between father and child. DNA from the selected trios was used to type 7 VNTRs (D2S44, D5S43, D5S110, D7S21, D7S22, D12S11, and D16S309) using the RFLP technique, and 52 SNPs using a PCR multiplex with 52 PCR primer pairs and two SBE multiplexes with 23 and 29 SBE primers, respectively (for details of the 52-SNP-plex, see SNPforID presention). PIs were calculated based on each set of loci (STRs, VNTRs and SNPs) and the results were compared.


P-038
Whole genome amplification of blood and saliva samples placed on FTAÒ cards

Børsting C1, Thacker C2, Syndercombe Court D2, Morling N1

1Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Denmark
2Centre for Haematology, ICMS, Barts and The London, Queen Mary's School of Medicine and Dentistry, UK

Cells that come in contact with FTAÒ cards (Whatman Bioscience) lyse. The DNA is released and irreversibly bound to the filter matrix, from where the DNA can be assayed directly. The GenomiPhiä DNA amplification kit (Amersham Biosciences) utilizes Phi29 DNA polymerase and random hexamer primers to exponentially amplify DNA by strand displacement amplification (SDA). We tested the GenomiPhi DNA amplification kit on 50 blood and 50 saliva samples placed on FTA cards. A 1.2 mm disk was punched out of the FTA cards using the BSD600-duet (BSD Robotics). The disk was washed three times with 150 l Milli-Q water using the THEONYX robotic system (MWG) and left overnight in 150 l Milli-Q water to remove all inhibitors of Phi29 polymerase. The disk was dried and used as target for the GenomiPhi DNA amplification kit. On average, the Phi29 polymerase produced 2 mg DNA (100 ng/l) with DNA fragment sizes ranging from a few hundred bp to 12 kbp. A total of 1-2 ng Phi29 amplified DNA was typed using the AmpFlSTRÒ SGM Plusä amplification kit (Applied Biosystems) and the resulting STR profiles analysed according to the guidelines of each of the two forensic laboratories.


P-039
Autosomal microsatellite analysis of the Azorean population

Branco CC1,2, Pacheco PR1,2, Cabral R1,2, de Fez L1,2, Peixoto BR1,2, Mota-Vieira L1,2

1 Molecular Genetic and Pathology Unit, Hospital of Divino Espírito Santo, São Miguel, Azores, Portugal
2 Instituto Gulbenkian de Ciência, Oeiras, Portugal

The knowledge of population history, demography and genetic structure has proven to be fundamental to address research in human genetics. Here, we describe the genetic diversity of Azorean population and its affinity with other populations by the analysis of 13 microsatellite loci (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51 and D21S11) in 222 unrelated blood donors. These short tandem repeat (STR) markers were typed by Polymerase Chain Reaction (PCR) with fluorescently labelled primers. Statistical analysis was performed using Arlequin v.2.0, and Nei’s genetic distance was calculated with DISPAN software and trees were constructed by Neighbor-Joining (NJ) using PHYLIP 3.63. To quantify the genetic contribution of Portuguese, African and European populations we calculated the admixture coefficient (mY) using Admix v. 2.0.
The analysis of microsatellite loci shows that the Azorean population presents an average gene diversity of 0.776. For each marker, gene diversity range between 0.661 for TPOX and 0.8812 for D18S51. Heterozigosity values calculated for each STR varies from 63.9% for TPOX to 89.2% for D18S51, although the majority of markers show values superior to 80%. In addition, the admixture coefficient reveals North Portuguese as the major contributors to the genetic background of the Azoreans. These results are corroborated by the dendrogram, in which Azores is closer to Belgians, Portuguese and Spanish, apart from Moroccans and Cabo Verdeans.
Taken together, these data indicate that the gene pool of the Azorean population is very diverse and are consistent with our previous results on Y-chromosome (Pacheco et al., Ann Hum Genet 69: 145-156, 2005). Moreover, no genetic differentiation between Azores and Portugal is observed.

Funded by DRCT (Azores).

P-040
Simultaneous versus serial DNA identification of related tsunami victims

Brenner CH

 School of  Public Health, UC Berkeley, California, USA

DNA has proven to be a major and essential tool for identification in recent mass fatality incidents including wars, bombings, airplane crashes, and the World Trade Center attack. It will surely prove to be so in dealing with the hundreds of thousands of victims of the 2004 Indian Ocean tsunami. Among the many mathematical complications characteristic of this sort of mass fatality is the prevalence of related victims. When several bodies are found that are suspected of being members of the same family and are to be identified through DNA profile comparison with to other, living, family members, the right method of analysis is to consider all the identities at once. Only a simultaneous approach takes full account of the power of the evidence, takes into account the extent to which each dead body’s identity is supported by its DNA similarity to the other dead bodies. By contrast, the serial method, which assigns the identities one at a time, thus letting each victim identity once established participate in the identification of the subsequent bodies, is superficially attractive but unfortunately it often understates the true value of the evidence. As an extreme example, imagine a father and daughter as the only two related victims of a small airplane crash. The two of them can probably be picked out and therefore identified from the DNA similarity even if no reference relatives are available, so simultaneous consideration of their types is almost infinitely better in this case. I will illustrate the “simultaneous method” with a realistic example and show how the logic and confidence of identification is stronger than using a serial identification approach.


P-041
Analysis of 29 Y-chromosome SNPs in a single multiplex useful to predict the geographic origin of male lineages

Brión M1, Sanchez JJ2, Balogh K3, Thacker C4,  Blanco-Verea A1, Børsting C2,  Stradmann-Bellinghausen B3, Bogus M3, Syndercombe-Court D4,  Schneider PM3, Carracedo A1, Morling N2, and the SNPforID Consortium

1 Institute of Legal Medicine, CEGEN, University of Santiago de Compostela, Spain
2 Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Denmark
3 Institute of Legal Medicine, University of Mainz, Germany
4 Barts and the London, Queen Mary’s School of Medicine and Dentistry, London, UK
www.snpforid.org

The European Consortium "High throughput analysis of single nucleotide polymorphisms for the forensic identification of persons – SNPforID", has performed a selection of candidate Y-chromosome SNPs (single nucleotide polymorphisms) for making inferences on the geographic origin of an unknown sample. From more than 200 SNPs compiled in the phylogenetic tree published by the Y Chromosome Consortium, and looking at the population studies previously published, a package of 29 SNPs has been selected for the identification of major population haplogroups.
A “Major Y chromosome haplogroup typing kit” has been developed, which allows the multiplex amplification of all 29 SNPs in a single reaction followed by a single base extension (SBE) reaction (minisequencing) and separation of the resulting extension products by capillary electrophoresis.
Validation of the kit was performed, firstly to check the accuracy and reproducibility of the 29-plex in different laboratories, and secondly to obtain haplogroup frequencies in samples from the major population groups. To compile the sample collections each of the participating groups reported the samples they had available in their labs. Among all the populations reported, a set of 1126 unrelated male samples distributed in 12 populations was selected. This selection was performed to obtain the best possible representation of the general worldwide distribution of populations. Selected population samples were distributed equally among the participating laboratories to perform the validation as a collaborative exercise.
The approach takes advantage of the specific geographic distribution of the Y-chromosome haplogroups and demonstrates the utility of binary polymorphisms to infer the origin of a male lineage.
Contact: brioniml@usc.es

P-042
Y-chromosomal and mitochondrial markers: a comparison between four population groups of Italy

Brisighelli F¹, Capelli C¹³, Álvarez-Iglesias V2, Arredi B¹, Baldassarri L¹, Boschi I¹, Dobosz M¹, Scarnicci F¹, Salas A2, Carracedo A2, Pascali VL¹

¹Forensic Genetics Laboratory, Istituto di Medicina Legale, Università Cattolica del S. Cuore, Roma Italy
² Instituto de Medicina Legal, Facultad de Medicina, Universidad de Santiago de Compostela, Galicia, Spain
³PromegaGenetic Identity Europe, Promega Corporation , Madison WI, USA

The investigation on the genetic diversity of humans has become fundamental to the complete understanding of the pre-history and history of populations, and it is presently addressing crucial issues of the human evolution that intersect with demographic, cultural and linguistic events. Numerous studies have been recently focused on the Italian Peninsula, and the current set of data regarding this country can now fit into a general frame in which local differences seems to emerge and be interpreted in the context of other cultural and historical knowledge. However, a comprehensive study based on multiple genetic systems and on extensive sampling is still missing. Here we report new data on the Y chromosome and mitochondrial DNA (mtDNA) over a significant larger Italian sample. In particular we address four geographic sites that in the past have been the theatre of significant events in the framework of Italy’s peopling: Latium (central-west), Piceno (central-east), Calabria (south-west) and Messapia (south-east). Concerning the Y haplotype, we based our study on STRs and SNPs polymorphisms in order to tackle populational events positioned at various stages of the evolutionary history of Italy, and to account of local differences. Much to the same purpose, mtDNA has been characterized for the complete sequence of the two hypervariable segments (HVS-I and HVS-II) and to a selection of informative mtDNA coding region SNPs. The availability of both sets of loci including slow- and fast-evolving markers has enabled us to undertake multiple-level comparisons. We paid special interest to the distribution of genetic variability across our populations and we aimed to compare the mainframe emerging from the haploid male and female inherited loci. Preliminary results provided us with some intriguing inference regarding the prehistory and history of Italy will be discussed.

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