P-261
Multiplex
typing of 5 Y-chromosomal SNPs
Schell D, Klein R, Miltner E, Wiegand P
Dept.
of Legal Medicine, University Hospital
of Ulm, Prittwitzstraße
6, 89073 Ulm
Within
the last years PCR based SNP (single nucleotide polymorphism) typing has become
more and more important in forensic DNA analysis. Many different methods have
been established for SNP detection and especially minisequencing has often been
used. In the year 2000 a new kit (SNaPshot Kit, Applied Biosystems, Darmstadt, Germany)
was introduced which is based on the principle of minisequencing and especially
designed for SNP detection using capillary electrophoresis. For our study we
have selected 5 Y-chromosomal SNPs (M9, M17, M45, M170, M173) based on the
degree of polymorphism. PCR primers were designed with the aim to get amplicon
lengths < 200 bp. The Y-SNPs were
optimized in singleplex reactions and then combined to multiplex approaches by
sequential optimization. Finally, we were able to analyse these 5 Y-chromosomal
SNPs in one PCR/SNaPshot reaction. To validate this method for forensic stain
analysis we investigated bones, bloodstains, cigarette butts and epithelial
cells. Additionally, we typed 50
unrelated Caucasian individuals to obtain our regional haplotype frequencies.
P-262
CYP2D6 polymorphism and methadone
metabolism –a pharmacogenetic study
Schmid D,
Anslinger K, Rolf B
Institute
of Legal
Medicine, Ludwigs-Maximilians-Universtiy of Munich, Munich
Germany
CYP2D6 is a highly polymorphic enzyme of the cytochrome P450 family
involved in the metabolism of many drugs. Due to the polymorphism of this
enzyme, pharmacokinetically distinct phenotypes can be distinguished that show
different enzymatic activity of the CYP2D6. For many alleles of the CYP2D6 gene,
the resulting phenotype can be predicted.
Methadone is often used for substitution therapy of heroin dependence.
The dose of methadone is highly variable between patients. This great
interindividual variability is probably based on a pharmacokinetic difference
in the phase I metabolism. It is known that the first step of methadone
degradation is catalyzed by several cytochrome P450 enzymes, among others
CYP2D6.
To determine the effect of CYP2D6 on the metabolism of methadone, we
analysed the most common CYP2D6 polymorphism in a sample of 96 heroine abusers
that were undergoing a substitution therapy with methadone.
In this sample, poor metaboliser (PM), intermediate metobolizer (IM),
extensive metabolizer (EM) and ultra rapid metabolizer (UM) were observed. The
phenotypes are compared to the methadone dose and the methadone concentration
in the blood of the patients.
Contact:
Burkhard.Rolf@med.uni-muenchen.de
P-263
Absolute DNA quantification of forensic casework
samples
Schulz I, Schneider PM,
Rothschild MA
Institute of Legal Medicine, University of Cologne, Germany
In forensic casework, identification systems deal with
routine DNA typing of minute amounts of DNA, characteristically found in small
blood stains, saliva stains, and other old or degraded biological material.
Fragment size analysis using multiplex short tandem repeat (STR) systems yields
excellent results from small quantities of DNA with high discrimination power.
However, validation studies on the STR systems have shown that the amount of
template DNA must be controlled to ensure optimal amplification and subsequent
identification of the PCR product. The STR systems have an optimum DNA range of
0.5 to 2.0 ng. Thus, it is essential to determine the concentration of
extracted human DNA prior to amplification. In addition to recently introduced
real-time PCR methods, routine quantification of forensic samples is still
predominantly done either by subjective assessment, by time- and labor-intensive
slot blot procedure, or by sample-consuming and often unreliable
UV-spectroscopy.
In this study, two DNA quantification methods were
validated and compared in their quantification efficiency using the ABI PRISM®
7000 Sequence Detection System (SDS, Applied Biosystems). In addition to the
QuantifilerTM Human DNA Quantification Kit (AB), a "home
made" Telomerase Assay was designed based on sequence information provided
by Applied Biosystems, and having a 98 bp target fragment. The assays are based
on fluorescence resonance energy transfer and the 5’-3’ exonuclease activity of
Taq DNA polymerase, respectively. Both assays detect a non-translated region of
the human telomerase reverse transcriptase gene (hTERT). In contrast to the
home made assay, the minor groove binder (MGB) probe of the QuantifilerTM
Human DNA Quantification Kit has a length of 62 bp, and is thus 36 bp shorter
than Telomerase Assay probe which is directly reflected in sensitivity
differences. After establishing the within- and between-run precision, the
range, the sensitivity limit and human specificity for both assays, a panel of
forensic casework samples (n = 1000) was quantified, compared and investigated
to assess the potential correlation with the subsequent typing results.
P-264
A novel method to quantify deleted
mitochondrial DNA in a real time PCR
Schwark T, Fisch-Kohl C, von Wurmb-Schwark N
Institute of Legal Medicine, University of Kiel, Kiel, Germany
The
quantification of deleted 4977 bp mitochondrial DNA (dmtDNA) may be of interest
for the forensic as well as clinical pathologist. However, the determination of
dmtDNA in two separate PCR reactions may lead to imprecise if not false results
due to pipetting inaccuracies, deviant PCR conditions, etc. A conventional
duplex PCR with subsequent fragment analysis yields only relative quantities of
dmtDNA based on the analysis of PCR end products. To eliminate these factors,
we established a duplex real time PCR using FAM- and VIC-labeled MGB probes.
The PCR was carried out on an ABI Prism 7000 Sequence Detection System using
standard chemistries. Amplicon sizes were 123 bp for deleted and 113 for total
mitochondrial DNA. Serial dilutions showed a detection limit of 10 copies for
both fragments.
In
our opinion, the presented duplex PCR is an efficient means to reliably and
easily quantify the mitochondrial 4977 bp deletion without the limitations of (conventional)
singleplex PCRs.
Contact: NvonWurmb@rechtsmedizin.uni-kiel.de
P-265
The development of three SNP-assays for
forensic casework
Senge T , Junge A , Madea B
Institute of Legal Medicine, University of Bonn, Germany
Several groups of forensic
researchers work on the development of SNPs as a new marker generation for DNA
classification.
The present work introduces
three novel validated SNP-assays and describes their applicability in forensic
casework. For this study three unlinked and non-coding SNPs named TSC0582423
(Chromosome 2), TSC0171847 (Chromosome 1) and TSC0741184 (Chromosome 3) with a
balanced allele distribution were selected from the database of the
SNP-consortium. SNP-detection was based on the 5'Nuclease-system from Applied
Biosystems. Before the use of this SNP-detection-system in the laboratory
routine, validation studies must be performed including determination of
SNP-genotypes by sequencing, sensitivity studies, population studies and
analysis of artificial stains. For each SNP DNA of eight unrelated persons was
sequenced by Taq Cycle Sequencing to determine their SNP-alleles. These
sequenced samples with known genotypes served as standard probes to establish
the 5´Nuclease-assays and were used as positive controls in all analyses. For
each SNP the genotype of each person determined by sequencing was identical
with that determined by the 5´Nuclease-assays. Sensitivity studies were carried
out with template DNA-amounts ranging from 5ng to 50pg. The test results show
that an amount of at least 250pg genomic DNA could be reproducibly typed. The
population studies, which were done with a group of 40 unrelated persons, show
an equated allelic distribution of 58% T-Allele to 42 % C-Allele for
TSC0582423, 40% T-Allele to 60% C-Allele for TSC0171847 and 59% T-Allele to 41%
C-Allele for TSC0741184. The allele frequencies were comparable with those
published by the SNP-consortium. The artificial stains consisted of two
cigarette butts, two chewed-on chewing gums, a bottle neck abrasion as well as
a scalp abrasion taken from under a fingernail. DNA extracted from the
stain-material gave the expected genotypes as determined by the analysis of the
corresponding saliva samples. In order to type stains with a low DNA content,
an upstream multiplex PCR was developed. For each SNP a singleplex PCR with the
5´Nuclease-primers was established to get the optimal conditions for the multiplex
PCR. Since only short PCR products -of about 100bp- are detected by SNP-typing
using the 5´Nuclease-method, this method could be needful to detect degraded
DNA. To assess the correctness of this hypothesis different approaches will be
applied in the future: e.g. DNAse I-digestion and environmental studies. Also a
further aspect of this work will be the comparison of the 5´Nuclease-system
with the so-called Minisequencing, another method for SNP-detection, to get
knowledge of the effectiveness and robustness of the two methods.
Contact: b.madea@uni-bonn.de
P-266
Y-STR loci multiplex amplification and
haplotype analysis in a Chinese Han population
Shi MS, Li YB, Wu J, Hou YP
Department of Forensic
Genetics, Sichuan
University (West China
University of Medical
Sciences), Chengdu,
P.R.China
We have developed a multiplex PCR assay dealing with simultaneously
amplifying 9 STR loci on Y chromosome to aid human identity testing. These Y-
STR markers included DYS434, Y-GATA-A10, DYS438, DYS439, DYS531, DYS557,
DYS448, DYS456 and DYS444. Efforts were made to design three sets of the tailed
primers to improve the efficiency of the multiplex PCR as well as close packing
of PCR product size ranges in order to keep all alleles less than 300 bp
through careful examination of known allele ranges. A total of 101 different
haplotypes was found among 120 unrelated males in the Chinese Han population by
using the Y-STR-9-plex system, 91 of them being unique. Gene diversity ranged
from 0.4394 at DYS434 to 0.7975 at DYS557. The haplotype diversity value
calculated from all nine loci combined was 0.9968. The minimum amount of input
DNA that could be used to obtain a full 9 Y-STR profile was 1 ng. For the
male/male mixtures, the minor component in the mixture could be identified to a
ratio of 1:9. In male/female DNA mixtures, the Y-STR-9-plex proved to be highly
specific for the Y chromosome in that no significant female DNA products were
observed up to 300 ng of female DNA. Our results revealed that the Y-STR-9-plex
system was useful for forensic analysis and paternity tests in the Chinese Han
population
P-267
Characterisation of Y
Chromosome SNPs Duplications
Silva MR1 , Serra S1 , Ribeiro T1
, Geada H2
1Forensic Genetics,
Lisbon Delegation, National Institute of Legal Medicine
2Faculty of Medicine,
University of Lisbon (hgeada@dlinml.mj.pt)
Single
nucleotide polymorphisms (SNPs) have become widely used for population
genetics. Multiple analysis of SNP markers can be important in future for human
identity testing, specially in parentage testing and forensic casework due to
low mutation rates of SNPs and analysis of 40-50 bps from heavily degraded DNA.
Automation of SNP technology is also key demand. The lack of recombination along most of the Y
chromosome makes it a useful tool in male population studies and in special
forensic situations, e.g., in cases of mixtures of DNA and unavailable alleged
father. Furthermore, Y- SNP markers offer additional information to that
obtained by STR typing. SNP typing method was based on multiplex PCR and
minisequencing reaction with SNaPshotTM Multiplex Kit followed by
capillary electrophoresis on an AB Prism 3100 Genetic Analyser. In a total of
102 unrelated South Portuguese males typed, 28 were from Faro District (Algarve) and 19
from Beja District. 20 Y-SNPs have been studied in three multiplex (MP)
reactions – MP1/M22, P25, 92R7, SRY1532, M173, M70, Tat, M213, M9; MP2/M170,
M62, M172, M26, M201; MP3/M34, M81, M78, M35, M96, M123. The most frequent
haplogoup was R1b* and the haplogoup diversity was 0.6720 and 0.8304,
respectively, in Faro and Beja populations. E3b1 haplogoup was only encountered
in these Portuguese population samples.
In
Forensic Genetics, it is important that SNP markers have only one polymorphic
site for results interpretation in forensic casework. However, two of the most
widely used Y-SNPs for population studies and for forensic purposes present two
signals – P25 and 92R7. These duplicate segments, called Paralogous Sequence
Variations (PSVs), occur in the same multiplex reaction (MP1), which at a first
glance could interfere in the results interpretation. P25 polymorphism is
considered to be a C-A transversion, while 92R7 is a G-A transition. These two
SNP markers are implicated in the definition of the P, Q or R haplogrups in the
phylogenetic tree of the Y-chromosome. Almost all samples studied presented
duplication in one of these SNP markers. Two situations have been well
characterised – 92R7GA, M173A, P25C for the ancient state in these SNP markers
and 92R7A, M173C, P25CA for defining haplogroup R1b*(0.4901 in the Portuguese
population). In very few samples characterising haplogroups R1* or R1a no
duplication was detected in the two Y-SNP loci (92R7A and P25C). The
polymorphism of the two Y-SNPs will be considered as C – CA for P25 and GA – A
for 92R7. No P25A or 92R7G has been detected in the Portuguese population.
However, in a latter African population study a 92R7G was detected, defining
the ancient state as 92R7G detected in association with M173A and P25C. Another
duplication was detected in the Y-SNP M78 when studying MP3. M78 polymorphism
was defined as a C-T transition. While in the ancient state (M78C) the
duplicated peak was in the M96 peak area, but when defining the E3b1 haplogroup
(M78T), a double T peak occurred. Singleplex defined the M78 polymorphism as CC
– TT. These different types of
polymorphims should be capped in mind when performing SNP typing, as by any
chance, a different genetic mechanism can occur, which enable a different
number of peaks in a SNP electropherogramme. Rather than an obstacle, these
problems should be a challenge when using this useful new methodology for SNP
typing.
Contact:
ARebelo@inml.mj.pt
P-268
Results of the 2005
Paternity Testing Workshop of the English Speaking Working Group
Simonsen
BT, Hallenberg C, Morling N
Department
of Forensic Genetics, Institute
of Forensic Medicine, University of Copenhagen, Denmark
Since 1991, The English
Speaking Working Group (ESWG) of the International Society of Forensic Genetics
(ISFG) has, once a year, offered an exercise involving genetic analysis in a
paternity case. In 2005, the laboratories were invited to calculate a paper
challenge in addition to performing paternity testing of a mother, two children
and an alleged father. Blood samples were sent to 67 laboratories together with
information about the paternity case. Also, a questionnaire concerning the
techniques and routines in the laboratories were distributed.
Here, we present the results
of the 2005 Paternity Testing Workshop. The evaluation includes
concordance/discordance in typing results, collation of the systems used by the
laboratories as well as methods used for DNA-typing. Furthermore, we present a
comparison of the requirements given by the laboratories to issue a report with
an excluded man and with a non-excluded man, respectively. As laboratories used
different systems for typing as well as different frequency-databases in
calculations, comparison of calculated PI-values in the performed paternity
test was not possible. Therefore, the paper challenge constituted a valuable
tool to compare calculations and to compare how laboratories deal with genetic
inconsistencies, silent alleles, rare alleles as well as Y-chromosomal
haplotypes. Laboratories were encouraged to treat results in the paper
challenge as they would do in a paternity case as well as in an immigration
case. The results of the paper challenge are presented and discussed.
[1] Syndercombe Court D, Lincoln P, in:
Carracedo A, Brinkmann B, Bär W (Eds.). A review of the 1991-1994 Paternity
Testing Workshops of the English Speaking Working Group. Adv Forensic Haemogenet 1996;6:683-685.
[2]
Bjerre A, Syndercombe
Court D, Lincoln P, Morling N. A report of the
1995 and 1996 Paternity Testing Workshops of the English Speaking Working Group
of the International Society of Forensic Haemogenetics. Forensic Sci Int 1997; 90(1-2):41-55.
[3]
Hallenberg C, Morling N. A report of the 1997, 1998
and 1999 Paternity Testing Workshops of the English Speaking Working Group of
the International Society of Forensic Genetics. Forensic Sci Int
2001;116(1):23-33.
[4]
Hallenberg C, Morling N. A report of the 2000 and 2001
Paternity Testing Workshops of the English Speaking Working Group of the
International Society of Forensic Genetics. Forensic Sci Int 2002;129(1):
43-50.
Contact:
Bo.Simonsen@forensic.ku.dk
P-269
Validation of multiplex STR
systems for the investigation of familial relationships in immigration cases
Sippel H, Hedman M, Sajantila A
Department of Forensic Medicine, University of Helsinki,
Finland
The purpose of this study was to evaluate the
efficiency of three multiplex STR PCR Kits: SGM Plus and Profiler from Applied
Biosystems and Penta BEC Multiplex from Promega in family reunion testing. The
SGM Plus Kit is composed of the sex marker Amelogenin and 10 STR systems:
D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA.
The second Kit, designed as Profiler combines Amelogenin and 9 STR loci:
D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820. Finally, the
third kit Penta BEC Multiplex combines three STR loci, Penta B, Penta E and
Penta C, and provides additional power to resolve difficult cases.
Practically any first-degree biological family
relationship can be established with the same technique as that for paternity
investigation. Conventional paternity testing usually assumes that the mother
is the true biological mother. However, in family reunion testing the aim is to
investigate whether the family is a true biological family, so there are
several potential constellations, including the maternity and sibship, to test.
The paternity, maternity,
sibling and avuncular indices and the likelihood ratios were calculated using
the DNA-view immigration program (Brenner C, Berkeley, USA),
and this calculation was based on a database constructed from the respective
ethnic group. For those situations where a specific population was not
available a Finnish database was used. For correcting for population
substructure, an inbreeding coefficient Θ = 0.01 was used.
On the basis of this study
the use of 18 STR loci is recommended for difficult familial relationship
cases. Often cases with mutations, single-parent paternity cases and sibling
testing without parents cannot be sufficiently resolved with SGM Plus and
Profiler systems alone.
Helmuth.Sippel@helsinki.fi
P-270
Molecular variation and genetic structure of variable
drug response in a worldwide population sample
Sistonen J1, Fuselli S1,
Barbujani G2, Sajantila A1
1Department of
Forensic Medicine, University
of Helsinki, Helsinki, Finland
2Department of Biology, University of Ferrara, Ferrara, Italy
Pharmacogenetics is the study
of genetically determined variation in drug response. The polymorphic
Cytochrome P450 2D6 (debrisoquine 4-hydroxylase) is one of the best
characterized genetic factors underlying abnormal responses to drugs. It
belongs to the group of drug-metabolizing enzymes and it is responsible for
about 25% of the metabolism of commonly used drugs belonging to classes like
antidepressants, neuroleptics, beta-blockers and antiarrhytmics. CYP2D6
is highly polymorphic and the phenotypic consequences are considerable: the
enzyme activity ranges from complete deficiency, possibly giving rise to
profound toxicity of medication, to ultrarapid metabolism, which can lead to
therapeutic failure with recommended drug dosages. SNPs and sequence
polymorphisms of the CYP2D6 gene have been studied earlier in several
populations but no systematic analysis of SNPs at the intercontinental level
has been attempted to date. Here, we present the first population-genetic study
of the CYP2D6 data collected in 1060 individuals from a worldwide sample
of 52 populations (HGDP-CEPH panel). In the whole set of individuals, we
genotyped 12 SNPs and two major rearrangements of the gene using a combination
of long PCR and multiplex single base extension reaction (SNaPshot). The data
was analysed to describe the geographic distribution of the gene diversity and
to identify a possible spatial structure. Analyses of genetic distances and
spatial autocorrelation suggest the absence of major genetic barriers and the
existence of a clinal pattern. The observed partition of molecular variance
shows that the percentage of global genetic variation due to intercontinental differences
agrees with the value commonly obtained for neutral markers. This variation
turned out to be sufficient to identify by means of a Bayesian analysis a
stable population structure. Considering the relevance of CYP2D6
variation in drug metabolism and the worldwide distribution of alleles coding
for enzymes with abnormal metabolic activity our results have applications in
clinical as well as in forensic medicine.
P-271
Mitochondrial DNA variability in
populations from East Timor (Timor
Leste)
Souto L1,
Rocha A M2, Pires A2, Ferreira E2,
Kayser M3, Amorim A4,5, Côrte-Real F6, Vieira
D N6
1Centro de Biologia Celular, Departamento de Biologia,
Universidade de Aveiro, Aveiro, Portugal
2Departamento de Biologia, Universidade de Aveiro,
Aveiro, Portugal
3Department of
Forensic Molecular Biology, Erasmus
University Medical
Centre Rotterdam, The Netherlands
Centre Rotterdam, The Netherlands
4IPATIMUP, Instituto de Patologia e Imunologia Molecular
da Universidade do Porto, Portugal
5Faculdade de Ciências, Universidade do Porto, Portugal
6 Delegação de Coimbra, Instituto Nacional de
Medicina Legal, Coimbra, Portugal and Faculdade de Medicina da Universidade de
Coimbra, Portugal.
East Timor (Timor Leste)
represents a region with high linguistic diversity where two major language
groups: Austronesian and non-Austronesian (or Papuan) languages are found. The
purpose of our study is to reveal whether the linguistic diversity is reflected
in the genetic diversity and whether genetic data can shed light into the human
population history of East Timor. In this
study we continue the genetic characterization of human populations from East Timor, as previously started for autosomal STRs and
Y STRs, with a preliminary report on mitochondrial DNA (mtDNA) diversity. A
sample of 110 individuals collected from all the districts of Timor
and representing 12 linguistic groups was studied. Analyses of the mtDNA
sequence from the hypervariable region 1 (HVS1) and the presence of the 9-bp
deletion (intergenic regions COII-tRNA lys) were performed.
These genetic data allowed us
to detect the presence of the P and Q haplogroups, typical of non-Austronesian
(Papuan) speaking populations from New Guinea, along with a significant frequency
of haplogroup B (namely B4a and B4b) generally associated with the expansion of
Austronesian-speaking groups from East Asia along with several M sub-branches
previously reported to the Southeast Asian region. Thus, our preliminary data
show a correlation between linguistic and genetic diversity in East Timor and further more detailed analyses will reveal
insights into the population history of the region. In addition our genetic
data (mtDNA, STRs) may serve as prerequisite for forensic genetic applications
in East Timor.
P-272
Y-chromosome haplotypes in East
Timor: evidences of population differentiation
Souto L1, Gusmão L2, Ferreira E3,
Pires A3, Rocha A M3, Amorim A2,4,
Côrte-Real F5,Vieira D N5
1Centro de Biologia Celular, Departamento de Biologia,
Universidade de Aveiro, Aveiro, Portugal
2IPATIMUP, Instituto de Patologia e Imunologia Molecular
da Universidade do Porto, Portugal
3Departamento de Biologia, Universidade de Aveiro,
Aveiro, Portugal
4Faculdade de Ciências, Universidade do Porto, Portugal
5 Delegação de Coimbra, Instituto Nacional de
Medicina Legal, Coimbra, Portugal and Faculdade de Medicina da Universidade de
Coimbra, Portugal.
The Y-chromosome
markers, due to their peculiar characteristics are considered useful tools in
detecting populations’ differentiation.
The population of East Timor is known to gather a high number of
ethnolinguistic groups. From linguistics and classical anthropological studies,
the different groups in east Timor are
generally assigned to Austronesian or Papuan ancestry.
We analysed a total
of 346 males from East Timor for 12
Y-chromosome specific STRs (DYS19, DYS389I and II, DYS390, DYS391, DYS392,
DYS393, DYS385, DYS437, DYS438 and DYS439). According to anthropological and
linguistics information, samples were classified in major linguistic subgroups:
Timoric (Timorese-Austronesian), which
includes Group
A (“Fabronic”, N=92) and Group A1 (“Ramelaic”, N= 130), and a third group
including languages that are related to a Trans-New Guinea phylum (Papuan) and supposed
to represent aboriginal (pre-austronesian) signature (Group B, N= 124).
The highest
haplotype diversity value was found in Group A, “Fabronic” (0.9931), while the
“Papuan” group B shows the lowest value (0.9882).
Comparison of the 3
samples suggests that the “Ramelaic” group differentiates from the “Fabronic”
and “Papuan” ones (Rst values of 0.02710, P= 0.00366 and Rst of 0.05839, P=
0.0000).
These genetic
results, while putting in evidence some accordance with the proposed
linguistics classification, and strengthening a high homogeneity of “Papuan”
speaking populations, they also suggest
that the Papuan/Austronesian linguistics criteria is not sufficient to
differentiate East Timor populations and deeper analyses at ethnolinguistic
groups and geographic districts should be considered.
Despite the
uniqueness of the vast majority of the haplotypes found in our east Timor sample,
in an extensive search in the international Y Chromosome Haplotype Reference
Database (YHRD – www.yhrd.org) most of the “matches” were produced with South
East Asia and Oceania samples, although “matches” with some European haplotypes
were also observed.
P-273
Genotyping of human
DNA recovered from mosquitoes found on a crime scene
S.Spitaleri, C. Romano, E. Ginestra and L.Saravo*
1 Laboratory of Molecular
Biology – Raggruppamento Carabinieri Investigazioni Scientifiche (RaCIS),
98128 Messina
– ITALY.
Many mosquitoes species are man parasitical
biters commonly found in the south of Italy by crime scene investigators.
Some authors already demonstrated the possibility of amplifying human DNA from blood-meals of Diptera
species using various methods for epidemiological issues. Here we decribe a
casework occurred in Sicily: a person was killed in a room where no traces were
found apart from a fresh mosquito blood-meal stain which could be probably
referred to who had been sleeping in that place during the previous nites.
Hence an optimized DNA extraction was carefully carried out in order to yield
deoxyribonucleic acid uncontaminated by
insect-specific molecules. PCR amplification and STRs profiling at 15
human genetic loci was then performed on the extracted DNA, using AmpFLSTR
Identifiler kit by Applied Biosystems. Additionally a DNA sample extracted from
a mosquito of the same genus was processed, with equal conditions, to assess
eventual unspecific detection. Results showed that it is possible to
successfully amplify and obtain a complete genetic profile even if DNA is
recovered from small and biologically-contamined traces. The applied analitical
strategy represented a powerful tool for the investigations and allowed to
address the profile to the major suspect of the murder.
Keywords: DNA STR typing;
Forensic casework; Identifiler, Mosquitos.
*Corresponding
author: rismebiologia@carabinieri.it
P-274
Single Human Telogen Hair Analysis: Multiplex Amplification of 8 STR loci
Spitzer E, Borck
C, Grosse R
Institute of Medical Molecular Diagnostics GmbH,
Schoenstrasse 90, 13086 Berlin,
Germany
Hairs in the
telogen phase represent the majority of trace hairs collected in connection
with criminal cases. However, until now nuclear DNA profiling has not yet
become routine in forensic casework because of PCR problems related to low
amount and high degradation of DNA isolated from telogen hairs. There are no
published procedures for efficient genotyping of single hair shafts.
Here we describe a
protocol for simultaneously amplifying 8 STRs in 3 triplex amplification
reactions. DNA has been purified from single hairs by phenol –chloroform
extraction yielding a total of 15μl of DNA.
The primers were designed to produce amplicons of 50-150 bp. SE 33 has
been excluded from triplexes due to its fragment size of 200 bp and more.
The three triplexes designated as MPX1, MPX2 and MPX3 consisted of the
STRs TPOX/THO1/D18, FGA/vWA/D3, and Amelo/D8/D21 respectively. Fragment analysis
has been performed by means of the 310 Genetic analyzer.
We show a number of examples to demonstrate usefulness and efficacy of
the multiplex approach for single hair DNA analysis. Also, we give examples
showing the necessity to perform STR typing of short DNA fragments in a special
clean area apart from laboratory.
Finally, we show that our approach is suitable for other stain analysis,
particularly if degraded DNA of low amounts is to be expected.
P-275
Molecular analysis of genomic low
copy number DNA extracted from laser-microdissected cells
N. Staiti (1), G. Giuffrè (2), D. Di Martino (1), A. Simone(2), G. Sippelli(2), G. Tuccari(2), and L . Saravo(1)*
1 Laboratory of Molecular Biology –
Raggruppamento Carabinieri Investigazioni Scientifiche (RaCIS), 98128 Messina –
ITALY.
2 Department of Human Pathology, University of Messina.
Tissue microdissection
techniques, allowing a correlation between the topologic organization of the
cells and the molecular analysis of nucleic acids, offer the chance to
characterize the several kinds of cells present both in pathologic and in
forensic samples cutting small tissue fragments as well as single cells by an
ultraviolet laser beam under direct microscopic visualization. The low number
of target cells among a wide spectrum of cell types in heterogeneous tissue
samples may complicate the analysis of biological residuals found on crime
scenes, while the possibility to perform a genomic analysis of low copy number
DNA from few cells harvested by laser microdissection represents a valid aid in
order to solve forensic problems.
In the present report we
evaluate the sensitivity of the above described method in order to perform
genomic analysis from low copy number DNA. Laser-microdissection was performed
using a Leica AS LMD system (Leica Microsystems, Germany) on fresh smears of aploid
(spermatozoa) and diploid (lymphocytes) cells, on sections of routinely
formalin-fixed and paraffin-embedded tissues, on cryostatic sections obtained
at surgery and post-fixed with cold methanol. The samples were stained with
specific procedures (Haematoxilin-Eosin, Giemsa, Papanicolau,
Picroindigocarmine-Nuclear fast red) and an increasing number of cells (from 1
to 100) was harvested from them in different PCR tubes. DNA extraction was
performed by Chelex™ 100 (Biorad), QIAmp DNA Micro Kit (Qiagen) and DNA IQ™ System (Promega Corp.). DNA extracted
from each sample was amplified to identify a specific genetic profile by the
most common microsatellite loci of pathologic and forensic interest. PCR
products were separated by capillary electrophoresis with 3100 AB Prism Genetic
Analyzer, and analyzed by Genemapper Software v 3.2 (Applera Europe B.V.).
A complete genotypic profile
was obtained down to 10 aploid and 5 diploid cells as to pathological and
forensic genetic markers; different results - concerning the integrity of the
extracted DNA - were achieved according to the kind of histological stainings
performed.
Keywords:
DNA STR typing; Forensic
casework; Mentypeâ, LMD, LCN.
*Corresponding author: rismebiologia@carabinieri.it
P-276
AZF deletions of the Y chromosome and failed amplification of commonly used Y-STRs
Stein B1, Willuweit
S1, Nagy M1, Vogt PH2, Roewer L1
1Institute of Legal
Medicine, Charité – University Medicine Berlin,
Hannoversche Str. 6, 10115 Berlin,
Germany
2Section of Molecular Genetics and Infertility,
Department of Gynecological Endocrinology and Reproductive Medicine, University of Heidelberg, Im Neuenheimer Feld 328,
D-69120 Heidelberg
Deletions of the Y chromosomal
azoospermia factor called AZFa, AZFb and AZFc have shown to be associated with impaired
spermatogenesis. AZFc deletions are the commonest of the deletions found
in infertile men, with a frequency estimated to be 1 in 4000. AZFa
deletions are particularly rare (< 1 : 100 000), whereas AZFb deletions are
intermediate in frequency. Sequence analysis of the complete Yq11 region
reveals a 1.5 Mb overlap of the AZFb and AZFc regions (AZFb+c).
Several widely used as well as new Y-STRs can be affected by the AZF deletions.
For example, the multicopy Y-STR DYS464 lies within the ampliconic r1-r4
repeats of the AZFc region. The locus DYS389 resides in the AZFa
region, and DYS385 and DYS392 in AZFb.
In a Y chromosomal “minimal”
haplotyping study of 48 German males deleted for AZFc, AZFb and AZFb+c
we found several incomplete haplotypes lacking PCR products at the loci DYS392
and DYS385. Two AZFc diagnosed male DNAs failed to amplify at the locus
DYS392. This points to a wrong AZF classification, an AZFb+c deletion is
more probable for this Y chromosome. One AZFb male lacks only the loci
DYS385. Two AZFb males lack both DYS392 and DYS385. All three AZFb+c
males show PCR failure at the loci DYS392 and DYS385. The remaining 41 AZFc
diagnosed males show complete minimal haplotypes.
Recently, King et al. report
deletions of DYS464 in three AZFc males (f = 3/3255) and 9 out of 19 Y-STR amplification failures for
an AZFa deleted chromosome(f = 1/5374).
The core set marker set of the Y-STR haplotype reference database (YHRD)
currently includes the markers DYS19, DYS389I+II, DYS390, DYS391, DYS392,
DYS393, DYS385, DYS438 and DYS439. All of these STRs are well established in
forensic and genealogical routine and are included in most commercial kits. Of
these STRs the markers DYS389, DYS392, DYS385, DYS438, DYS439 could fail to be
amplified due to AZF deletions, thus generating incomplete haplotypes. King et
al. have recommended to skip DYS464 testing for genealogical and forensic
routine because of the relative high frequency of the AZFc deletion and
its association with infertility. Probably because of its rarity, incomplete
haplotypes pointing to the AZFa and AZFb deletions have not yet
been reported to the YHRD currently comprising about 28.650 minimal and 6.281
extended haplotypes.
A consequence of inadvertent
diagnosis of male infertility through genealogical or forensic Y-STR
haplotyping should be information and possibly warning of customers by
companies and institutions using Y-STR markers for identity testing.
Contact: lutz.roewer@charite.de
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