P-277
TSUNAMI-Disaster: DNA typing of Sri Lanka
victim samples and related AM matching procedures
Steinlechner M, Parson W, Rabl W, Grubwieser P,
Scheithauer R
Institute of Legal Medicine, Innsbruck Medical University, Austria
More than 30.000 individuals
of varied racial and ethnic background lost their lives when Sri Lanka was
affected by the South Asian tsunami. DVI-Teams from many countries were
involved in examining victims, taking DNA-samples from victims for
identification procedures. The principal role of our laboratory in this process
was to provide barcoded sampling kits, to perform DNA typing of human remains
and ante mortem (AM)/kinship samples and to accomplish DNA matching services to
support DNA-based identifications.
Until now (22.04.2005) more
than 400 samples related to 101 victims were sent to our laboratory. The
barcoded sampling kits allowed for the collection of 2 swabs (from intact inner
surfaces like muscle, urinary bladder etc.), 1 4 cm piece of long bone and 1-2
teeth, which were all shipped on ice.
A high throughput DNA
extraction and STR analysis procedure for the swabs were developed in order to
provide STR profiles within 24 hours. For the skeletal remains and teeth a
highly sensitive DNA extraction procedure was set up in order to maximise DNA
recovery. Both processes included electronic accessioning to maintain the
numbering system of the DVI team in Sri Lanka and the data exchange via
Interpol and PLASS Data Software.
AM DNA profiles related to
missing persons from Sri
Lanka and Austria were generated in our
laboratory, AM DNA profiles related to missing persons from other countries
were transmitted via Interpol or directly sent to our laboratory for matching
purposes. DNA matching procedures were facilitated by using the Mass Disaster
Matches option of Charles Brenners DNAVIEW software.
In all, the established DNA
typing procedures proved to be highly efficient. The swab typing procedure
produced successful results in 47% of the completed cases (n=81). For the
remaining cases skeletal remains had to be typed via the sensitive DNA
extraction procedure and produced useful results in all investigated samples so
far, obtaining 34% full profiles (AmpFLSTR® Identifiler® PCR Amplification Kit)
and 66% partial profiles (>=7 STR loci). In nine cases the DNA investigations
were stopped due to successful dental identifications. The analyses of the
remaining eleven cases are currently underway.
Until now, the DNA matching
procedures enabled 22 DNA-based identifications of victims from eight
countries.
P-278
Norwegian population data for 2
autosomal STR loci; D12S392 and D17S906
Stenersen M, Perchla D, Dupuy
BM
Institute of Forensic Medicine, University of Oslo,
Norway
Autosomal STR
polymorphisms at 2 loci (D12S392 and D17S906) are presented. Samples from
current paternity analysis (n=713) were analysed. The observed heterozygosities
were 0.891 (D12S392) and 0.934 (D17S906). No significant deviation from
Hardy-Weinberg equilibrium was observed. 19 different alleles were observed at
D12S392 whereas a total of 56 different alleles were observed at D17S906. The
repeat numbers ranged from 15 to 27 at D12S392. For D17S906 the allele sizes
ranged from 331 to 437 bases. Sequence variation at both loci will be presented
as well as frequency databases and other relevant forensic genetic parameters.
Corresponding
author: Margurethe Stenersen
Mailing address: Rettsmedisinsk
Institutt
Rikshospitalet
N-0027 Oslo
Norway
Telephone
number: 00 47 23 07 13 17
Fax
number: 00 47 23 07
13 18
E-mail: margurethe.stenersen@labmed.uio.no
P-279
Norwegian population data for 15
autosomal STR loci: PowerPlex 16
Stenersen M, Perchla D, Dupuy
BM
Institute of Forensic Medicine, University of Oslo,
Norway
Autosomal STR
polymorphisms at 15 loci (D3S1358, THO1, D21S11, D18S51, Penta E, D5S818,
D13S317, D7S820, D16S539, CSF1PO, Penta E, vWA, D8S1179, TPOX and FGA) are presented.
Samples from current paternity analysis (n=1380) were analysed. The observed
heterozygosities ranged from 0,610 (TPOX) to 0,896 (Penta E). One significant
deviation from Hardy-Weinberg equilibrium was observed at Penta D. The number
of observed alleles ranged from 7 (THO1 and TPOX) to 19 (D21S11 and FGA). The
shortest tandem repeat observed was 2.2 at Penta D and the largest 44.2 at FGA.
The power of discrimination and exclusion ranged from 0.787 (TPOX) to 0.967
(FGA) and from 0.303 (TPOX) to 0.788 (Penta E) respectively. Frequency
databases and other relevant forensic genetic parameters will be presented in
detail.
Corresponding
author: Margurethe Stenersen
Mailing address: Rettsmedisinsk
Institutt
Rikshospitalet
N-0027 Oslo
Norway
Telephone
number: 00 47 23 07 13 17
Fax
number: 00 47 23 07
13 18
P-280
A Comparison of
various methods used in extraction of DNA in Sexual assault cases
Student B 2, Fox S 1
1LGC, Queens Rd, Teddington, TW11 0LY,
UK
2 King's College London, London
WC2R 2LS, UK
For the past two decades the method of choice for the enrichment of sperm
cells from sexual assault cases has been that documented by Gill et al (1) in 1985, the preferential
extraction. Following a sexual assault
DNA evidence is usually comprised of vaginal swabs or semen stains. The problem with these samples is that the
spermatozoa are mixed in with vaginal epithelial cells from the victim. Separating spermatozoa from epithelial cells
results in more simple interpretation and increased success of
individualisation. The traditional
method is relatively inefficient and often does not produce complete separations
especially when there is low numbers of spermatozoa which is often the case in
sexual assault samples. This study has
considered different techniques of separating the spermatozoa from epithelial
cells. The first one is the Differexä system by Promega which
essentially uses the same chemistry as the traditional method but employs a
more sophisticated method of separation based on a combination of phase
separation and differential centrifugation.
Further consideration has been given to filtration methods based on the
differences in size and shape of the two cell types.
Reference: Gill, P. Jeffreys, A.J. and Werret,
D. J. (1985) Forensic Application of DNA ‘fingerprints’. Nature 318, 577-9
P-281
Coding region mtDNA analysis
for increased forensic discrimination using
Pyrosequencing technology
Styrman H, Andréasson H,
Nilsson M, Allen M
Department of Genetics and
Pathology, Rudbeck laboratory, Uppsala
University, Uppsala, Sweden
Analysis of mitochondrial DNA (mtDNA) is very useful
for samples where a nuclear DNA analysis fails due to degradation or
insufficient DNA amounts. However a drawback of mtDNA analysis is its limited
discrimination power. The D-loop sequencing performed in routine analysis today
might therefore result in different individuals showing identical HVI and II
sequences. In order to resolve identical mtDNA types from different individuals,
additional discrimination might be achieved by analysis of coding region
variation as a complement to the sequencing of the HVI and II regions. We have previously developed a
Pyrosequencing-based system for both mtDNA control region and coding region
analysis. Pyrosequencing is a fast, non-electrophoretic DNA sequencing
technique that uses PCR products as templates and is based on a four enzyme
reaction to monitor DNA synthesis. In this study, the coding region assay has
been further expanded to cover additional informative positions for increased
discrimination. The entire coding region analysis comprises 17 pyrosequencing
reactions on 15 PCR fragments. Coding region analysis was performed on all 15
fragments in 135 samples, with an average read length of 83 nucleotides. A
total of 52 SNPs with frequencies ranging from 1% to 47% were identified. Of these, 18
variants were found in a single individual, illustrating the potential to
detect highly informative SNPs. The revised Cambridge
reference sequence (rCRS) has the most common HVI/HVII mtDNA sequence and belongs
to haplogroup H. As the rCRS HVI/HVII sequence is very common in Caucasians,
this sequence is often observed in cases with unresolved sequences samples from
different individuals. In a group of 60 samples with identical sequences to
rCRS or with a single difference in the D-loop, only 12 samples could not be
resolved by at least two differences using pyrosequencing analysis of these
coding region fragments. Thus, the use of this pyrosequencing mtDNA coding
region analysis system has the potential to increase the discriminatory power
of mtDNA analysis. Coding region analysis proved very useful in a case involving the bishop
election in Sweden
in 1952. Prior to the election, approximately 500 anonymous letters were sent
to the 190 voting priests. The letters
contained propaganda for one of the five candidates, Dick Helander and slander
about the other candidates. Soon after Helander was assigned as bishop by the
Swedish government, the police initiated an investigation regarding the
letters. Helander became a prime suspect and was convicted on a chain of
circumstantial evidence. DNA from the sealing of a total of four anonymous
letters were analysed by sequencing of HVI and HVII. The results were compared
to DNA from four official letters written by Dick Helander, resulting in a
single C/T difference at position 16239. Due to this inconclusive result,
further analysis of coding region variation was performed. All 15 PCR fragments
were amplified and three additional variable positions (3010, 7028 and 16519)
were identified by pyrosequencing analysis. In conclusion, the use of this
mtDNA coding region analysis system has demonstrated the potential to reduce
the number of unresolved individuals with similar or identical HVI/HVII
sequences by 80%. Furthermore, this pyrosequencing-based system was shown to be
informative in resolving samples with a single difference in the HVI/HVII
sequences in an old forensic investigation. The assay is very easy to use,
rapid and highly flexible, facilitating analysis of different target
combinations. Analysis of the mitochondrial coding region by pyrosequencing can
provide a useful tool in cases where different individuals share identical
HVI/II sequences or when only a single difference is detected between samples.
P-282
STR sequence variants revealed
by Pyrosequencing technology
Styrman H, Divne A-M, Allen M
Department of Genetics and
Pathology, Rudbeck Laboratory, Uppsala
University, Uppsala, Sweden
Pyrosequencing has proven to be useful to evaluate
allelic variants of STR repeats. Pyrosequencing is a fast real time
non-electrophoretic sequencing-by-synthesis method, based on a cascade of four
enzymatic reactions, which enables nucleotide incorporation and release of
pyrophosphate (PPi), to yield detectable light. The produced light is
proportional to the number of incorporated nucleotides and shown as peaks in a
pyrogram. As the actual sequence is determined rather than the fragment length
in pyrosequencing variant alleles resulting in the same fragment length can be
revealed in addition to length variation.
In a population study of 10 autosomal markers (CSF1PO,
THO1, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539 and Penta E)
several allelic variants were detected. At the THO1 locus the
incomplete allele 9.3 was detected in 61 genotypes of which 19 were homozygous.
Furthermore, additional variation was found at loci D13S317, D7S820, D5S818 and
D8S1179. At locus D13S317, we found alleles carrying a T/A SNP in the last
repeat, resulting in the repeat unit AATC. The
possibility to detect this variant allele increases the resolution. For
instance, four different combinations of alleles (12/13, 11AATC/13,
11AATC/12AATC and 12/12AATC) were observed that all generate the same fragment
length. At
the DYS720 locus a C/T SNP in the first sequenced upstream flanking nucleotide (C/TGAACTAAC[GATA]n)
was observed in 9/114 genotypes (8%). The SNP was found in the flanking region
of allele 8 (8 out of 9 samples) and allele 9 (1 of 9 samples). At the D5S818 locus a four base
pair deletion, involving a CTCT motif next to the ATCT repeat was observed in 45/114
genotypes. Finally, at the D8S1179 locus a variant allele, due to a G/C SNP,
resulted in the repeat structure [TCTA][TCTG][TGTA][TCTA]11 in four genotypes.
Furthermore, in a population study of eight Y
chromosome markers (DYS19, DYS389 I-II, DYS390, DYS391, DYS392, DYS393 and
DYS438) variants were detected at four loci. At the DYS391 locus a G/A SNP was
observed in one out of 70 individuals. One nucleotide upstream the repeat, TCTA
was seen resulting in TCTG. At the DYS390 locus a G/A SNP was observed
in one individual, upstreams of the first repeat unit. At the DYS393 locus a
A/C SNP was observed in the first repeat unit, converting the repeat from AGAT
to CGAT in 10/70 individuals. Finally, at the DYS389 II locus an A/G SNP
resulted in (TCTG)6 instead of (TCTG)4-5 in one genotype.
All detected autosomal and Y-chromosome variants were
confirmed by Sanger sequencing. In conclusion, pyrosequencing is a useful tool
for rapid compilation of population data with higher resolution for some
markers. As allelic variants were easily detected in most cases, pyrosequencing
is a suitable method for sequence evaluation of known or novel markers of less
complex nature.
contact: hanna.styrman@genpat.uu.se
P-283
Length Heteroplasmy in the HVI Control Region
Sucena A1 , Ribeiro T1 , Geada H2
1Forensic Genetics,
Lisbon Delegation, National Institute of Legal Medicine
2Faculty of Medicine,
University of Lisbon (hgeada @dlinml.mj.pt)
MtDNA is generally accepted as
a tool for forensic identity testing and evolutionary studies. It is used to
analyse biological samples where the quality and/or quantity of nuclear DNA
content is low. The evaluation of results obtained by mtDNA typing is
continuously subjected to scientific scrutiny and some genetic issues have to
be considered when establishing an effective methodology. In order to properly
interpret results, one needs to appreciate mtDNA’s features such as
nomenclature, heteroplasmy, recombination and haploid maternal inheritance.
Forensic community has adopted a common language to describe the variation
observed in human populations naming mtDNA sequences by referring to a standard
sequence, CRS. In a forensic case, the weight of evidence is primarily based on
the number of times a profile is observed in a reference data set. The relevance
of these databases should be considered for forensic applications.
HVI
and HVII mtADN regions have two citosine segments (np16184-16193 and np303-315,
respectively), which in accordance with CRS possessed a timine at np16189 and
np310. In our population data, 15% of mtADN sequences have a transition T - C
at np16189. In 20% of these sequences occurs a C – T transition at np16186 (2
samples) and at np16187 (1 sample). In five samples besides a 16189T, there
were also a 16188T (1sample), a 16193T (2samples) and a 16192T (2samples).
Sequence variability was referred by the number and position of Cs and Ts (CxTx).
The
variability in np16184-16193 HVI region presents two different types: sequence
variability detected in eight samples representing six distinct timine
positions, not interfering with results and length variability with length
heteroplasmy with no timine position in this region. The different types of
length heteroplasmy were identified as Lh1I (A3C10), Lh2I (A3C11), Lh3I
(A3C12), Lh4I (A3C13), Lh5I (A2C11), Lh6I (A2C12) and Lh7I (A2C13), considering
also adjacent np16182 and 16183. Twenty-seven samples in our studied have been
detected with length heteroplasmy.
For
forensic purposes, the HVI homopolimeric regions should be well defined with a
correct classification of the homopolimeric track defined by As and Cs
encountered in the electropherogrammes and the various population types
involved identified by the three nuclear adjacent positions to the
homopolimeric track.
Contact:
ARebelo@inml.mj.pt
P-284
Genetic studies of seventeen X –STR in the
Japanese population
Tamura A, Iwata M, Takase I,
Fukunishi S, Takagi T, Tsuboi K, Miyazaki T, Nishio H, Suzuki K
Department of Legal Medicine, Osaka Medical College, Japan
Introduction Short tandem repeat (STR)
loci are useful for personal identification and paternity testing because the
number of repeats within STRs tends to be highly variable and these STR
polymorphisms can be rapidly analyzed using PCR. Autosomal STR makers are widely applied to
personal identification and paternity testing.
However, in some kinship or deficiency paternity cases typing of sex
chromosomal STRs may be more informative than autosomal STRs. Typing of X chromosomal STRs
(X-STRs) could be of great usefulness to the paternity where the child is
female because fathers transmit their X-STRs to all their daughters. We studied 17 X-STR (DXS101, DXS6789, DXS6800,
DXS6803, DXS6807, DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378,
DXS9895, DXS9898, DXS10011, HUMARA, HPRTB, GATA172D05) polymorphism in 99
unrelated Japanese individuals (55 males, 44 females) and mutation rate in 22
true biological trio families.
Materials and Methods Genomic DNAs were recovered
from buffy coat by proteinase K digestion followed by sodium iodide
extraction. The
17 X-STRs were separately amplified using each primer set as previously
reported.
Results and discussion Power of discrimination (PD)
of the 17 X-STRs ranged from 0.331 to 0.933 (male) and from 0.447 to 0.997
(female). Allele frequencies and number
of alleles were 0.007-0.853 and 4-35, respectively. High PD values were observed at DXS101 (0.822
male, 0.933 female), DXS6789 (0.799 male, 0.956 female), DXS8377 (0.888 male,
0.981 female), DXS9895 (0.748 male, 0.902 female), DXS10011 (0.930 male, 0.997 female), HUMARA (0.881 male, 0.975 female), and GATA172D05 (0.715 male, 0.912
female). DXS10011 showed the highest PD
value among them.
We have detected two de novo alleles
at the DXS10011 locus in two paternity trio cases (probabilities:
>0.999999). No alleles of the mothers
were found to be shared with the daughter in one case and with the son in the
other case. The mutation events seem to
have occurred during oogenesis, probably by replication slippage. Further studies are needed to evaluate the
mutation rate at the locus.
P-285
Mutation
analysis in fatal pulmonary thromboembolism
- Postmortem validation study and beyond
Tang, Y.1, Kim, Y.1,
Jeudy, S.1, Roman, K.2, Sansone, M.1, Shaler,
R.1
1Department of
Forensic Biology, 2Department of Forensic Pathology, Office of Chief
Medical Examiner, New York,
NY
Sudden fatal pulmonary
thromboembolism (PE) is a common finding in forensic pathology practice,
usually presented as a complication of deep venous thrombosis (DVT). The
current view of the aetiologies of DVT is multifactorial, where inherited
genetic predisposition interplays with acquired risk factors, such as surgery,
pregnancy, inactivity of any cause, malignancy, obesity. There are several genetic risk
factors involved in the predisposition of individuals to develop DVT. The most
common mutation in the Caucasian population, but less in other ethnic groups,
is the Factor V Leiden (G1691A) mutation. Heterozygotes for the Factor V Leiden
(G1691A) mutation have an approximately 4-fold to 7- or 8-fold increased risk
for DVT as compared to individuals without the mutation. Homozygotes for the
Factor V Leiden (G1691A) mutation have an approximately 80-fold increased risk
for DVT as compared to individuals without the mutation. The second most common
mutation is the G20210A mutation in the prothrombin (Factor II) gene.
Heterozygotes for the prothrombin G20210A mutation have an estimated 2 to
4-fold increased risk for DVT as compared to individuals without the mutation.
In addition, individuals carrying both the Factor V Leiden and the prothrombin
G20210A mutations have a 20 fold more likely chance of having DVT than
individuals without either mutation. Another risk factor for DVT is associated
with the homozygous state of a nucleotide variant (C677T) in the
methylenetetrahydrofolate reductase (MTHFR) gene.
Molecular testing for these common mutations is one of
the most frequently ordered laboratory tests in a clinical setting, but is not
routinely done by forensic pathologists. Molecular testing for fatal PE can
confirm autopsy results, provide insights regarding disease effects and provide
knowledge for genetic counseling of family members. Clinical diagnostic testing
methodologies for the common mutations associated with DVT are well established
but there are limited data about the reliability of these procedures for
various post mortem samples. The aim of this study was to establish and
validate a genetic test for DVT in a forensic setting.
To investigate the genetic risk
factors in the fatal PE cases, we focussed the validation work on the three common
mutations described above. The in-house molecular testing methodology is based
on the polymerase chain reaction (PCR) and automated DNA sequencing
technologies. The method was validated for postmortem tissue samples, such as
heart, spleen, and liver. Tissues are stored in RNAlaterâ solution for up to
two years. The method was also validated for blood specimens collected in tubes
with or without anticoagulant. Blood was spotted on staincards, dried and
stored at room temperature for up to two years. All samples were extracted
using a magnetic bead capture and release chemistry. We obtained results for
all tested specimens, including those displaying varying extents of
decomposition.
One of
the first cases, submitted less than a month after the DVT mutation assay had
been approved for routine casework, involved a 63 years old Caucasian woman who
was presented as a sudden death and PE at autopsy. She was diagnosed as a
carrier for the homozygous MTHFR C677T mutation. Given the potential benefits
of the test results to the surviving family members, pursuing molecular testing
to investigate the genetic cause of fatal PE should be a common practice in
forensic pathology.
Contact: PRINZ@ocme.nyc.gov
P-286
The Effect of
Whole Genome Amplification on Samples Originating From More Than One Donor
Thacker CR1, Balogh
K2, Børsting C3,
Ramos E4,Sanchez-Diz P4, Carracedo A4, Morling N3, Schneider P2,
Syndercombe Court D1
SNPforID Consortium
1Centre for Haematology, ICMS, Barts and The London, Queen Mary's School of Medicine
and Dentistry, UK
2Institute of Legal Medicine, University
of Mainz, Germany
3Department of Forensic Genetics, Institute of Forensic
Medicine, University
of Copenhagen, Denmark
4Institute of Legal Medicine, University of Santiago de
Compostela, Galicia, Spain
Limited starting material is a common problem in
forensic science. Samples are often
compromised in terms of quality or quantity (sometimes both) and the
possibility of contribution by more than one donor is a necessary and frequent
consideration. Whole genome
amplification (WGA) offers the opportunity to create a ‘stock’ of starting
substrate on which to perform subsequent testing and provides an interesting
avenue of investigation for the forensic scientist. Its potential to deal with mixed samples is
of particular interest and the research presented here looks at the ability of
WGA (using the GenomiPhi™ DNA Amplification Kit, Amersham Biosciences) to cope
with samples originating from more than one individual.
Blood samples were taken from four individuals (A,
B, C and D) and DNA extracted using the QIAamp® DNA Mini Kit
(Qiagen). The extracts were quantified
(in duplicate) using the Quantifiler™
Human DNA Quantification Kit (Applied Biosystems) on the ABI PRISM®
7700. Following quantification, the
extracts were normalised and extract A was mixed with B whilst extract C was
mixed with D. In each case the samples
were combined in the ratios 1:1, 1:3, 1:7 and 1:15. The mixture
proportions were verified by performing routine amplifications using both the AmpFLSTR® SGM Plus®
PCR Amplification Kit (Applied Biosystems) and the PowerPlex® 16 System
(Promega). Peak areas were used to
calculate observed ratios. WGA was
performed by adding the minimum concentration of starting material recommended
by the manufacturer (1ng/ µL). The
reaction was also performed by adding DNA at concentrations known to exceed
this minimum value. The remainder of the protocol was performed according to
manufacturer’s guidelines.
Relative proportions were found to be maintained in
the 1:1 and 1:3 ratios following WGA; the observed peak ratios were found to
match the expected peak ratios regardless of the starting concentration of
DNA. With samples mixed in the ratio of
1:7 and 1:15, and when the
concentration of starting material was at the lower limit, too few minor
component peaks were found to allow for statistical analysis. With an initial template exceeding 1ng/ µL
there was an increase in problems associated with profile interpretation but
the results obtained indicated that mixture proportions could be quantifiably
maintained. To check the reproducibility
of these findings, initial mixture preparations were shipped to collaborating
laboratories for WGA. The results of
these extra replicates are presented and the findings discussed.
Address for
Correspondence:
Catherine R Thacker, Centre for Haematology, Institute of Cell and Molecular Science, Barts and
The London
Queen Mary’s School of Medicine
and Dentistry, 4 Newark Street, London
E1 2AT, UK
E-mail: c.r.thacker@qmul.ac.uk
P-287
An Investigation into Methods
to Produce Artificially Degraded DNA
Thacker CR, Oguzturun C, Ball
KM, Syndercombe Court D
Centre for Haematology, ICMS,
Barts and The London,
Queen Mary's School
of Medicine and
Dentistry, UK
DNA samples recovered from a crime scene are often
subjected to detrimental environmental conditions before they can be collected
for analysis. Environmental sources of
degradation, which can include heat, light and bacterial decomposition, are by
their very nature random in the effect they have on the DNA deposited at the
scene. These effects further test the
scientist’s ability to produce an evidentially valuable profile from a sample already
compromised in terms of quantity. The
facility to produce a ‘stock’ of degraded DNA on which to optimize existing
protocols would go some way to help in the preparation of standard practices to
follow when faced with an environmentally degraded sample. The knowledge gained from the preparation of
such stocks has the potential to benefit those asked to give their Expert
opinion in a court of law. Experience
gained on the behaviour of DNA stored in a variety of hostile conditions
(albeit in controlled environments) could help with the interpretation of
results produced from degraded samples and may also be useful if asked to
consider storage conditions of that sample prior to generating the resultant
profile. Blood samples were collected from volunteers and blood stains on
cotton cloth squares prepared. The
prepared stains proved difficult to process in terms of laboratory space
required for drying and maintaining sterility whilst monitoring degradation
over an extended time frame. An
alternative sampling source was found and subsequent experiments were performed
using blood stained Salivettes® (Sarstedt). Cigarette ends and chewing gum were also
collected for examination. Control
samples (Day 0) were taken from each ‘exhibit’ and the DNA extracted.
Throughout the course of the work a number of different extraction techniques
were investigated: Chelex® 100 (Sigma); Charge Switch™ (Invitrogen);
Invisorb® Forensic Kit I (Invitek) and
Qiagen. DNA profiles were generated
using the AmpFLSTR® SGM Plus® PCR Amplification Kit
(Applied Biosystems) run under standard conditions. A previous study used sonication and DNAse I treatment
to artificially degrade DNA (1). Our aim
was to mimic as closely as possible environmental conditions and as a
consequence UV light, humidity and
temperature were investigated as degradation agents.
In the
case of chewing gum, prepared samples left outside in direct sunlight were also
analysed. Sections were taken from the samples at timed
intervals throughout the period of degradation.
The DNA was extracted and amplified.
The resultant electropherogram was analysed and, if necessary,
amplification was repeated with slight modifications to improve the quality of
the profile. UV light caused a clear ‘drop-out’ of heavier alleles. This increased as exposure to UV light
increased. Cigarette analysis yielded
inconsistent results but partial profiles were produced that could assist in
excluding a suspect. Chewing gum was an
excellent material for obtaining profiles.
Full profiles were obtained even after exposure to 30 hours of
sunlight. Humidity degradation
experiments seemed to produce the most controlled method of degradation. The
performance of different extraction techniques varied according to the extent
of degradation.
(1)
Bender K et al. Preparation of
degraded human DNA under controlled conditions.
Forensic Science International
139 (2004) 135-140
Catherine R Thacker, Centre for Haematology, Institute of Cell and Molecular Science, Barts and
The London Queen Mary’s School
of Medicine and
Dentistry, 4 Newark Street,
London E1 2AT,
UK
P-288
Population genetics of Y-chromosomal STRs in Amharic
males from Ethiopia
Thiele K1, Reißig D2,
Assegedech B2, Yared W2, Edelmann J1, Lessig R1
1Institute of Legal
Medicine, University of Leipzig,
Germany
2Department of
Anatomy, University of Gondar,
Ethiopia
The
Y-STRs are well established in the forensic routine case work. The
investigation of Y-STRs in different populations is very important to get
informations about the distribution of the haplotypes especially in relatively
closed populations worldwide. So the main population in Gondar (Ethiopia) are the Amharics.
Samples
from 173 unrelated males of this population were analysed. The samples were
typed using the Y-PowerPlex-Kit (Promega) containing the markers of the so
called “minimal haplotpye” and additional the STRs DYS437, DYS438 and DYS439.
For allele typing a denaturing PAG and the ABI PRISM™ 377
DNA Sequencer were used.
The
allele and haplotpye frequency data, the exclusion power of the STRs according
to Nei and the haplotype diversity index to Takayama were calculated.
The most
frequent haplotypes are obtained with a frequency of 0.0231. The
haplotype diversity was estimated with 0.99 and the power of discrimination
with 0.99. The allele diversity of the analysed markers differs between 0.068
and 0.747.
contact: yp2@gmx.de
P-289
Usefulness
of X-chromosome markers in resolving relationships among females,
with
reference to a deficiency case involving presumed half sisters
1Unit of Legal Medicine, School
of Medicine, University of Pisa, Italy
2Center of Statistical Genetics, University
of Pisa, Italy
The use of X-chromosome (Chr-X) markers in forensic practice has played
a minor role so far, probably because of its peculiar transmission rules, which
reduce their potential use in forensic analyses to cases involving females
only. However, the probability of excluding a false father in standard trios is
higher for Chr-X markers than for autosomal loci with comparable values of
polymorphic information content, and there are special circumstances in which
they may resolve cases with deficiencies more efficiently than conventional
loci. Therefore,
Chr-X genotyping can efficiently complement the analysis of other genetic
markers, and may resolve cases that otherwise would remain inconclusive. We
were interested in the probability that two women with deceased parents were
half sisters rather than unrelated. We
first typed 16 autosomal markers commonly used in forensic practice, and
obtained a cumulative likelihood ratio (LR) of 701.3, in favour of the
hypothesis that they were half sisters, corresponding to a P value of 99.86%
(assuming equal priors). As we usually present more compelling evidence in
court cases, we typed the four unlinked Chr-X markers DXS101, HPRTB, STRX1, and
DXS8377. Formulas needed for calculating likelihood ratios were obtained by
Bayesian analysis (see Table below).
We compared the power of
discriminating relationships between Chr-X and autosomal markers of equivalent
informativeness in relation to the case at hand. All possible genotype
configurations of any two individuals were listed for each marker, and for each
of these configurations the LR that they were half sisters rather than
non-relatives was obtained, using both the autosomal and the Chr-X formulas.
LRs were converted into probabilities as usual [P = LR/(LR+1)], and the mean
value of these probabilities was computed separately for the autosomal and the
Chr-X cases. The ratio of the two mean values was chosen as a measure of the
relative power of discrimination. The following ratios Chr-X/Autosomal were
obtained: HPRTB, 1.19; STRX-1, 1.26; DXS8377, 1.37;DXS101, 1.33. In fact, the
LR computed using these four markers in the casework was 495.8, not much lower
than that obtained with 16 autosomal markers (701.3). The final (combined)
probability value was 99.9997%, thus providing sufficient proof.
|
|
Autosomal markers
|
Chr-X markers
|
|
Genotype
configuration
|
LR
|
LR
|
|
AA,AA
|
½ + 1/ 2pA
|
1/ pA
|
|
AA,AB
|
½ + 1/ 4pA
|
1/ 2pA
|
|
AA,BB
|
½
|
0
|
|
AB,AB
|
½ + 1/ 8pA + 1/ 8pB
|
1/ 4pA + 1/ 4pB
|
|
AA,BC
|
½
|
0
|
|
AB,AC
|
½ + 1/ 8pA
|
1/ 4pA
|
|
AB,CD
|
½
|
0
|
Table
1. Formulas used for calculating the likelihood ratios (LR) that two females
are half-sisters rather than unrelated
Contact:
sprex@biomed.unipi.it
P-290
Variability
in the detection of mixed profiles in four commercial autosomic STR
multiplexes.
Torres Y, Sanz P
Instituto
de Toxicología y Ciencias Forenses. Sevilla. Spain.
In some samples of forensic casework autosomic
STR allelic mixtures can be not detected depending of the commercial kit used.
Since it consumes time and money, the reference samples can be typed with one
multiplex, but each evidence must be analyzed by duplicate with two different
multiplexes, resulting in the confirmation of the results upon some markers.
The
variability in the detection of mixed profiles with four multiplexes of a same
manufacturer in the same DNA extracts of casework evidences were analyzed. DNA
extracts of 55 evidences in forensic cases previously typed and reported as
allelic mixtures were amplified by AmpFlSTR® Profiler PlusTM,
CofilerTM, IdentifilerTM and SGMPlusTM (Applied
Biosystems), electrophoresed in an ABIPrimsTM 310 and analysed with Genotyper® software v2.5.2 and GeneScan
Analysis software 3.1. Re-injections of 15-20 second were made in the same tube
when it was considered necessary. Only alleles over 100 r.f.u. were considered.
SGMPlusTM
has been the multiplex that has identified allelic mixtures in a greater number
of casework samples. With IdentifilerTM we detected a lack of
detection of 21% in the same DNA extracts. The presence/absence of some markers
seems to be most decisive in the mixtures detection that the number of markers
included in each commercial kit. The greater percentages of three or more
allele determinations in the 55 samples tested were found in D8S1179 and VWA.
CSF1PO, D2S1338, D7S820, TPOX and D13S317 showed the lower capacity of
detection of mixed profiles.
contact: yolanda.torres@mju.es
P-291
The inclusion of
profiles of evidence of sexual aggressions in DNA databases: The viewpoint of a
forensic genetics laboratory
Torres Y1, Gamero JJ2 , Sanz P1, Romero JL2
1Institute of Toxicology and Forensic Sciences. Sevilla. Spain
2Faculty of Medicine. University of Cádiz. Fragela s/n,
Cádiz 11003. Spain.
In
Spain, although there exist norms that will enable the elaboration of future
regulations regarding DNA profile databases, no text has yet appeared that
could give those institutions directly involved in identification processes
using DNA profiles some indication of the imminent coming into force of such
regulations
We
consider that it is necessary to examine and define confronting social and
individual interests in order to obtain a legislative answer that would bring
about the regulation of said databases, before an agreement on the final text
is reached. In this sense, any future law that may come into force should be
drawn up taking the following criteria into account: the way society conceives
and values the subject, specialist advice that is obtained on the subject
(based on prevailing scientific knowledge regarding forensic genetics), and
finally the law itself
In this paper the possibilities, advantages and
inconveniences of the inclusion in DNA profile databases of the results of the
casework of sexual aggression obtained in our laboratory are analysed. The aim
would be to provide objective data that may serve to aid the drawing up of
future database regulations in Spain.
Keywords: National DNA database; DNA profile; sexual aggression; Forensic genetics; Ethics.
P-292
Genetic variability of 17 Y
chromosome STRs in two Native American populations from Argentina
aToscanini U, bGusmao L, aBerardi
G, bcAmorim A, dCarracedo A, dSalas A, aRaimondi
E
aPRICAI-FUNDACIÓN FAVALORO, Buenos Aires, Argentina
bIPATIMUP, Instituto de Patología e Imunología Molecular da Universidade do Porto, Portugal
cFaculdade
de Ciências, Universidade do Porto, Portugal
dInstituto de Medicina Legal, Facultad de Medicina,
Universidad de Santiago de Compostela, Spain
Seventeen Y-STRs (DYS19, DYS389I, DYS389II,
DYS389I, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATA H4) were analyzed in two Native American populations, namely Tobas
(N = 47) and Collas (N = 28), settled in
the north and northwest regions of Argentina respectively. Standards diversity
indices and haplotype frequencies were estimated. Genetic distance between both
population was estimated by mean of Fst
(Rst) test. Statistical tests were performed using Arlequin software Ver 2.000. Thirty three and fifteen different
complete haplotypes were observed for the Tobas and Collas respectively.
Haplotype diversity was 0.9769 +/- 0.01
for Tobas, and 0.9497 +/- 0.02 for
Collas. These values are lower than those observed in other populations. A new
variant, present in thirteen haplotypes was identified at DYS385 loci in Tobas.
Two alleles were found in two samples from Toba population and in one sample
from Collas at DYS448. No shared haplotypes were found between the two
populations. A significant Fst value of 0.1466 was obtained at the pairwise
comparison between the two populations (P = 0.00 +/- 0.0).
P-293
Forensic
considerations on STR databases in Argentina
aToscanini U, aBerardi G, bcAmorim
A, dCarracedo A, dSalas A, bGusmao L, aRaimondi
E
aPRICAI-FUNDACIÓN
FAVALORO, Buenos Aires, Argentina; bIPATIMUP, Instituto de Patología e Imunología Molecular
da Universidade do Porto, Portugal; cFaculdade
de Ciências, Universidade do Porto, Portugal; dInstituto de Medicina Legal, Facultad de Medicina,
Universidad de Santiago de Compostela, Galicia, Spain
A
genetic comparison study was conducted between populations from different
regions of Argentina
in order to determine if a pooled population STR database could be used for
general forensic purposes. Samples were from urban populations of six
geographically distant provinces of Argentina, namely, Tucumán (N = 51), San Luis (N = 42), La Pampa (N =
147), Buenos Aires
(N = 879), Neuquén (N = 355) and Santa Cruz (N=82), and two Native American
populations from the North and northwest region of the country, namely, Tobas
(N = 129) and Collas (N = 43). A
total of fifteen autosomal markers (D3S1358, TH01, D21S11, D18S51, PENTA E,
D5S818, D13S317, D7S820, D16S539, CSF1PO, PENTA D, vWA, D8S1179, TPOX, and FGA)
were analyzed. Exact tests P-values
did not show deviation from Hardy-Weinberg equilibrium for both, urban and
Native American populations (0.005 < P
< 0.987, SD < 0.016). Regarding population differentiation, low Fst
values were observed for the population pairwise comparisons; however, only
significant differences were found when comparing Buenos Aires with Neuquén,
and Santa Cruz (P = values between
0.000 and 0.024). Concerning Native American populations, Fst P-values
were statistically significant when comparing Toba and Collas with every urban
populations (P = 0.000 ± 0.000). Furthermore, the two Native American
populations themselves appeared to be significantly different (P = 0.000
± 0.000). Single locus comparisons showed some significant differences when
comparing Neuquén and Buenos Aires,
namely at D5S818, FGA and Penta D (0.000 < P < 0.002). However, no significant differences were found
between the four remaining urban samples. When comparing urban populations with
Amerindians and European populations, significant P-values were observed at 12 to 15 locus comparisons (0.000
< P
< 0.0498). The four non differentiated urban populations studied were
pooled in a single population database (N = 322). Exact test for Hardy-Weinberg
equilibrium, frequencies estimates and forensic parameters were computed for
the pooled sample as well as for Buenos
Aires and Neuquén. P-values showed no deviation
for Hardy-Weinberg equilibrium in the global sample (0.057 < P <
0.991). The combined matching probability and a priori chance of exclusion were 2.0 x 10-18 and
0.9999995, respectively. These results suggest that it would be possible to use
a combined database for Tucumán, San Luis, La Pampa and Santa Cruz, provided that no significant
differences were found between any of these populations. Caution should be
taken concerning small isolates where Native American component could be much
more relevant. In addition, it is
remarkable that when comparing Argentina
urban population with two Iberian samples (a major population source of the
country European stock), some significant differences were found. Therefore, an
Iberian database, might not adequately represent the Argentinean genetic
makeup, although the real impact in forensic casework would require further
investigation. utoscanini@ffavaloro.org
P-294
Chromosome Y Haplotypes Database in a Venezuelan
Population
Tovar F1, Chiurillo MA2, Lander N1, and
Ramírez JL1
1.Centro de Biotecnología, Fundación Instituto de Estudios
Avanzados - MCT, Caracas; Venezuela.
2Decanato de Medicina, Universidad
Centro Occidental Lisandro Alvarado, Barquisimeto; Venezuela.
Tidak ada komentar:
Posting Komentar