P-043
A comparative study between
Brazilian, Iberian and African populations in an evolutionary perspective
Brito P1, Carvalho M1,
Lopes V1, Andrade L1, Anjos MJ1, Serra A1,
Balsa F1, Oliveira AC1, Oliveira C1, Batista L1,
Gamero JJ2, Romero JL2, Corte-Real F3, Vieira
DN3, Vide MC1
1Forensic Genetic Service.
National Institute of Legal Medicine. Largo da Sé Nova, 3000 Coimbra. Portugal
2Departament of Legal Medicine,
Faculty of Medicine, University of
Cádiz, Spain
3National Institute of Legal
Medicine. Largo da Sé
Nova, 3000 Coimbra. Portugal
The
STR’s study of the Y chromosome has a great importance on Forensic Genetics,
namely, in parentage investigations and biological crime evidence
investigations. These markers pass from father to son without suffering
recombination and with a very low occurrence of mutations. These
characteristics combined with the high level of Y chromosome polymorphisms,
made it in one of the main elements of study in forensic genetics, as like as
in population genetics, allowing the study of lineages and the origin of a
certain population.
Basing
on the minimum haplotype of the Y chromosome STR’s (DYS19, DYS385, DYS389 I,
DYS389 II, DYS390, DYS391, DYS392, DYS393), the haplotype diversity was
calculated according to Nei (1973), in Brazilian, African and Iberian
populations. The analysis of Molecular Variance (AMOVA) was determinate by
Markov test using the Arlequin Software (ver. 2.000). The distance matrix
between populations was obtained by the genetic differences between haplotypes.
Contact: geneforense@dcinml.mj.pt
P-044
Analysis of 16 Y-chromosomal
STRs in a Valle (Colombia)
population sample
Builes JJ1,2, Castañeda
SP3, Bravo MLJ1, Espinal CE1, Gómez MV4,
Moreno MA1,2
1 GENES Ltda., Laboratorio
de Genética Forense y Huellas Digitales del DNA. Medellín – Colombia.
2 Instituto de Biología.
Universidad de Antioquia. Medellín – Colombia.
3 Facultad de Ciencias.
Universidad Nacional de Colombia. Medellín – Colombia.
4 INMUNOGEN Ltda. Cali –
Colombia.
The object of this work was to examine a set of 16
Y-STR systems in a population sample from Valle (Colombia) to create a
database. In the present study, 150 DNA
samples taken from unrelated males were analyzed and PCR amplification of
DYS19, DYS385, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438,
DYS439, DYS460, DYS461, GATA-A10, GATA-H4 and DYS635 was performed. PCR products were separated in 4% acrylamide-bis-acrylamide
denaturing gels followed by silver staining.
Allele size determination and genotyping were performed according to
recommendations of the DNA Commission of the International Society of Forensic
Genetic using the allelic ladder manufactured at home. Gene frequencies, gene and haplotype
diversity and AMOVA for 16 Y-specific STR loci were calculated using ARLEQUIN
version 2000.
One
hundred forty six different haplotypes were found, 142 haplotypes of them were
found to be unique and the others were shared by two persons. The haplotype diversity was 0.9996. Regarding the minimal haplotype, one hundred
twenty four different haplotypes were found (haplotype diversity 0.9970), and
one hundred thirty two different haplotypes were found with the GEPY system
(haplotype diversity 0.9977). Twenty
seven percent of this haplotypes do not match any sample in the Y-STR Haplotype
Reference Database which assigned specific region characteristic to these
population samples. We compared our
data whit a Spain
population and another Colombian populations.
The AMOVA results show that the percentage of variation is mainly within
populations (99.95%) in agreement with previous results in European
populations.
By combining the allelic states of the 16
Y-chromosomal STRs we could construct highly informative haplotypes that
allowed the discrimination of 94.7% (142 out of 150) of the samples tested.
This approach represents a very powerful tool for individual identification and
paternity testing in forensic medicine.
contact: genforense@epm.net.co
P-045
Analysis of 16 Y-chromosomal
STRs in a Córdoba (Colombia)
population sample
Builes JJ1,2, Castañeda SP3,
Espinal CE1, Moreno MA1,2, Gómez JR4,
Bravo MLJ1
1 GENES Ltda., Laboratorio
de Genética Forense y Huellas Digitales del DNA. Medellín – Colombia.
2 Instituto de Biología.
Universidad de Antioquia. Medellín – Colombia.
3 Facultad de Ciencias.
Universidad Nacional de Colombia. Medellín – Colombia.
4 INGEIN Ltda. Medellín –
Colombia.
The object of this work was to examine a set of 16
Y-STR loci in a population sample from Córdoba (Colombia) to create a population
database. In the present study, 123 DNA
samples taken from unrelated males were analyzed and PCR amplification of
DYS19, DYS385, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438,
DYS439, DYS460, DYS461, GATA-A10, GATA-H4 and DYS635 was performed. PCR products were separated in 4% acrylamide-bis-acrylamide
denaturing gels followed by silver staining.
Allele size determination and genotyping were performed according to
recommendations of the DNA Commission of the International Society of Forensic
Genetic using the allelic ladder manufactured at home. Gene frequencies, gene and haplotype
diversity and AMOVA for 16 Y-specific STR loci were calculated using ARLEQUIN
version 2000..
One hundred thirteen different haplotypes were found,
103 haplotypes of them were found to be unique and the others were shared by
two men. The haplotype diversity was
0.9987. Regarding the minimal haplotype,
one hundred different haplotypes were found (haplotype diversity 0.9896), and
one hundred two different haplotypes were found with the GEPY system (haplotype
diversity 0.9959). Thirty six percent of
this haplotypes do not match any sample in the Y-STR Haplotype Reference
Database which assigned specific region characteristic to these population
samples. We compared our data whit a Spain
population and another Colombian populations.
The AMOVA results show that the percentage of variation is mainly within
populations (99.95%) in agreement with previous results in European
populations.
By combining the allelic states of the 16 Y-chromosomal STRs we could
construct highly informative haplotypes that allowed the discrimination of
83.7% (103 out of 123) of the samples tested. This approach represents a very
powerful tool for individual identification and paternity testing in forensic
medicine.
contact: genforense@epm.net.co
P-046
Analysis of 16 Y-chromosomal
STRs in a Cartagena
(Colombia)
population sample
Builes JJ1,2, Gómez A2,
Bravo ML1, Espinal C1, Aguirre D1, Montoya
A2, Caraballo L3, Martínez B3, Moreno M1,2
1 GENES Ltda., Laboratorio
de Genética Forense y Huellas Digitales del DNA. Medellín – Colombia.
2 Instituto de Biología.
Universidad de Antioquia. Medellín – Colombia.
3Instituo de
Investigaciones Inmunológicas, Universidad de Cartagena. Cartagena-Colombia
Whit this work we stablished a data base of Y-STR,
some parameters of forensic importance were calculated. We studied 16 Y-STR (DYS19, DYS385,
DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460,
DYS461, GATA-A10, GATA-H4 and DYS635) in a population of 173 unrelated males of
Cartagena (Colombia). PCR products were separated in 4% acrylamide-bis-acrylamide
denaturing gels followed by silver staining.
Allele size determination and genotyping were performed according to
recommendations of the DNA Commission of the International Society of Forensic
Genetic using the allelic ladder manufactured at home. Gene frequencies, gene and haplotype
diversity and AMOVA for 16 Y-specific STR loci were calculated using ARLEQUIN
version 2000.
All
men presented different haplotypes. The
haplotype diversity was 1.000 +/- 0.0006.
Regarding the minimal haplotype, one hundred fifty seven different
haplotypes were found (haplotype diversity 0.9974), and one hundred forty
different haplotypes were found with the GEPY system (haplotype diversity
0.9952). Forty one percent of this
haplotypes do not match any sample in the Y-STR Haplotype Reference Database
which assigned specific region characteristic to these population samples. We compared our data whit a Spain
population and another Colombian populations.
The AMOVA results show that the percentage of variation is mainly within
populations (99.95%) in agreement with previous results in European
populations.
By combining the allelic states of the 16
Y-chromosomal STRs we could construct highly informative haplotypes that
allowed the discrimination of 100% (173 out of 173) of the samples tested. This
approach represents a very powerful tool for individual identification and
paternity testing in forensic medicine.
contact: genforense@epm.net.co
P-047
Peruvian
population study with 16 Y-STR loci
Builes JJ1,2, Hau J3, Bravo MLJ2,
Rodríguez J1,2, Montoya A2,3, Izarra F3, Ochoa
O3, Pérez L3
1 GENES Ltda., Laboratorio
de Genética Forense y Huellas Digitales del DNA. Medellín – Colombia.
2 Instituto de Biología.
Universidad de Antioquia. Medellín – Colombia.
3 Laboratorio de Biología
Molecular-ADN Forense, Dir. de Criminalística de la Policía Nal. de Perú, Lima,
Perú.
The
aim of this study was to present the first report of a Peruvian Population
Database of 77 samples studied with 16 Y-STR loci including the eight minimal
Y-STR haplotype (DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391,
DYS392 and DYS393) and other Y-STR loci (DYS437, DYS438, DYS439, DYS460,
DYS461, GATA A10, GATA C4 and GATA H4).
PCR products were separated in 4% acrylamide-bis-acrylamide
denaturing gels followed by silver staining.
Allele size determination and genotyping were performed according to
recommendations of the DNA Commission of the International Society of Forensic
Genetic using the allelic ladder manufactured at home. Gene frequencies, gene and haplotype
diversity and AMOVA for 16 Y-specific STR loci were calculated using ARLEQUIN
version 2000.
Seventy
six different haplotypes were found, seventy five haplotypes of them were found
to be unique and only one was detected in two men. The haplotype diversity was 0.9997.
By
combining the allelic states of the 16 Y-chromosomal STRs we could construct
highly informative haplotypes that allowed the discrimination of 97.4% (75 out
of 77) of the samples tested. This approach represents a very powerful tool for
individual identification and paternity testing in forensic medicine.
P-048
Detection of a 1% to 2%
Contributor in a DNA Sample Mixture from Human Milk
Burger, MF, Schumm, JW
The Bode Technology Group, 7364 Steel Mill Rd.;
Springfield, VA 22150; USA.
We describe a method to detect very small amounts of
DNA in a mixed sample using commercially available multiplexes. We have
received a number of breast milk samples from human donors and have been asked
by our supplier to determine whether pooled milk samples originate from one
donor or from multiple donors.
We determined that it is possible to extract DNA from
whole milk samples using the QIAamp® DNA Blood Mini Kit. Total DNA
yields from 200 ml of milk were measured using the BodeQuant LCN
method described in the accompanying work and ranged from 6.5 ng to
205 ng. The significant observed variability could be due to many causes
such as sample age, care in handling by the original donor, or method of
shipment to us. The primary cause of variability is likely differences in the
numbers of cells shed into the milk by different source individuals. However,
in each case, enough DNA was obtained to generate a DNA profile with the AmpFlSTR®
Identifiler® kit.
We then created volume/volume mixtures of milk samples
in ratios of 98:2, 96:4, 92:8, and 88:12 to determine the minimum amount of the
minor component that could be detected. Using modified amplification conditions
and interpretation guidelines, we can detect the presence of a mixture
containing 2% or less of the total DNA content from the minor contributor.
Thus, so long as the two donors provide equivalent DNA mass per milliliter of
milk the minor component can be scored with as little as one part in 50
contributions.
However, we learned in our initial evaluations that
the DNA yield per milliliter of milk varies significantly from sample to sample
so that the volume: volume ratio does not always reflect the DNA mass:DNA mass
ratio in the sample. In practice, we can generally still detect the minor
component of a mixture even when this sample is mixed with 6 other samples and
even when the minor component has a lower DNA yield per milliliter of milk.
We will discuss the methods that allow detection of
mixtures at these low levels and how these results relate to evaluation of
blood mixtures of similar imbalance.
P-049
Probability distribution of
sibship determination with ABI Identifiler multiplex system using different
software
Caenazzo L. 1 , Cerri N.2, Ponzano E. 1, Sbrignadello S. 1, Benciolini P. 1Verzeletti A. 2, De Ferrari F. 2, Presciuttini S. 3
1Dept. of
Environmental Medicine and Public Health - University of Padua, Italy
2Dept. of Forensic
Medicine - University of Brescia,
Italy
3 Center of Statistical Genetics
- University of Pisa, Italy
Forensic laboratories may be
asked to provide genetic evidence that two persons are or are not related, when
no other relatives are available for study.
Sibship analysis of the
autosome polymorphisms are more complicated since there are no obligatory
alleles between siblings that make it possible to exclude a biological
relationship with absolute certainty.
In our work forty full-sib pairs were
genotyped using the AmpFLSTR Identifiler PCR Amplification kit. All subjects
belonged to families that included mother, two or three children, and an
alleged father, in which neither the mother nor the alleged father were
excluded as biological parents, and no mutational event was observed. In
addition, the Y chromosome was investigated to provide further support for the
relationship. The probability that each pair was composed of full sibs rather
than non-relatives was calculated by standard formulas, and was verified using
different published software. The distribution of these probability values was
used to ascertain the statistical power of the Identifiler kit to resolve sibship
relationships to forensic purposes.
P-050
Genetic identification of
forensically important Calliphoridae in Portugal
Cainé L1, Corte Real F2,3,
Lima G1, Pontes L1, Abrantes D1, Pinheiro MF1,4
1Instituto
Nacional de Medicina Legal – Delegação do Porto
2Instituto
Nacional de Medicina Legal
3Faculdade
de Medicina – Universidade de Coimbra
4Faculdade
de Ciências da Saúde – Universidade Fernando Pessoa
Medico-legal
Entomology, one area in the broad field of Entomology, is routinely involved in
forensic applications. Identifying species is an important first step in the
investigation, but morphological identification of immature stages can be
difficult and sometimes impossible, due to the similarity between different
species, and the possible dead of the insects. The genetic identification
provides a rapid and accurate determination of species.
Species from Calliphoridae
family are among the first insects to discover and colonize human remains and
they give information relating to the estimation of the postmortem interval
(PMI). They are attracted to carrion and a large number of eggs are commonly
placed in natural body openings and wounds that are exposed. To date many
geographical regions were studied, but Portugal presents a total lack of
genetic data collected on the main species of forensic interest. The main goal
of this study is to improve the genetic data knowledge of cadaveric entomofauna
in Portugal.
Maggots were collected from different human bodies during autopsy procedures in
the National Institute of Legal Medicine. DNA was extracted using two methods:
DNeasy® Tissue Kit (Qiagen) or BioRobot® EZ1 workstation
(Qiagen). The obtained sequences were used to identify species; they were
aligned to the gene sequences entries, using the online BLAST search engine of
the National Center for Biotechnology Information
(NCBI). The sequences are included in the database of GenBank and the maximum
scoring segment pair (MSP) was found. The information content within the
nucleotide sequence of the gene enabled the identification of all species used
in this study. This study doesn’t include all insect species that an investigator
might find during autopsy, but it represents their general appearance.
P-051
Species identification by Cytochrome
b gene: casework samples
Cainé L1, Lima G1,
Pontes L1, Abrantes D1, Pereira MJ1, Pinheiro
MF1,2
1
Instituto Nacional de Medicina Legal – Delegação do Porto
2Faculdade
de Ciências da Saúde – Universidade Fernando Pessoa
In routine casework (paternity tests, criminal
cases and human remains identification), sometimes is necessary to do the
discrimination between human and non-human samples, by identifying the exact
specie of the sample. The specie determination can change the overall direction
of the investigation. This study presents the determination of the biological
origin of unknown casework samples involved in criminal investigations, where
the forensic evidence was important to solve the cases. Species identification
was carried out by nucleotide sequence analysis of the citochrome b (cyt b)
gene, which contains species-specific information. The DNA was extracted using
the phenol-chloroform procedure and a fragment of 358 pb was amplified.
Sequence determination of PCR products was performed using the PCR primers
separately. The electrophoretic separation and detection of the sequencing
reaction products were performed using an ABI PRISM® 310 Genetic
Analyzer (Applied Biosystems). The sequences obtained were used to identify the
biological origin of the samples by aligning to the cyt b gene sequences entries
using the online BLAST search engine of the National Center
for Biotechnology Information (NCBI). The information content within the
nucleotide sequence of the cyt b gene enabled the identification of the samples
species of the investigated cases.
P-052
Haplogroup H in prehistoric
osseous remains from the Basque Country as a genetic marker to study the
resettlement of Europe
Cardoso S1 , Amory
S2 , Álvarez M1, Gómez A3, Keyser-Tracqui C2
, Ludes B2 , Fernández J3, Martínez de Pancorbo M1
1Servicio de Genómica:
Banco de ADN, Universidad del País Vasco, Basque Country, Spain
2Laboratoire d’Anthropologie
Moléculaire, Institut de Médecine Légale, Strasbourg Cedex, France
3Facultad de Geografía e Historia, Universidad del País Vasco, Basque Country, Spain
Wide
studies have been done on how the resettlement of Europe
took place after the end of the Last Glacial Maximum (LGM), approximately
15.000 years before the present. The Basque Country is said to have played a
major role as a refuge during the LGM and as an expansion focus during the
resettlement of the continent (Torroni et al. 1998, 2001; Achilli et al. 2004).
These studies have been carried out using mainly mitochondrial DNA data from
modern populations. However, these data are influenced by some aspects such as
the variation generated along the generations by genetic drift. To overcome
these problems it is of great value to use ancient DNA. Working with ancient
osseous remains to obtain DNA requires extremely careful manipulation and is
not always successful. However, the possibility of analysing ancient
mitochondrial DNA it is of great interest for this study, as the variations
localized in the control and coding regions would help to understand the
movements of the populations along the history.
In this paper we present the
preliminary results of our project, based on ancient DNA analysis. The aim of
the project is to establish a theory about the importance of haplogroup H and
its subhaplogroups in the migratory movements occurred in Europe
by comparing data from ancient samples with those from modern populations.
All the samples already analysed belong to the site of Las Yurdinas II,
located in the south part of the province
of Álava (Basque
Country). These samples yielded a radiocarbon date of 4350 +/- 50 years, thus
belonging to the Calcolithic period. DNA was extracted from both ulna bones and
teeth. Although the region HVI of the mtDNA was successfully sequenced for all
the samples, we concluded that the DNA was better preserved in the dental
remains. All the samples were classified as belonging to haplogroup H. In order
to prevent contamination the samples were processed in specific laboratory for
ancient DNA and negative controls for all the steps were included. Moreover,
all the persons involved in the processing of the samples were typed. Thus it
was possible to discard any false positive result caused by external
contamination.
P-053
Analysis of the maternal and
paternal lineages of Azores islands population
Carvalho M1, Brito P1,
Balsa F1, Antunes H1, Anjos MJ1, Andrade L1,
Lopes V1, Serra A1, Oliveira C1, Gamero JJ2,
Romero JL2, Corte-Real F3, Vieira DN3, Vide MC1
1Forensic Genetic
Service. National Institute of Legal Medicine. Largo da Sé Nova, 3000 Coimbra. Portugal
2Faculty of Medicine. University of Cadiz. Fragela s/n,
Cádiz 11003. Spain
3National Institute of Legal Medicine. Largo da Sé
Nova, 3000 Coimbra. Portugal
The aim of this study was the
analysis of the origin of maternal lineage (mithocondrial DNA) and paternal
lineage (Y Short Tandem Repeats) of Azores
Islands population comparing
our data with other populations from Europe
and Africa.
The polymorphism of the two hypervariable segments
(HVI and HVII) of control region of mtDNA was analyzed in unrelated individuals
from Azores Islands, using a amplification method
with primers refered by Wilson et al.(1995). Sequences have been
obtained with ABI PRISM Big Dye Terminator and dRhodamine Terminator Cycle
Sequencing Ready Reaction Kit s, with amplitaq DNA polymerase FS, and have
been detected with ABI 3100 Avant sequencer. We will describe the number of
different sequences for HVI and HVII regions in our population data and the
polymorphic sites.
The Y-chromosomal haploptype was defined by 17 Y- STRs
(DYS19, DYS385, DYS389 I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437,
DYS438, DYS439, DYS460, DYS461, GATA A10, GATA C4 and GATA H4) in a sample
of unrelated individuals of Azores
islands. The minimal haplotype was carried out according with the primers and conditions of the PowerPlexY
PCR Amplification Kit, de Promega and the other YSTR were amplified with two
tetraplexs reactions (GEPY I and GEPY II), using the protocol according to
Sanchez-Diz et al.(2003). We will describe the most common haplotype in this
population and how many haplotypes will be unique.
The comparison of maternal and paternal lineages from Azores Islands
with other lineages from Europe and Africa was performed using the Arlequin software version
2.000.
P-054
In-house validation of the PCR
amplification kit « Mentype® Argus X-UL »
Castella V, Dimo-Simonin N,
Morerod M-L and Mangin P
Laboratoire de Génétique
Forensique, Institut Universitaire de Médecine Légale, rue du Bugnon 21, 1005
Lausanne, Switzerland
X
chromosome-specific short tandem repeats (STRs) may complement the analysis of
conventional genetic markers (autosomal STRs, Y-STRs or mitochondrial DNA),
especially when complex relatedness testing cases are analyzed. The Mentype®Argus X-UL PCR
amplification kit contains 4 X-STRs : DXS8378, HPRTB, DXS7423 and
DXS7132 plus the gender marker Amelogenin as an amplification control. In this
study, 50 females and 50 males of Swiss Caucasian origin were analyzed in order
to validate the forensic utilization of this kit and to determine some
population statistics. Preliminary tests have shown that the diminution of PCR
reaction volume from 25.0 to 12.5 l increased the sensitivity of the kit. With
the smaller volume, full profiles were obtained with ≥ 100 pg DNA template.
Female/male mixtures produced full profiles from the minor contributor with
10-20-fold excess of the major contributor. Intra and inter-day reproducibility
of allele sizing, stutter height and heterozygous balance were comparable to
those observed with other amplification kits used by the forensic community.
Allelic frequencies and forensic characteristics of individual markers (polymorphism,
discrimination power, etc.) are presented in the poster. At the population
level, the sample of 50 females enabled to verify that the Hardy-Weinberg
equilibrium was respected and that the 4 X-STRs were statistically unlinked.
Finally, 4 relatedness testing cases were performed in order to evaluate the
efficiency of the Mentype® Argus X-UL kit for solving deficiency
cases.
P-055
Low diversity in Cannabis sativa from Brazil and Paraguay
illegal plantations accessed through fluorescent multiplex STRs
1,2Castro J,
Grattapaglia, D1,3 and Pereira RW1
1Graduate Program in Genomic Science and Biotechnology, Catholic University
of Brasilia, Brazil; 2Brazilian Department of Federal Police; 3Heréditas
Tecnologia em Análise de DNA Ltda.
The Cannabis sativa has a long history in the human
cultural evolution, starting around 6500 from now, somewhere in the central Asia. Along this relationship the C. sativa have been
cultivated as source of fiber, oils, medicines and also as source of a
recreational psychoactive. The cannabinoid D9-tetrahydrocannabinol (THC) is
the main component responsible for the psychoactive effects. Many countries
around the world, faced with the increasing use of C. sativa (marijuana) as a
recreational drug, take the decision to turn it illegal, strongly repressing
their dealing. But, in some countries the agricultural use of C. sativa
is allowed. However, the varieties used in this case are THC poor. Methods to
differentiate legal from illegal crops have been widely explored, mainly based
on THC identification and quantification. The Brazil legal system does not allow
any use of C. sativa and all levels of our security system repress it.
The marijuana that is consumed in Brazil comes mainly from local and
from Paraguay
plantations. The Brazilian marijuana combat program has the Federal Police as
the major action planner. The C. sativa banish program effectiveness
involves investigation, intelligence actions and effective operations carried
out by specialized police task forces aiming the shipping interception and
plantations destruction. Any technology that could improve the success rate in
the law enforcement is welcome. In this regard, the characterization of
polymorphic STRs in C. sativa showed up as a potential toll to establish the
geographical origin of marijuana and the identification of marijuana produced
clonally, helping in the disruption of criminal network established around the
illegal dealing of marijuana. The Brazilian Federal Police and the Graduate
Program in Genomic Science and Biotechnology from Catholic University
from Brasilia
starts a project to develop a multiplex system based on C. sativa STRs
characterized by others groups and investigate de genetic diversity in
different plantations from the main source areas in Brazil and Paraguay. The
fluorescent multiplex standardization starts with 13 STRs. The forward primer
to amplify the ANUCS304, the C08-CANN2, the ANUCS201, the H11-CANN1 and the
B01-CANN1 STRs loci were labeled with 6-FAM. The forward primer to amplify the
ANUCS302, the ANUCS305, the B05-CANN1 and the H06-CANN2 were labeled with
5-HEX. The last four loci, the ANUCS302, the ANUCS202, the H09-CANN2 and the
ANUCS301 were labeled with NED. New primers were designed to seven out of the
13 loci listed above. By the end of the optimization we end up with two
multiplex set based on primers annealing temperature and with nine microsatellites
amplified. Four of them failed to be optimized. These multiplex sets were used
to amplify 48 DNA samples from twelve plantations distributed as follow: six
plantations from Paraguay,
two plantations from Maranhão (Brazil)
and four plantations from Pernambuco (Brazil). The PCR products were
analyzed in the ABI Prism 377. The sample files were analyzed using Genescan
and Genotyper software. The basic diversity indices were computed using
Arlequin package. The overall results analysis clearly showed a low diversity
in Brazilian and Paraguay
plantation. Only the C08-CANN2 and H09-CANN2 had heterozigosity higher than
0.5, respectively 0.8 and 0.56. The ANUCS305 and H06-CANN2 showed only one
allele. The fail to optimize all loci may be explained by sequence divergence
among our samples and the original sequence from Genbank. All these loci had
previously showed to be high polymorphic, mainly in fiber varieties. The low
heterozigosity to the majority of the loci we studied shows that more
investigation in the classification of new high polymorphic C. sativa
microsatellites is necessary if we would like to continue testing this tool. contact: rinaldo@pos.ucb.br
P-056
Evaluation
of reliability of STR typing for forensic purposes in different types of
cancerous tissues
S. Ceccardi1, M. Alù,
F2. Lugaresi1, G. Ferri1, C. Bini1,
T. Balbi1, F. Ingravallo1, S. Pelotti1
1Department of
Medicine and Public Health, Section of Legal Medicine, University of Bologna,
Bologna, Italy
2Department of Pathological Anatomy and Legal
Medicine, Section of Legal Medicine, University
of Modena and Reggio
Emilia, Modena, Italy
Forensic DNA testing by STR
kits validated to have reliable and robust results, might be questionable
when cancerous tissues are forcibly
used for forensic purposes.
Several studies were performed
to elucidate the mechanism underlying gene-environment interaction in
carcinogenesis, investigating short tandem repeats. One of the most
investigated STR in cancer is the CAG repeats in the androgen receptor gene
(AR) used also in forensic application, even if recently Szibor et al.
recommended the forensic Community to refrain from its use for the link with
disease risks.
In recent studies a number of
findings demonstrating DNA instability in tumor DNA also at STRs used in
forensic casework, was detected. Partial loss of one allele, complete loss of
one allele and microsatellite instability (MSI) were described in esophageal,
gastrointestinal, lung, oral, head and squamous cancers and cervical carcinoma.
We analyze 68 sporadic primary
tumor samples, including gastrointestinal, urogenital and oral carcinomas, in
parallel with near non cancerous tissues for 15 STR loci including in a
commercial kit.
To avoid the problem of DNA
degradation in paraffin embedded specimens as source of mixture of fragments of
diverse length that can lead to misinterpretation of instability; we analyze
frozen cancerous tissues compared to frozen normal tissues surgically
collected.
The problem of stuttering and
complex artefacts in the context of MIN is considered to compare the results
and to avoid a false positive diagnosis of MIN. The adopted criteria to
classify a sample as MIN positive are those suggested in assessing
microsatellite instability (Sobrido M.J. et al. Electrophoresis 2000, 21,
1471-1477).
Besides, the relationship
between the pathological stage of cancers and their respective allelic
alteration patterns is presented.
Finally, our study may
contribute to look for the uniform panel of microsatellites suggested by
Sobrido et al. suitable for cancerous tissues analysis.
Contact: susi.pelotti@unibo.it
P-057
Incest by father or by brother? A case report
Cerri N1, Presciuttini S2,
Notarangelo L3, Verzeletti A1, De Ferrari F1
1Department of
Forensic Medicine, University
of Brescia, Brescia, Italy
2Center of
Statistical Genetics, University
of Pisa, Pisa, Italy
3Department of
Pediatric, University
of Brescia, Brescia, Italy
Ten years ago a 16 years old
girl gave birth to a child who deceased five days later into the hospital. The
girl reported to the Prosecutor that she had been raped by a schooolmate.
The molecular analysis to
identify cistyc fibrosis mutations in the child, as a screening performed in
all new-borns in Italy,
allowed to identify the homozigosity status for the mutation N1303K. This
mutation is quite rare in Italy
(4% of all mutations regarding this disease) so the clinicians suspected that
the father could be a member of the girl’s family. In fact, the analysis
performed on the girl’s father, confirmed the presence of the same mutation.
The Prosecutor asked a genetic
analysis on the dead child, on the girl and on her father and mother. At the
investigation time, only traditional markers such as DQalpha, D1S80, LDLR,
GYPA, HBGG, D7S8, GC, TPOX, F13A01, AR, APOB were investigated. Only some years
later a genetic profile was obtaind using the commercial kit Profiler Plus (Applera,
Foster City, CA, USA).
The DNA for the analysis was
obtained from the child’s whole blood collected during autopsy and from whole
blood from the girl, her father and her mother. DNA was extracted using
Phenol/Chloroform method. The amplification of the VNTR was permormed according
to the protocols present in Literature and the amplification for the Profiler
Plus Kit was performed according to manufacturer’s recommendations in a GeneAmp
PCR System 2400 (PE).
All markers investigated were
consistent with a relationship father/girl except for the APOB and D8S1179
loci.
A research in the Literature
regarding the mutation rate at these loci showed no relevant mutation rate,
above all for D8S1179. So two alternative hypotesis were considered: a) the
girl’s father wasn’t the child’s father; b) the girl’s father was the child’s
father and the two incompatibility were due to new mutations. Using the
software for genetyc analysis “Familias”, this second hypothesis was excluded
because of the inconsistency of the probabilty of two mutations in the two
systems considered. Considering another hypothesis, i.e. a a girl’s brother as
the child’s father, the probability index was very strong.
In fact the investigations led
to the discovery of a girl’s brother who admitted the crime later.
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