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Gene
journal homepage: www.elsevier.com/locate/gene
a r t i c l e
i n f o
Article history:
Received 30 May 2014
Received in revised form 9 July 2014
Accepted 11 July 2014
Available online 15 July 2014
Keywords:
Short Tandem Repeats
Identiler
Pakistan
Forensic
Trisomy
Karyotyping
a b s t r a c t
Purpose: Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular
diagnostics and molecular genetics research. AmpFlSTR Identiler PCR amplication kit is a multiplex system
for co-amplication of 15 STR markers used worldwide in forensic investigations. This study attempts to assess
forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies.
Methodology: Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for
forensic parameters using Identiler STRs and 74 patients' samples with different aneuploidies were evaluated
for diagnostic strengths of these markers.
Results: All STRs hold sufcient forensic applicability in Pakistani population with paternity index between 1.5
and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power 0.9 (except TPOX
locus). Variation from HardyWeinberg equilibrium was observed at some loci reecting selective breeding
and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable
while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using
Identiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients.
Conclusion: The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick
and simultaneous identication of some common trisomic conditions. Adding more sex chromosome specic
STR markers can immensely increase the diagnostic and forensic potential of this system.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Molecular genotyping using the Short Tandem Repeat (STR)
markers is used frequently in a broad spectrum of scenario in molecular
diagnostics, DNA forensics and in molecular genetics research. STRs are
employed for checking sample contamination in forensic (Schwark
et al., 2008) and fetal/maternal (Messaoud et al., 2013; Wapner et al.,
2012) samples, forensic identications using DNA evidences (Budowle
et al., 2005; Chen et al., 2010), aneuploidy detection in pre- (Sohrab
et al., 2010) and post-natal samples (Donaghue et al., 2010; Gekas
et al., 2011) and genetic linkage analysis of various diseases (Shi
et al., 2010). The advantages of using STRs in molecular studies and
in detection of major chromosomal abnormalities are the ability for
multiplexing i.e. simultaneous amplication of a number of STRs,
Abbreviations: CODIS, Combined DNA Index System; STR, Short Tandem Repeat; PCR,
polymerase chain reaction; POC, product of conception; MP, matching probability; PD,
power of discrimination; PE, power of exclusion; PIC, polymorphic information content;
TPI, typical paternity index; OH, observed heterozygosity; EH, expected heterozygosity;
HWE, HardyWeinberg equilibrium; DNA, deoxyribonucleic acid.
Corresponding author at: Institute of Biomedical & Genetic Engineering, 24-Mauve
Area, G-9/1, Islamabad, Pakistan.
E-mail address: ahameed0786@hotmail.com (A. Hameed).
http://dx.doi.org/10.1016/j.gene.2014.07.035
0378-1119/ 2014 Elsevier B.V. All rights reserved.
218
(Furtado et al., 2013b), prenatal diagnosis of chromosomal abnormalities (Mann et al., 2004) and in tracing the etiology of various cancers
(Heaphy et al., 2007a). Common chromosomal aneuploidic conditions
include trisomy 21 (Down syndrome), trisomy 18 (Edward syndrome),
trisomy 13 (Patau syndrome), trisomy 8 (Warkany syndrome 2), Monosomy X0 (Turners syndrome) and XXY (Klinefelter syndrome). The diagnosis of these fetal and postnatal aneuploidies is routinely carried
out by traditional karyotyping approach that takes a long time period
up to 14 days to reach a diagnosis (Atef et al., 2011; Furtado et al.,
2013b). Multiplex STR genotyping using identiler kit and/or similar
multiplex PCR amplication systems has been proposed as a rapid, convenient and cost effective alternate to this traditional approach (Chen
et al., 2010; Furtado et al., 2013b; Heaphy et al., 2007a, 2007b; Li et al.,
2011) with an added advantage of ability to trace the parental origin
of aneuploidy (An et al., 2005; Kirby et al., 2010).
Intended for Pakistan, that has the sixth largest population of the
world (Anon., 2013), a forensic DNA database that covers major groups
of Pakistani population is still awaited. In the prospective study, we attempt to investigate the forensic applicability of Identiler STR markers
in Pakistani population along with an attempt to contribute towards
development of DNA database covering population representation
across the country. Alongside, the same genetic marker set in identiler
is evaluated for the applicability in rapid and simultaneous diagnosis of
various common chromosomal aneuploidies and tracing their parental
origin.
software to assign allele calls to electropherograms, using the allelic ladder provided in the AmpFlSTR Identiler PCR amplication kit as a
reference (Applied Biosystems, Inc., Foster City, CA).
For aneuploidy detection, the presence of an extra chromosome was
detected from the electropherogram with peak heights or peak number
other than the normal 1:1 allelic ratio at any locus. A pattern of 1:1:1
was observed in cases of trisomic triallelic pattern having 3 peaks at
the concerned locus (Fig. 1), or the double dose of one of the two alleles
i.e. 2:1 or 1:2 ration, in cases where two of the three concerned chromosome carried similar allele i.e. trisomic diallelic pattern (Fig. 1).
2.4. Data analysis
Allele frequencies for each locus were calculated from the genotype
counts in the sample population using the gene count method. For
the forensic statistical analysis, Power-Stats v1.2 software package
(Promega, UK) was used to calculate allele frequencies, matching probability (MP), power of discrimination (PD), power of exclusion (PE),
polymorphic information content (PIC) and typical paternity index
(TPI). Observed heterozygosity (OH) and expected heterozygosity
(EH) were computed using the Microsatellite Toolkit v3.1.1 (Park,
2001). GenePop v4.0.10 (Rousset, 2008) was used to test the Hardy
Weinberg equilibrium (HWE) and to obtain exact genotype probability
for each marker. The results of genotyping for suspected aneuploidy
were cross-checked with karyotyping. The DNA proles of the aneuploidy patients were matched with those of their parents to determine the
source of extra chromosome(s).
3. Results
219
Table 1
Summary of trisomic cases detected using Identiler STR markers along with determination of parental origin of extra chromosome.
Condition
No. of samples
Paternal
Not determined
44
13
9
1
44
13
9
1
N99%
N99%
N99%
N99%
31
8
3
0
10
5
4
1
3
0
2
0
Table 2
Use of STR prole for the determination of parental source of extra chromosome in trisomy
21 patients.
Case 1
Case 2
Case 3
Case 4
Hypothetical case
Mother's
genotype
Father's
genotype
Down syndrome
child's genotype
Origin of extra
chromosome 21
28, 31.2
29, 30
30, 31
31.2, 31.2
28, 28
29, 31
28, 33.2
29, 31
29, 31.2
28, 28
Paternal
Maternal
Not determined
Not determined
Not determined
4. Discussion
The AmpFlSTR Identiler PCR amplication kit is frequently used
across the globe for analysis of DNA evidences in forensic investigations
by the law enforcement agencies and the forensic science researchers.
These DNA evidences carry much importance in cases like murder,
rape and paternity/maternity testing, in addition to the identication
of severely damaged or burnt bodies and body parts of victims of mass
disasters, bomb blasts or air plane crashes. Pakistan has witnessed
many incidences during the last decade that highlighted the importance
of the use of DNA in forensic investigations. The 15 autosomal STR
markers in the AmpFlSTR Identiler kit provide signicant approach
to generate an individual's DNA prole for use in forensic case work.
The current study was conducted with aim to assess the medical
diagnostic applicability of identiler kit in parallel with its forensic
application.
In our studied population, the 15 autosomal STR markers carry PD in
the range of 0.84 (for TPOX) to 0.97 (for D2S1338) and random match
probability (MP) from 0.03 (for D2S1338) to 0.16 (for TPOX). These differences in observed and expected heterozygosity (Table 3) indicate
high genetic diversity in Pakistani population. These values show a
great suitability of the studied markers for forensic investigation in the
Pakistani population. The results of our study are in line with the previous studies conducted on Pakistani population in 2009 by Clark et al.
(2009) and Allah-Rakha et al. (2009). However the deviation from
HWE noticed at different loci in the present study can be attributed to
high rate of inbreeding and consanguinity commonly practiced in
Pakistan. The present study coupled with the studies referred above
(Allah-Rakha et al., 2009; Clark et al., 2009), further contributes towards
development of a reference database for Pakistani population.
Besides evaluating its forensic application, this study presents
identiler as a valuable commercially available tool with potential to
be employed for screening of various common chromosomal aberrations associated with 13 different autosomes. Success rate was over
99% in detection of various autosomal trisomic conditions. Similar ndings are reported by Li et al. (2011) where they were able to detect 4
cases of trisomy from 120 prenatal samples, using identiler. However,
in our study, 74 samples already reported to possess different aneuploidies were included to check out capability of Identiler STRs in
detecting those aneuploidies and reproduce the results of karyotyping
in short time. Similarly, Heaphy et al. (2007a, 2007b) successfully
Table 3
Summary of statistical analysis of the forensic parameters for 15 autosomal STR loci in Pakistani population.
Markers
CSF1PO
FGA
TH01
TPOX
vWA
D2S1338
D3S1358
D5S818
D7S820
D8S1179
D13S317
D16S539
D18S51
D19S433
D21S11
N
MP
PD
PIC
PE
TPI
P
EH
OH
13
0.1
0.90
0.74
0.4
1.6
b0.001
0.8
0.68
15
0.04
0.96
0.85
0.71
3.5
0.513
0.9
0.86
12
0.08
0.92
0.76
0.54
2.2
0.063
0.8
0.77
8
0.16
0.84
0.63
0.4
1.5
0.008
0.7
0.67
11
0.07
0.93
0.78
0.55
2.2
0.020
0.8
0.77
13
0.03
0.97
0.87
0.66
3.0
b0.001
0.9
0.83
11
0.09
0.91
0.74
0.44
1.7
b0.001
0.8
0.71
10
0.11
0.90
0.71
0.46
1.8
b0.001
0.76
0.72
12
0.06
0.94
0.79
0.52
2.1
0.007
0.8
0.76
13
0.05
0.95
0.83
0.61
2.6
0.001
0.85
0.81
10
0.07
0.93
0.76
0.45
1.7
0.190
0.8
0.7
11
0.07
0.93
0.78
0.55
2.2
0.011
0.8
0.77
20
0.04
0.96
0.85
0.50
2.0
b0.001
0.87
0.75
16
0.04
0.96
0.82
0.55
2.2
0.305
0.84
0.8
16
0.04
0.96
0.84
0.67
3.0
0.495
0.86
0.84
N = number of alleles observed; MP = matching probability; PD = power of discrimination; PIC = polymorphic information content; PE = power of exclusion; TPI = typical paternity index; P = P value for HardyWeinberg equilibrium; EH = expected heterozygosity; OH = observed heterozygosity.
220
extra chromosome. At some instances, the allelic combination in parents prevents the tracing of this origin as described in Table 2 including
a hypothetical case that we did not encounter in our study, where both
mother and father are homozygous at the concerned locus. Tracing the
parental origin can help in counseling of the parents, in taking the appropriate measures for avoiding the aneuploidy in following pregnancy
Fig. 2. (a, b, c) Allele frequency distribution at 15 autosomal STR loci in Pakistani population and their relative prevalence among the Pathan, Sindhi, Hazara, Punjabi and Balochi ethnic
groups.
221
Fig. 2 (continued).
event(s) and in conduct of studies correlating the incidences of aneuploidy to possible contributory factors like maternal/paternal age, radiation exposure, maternal health status and medication history, etc.
Our study has indicated insufciency of amelogenin marker for the
detection of sex chromosomal aneuploidies. None of the seven samples
was clearly detectable to reach nal diagnosis of the underlying abnormal number of X and Y chromosomes that were evident in the individual karyotyping results. Moreover, the amelogenin used as the only
marker for gender identication in identiler and the similar systems,
is insufcient to fulll the requirement. As reported in a recent study
(Laverde, 2013), in certain forensic cases like rape, missing person
identication or cases involving sexual identity, amelogenin marker
can occasionally furnish false positive or negative results and shall be
interpreted with extra care. These false results can be attributed to hermaphroditism, aneuploidies, difference in genetic and physical gender,
mutations in the amelogenin sex marker or in their primer annealing regions (Laverde, 2013). Considering these limitations with amelogenin, it
is proposed that some other STR markers on X and Y chromosomes be
added to the multiplex system for forensic uses. Furthermore, adding
X/Y chromosomal STR markers can help in accurate detection of aneuploidies of the sex chromosomes.
5. Conclusions
Our study has presented the Identiler multiplex assay kit for STR
markers as a powerful tool in DNA based forensics, holding promising
applicability in Pakistani population along with its potential validity in
rapid detection of common aneuploidies. This system can be taken as
222
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