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Gene 548 (2014) 217222

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Gene
journal homepage: www.elsevier.com/locate/gene

Application of Short Tandem Repeat markers in diagnosis of


chromosomal aneuploidies and forensic DNA investigation in Pakistan
Hafsah Muhammad Chishti a,b, Muhammad Ansar a, Muhammad Ajmal b, Abdul Hameed b,
a
b

Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan


Institute of Biomedical and Genetic Engineering, Islamabad, Pakistan

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.

sensitivity, specicity, prompt analysis and high throughput of samples


at low cost and with less labor (Atef et al., 2011). Considering these
utilities and advantages, many commercial kits and systems have been
developed by different companies with different combinations of STR
markers. One such multiplex assay is the AmpFlSTR Identiler PCR
amplication kit, that is widely used across the world in DNA based
forensic investigations. 15 tetranucleotide repeat loci located on
13 different autosomes and a gender determining marker amelogenin
located on X and Y chromosomes comprise the identiler kit. Autosomal
markers include TPOX (2p232pter), D2S1338 (2q3537.1), D3S1358
(3p21.31), FGA (4q28), CSF1PO (5q33.334), D5S818 (5q2131),
D7S820 (7q11.2122), D8S1179 (8q24.13), THO1 (11p15.5), VWA
(12p12-pter), D13S317 (13q2231), D16S539 (16q24-qter), D18S51
(18q21.3), D19S433 (19q1213.1) and D21S11 (21q11.2q21). This
multiplex system has been used to develop the DNA database of many
populations. The well-known Combined DNA Index System (CODIS) of
the United States consists of 13 of the autosomal 15 STRs included in
identiler. These DNA databases are routinely used in forensic investigation across the world. Moreover, this system of multiplex PCR with
the theme of utilizing STR markers for aneuploidy identication have
been employed by many researchers in nding cause of miscarriage in
Products of Conception (PoC) (Furtado et al., 2013a), molar pregnancies

218

H.M. Chishti et al. / Gene 548 (2014) 217222

(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

2. Materials and methods

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).

2.1. Sample collection

3. Results

In the present study, 554 healthy unrelated individuals belonging to


the Punjabi, Sindhi, Balochi, Pathan and Hazara ethnicities were investigated. Collection of blood samples was carried out during 20112012,
from different villages, towns and small cities all over Pakistan. Only
the individuals with their mother and father belonging to the same ethnicity were included. 5 to 8 ml blood samples were collected in BD
Vacutainer tubes (BD Franklin Lakes NJ, USA) and labeled with appropriate alphanumeric codes to keep the individual identity anonymous.
Informed written consent was obtained from all the participants before
their enrollment in the study. Blood samples of 74 patients with different chromosomal aneuploidies, reported during 20102013 were
obtained from karyotyping lab of the Institute of Biomedical and Genetic
Engineering, Islamabad. Blood samples from both parents of these
patients were also taken with written informed consent to tract the
parental source of aneuploidy. The study was approved by the ethical
committee of the Institute of Biomedical and Genetic Engineering,
Islamabad.

3.1. Chromosomal aneuploidy detection


From the 74 samples of suspected chromosomal aneuploidies, 44
cases of trisomy 21 (Down syndrome), 13 cases of trisomy 18 (Edward
syndrome), 9 samples of trisomy 13 (Patau syndrome), and 1 case of
trisomy 8 (Warkany syndrome 2) were successfully detected using
the AmpFlSTR Identiler kit. Abnormal allelic patterns were observed at locus D21S11 for Down syndrome patients, at locus D18S51
for Edward syndrome patients, at locus D13S317 for Patau patients
and at locus D8S1179 for trisomy 8 patient. All these results were
cross-checked with the karyotyping results.

2.2. Genomic DNA extraction and PCR amplication of STR markers


Genomic DNA extracted from the blood samples following the standard organic DNA extraction protocol (Sambrook and Russell, 2001),
was quantied by measuring absorbance at 260 nm & 280 nm using a
spectrophotometer (Hitachi, 3020, Japan). Approximately 2 ng of each
target genomic DNA was used for simultaneous amplication of 15
STR markers plus the gender determination marker, amelogenin (multiplex PCR). This amplication was performed by using the AmpFlSTR
Identiler PCR amplication kit according to the user's manual recommendations (Applied Biosystems, Foster City, CA, USA).
2.3. Genotyping
Samples were genotyped using the ABI Prism 3130 Genetic
Analyzer, following the manufacturer's instructions, using referenced
sequenced ladders (Applied Biosystems, Foster City, CA). Alleles were
assigned from the electropherograms using the Genotyper Version 3.2

Fig. 1. Electropherogram image at locus D21S11of Down syndrome patients, revealing a


trisomic triallelic pattern (1:1:1) in upper and trisomic diallelic (2:1) pattern in lower
electropherogram.

H.M. Chishti et al. / Gene 548 (2014) 217222

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

Case detected using identiler

Success rate of Identiler

Source of extra chromosome


Maternal

Paternal

Not determined

Trisomy 21 (Down syndrome)


Trisomy 18 (Edward syndrome)
Trisomy 13 (Patau syndrome)
Trisomy 8 (Warkany syndrome 2)

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

29, 31, 31.2


28, 29, 30
29, 30, 31
29, 31.2, 31.2
28, 28, 28

Paternal
Maternal
Not determined
Not determined
Not determined

Outcomes for origin of each investigated condition are summarized


in Table 1. The parental origins of the 92.54% autosomal aneuploidy
cases were determined by comparing the DNA proles and electropherograms of investigated patients with their parents'. In 67.7% cases the
extra chromosomes were of maternal origin as exemplied in case 1
of Table 2 and 32.3% came from paternal source as in case 2 of Table 2.
In the remaining 7.46% patients, parental origin was not determined
(Table 1) mainly because of allelic combination at concerned loci as exemplied by cases 3 and 4 in Table 2.
From the samples involving sex chromosomal aneuploidies, 1 case of
XYY, 1 case of XXX (Triple X syndrome), 1 case of tetra X, 2 cases of XXY
(Klinefelter syndrome), and 2 cases of monosomy X0 (Turner Syndrome) were not clearly detectable. False negative results were obtained using identiler; however, the aneuploidic conditions were evident
in the karyograms for each of these patients.

3.2. Forensic parameters


The forensic statistical parameters calculated for the 15 STR markers
in the Pakistani population are summarized in Table 3. The assigned alleles with their frequency distribution among the different Pakistani
ethnicities are illustrated in Fig. 2. D18S51 carried the highest number
of alleles. Among all the 15 STR markers studied, D2S1338 was forensically the most valuable marker observed carrying the highest PD (0.97)
and the lowest MP (0.03) whereas TPOX emerged to be the least polymorphic and least discriminative marker. The OH values ranged from
0.6 to 0.86 and the EH values ranged from 0.6 to 0.9. Signicant deviation from HWE expectations was observed at various loci (Table 3).

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.

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H.M. Chishti et al. / Gene 548 (2014) 217222

utilized identiler system for detection of allelic imbalances in a variety


of frozen and archival tissues including cancerous tissues. Identiler
system has value added advantage over the traditional karyotyping approach, of the potential to trace the parental origin of the extra chromosomes in aneuploidic conditions. In most of the cases, comparison of
both parent's prole with that of the offspring's reveals the source of

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.

H.M. Chishti et al. / Gene 548 (2014) 217222

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

a model for development of similar system with larger number of STR


markers from all 23 chromosomes, for more accurate diagnosis of
aneuploidies.
Acknowledgments
We thankfully acknowledge theHigher Education Commission
(HEC) of Pakistan for support and funding to conduct this research
project.
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