DOI 10.14456/tjg.2014.14 Thai J. Genet.

2014, 7(2) : 133–138

Analysis of paternity testing results by Identifiler™ system in Thailand

Nattapon Plodthong1, Takol Chareonsirisuthigul2, Putthapoom Lumjiaktase3, Budsaba

Rerkamnuaychoke2*
1
Master Degree in Clinical Pathology, Department of Pathology, Faculty of Medicine Ramathibodi Hospital,
Mahidol University, 10400 Thailand
2
Human Genetics Laboratory, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol
University, 10400 Thailand
3
Immunology Laboratory, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol
University, 10400 Thailand

*Corresponding author: budsaba.rer@mahidol.ac.th

ABSTRACT Keywords: paternity testing; autosomal STR markers;

One of the applications of Identifiler™system combined paternity index; power of exclusion;
is to use for paternity testing. This study aims to Thailand
analyze the paternity testing results of trio and duo
cases. A total of 500 paternity cases were divided INTRODUCTION
into 120 trio and 380 duo cases. Combined paternity A commercial DNA typing kit, AmpFlSTR®
index (CPI) and power of exclusion (PE) were Identifiler® PCR Amplification is a short tandem
calculated for inclusion and exclusion results. Based repeat (STR) multiplex assay that simultaneously
on statistical analysis, the lowest CPI value obtained amplifies 15 tetranucleotide repeat loci in a single
from trio cases was 1,340.89 with a probability of tube (Butler, 2012). This kit is composed of 13 DNA
paternity more than 99.92%. In duo cases, the markers recommended by the Combined DNA Index
lowest value of CPI was 32.18 with a probability of System (CODIS), which are CSF1PO, FGA, TH01,
paternity higher than 96.98%. Moreover, we noticed TPOX, VWA, D3S1358, D5S818, D7S820,
that approximately 50% of the CPI values in trio and D8S1179, D13S317, D16S539, D18S51, D21S11and
duo cases were in a range of 106–108 and 104–106, 2 additional DNA markers (D2S1338 and D19S433)
respectively. For exclusion results, D2S1338 had the (Miller et al., 2003; Butler, 2006). In addition, the
highest PE value in trio cases (81.20%). In duo Amelogenin is used as a control marker for
cases, D8S1179 showed the highest PE value genderdetermination (Butler, 2005). The application
(75.80%). TPOX had the lowest PE value in both trio of this kit is for paternity testing to determine the
and duo cases (29.10% and 24.60%). The minimal biological father of a child based on the variability in
numbers of excluding loci were 7 and 3 in trio and STR regions, which can be used to distinguish one’s
duo cases, respectively. In addition, this study DNA profile from another’s (Jacewiczet al., 2004;
showed that a threshold value for CPI should be Babol-Pokoraet al., 2006; El-Alfy and El-Hafez,
greater than or equal to 1,000. 2012). There are two possible results of paternity
134 Thai J. Genet. 2014, 7(2) : 133–138 Nattapon Plodthong et al.

testing. One is a negative paternity result paternity testing results were divided into 2 major
(exclusion), a mismatch in 3 or more gene regions groups which were 120 trio cases and 380 duo
between the alleged father and the child which cases. For trio cases, there are 100 inclusion
means the alleged father is not the biological father paternity cases and 20 exclusion cases, while duo
(Ayadiet al., 2007).The other, a positive paternity cases include 300 inclusion paternity cases and 80
result (inclusion), a complete matching of DNA exclusion cases.
profile between the alleged father and the child
which means the alleged father has greater than a Statistical analysis
99.999% chance of being the biological father For all 15 autosomal STR loci, the
(Jacewiczet al., 2004;Tug and Akduman, 2009). For combined paternity index (CPI) was determined for
statistical analysis, the combined paternity index inclusion paternity cases by Rapid DNA program
(CPI) and power of exclusion (PE) are routinely based on John Buckleton’srecommendation. The power
reported for inclusion paternity cases and exclusion of exclusion (PE) was determined for exclusion
paternity cases, respectively. In paternity testing, paternity cases, which was calculated by a modified
mutation of STR alleles is an important issue version of PowerStats software package Version 1.2.
because a complete matching between a child and
alleged father cannot be made when a mutation is Paternity Index (PI)
present. Mainly, mutation of STR alleles occurs as a Paternity Index (PI) is the relative probability
result of a gain or loss of a single repeat unit (Butler, that the alleged father and not an unrelated, randomly
2005). A high mutation rate for STR marker could selected male of the same ethnic background
result in a false exclusion at the particular locus. It transmitted the obligate allele to the child. This is a
was previously reported that D21S11, FGA, D7S820, likelihood ratio and is presented in the following
D16S539, and D18S51 have the highest mutation formula, PI = X/Y, where X is probability that the
rates with the most polymorphic genotypes and tested man is the father. Y is probability that a
possess the highest number of observed alleles, random man is the father (Gjertsonet al., 2007).
which is useful in human identity investigation Therefore, the combined paternity index (CPI) is
(Butler, 2005). The purpose of this study is to calculated by multiplying the individual PIs for each
analyze and compare the paternity testing results STR locus, CPI = PI1 * PI2 * ... * PIN, (Stephenson,
between trio cases and duo cases. 2010). CPI is ratio that indicates the likelihood of the
alleged father being the biological father in
MATERIALS AND METHODS comparison to the likelihood of a random, unrelated
Population man in the population being the father.
In this study, all 500 paternity testing results
determined by AmpFlSTR®Identifiler® PCR amplification The power of exclusion (PE)
kit with 15 DNA markers (Life Technology, USA) The power of exclusion (PE) is the
during 2012–2013 were collected. The data were parameter evaluating the loci or system efficiency in
obtained from Human Genetics Laboratory, Department excluding a non-related individual in paternity
of Pathology, Faculty of Medicine Ramathibodi investigation (Oliveira et al., 2006). The PE has been
Hospital, Mahidol University, Bangkok, Thailand. The commonly presented by the formula, PE = h2 (1-
Nattapon Plodthong et al. Thai J. Genet. 2014, 7(2) : 133–138 135

2*h*H2), where h is the proportion of heterozygous from this study were lower than the results of Babol-
individuals and H the proportion of homozygous Pokoraet al., (2006). Their results showed that 50%
individuals in the population sample (Brenner, 1990; of CPI value in duo cases were lower than 99.999%
Oliveira et al., 2006; SWGDAM, 2010). which were not sufficient to ascertain fatherhood
status according to Polish Forensic Genetic
RESULTS AND DISCUSSION Commission (Babol-Pokoraet al., 2006). According
The comparison of CPI values between 100 to the legal threshold for the presumption of
trio and 300 duo cases which have inclusion paternity through genetic testing,a minimum CPI of
paternity results was shown in Figure 1. The lowest 1,000 or a probability of paternity of 99.9% of men
value of CPI obtained from trio cases was 1,340.89 in the populationis required.Therefore, a threshold
with a probability of paternity greater than 99.93%. In value for CPI in this study should be greater than
duo cases, the lowest value of CPI was 32.18 with a or equal to 1,000 (Pankeet al., 2001; Barbaro,
probability of paternity greater than 96.99%. In 2012). Moreover, from our study we noticed that
addition, the highest value of CPI from trio cases was approximately 50% of the CPI values in trio and
524,455,803,620.27 with a probability of paternity duo cases were in a range of 106-108 and 104-106,
greater than 99.9999999998%. In duo cases, the respectively. Practically, when the statistic value
highest value of CPI was 35,433,401,625.84 with a does not meet the requirement, additional
probability of paternity greater than 99.999999997%. laboratory testing is needed.
The minimal CPI value in both trio and duo cases

Figure 1 Comparison of combined paternity index (CPI) values of 100 trio and 300 duo cases with inclusion
paternity results. The lowest value of CPI obtained from trio cases was 1,340.89 with a probability of paternity
greater than 99.93%. In duo cases, the lowest value of CPI was 32.18 with a probability of paternity greater
than 96.99%. In addition, the highest value of CPI from trio cases was 524,455,803,620.27 with a probability
of paternity greater than 99.9999999998%. In duo cases, the highest value of CPI was 35,433,401,625.84
with a probability of paternity greater than 99.999999997%.
136 Thai J. Genet. 2014, 7(2) : 133–138 Nattapon Plodthong et al.

Power of exclusion values for each STR addition, Shotivaranonet al. (2009) reported that FGA
locus were shown in Figure 2. In trio cases, D2S1338 had the highest PE value at 73.60%, which was
had the highest PE value of 81.20%. In duo cases, different from our study and their results showed
D8S1179 had the highest PE value of 75.80%. On TPOX with the lowest PE value of 25.70%, which
the other hand, D2S1338 was reported to have the was concordant with this study (Shotivaranonet al.,
highest PE value in both trio and duo cases in Central 2009). The discrepancy of these results may be due
Poland population (Babol-Pokoraet al., 2006). For the to the difference in sample size: n=500 (in this
lowest PE value, our results were consistent with the study), n=80 (Chanpreechayaet al., 2009) and n=929
report fromBabol-Pokoraet al., (2006), which showed (Shotivaranonet al. 2009). Another study of PE value
that TPOX had the lowest PE value in both trio and was reported from analysis of a population in the
duo cases at 29.10% and 24.60%, respectively. In Middle - West region of Brazil,showing that the
Thai population, Chanpreechayaet al., (2009) reported combination of 18 STR loci of 3 multiplex
that CSF1PO and TPOX had the lowest PE values, amplification systems gave the higher PE value and
whereas D13S317 and FGA had the highest PE was efficient and enough for the analysis of paternity
values (Chanpreechaya and Taechowisan, 2009). In cases (Oliveira et al., 2006).

Figure 2 Power of Exclusion (PE) values for each DNA marker between 20 trio and 80 duo cases with
exclusion paternity results. In trio cases, D2S1338 had the highest PE value (81.20%). In duo cases,
D8S1179 had the highest PE value (75.80%).TPOX had the lowest PE value in both trio and duo cases,
which are29.10% and 24.60%, respectively.
Nattapon Plodthong et al. Thai J. Genet. 2014, 7(2) : 133–138137

The minimal numbers of excluding STR In summary, this study showed that
loci in trio and duo cases were 7 and 3, D2S1338 and D8S1179 were the most effective
respectively, which was different from the data of genetic markers for determining individuals who are
Central Poland (Figure 3). There were 4 loci in not real biological fathers in trio and duo cases,
trio cases and 1 or 2 of excluding loci in duo respectively. On the contrary, TPOX was the lowest
cases (Babol-Pokoraet al., 2006). The sample powerful STR in both trio and duo cases. A
size in this study included only 80 exclusion duo threshold value for CPI should be greater than or
cases and 20 exclusion trio cases but the report equal to 1,000. Moreover, there was no mutation
of Central Poland consisted of 150 exclusion event detected and the lowest number of non-
cases in both duo and trio. In addition, the matching genetic markers in duo cases was 3 loci,
lowest number of excluding loci in Egypt which was enough for paternity conclusion.
population was 5 to 7 from 20 excluded paternity However, if one or two mutations occur, additional
cases (El-Alfy and El-Hafez, 2012). DNA tests are recommended to confirm the results.

Figure 3 Comparison of the number of excluding STR loci between 20 trio and 80 duo cases with exclusion
paternity results. The minimal numbers of excluding STR loci in trio and duo cases were 7 and 3, respectively.

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