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Assessing the forensic efficiency of the GlobalFiler STR loci among the genetically isolated Chechen subpopulation in Jordan
Gene 720 (2019) 144078
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Research paper
Assessing the forensic efficiency of the GlobalFiler STR loci among the genetically isolated Chechen subpopulation in Jordan
T
⁎
Laith N. AL-Eitana,b, , Nizar N. Darwisha, Nancy M. Hakoozc, Rana B. Dajanid,e a
Department of Applied Biological Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan Department of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, Irbid 22110, Jordan c Department of Biopharmaceutics and Clinical Pharmacy, School of Pharmacy, University of Jordan, Amman 11942, Jordan d Department of Biology and Biotechnology, Hashemite University, Zarqa 13133, Jordan e Radcliffe Institute for Advanced Studies, Harvard University, Cambridge, 02138, MA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: STR Jordan Chechen GlobalFiler
Short tandem repeats (STRs) are a widely utilized tool in forensic applications, the latter of which range from human identification and paternity testing to population analysis. The GlobalFiler STR loci, which includes 21 autosomal STRS, were analyzed in the Chechen subpopulation of Jordan. Whole blood samples were withdrawn from 159 Jordanian Chechen individuals, and genomic DNA was extracted from each sample. The GlobalFiler™ kit PCR Amplification Kit amplified and analyzed the STR loci on the 3130xl Genetic Analyzer using GeneMapper ID-X software. The combined match probability for the 21 autosomal STR loci was calculated to be 1.06 × 10−24, a number that is highly discriminatory and informative. The SE33 (0.983) and TPOX (0.806) loci exhibited the highest and lowest powers of discrimination, respectively. Conclusively, the current study indicates that the GlobalFiler loci have a high utility in the Jordanian Chechen population, possibly paving the way for the future establishment of a reference population database in Jordan.
1. Introduction Jordan is a Middle-Eastern country located on the Jordan River's east bank, and it lies between the northern part of the Arabian Peninsula and the southern part of the Levant. Based on the 2015 census, Jordan has a population of 9.5 million, 60% of which are Jordanians and the remainder of which are Syrians (1.265 million), Egyptians (0.636 million), Palestinians (0.634 million), Iraqis (130,911), Yemenis (31,163), Libyans (22,700), and other nationalities (197,385) (DE BEL-AIR F, 2016). Among Jordanians, there exists a level of genetic diversity that came about due to historical and contemporary movements of subpopulations into and out of Jordan, resulting in the Jordanian population being made of Arabs, Bedouins, Armenians, Circassians, and Chechens (Zanetti et al., 2014; Al-Eitan et al., 2017). The Chechens are Sunni Muslims of Caucasian origin that arrived in Jordan in a series of waves in the early 20th century, forming a distinct subpopulation within the Jordanian Arab majority (Dweik, 2000; Shishani
et al., 2013). In the forensic sciences, DNA profiling is an integral technique that allows the identification of individuals based on their unique genetic backgrounds (Jobling and Gill, 2004). Repeated DNA sequences are abundant in eukaryotic genomes, and vary in size between individuals without affecting their genetic health (Ellegren, 2004). Despite being harmless from a clinical perspective, inter-individual variation in the length of repeated sequences has much forensic practicality. In fact, short tandem repeats (STRs), which are non-coding DNA regions with repeated 2-to-6-bp units, are routinely used in DNA profiling systems (Butler, 2006; Butler, 2007). > 100,000 independent STR markers are available to forensic investigators to choose from, but expanding the number of examined loci increases the probability that two unrelated individuals will have distinct DNA profiles (Weir, 2007). New STR markers are constantly being identified, and each marker has different powers of discrimination and overall forensic efficiency depending on the ethnic group or population in question (Yang et al., 2017; Liu et al.,
Abbreviations: Bp, base pair; CDP, combined discrimination power; CEP, combined exclusion probability; CMP, combined matching probability; CODIS, combined DNA index system; CPI, combined paternity index; CSF1PO, c-fms proto-oncogene for CSF-1 receptor gene; DNA, deoxyribonucleic acid; FGA, fibrinogen alpha chain gene; He, expected heterozygosity; HLA, human leukocyte antigen; Ho, observed heterozygosity; HWE, Hardy Weinberg Equilibrium; NA, not available; PCR, polymerase chain reaction; PD, power of discrimination; PE, probability of exclusion; PIC, polymorphism information content; STR, short tandem repeats; TH01, human tyrosine hydroxylase gene; TPI, typical paternity index; TPOX, thyroid peroxidase gene; vWA, von Willebrand factor gene ⁎ Corresponding author at: Department of Applied Biological Sciences, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan. E-mail address: [email protected] (L.N. AL-Eitan). https://doi.org/10.1016/j.gene.2019.144078 Received 17 June 2019; Received in revised form 22 August 2019; Accepted 23 August 2019 Available online 29 August 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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2013; Butler, 2015; Alshamali et al., 2005). Few published studies have incorporated STR analysis in the genetic exploration of different Jordanian subpopulations. Al-Eitan and Tubaishat (2018) evaluated the forensic efficiency of 22 autosomal STR loci in a Jordanian-Arab population sample using the PowerPlex® Fusion (PPF) system (Al-Eitan and Tubaishat, 2018). Likewise, the allelic frequencies and other forensic efficiency parameters for 15 STR markers were evaluated in the African-Jordanian population (Yasin et al., 2005). Lastly, a Jordanian population sample was profiled in order to determine the forensic efficiency parameters of five STR markers (CSF1PO, D16S539, Penta D, Penta E, and TPOX) (Yasin et al., 2005). To the authors' knowledge, there are no previous reports regarding the frequency of STRs in the Jordanian-Chechen community. Therefore, this study aims to determine the degree of forensic efficiency of 21 autosomal STR markers in the Chechen subpopulation of Jordan.
Table 1 Linkage disequilibrium for three sets of STR loci in Jordanian Chechens (n = 159). STR loci set
P-value
CSF1PO/D5S818 D12S391/vWA D2S1338/D2S441 TPOX/D2S1338 TPOX/D2S441
0.1045 0.1503 0.2937 0.1173 0.1038
2.4. Data availability The entirety of the data produced during this study is available upon request from the corresponding author. 3. Results
2. Materials and methods
3.1. Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD)
2.1. Sample collection and DNA extraction
Out of the investigated STR markers, the D19S433, SE33, and D1S1656 loci exhibited departure from HWE (p-value < 0.05). However, after Bonferroni correction was applied, no significant departure from HWE was observed for the 21 autosomal loci, allowing HWE to be employed in the estimation of the genotypic frequencies. Moreover, conducting the linkage equilibrium exact test on the 21 STR loci showed that there was no significant linkage disequilibrium (pvalue > 0.05), ensuring stability of the calculations concerning genotypic frequencies (Table 1).
Ethical approval to conduct this study was granted by the Institutional Review Board (IRB) at Jordan University of Science and Technology. 159 healthy and unrelated individuals were recruited from the Chechen subpopulation in Jordan. Subjects completed a survey to confirm their Chechen lineage. After obtaining written informed consent, 3–5 ml samples of whole blood were collected from the participants. DNA was extracted from each sample using the Gentra PureGene Blood Kit (Qiagen, Germany) according to the manufacturer's protocol. The quantity and quality of the extracted DNA samples were determined using the Nano-Drop ND-1000 (Thermo Scientific, USA), after which the quantified DNA samples were diluted to achieve final concentrations of 0.5–1 ng per 15 μl.
3.2. Genotypic and allelic frequencies The genotypes (DNA profiles) of the 159 participants were successfully determined without any PCR or STR scanning artifacts. No offladder alleles or tri-allelic patterns were detected as all of the determined allelic genotypes were previously reported on STR Base. Therefore, all of the obtained allelic genotypes were used in the allelic frequency and forensic efficiency calculations. Tables 2a and 2b lists the allelic frequencies of the 21 autosomal STR loci that were investigated in this study. However, a total of 8 loci exhibited incomplete repeat units of microvariant alleles, the majority of which were exhibited by the SE33 locus (51.8%). Microvariant allelic frequencies of the investigated STR markers are displayed in Table 3. With regard to the sex-determining STR marker Amelogenin, the X/ X genotype (female) was observed 72 times, while the X/Y genotype (male) was detected 87 times. The frequency of the Y allele of Amelogenin was calculated to be 0.273, and the frequency of the X allele for the same locus was 0.726. Table 4 shows the genotypes and allelic frequencies of the Amelogenin marker in Jordanian Chechens.
2.2. Multiplex PCR and capillary electrophoresis Based on the manufacturer's guidelines, the GlobalFiler™ PCR Amplification Kit (Life Technologies, USA) was utilized to amplify the sex-determining marker Amelogenin, 1 Y STR locus, 1 Y insertion/deletion (Y indel) locus, and 21 autosomal STR loci, namely CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX, and vWA. Amplification was performed on the GeneAmp™ PCR System 9700 Fast Thermal Cycler (Life Technologies, USA). STR typing of the amplified DNA samples was carried out via capillary electrophoresis, the latter of which was completed on the Applied Biosystems 3130XL Genetic Analyzer (Life Technologies, USA). GeneScan™ - 600 LIZ®, an internal lane size standard, was included with every sample, and an allelic ladder was used to facilitate the automatic sizing of alleles by the GeneMapper™ ID-X Software (Life Technologies, USA).
3.3. Allelic variability and possible genotypes (PG) Table 5 depicts the observed alleles as well as the possible genotypes for the 21 autosomal STR markers investigated in the Chechen population sample. Based on these figures, the total number of observed alleles among the investigated population sample was found to be 203, indicating high allelic polymorphism. The total number of possible genotypes (PG) was calculated according to the following equation: If n alleles exist for an STR marker then the number of possible genotypes equals n (n + 1)/2. Among the investigated STR makers, SE33 exhibited the highest number of alleles (n = 27) with the highest PG, making it the most polymorphic and informative STR marker among the Jordanian Chechen subpopulation. To the contrary, the lowest number of observed alleles was reported for D22S1045 (n = 5) and TPOX (n = 6). However, the TPOX marker can be considered to be the least
2.3. Statistical analysis The PowerStats 1.2 software (Promega, USA) was used to calculate the allelic frequencies as well as the following forensic efficiency parameters: matching probability (MP), power of discrimination (PD), polymorphism information content (PIC), observed heterozygosity (OH), and paternity-related data (probability of exclusion (PE) and typical paternity index (TPI)). The Arlequin 3.5 software was employed to determine any possible departures of the Chechen sub-population from the Hardy-Weinberg equilibrium (HWE) and to calculate linkage disequilibrium (LD). Bonferroni's correction for multiple testing was applied in order to reduce the level of significance of the HWE from α = 0.05 to α = 0.002, which was achieved by dividing the p-value cutoff (α = 0.05) by the number of tested autosomal loci (n = 21). 2
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Table 2a Calculated allelic frequencies for 21 autosomal STR loci in Jordanian Chechens (n = 159). Allele
CSF1PO
D16S539
D18S51
D19S433
D21S11
D2S441
D3S1358
D8S1179
D10S1248
D12S391
5.3 6 7 8 9 9.3 10 11 11.3 12 12.2 12.3 13 13.2 14 14.2 15 15.2 15.3 16 16.2 16.3 17 17.2 17.3 18 18.3 19 19.2 19.3 20 20.3 21 21.2 22 22.2 23 23.2 24 24.2 25 25.2 26 26.2 27 27.2 28 28.2 29 29.2 30 30.2 31 31.2 32 32.2 33.2 34 34.2 35
– – – – 0.016 – 0.368 0.164 – 0.362 – – 0.066 – 0.025 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – 0.009 0.116 – 0.138 0.28 – 0.223 – – 0.217 – 0.016 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – 0.028 – 0.088 – – 0.126 – 0.17 – 0.208 – – 0.126 – – 0.066 – – 0.057 – 0.088 – – 0.031 – – – 0.013 – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – 0.094 0.006 – 0.252 0.006 0.371 0.06 0.151 0.016 – 0.028 0.006 – 0.006 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.013 – – – 0.019 – 0.091 – 0.258 – 0.186 0.028 0.028 0.11 0.047 0.107 0.063 – 0.05 –
– – – – – – 0.258 0.264 0.091 0.066 – 0.003 0.019 – 0.286 – 0.013 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – 0.038 – 0.245 – – 0.264 – – 0.217 – – 0.233 – 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – 0.013 0.003 – 0.066 0.063 – 0.148 – – 0.425 – 0.189 – 0.094 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – 0.003 – – 0.179 – 0.415 – 0.274 – – 0.094 – – 0.035 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – 0.016 – – 0.047 – – 0.069 – – 0.094 0.053 0.104 – – 0.214 – 0.113 – 0.11 – 0.113 – 0.031 – 0.035 – – – – – – – – – – – – – – – – – – –
Statistical analyses EH 0.702 OH 0.704 MP 0.142 P 0.534 PD 0.858 PE 0.44 PIC 0.65 TPI 1.69
0.792 0.824 0.084 0.147 0.925 0.595 0.76 2.84
0.871 0.918 0.037 0.289 0.97 0.746 0.86 6.12
0.763 0.811 0.094 0.013* 0.911 0.549 0.73 2.65
0.856 0.862 0.041 0.113 0.965 0.719 0.84 3.61
0.769 0.78 0.099 0.323 0.909 0.553 0.73 2.27
0.767 0.748 0.099 0.243 0.905 0.548 0.73 1.99
0.745 0.73 0.093 0.098 0.904 0.524 0.71 1.85
0.711 0.673 0.136 0.221 0.869 0.458 0.66 1.53
0.885 0.868 0.029 0.092 0.976 0.772 0.87 3.79
EH: expected heterozygosity; OH: observed heterozygosity; MP: matching probability; P: p-value of the HWE exact test; PD: power of discrimination; PE: probability of exclusion; PIC: polymorphic information content; TPI: typical paternity index.
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Table 2b Calculated allelic frequencies for 21 autosomal STR loci in Jordanian Chechens (n = 159). Allele
D13S317
D1S1656
D22S1045
D2S1338
D5S818
D7S820
FGA
SE33
TH01
TPOX
vWA
5.3 6 7 8 9 9.3 10 11 11.3 12 12.2 12.3 13 13.2 14 14.2 15 15.2 15.3 16 16.2 16.3 17 17.2 17.3 18 18.3 19 19.2 19.3 20 20.3 21 21.2 22 22.2 23 23.2 24 24.2 25 25.2 26 26.2 27 27.2 28 28.2 29 29.2 30 30.2 31 31.2 32 32.2 33.2 34 34.2 35
– – – 0.066 0.119 – 0.047 0.274 – 0.333 – – 0.148 – 0.009 – – – – 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – 0.145 – 0.113 – – 0.05 – 0.094 – 0.208 – 0.003 0.142 – 0.031 0.066 – 0.085 – 0.038 0.009 – 0.003 0.006 0.006 – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – 0.176 – – – – – – 0.116 – 0.387 – – 0.261 – – 0.06 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – 0.044 – – 0.198 – – 0.101 – 0.217 – – 0.075 – 0.006 – 0.047 – 0.119 – 0.091 – 0.075 – 0.006 – 0.019 – – – – – – – – – – – – – – –
– – – – 0.072 – 0.035 0.336 – 0.358 – – 0.142 – 0.05 – 0.006 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – 0.003 0.182 0.101 – 0.255 0.333 – 0.091 – – 0.035 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – 0.006 – 0.079 – – 0.028 – 0.097 0.003 0.186 0.006 0.255 0.003 0.157 – 0.041 – 0.085 – 0.053 – – – – – – – – – – – – – – –
– – – – – – – 0.003 – – – – 0.009 – – 0.003 0.016 – – 0.041 – – 0.006 – – 0.072 – 0.167 0.003 – 0.063 – 0.006 0.009 – 0.038 – 0.022 – 0.019 – 0.05 – 0.079 0.003 0.053 0.003 0.195 – 0.075 – 0.025 – 0.009 – – 0.006 0.003 – 0.019
0.003 0.355 0.17 0.16 0.126 0.176 0.009 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – 0.566 0.094 – 0.066 0.226 – 0.044 – – 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – 0.145 – 0.097 – – 0.255 – – 0.167 – – 0.255 – 0.066 – – 0.006 – 0.009 – – – – – – – – – – – – – – – – – – – – – – – – – – –
0.878 0.881 0.034 0.025* 0.973 0.758 0.87 4.18
0.734 0.717 0.115 0.097 0.887 0.496 0.69 1.77
0.865 0.862 0.034 0.203 0.968 0.734 0.85 3.61
0.729 0.736 0.119 0.456 0.883 0.488 0.69 1.89
0.771 0.774 0.088 0.078 0.913 0.56 0.74 2.21
0.848 0.881 0.051 0.125 0.96 0.702 0.83 4.18
0.902 0.881 0.025 0.008* 0.983 0.807 0.89 4.18
0.772 0.723 0.085 0.071 0.915 0.563 0.74 1.81
0.613 0.585 0.198 0.134 0.806 0.332 0.57 1.2
0.808 0.742 0.063 0.431 0.936 0.625 0.78 1.94
Statistical analyses EH 0.771 OH 0.774 MP 0.092 P 0.645 PD 0.914 PE 0.561 PIC 0.74 TPI 2.21
EH: expected heterozygosity; OH: observed heterozygosity; MP: matching probability; P: p-value of the HWE exact test; PD: power of discrimination; PE: probability of exclusion; PIC: polymorphic information content; TPI: typical paternity index.
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Table 3 Numbers and percentages of observed microvariant alleles in Jordanian Chechens (n = 159). STR loci
Observed microvariant
Percentage (%)
D12S391 D19S433 D1S1656 D21S11 D2S441 FGA SE33 TH01
1/12 6/12 6/15 5/12 2/8 3/13 14/27 2/7
8.3 50 40 41.7 25 23.1 51.8 28.6
Table 6 The two most common observed alleles and their frequencies as well as the most common genotypic frequencies in Jordanian Chechens (n = 159).
Table 4 Phenotypes and allelic frequencies of the Amelogenin locus in Jordanian Chechens (n = 159). Allele
Genotype
Phenotype
Observed genotype
Genotype frequency
Allele frequency
Y X
X/Y X/X
Male Female
87 72
0.547 0.453
0.273 0.726
Table 5 Number of observed alleles and possible genotypes in Jordanian Chechens (n = 159). STR loci
Observed alleles
Possible genotypes n(n + 1)/2
CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vWA Total
6 6 12 8 7 11 12 15 12 5 12 8 6 7 7 8 13 27 7 6 8 203
21 21 78 36 28 66 78 120 78 15 78 36 21 28 28 36 91 378 28 21 36 3.037 × 1034
STR loci
Allele 1
Allele 2
Allele 1 frequency (p)
Allele 2 frequency (q)
Genotype frequency (2pq)
CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vWA
10 14 20 11 11 14 13 11 29 15 17 11 15 11 10 13 22 19 6 8 16,18
12 15 21,23 12 12 15 14 15 30 16 19 14 16 12 11 14 33 28.2 9.3 11 17
0.368 0.415 0.214 0.274 0.28 0.17 0.252 0.145 0.258 0.387 0.198 0.264 0.245 0.336 0.255 0.425 0.186 0.167 0.355 0.566 0.255
0.362 0.274 0.113 0.333 0.223 0.208 0.371 0.208 0.186 0.261 0.217 0.286 0.264 0.358 0.333 0.189 0.255 0.195 0.176 0.226 0.167
0.266 0.227 0.048 0.182 0.125 0.071 0.187 0.060 0.096 0.261 0.086 0.151 0.129 0.241 0.169 0.161 0.095 0.065 0.125 0.256 0.085
Table 7 Polymorphism Information Content (PIC) values in Jordanian Africans, Arabs, and Chechens. STR loci
Chechen Jordanians (n = 159)
Arab Jordanians (n = 159)
African Jordanians (n = 159)
CSF1PO D13S317 D16S539 D18S51 D21S11 D3S1358 D5S818 D7S820 D8S1179 FGA TH01 TPOX VWA Average
0.65 0.74 0.76 0.86 0.84 0.73 0.69 0.74 0.71 0.83 0.74 0.57 0.78 0.74
0.68 0.75 0.74 0.88 0.84 0.75 0.76 0.76 0.83 0.87 0.77 0.76 0.78 0.78
0.74 0.65 0.79 0.88 0.79 0.72 0.67 0.69 0.77 0.89 0.73 0.71 0.8 0.75
Table 8 Observed heterozygosity (OH) in Jordanian Africans, Arabs, and Chechens.
polymorphic and informative marker among Jordanian Chechens due to a marked imbalance in its allelic distribution. The most common allele among Jordanian Chechens was allele 8 of the TPOX marker, as it was detected in 57% of sampled individuals (n = 159 or 318 measured chromosomes). Table 6 illustrates the two most commonly observed alleles for each STR marker as well as their allelic and genotypic frequencies.
3.4. Forensic efficiency parameters 3.4.1. Matching probability (MP) The maximum and minimum MP values were calculated for the TPOX (0.198) and SE33 (0.025) markers, respectively, while the average MP value for all 21 investigated autosomal loci was 0.084. Despite having a higher number of alleles, TPOX had a poorer MP value than D22S1045, indicating that the former had a more balanced allelic distribution. The combined matching probability (CMP) was 5
STR loci
Chechen Jordanians (n = 159)
Arab Jordanians (n = 159)
African Jordanians (n = 159)
CSF1PO D13S317 D16S539 D18S51 D21S11 D3S1358 D5S818 D7S820 D8S1179 FGA TH01 TPOX VWA Average
0.704 0.774 0.824 0.918 0.862 0.748 0.736 0.774 0.73 0.881 0.723 0.585 0.742 0.77
0.695 0.736 0.776 0.749 0.64 0.779 0.689 0.76 0.735 0.824 0.749 0.623 0.802 0.74
0.811 0.695 0.768 0.874 0.842 0.653 0.737 0.726 0.8 0.947 0.779 0.768 0.905 0.79
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Table 9 Studies incorporating GlobalFiler in the STR analysis of different populations. Population
Sample size
STR with highest PD (%)
STR with lowest PD (%)
CMP
Chinese Kazakh (Zhang et al., 2016a) Chinese Uyghur (Zhang et al., 2016b) Emirati Arab (Ali Alhmoudi et al., 2015) Japanese (Fujii et al., 2015) Jordanian Chechen Korean (Park et al., 2016) Mongolian (Choi et al., 2017) Polish (Ossowski et al., 2017) Saudi Arabian (Alsafiah et al., 2017)
748 1962 519 1501 159 1000 267 600 500
SE33 SE33 SE33 SE33 SE33 SE33 SE33 SE33 SE33
TPOX TPOX TPOX TPOX TPOX TPOX TPOX TPOX TPOX
3.203 × 10−26 1.254 × 10−26 6.2468 × 10−27 1.84 × 10−25 1.06 × 10−24 2.937 × 10−25 1.139 × 10−24 1.8 × 10−26 1.42091 × 10−26
(99.32) (99.15) (99.3) (99.28) (98.3) (99.2) (99) (99.4) (99.3)
determined to be 1.06 × 10−24 or 1 in 1 × 1024 (septillion), which means that the probability that two randomly selected Jordanian Chechens will match at all of the 21 loci is impossible. Moreover, the CMP value for the most common DNA profile was calculated to be 2.43 × 19−19. Tables 2a and 2b details the CMP and MP values for all of the tested STR markers, while Table 6 shows the frequencies of the most common DNA profiles and their CMP values.
(79.87) (82.34) (85) (81.67) (80.6) (78.4) (79.8) (78.5) (84)
values for Jordanian Chechens obtained in the current study as well as previously published PIC values for Jordanian Arabs and Africans. In addition, Table 8 shows the observed heterozygosity (OH) values determined for Chechen Jordanians as well as those that were previously published for Jordanian Arabs and Africans. Apart from the TPOX locus in Chechens and the TPOX and D21S11 loci in Arab Jordanians, the vast majority of the 13 overlapping CODIS core STR markers were higher than 70%, meaning that they have a sufficiently high discriminating power and are suitable for use in human identification (Butler, 2005). Afro-Jordanians exhibited the highest OH (79%) followed by Chechens (77%) and Arab Jordanians (74%). With regard to non-Jordanians, the GlobalFiler™ PCR Amplification Kit has also been investigated in the Emirati (Ali Alhmoudi et al., 2015), Japanese (Fujii et al., 2015), Korean (Park et al., 2016), Mongolian (Choi et al., 2017), Polish (Ossowski et al., 2017), Saudi Arabian (Alsafiah et al., 2017), Chinese Kazakh (Zhang et al., 2016a), and Chinese Uyghur (Zhang et al., 2016b) populations. As Table 9 illustrates, our findings are similar to those found in other populations in terms of CMP and PD, among other forensic parameters. From these results, it can be concluded that the genetic efficiency of the 21 tested polymorphic autosomal STR markers is suitable for criminal and forensic investigations, paternity testing, and human identification studies for the Chechen subpopulation of Jordan.
3.4.2. Power of discrimination (PD) The maximum and minimum PD values for the investigated STR markers were found for the SE33 (0.983) and TPOX (0.806) loci, respectively. Additionally, the average PD value was found to be 0.920, and, apart from the TPOX locus, the PD values of the rest of the investigated STR loci were close to their average. The combined power of discrimination (CPD) for all the 21 investigated autosomal STR markers was calculated to be 0.9999997, which is high enough to allow the employment of these loci in several forensic applications. Tables 2a and 2b displays the calculated PD values for all 21 investigated autosomal STR loci. 3.4.3. Polymorphism information content (PIC) The PIC values for the 21 autosomal loci were found to be > 0.5 among Jordanian Chechens and had an average value of 0.759 (Tables 2a and 2b). Among Jordanian Chechens, the TPOX locus possessed the least PIC value (57%), while the SE33 locus exhibited the highest PIC value (89%). These PIC values indicate that the 21 investigated STR markers are highly polymorphic in the Jordanian-Chechen subpopulation.
Declaration of competing interest The authors have declared that no competing interest exists. Acknowledgements
3.4.4. Observed heterozygosity (OH) The maximum and minimum OH values were observed for the D18S51 (0.918) and TPOX (0.585) loci, respectively, and the average OH value was equal to 0.784. For both observed and expected heterozygosity, the average levels were above 78% (Tables 2a and 2b).
This study was funded by the Deanship of Research (RN: 244/2017), Jordan University of Science and Technology. Ethics committee approval and patient consent All procedures performed in this study involving human participants were in accordance with the ethical standards of the Institutional Review Board (IRB) at Jordan University of Science and Technology with ethical code number 18/107/2017. Written informed consent was obtained from all individual participants included in the study.
4. Discussion The present study is the first to investigate allelic frequencies and other forensic efficiency parameters of 21 autosomal STR markers in the Jordanian Chechen subpopulation. Few studies have carried out STR analysis in the Jordanian populations, and those that did employed different commercial STR kits in their investigations, making it difficult to directly compare their results with the current findings. However, the 13 CODIS core STR markers overlapped between the aforementioned studies on Arab and African Jordanians and the present study on Chechen Jordanians (Al-Eitan and Tubaishat, 2018; Yasin et al., 2005). Among the 13 overlapping STR markers, the D18S51 STR locus exhibited the highest PIC value in Chechen and Arab Jordanians, while the FGA STR locus exhibited the highest PIC value followed by D18S51 in African Jordanians. The TPOX locus had the least PIC value in Chechens, but the CSF and D13S317 loci exhibited the least PIC values in Arab and African Jordanians, respectively. Table 7 depicts the PIC
References Al-Eitan, L.N., Tubaishat, R.R., 2018. Evaluation of forensic genetic efficiency parameters of 22 autosomal STR markers (PowerPlex® Fusion system) in a population sample of Arab descent from Jordan. Aust J Forensic Sci 50 (1), 97–109. https://doi.org/10. 1080/00450618.2016.1212401. Al-Eitan, L.N., Nassar, A.M., Dajani, R.B., Almomani, B.A., Saadeh, N.A., 2017. Diabetes mellitus in two genetically distinct populations in Jordan. A comparison between Arabs and Circassians/Chechens living with diabetes. Saudi Med J 38 (2), 163–169. https://doi.org/10.15537/smj.2017.2.17910. Ali Alhmoudi, O., Jones, R.J., Tay, G.K., Alsafar, H., Hadi, S., 2015. Population genetics data for 21 autosomal STR loci for United Arab Emirates (UAE) population using next generation multiplex STR kit. Forensic Sci Int Genet 19, 190–191. https://doi.org/10. 1016/j.fsigen.2015.07.009.
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Alsafiah, H.M., Goodwin, W.H., Hadi, S., Alshaikhi, M.A., Wepeba, P.-P., 2017. Population genetic data for 21 autosomal STR loci for the Saudi Arabian population using the GlobalFiler® PCR amplification kit. Forensic Sci Int Genet 31, e59–e61. https://doi.org/10.1016/j.fsigen.2017.09.014. Alshamali, F., Alkhayat, A.Q., Budowle, B., Watson, N.D., 2005. STR population diversity in nine ethnic populations living in Dubai. Forensic Sci. Int. 152 (2–3), 267–279. https://doi.org/10.1016/j.forsciint.2004.09.133. Butler, J.M., 2005. John M. Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers. Elsevier Academic Press. Butler, J.M., 2006. Genetics and genomics of core short tandem repeat loci used in human identity testing. J. Forensic Sci. 51 (2), 253–265. https://doi.org/10.1111/j.15564029.2006.00046.x. Butler, J.M., 2007. Short tandem repeat typing technologies used in human identity testing. Biotechniques 43 (4), Sii–Sv. https://doi.org/10.2144/000112582. Butler, J.M., 2015. The future of forensic DNA analysis. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 370 (1674). https://doi.org/10.1098/rstb.2014.0252. Choi, E.-J., Park, K.-W., Lee, Y.-H., et al., 2017. Forensic and population genetic analyses of the GlobalFiler STR loci in the Mongolian population. Genes Genomics 39 (4), 423–431. https://doi.org/10.1007/s13258-016-0511-6. DE BEL-AIR F, 2016. Migration Profile: Jordan. https://doi.org/10.2870/367941. Dweik, B.S., 2000. Linguistic and cultural maintenance among the Chechens of Jordan. Lang. Cult. Curric. 13 (2), 184–195. https://doi.org/10.1080/07908310008666598. Ellegren, H., 2004. Microsatellites: simple sequences with complex evolution. Nat Rev Genet 5 (6), 435–445. https://doi.org/10.1038/nrg1348. Fujii, K., Watahiki, H., Mita, Y., et al., 2015. Allele frequencies for 21 autosomal short tandem repeat loci obtained using GlobalFiler in a sample of 1501 individuals from the Japanese population. Legal Med. 17 (5), 306–308. https://doi.org/10.1016/J. LEGALMED.2015.08.007. Jobling, M.A., Gill, P., 2004. Correction: encoded evidence: DNA in forensic analysis. Nat Rev Genet 5 (10), 739–751. https://doi.org/10.1038/nrg1455. Liu, Q.-L., Wang, J.-Z., Quan, L., et al., 2013. Allele and haplotype diversity of 26 X-STR loci in four nationality populations from China. Mokrousov I. PLoS One 8 (6),
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Assessing the forensic efficiency of the GlobalFiler STR loci among the genetically isolated Chechen subpopulation in Jordan
T
⁎
Laith N. AL-Eitana,b, , Nizar N. Darwisha, Nancy M. Hakoozc, Rana B. Dajanid,e a
Department of Applied Biological Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan Department of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, Irbid 22110, Jordan c Department of Biopharmaceutics and Clinical Pharmacy, School of Pharmacy, University of Jordan, Amman 11942, Jordan d Department of Biology and Biotechnology, Hashemite University, Zarqa 13133, Jordan e Radcliffe Institute for Advanced Studies, Harvard University, Cambridge, 02138, MA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: STR Jordan Chechen GlobalFiler
Short tandem repeats (STRs) are a widely utilized tool in forensic applications, the latter of which range from human identification and paternity testing to population analysis. The GlobalFiler STR loci, which includes 21 autosomal STRS, were analyzed in the Chechen subpopulation of Jordan. Whole blood samples were withdrawn from 159 Jordanian Chechen individuals, and genomic DNA was extracted from each sample. The GlobalFiler™ kit PCR Amplification Kit amplified and analyzed the STR loci on the 3130xl Genetic Analyzer using GeneMapper ID-X software. The combined match probability for the 21 autosomal STR loci was calculated to be 1.06 × 10−24, a number that is highly discriminatory and informative. The SE33 (0.983) and TPOX (0.806) loci exhibited the highest and lowest powers of discrimination, respectively. Conclusively, the current study indicates that the GlobalFiler loci have a high utility in the Jordanian Chechen population, possibly paving the way for the future establishment of a reference population database in Jordan.
1. Introduction Jordan is a Middle-Eastern country located on the Jordan River's east bank, and it lies between the northern part of the Arabian Peninsula and the southern part of the Levant. Based on the 2015 census, Jordan has a population of 9.5 million, 60% of which are Jordanians and the remainder of which are Syrians (1.265 million), Egyptians (0.636 million), Palestinians (0.634 million), Iraqis (130,911), Yemenis (31,163), Libyans (22,700), and other nationalities (197,385) (DE BEL-AIR F, 2016). Among Jordanians, there exists a level of genetic diversity that came about due to historical and contemporary movements of subpopulations into and out of Jordan, resulting in the Jordanian population being made of Arabs, Bedouins, Armenians, Circassians, and Chechens (Zanetti et al., 2014; Al-Eitan et al., 2017). The Chechens are Sunni Muslims of Caucasian origin that arrived in Jordan in a series of waves in the early 20th century, forming a distinct subpopulation within the Jordanian Arab majority (Dweik, 2000; Shishani
et al., 2013). In the forensic sciences, DNA profiling is an integral technique that allows the identification of individuals based on their unique genetic backgrounds (Jobling and Gill, 2004). Repeated DNA sequences are abundant in eukaryotic genomes, and vary in size between individuals without affecting their genetic health (Ellegren, 2004). Despite being harmless from a clinical perspective, inter-individual variation in the length of repeated sequences has much forensic practicality. In fact, short tandem repeats (STRs), which are non-coding DNA regions with repeated 2-to-6-bp units, are routinely used in DNA profiling systems (Butler, 2006; Butler, 2007). > 100,000 independent STR markers are available to forensic investigators to choose from, but expanding the number of examined loci increases the probability that two unrelated individuals will have distinct DNA profiles (Weir, 2007). New STR markers are constantly being identified, and each marker has different powers of discrimination and overall forensic efficiency depending on the ethnic group or population in question (Yang et al., 2017; Liu et al.,
Abbreviations: Bp, base pair; CDP, combined discrimination power; CEP, combined exclusion probability; CMP, combined matching probability; CODIS, combined DNA index system; CPI, combined paternity index; CSF1PO, c-fms proto-oncogene for CSF-1 receptor gene; DNA, deoxyribonucleic acid; FGA, fibrinogen alpha chain gene; He, expected heterozygosity; HLA, human leukocyte antigen; Ho, observed heterozygosity; HWE, Hardy Weinberg Equilibrium; NA, not available; PCR, polymerase chain reaction; PD, power of discrimination; PE, probability of exclusion; PIC, polymorphism information content; STR, short tandem repeats; TH01, human tyrosine hydroxylase gene; TPI, typical paternity index; TPOX, thyroid peroxidase gene; vWA, von Willebrand factor gene ⁎ Corresponding author at: Department of Applied Biological Sciences, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan. E-mail address: [email protected] (L.N. AL-Eitan). https://doi.org/10.1016/j.gene.2019.144078 Received 17 June 2019; Received in revised form 22 August 2019; Accepted 23 August 2019 Available online 29 August 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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2013; Butler, 2015; Alshamali et al., 2005). Few published studies have incorporated STR analysis in the genetic exploration of different Jordanian subpopulations. Al-Eitan and Tubaishat (2018) evaluated the forensic efficiency of 22 autosomal STR loci in a Jordanian-Arab population sample using the PowerPlex® Fusion (PPF) system (Al-Eitan and Tubaishat, 2018). Likewise, the allelic frequencies and other forensic efficiency parameters for 15 STR markers were evaluated in the African-Jordanian population (Yasin et al., 2005). Lastly, a Jordanian population sample was profiled in order to determine the forensic efficiency parameters of five STR markers (CSF1PO, D16S539, Penta D, Penta E, and TPOX) (Yasin et al., 2005). To the authors' knowledge, there are no previous reports regarding the frequency of STRs in the Jordanian-Chechen community. Therefore, this study aims to determine the degree of forensic efficiency of 21 autosomal STR markers in the Chechen subpopulation of Jordan.
Table 1 Linkage disequilibrium for three sets of STR loci in Jordanian Chechens (n = 159). STR loci set
P-value
CSF1PO/D5S818 D12S391/vWA D2S1338/D2S441 TPOX/D2S1338 TPOX/D2S441
0.1045 0.1503 0.2937 0.1173 0.1038
2.4. Data availability The entirety of the data produced during this study is available upon request from the corresponding author. 3. Results
2. Materials and methods
3.1. Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD)
2.1. Sample collection and DNA extraction
Out of the investigated STR markers, the D19S433, SE33, and D1S1656 loci exhibited departure from HWE (p-value < 0.05). However, after Bonferroni correction was applied, no significant departure from HWE was observed for the 21 autosomal loci, allowing HWE to be employed in the estimation of the genotypic frequencies. Moreover, conducting the linkage equilibrium exact test on the 21 STR loci showed that there was no significant linkage disequilibrium (pvalue > 0.05), ensuring stability of the calculations concerning genotypic frequencies (Table 1).
Ethical approval to conduct this study was granted by the Institutional Review Board (IRB) at Jordan University of Science and Technology. 159 healthy and unrelated individuals were recruited from the Chechen subpopulation in Jordan. Subjects completed a survey to confirm their Chechen lineage. After obtaining written informed consent, 3–5 ml samples of whole blood were collected from the participants. DNA was extracted from each sample using the Gentra PureGene Blood Kit (Qiagen, Germany) according to the manufacturer's protocol. The quantity and quality of the extracted DNA samples were determined using the Nano-Drop ND-1000 (Thermo Scientific, USA), after which the quantified DNA samples were diluted to achieve final concentrations of 0.5–1 ng per 15 μl.
3.2. Genotypic and allelic frequencies The genotypes (DNA profiles) of the 159 participants were successfully determined without any PCR or STR scanning artifacts. No offladder alleles or tri-allelic patterns were detected as all of the determined allelic genotypes were previously reported on STR Base. Therefore, all of the obtained allelic genotypes were used in the allelic frequency and forensic efficiency calculations. Tables 2a and 2b lists the allelic frequencies of the 21 autosomal STR loci that were investigated in this study. However, a total of 8 loci exhibited incomplete repeat units of microvariant alleles, the majority of which were exhibited by the SE33 locus (51.8%). Microvariant allelic frequencies of the investigated STR markers are displayed in Table 3. With regard to the sex-determining STR marker Amelogenin, the X/ X genotype (female) was observed 72 times, while the X/Y genotype (male) was detected 87 times. The frequency of the Y allele of Amelogenin was calculated to be 0.273, and the frequency of the X allele for the same locus was 0.726. Table 4 shows the genotypes and allelic frequencies of the Amelogenin marker in Jordanian Chechens.
2.2. Multiplex PCR and capillary electrophoresis Based on the manufacturer's guidelines, the GlobalFiler™ PCR Amplification Kit (Life Technologies, USA) was utilized to amplify the sex-determining marker Amelogenin, 1 Y STR locus, 1 Y insertion/deletion (Y indel) locus, and 21 autosomal STR loci, namely CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX, and vWA. Amplification was performed on the GeneAmp™ PCR System 9700 Fast Thermal Cycler (Life Technologies, USA). STR typing of the amplified DNA samples was carried out via capillary electrophoresis, the latter of which was completed on the Applied Biosystems 3130XL Genetic Analyzer (Life Technologies, USA). GeneScan™ - 600 LIZ®, an internal lane size standard, was included with every sample, and an allelic ladder was used to facilitate the automatic sizing of alleles by the GeneMapper™ ID-X Software (Life Technologies, USA).
3.3. Allelic variability and possible genotypes (PG) Table 5 depicts the observed alleles as well as the possible genotypes for the 21 autosomal STR markers investigated in the Chechen population sample. Based on these figures, the total number of observed alleles among the investigated population sample was found to be 203, indicating high allelic polymorphism. The total number of possible genotypes (PG) was calculated according to the following equation: If n alleles exist for an STR marker then the number of possible genotypes equals n (n + 1)/2. Among the investigated STR makers, SE33 exhibited the highest number of alleles (n = 27) with the highest PG, making it the most polymorphic and informative STR marker among the Jordanian Chechen subpopulation. To the contrary, the lowest number of observed alleles was reported for D22S1045 (n = 5) and TPOX (n = 6). However, the TPOX marker can be considered to be the least
2.3. Statistical analysis The PowerStats 1.2 software (Promega, USA) was used to calculate the allelic frequencies as well as the following forensic efficiency parameters: matching probability (MP), power of discrimination (PD), polymorphism information content (PIC), observed heterozygosity (OH), and paternity-related data (probability of exclusion (PE) and typical paternity index (TPI)). The Arlequin 3.5 software was employed to determine any possible departures of the Chechen sub-population from the Hardy-Weinberg equilibrium (HWE) and to calculate linkage disequilibrium (LD). Bonferroni's correction for multiple testing was applied in order to reduce the level of significance of the HWE from α = 0.05 to α = 0.002, which was achieved by dividing the p-value cutoff (α = 0.05) by the number of tested autosomal loci (n = 21). 2
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Table 2a Calculated allelic frequencies for 21 autosomal STR loci in Jordanian Chechens (n = 159). Allele
CSF1PO
D16S539
D18S51
D19S433
D21S11
D2S441
D3S1358
D8S1179
D10S1248
D12S391
5.3 6 7 8 9 9.3 10 11 11.3 12 12.2 12.3 13 13.2 14 14.2 15 15.2 15.3 16 16.2 16.3 17 17.2 17.3 18 18.3 19 19.2 19.3 20 20.3 21 21.2 22 22.2 23 23.2 24 24.2 25 25.2 26 26.2 27 27.2 28 28.2 29 29.2 30 30.2 31 31.2 32 32.2 33.2 34 34.2 35
– – – – 0.016 – 0.368 0.164 – 0.362 – – 0.066 – 0.025 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – 0.009 0.116 – 0.138 0.28 – 0.223 – – 0.217 – 0.016 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – 0.028 – 0.088 – – 0.126 – 0.17 – 0.208 – – 0.126 – – 0.066 – – 0.057 – 0.088 – – 0.031 – – – 0.013 – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – 0.094 0.006 – 0.252 0.006 0.371 0.06 0.151 0.016 – 0.028 0.006 – 0.006 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.013 – – – 0.019 – 0.091 – 0.258 – 0.186 0.028 0.028 0.11 0.047 0.107 0.063 – 0.05 –
– – – – – – 0.258 0.264 0.091 0.066 – 0.003 0.019 – 0.286 – 0.013 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – 0.038 – 0.245 – – 0.264 – – 0.217 – – 0.233 – 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – 0.013 0.003 – 0.066 0.063 – 0.148 – – 0.425 – 0.189 – 0.094 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – 0.003 – – 0.179 – 0.415 – 0.274 – – 0.094 – – 0.035 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – 0.016 – – 0.047 – – 0.069 – – 0.094 0.053 0.104 – – 0.214 – 0.113 – 0.11 – 0.113 – 0.031 – 0.035 – – – – – – – – – – – – – – – – – – –
Statistical analyses EH 0.702 OH 0.704 MP 0.142 P 0.534 PD 0.858 PE 0.44 PIC 0.65 TPI 1.69
0.792 0.824 0.084 0.147 0.925 0.595 0.76 2.84
0.871 0.918 0.037 0.289 0.97 0.746 0.86 6.12
0.763 0.811 0.094 0.013* 0.911 0.549 0.73 2.65
0.856 0.862 0.041 0.113 0.965 0.719 0.84 3.61
0.769 0.78 0.099 0.323 0.909 0.553 0.73 2.27
0.767 0.748 0.099 0.243 0.905 0.548 0.73 1.99
0.745 0.73 0.093 0.098 0.904 0.524 0.71 1.85
0.711 0.673 0.136 0.221 0.869 0.458 0.66 1.53
0.885 0.868 0.029 0.092 0.976 0.772 0.87 3.79
EH: expected heterozygosity; OH: observed heterozygosity; MP: matching probability; P: p-value of the HWE exact test; PD: power of discrimination; PE: probability of exclusion; PIC: polymorphic information content; TPI: typical paternity index.
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Table 2b Calculated allelic frequencies for 21 autosomal STR loci in Jordanian Chechens (n = 159). Allele
D13S317
D1S1656
D22S1045
D2S1338
D5S818
D7S820
FGA
SE33
TH01
TPOX
vWA
5.3 6 7 8 9 9.3 10 11 11.3 12 12.2 12.3 13 13.2 14 14.2 15 15.2 15.3 16 16.2 16.3 17 17.2 17.3 18 18.3 19 19.2 19.3 20 20.3 21 21.2 22 22.2 23 23.2 24 24.2 25 25.2 26 26.2 27 27.2 28 28.2 29 29.2 30 30.2 31 31.2 32 32.2 33.2 34 34.2 35
– – – 0.066 0.119 – 0.047 0.274 – 0.333 – – 0.148 – 0.009 – – – – 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – 0.145 – 0.113 – – 0.05 – 0.094 – 0.208 – 0.003 0.142 – 0.031 0.066 – 0.085 – 0.038 0.009 – 0.003 0.006 0.006 – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – 0.176 – – – – – – 0.116 – 0.387 – – 0.261 – – 0.06 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – 0.044 – – 0.198 – – 0.101 – 0.217 – – 0.075 – 0.006 – 0.047 – 0.119 – 0.091 – 0.075 – 0.006 – 0.019 – – – – – – – – – – – – – – –
– – – – 0.072 – 0.035 0.336 – 0.358 – – 0.142 – 0.05 – 0.006 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – 0.003 0.182 0.101 – 0.255 0.333 – 0.091 – – 0.035 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – 0.006 – 0.079 – – 0.028 – 0.097 0.003 0.186 0.006 0.255 0.003 0.157 – 0.041 – 0.085 – 0.053 – – – – – – – – – – – – – – –
– – – – – – – 0.003 – – – – 0.009 – – 0.003 0.016 – – 0.041 – – 0.006 – – 0.072 – 0.167 0.003 – 0.063 – 0.006 0.009 – 0.038 – 0.022 – 0.019 – 0.05 – 0.079 0.003 0.053 0.003 0.195 – 0.075 – 0.025 – 0.009 – – 0.006 0.003 – 0.019
0.003 0.355 0.17 0.16 0.126 0.176 0.009 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – 0.566 0.094 – 0.066 0.226 – 0.044 – – 0.003 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – 0.145 – 0.097 – – 0.255 – – 0.167 – – 0.255 – 0.066 – – 0.006 – 0.009 – – – – – – – – – – – – – – – – – – – – – – – – – – –
0.878 0.881 0.034 0.025* 0.973 0.758 0.87 4.18
0.734 0.717 0.115 0.097 0.887 0.496 0.69 1.77
0.865 0.862 0.034 0.203 0.968 0.734 0.85 3.61
0.729 0.736 0.119 0.456 0.883 0.488 0.69 1.89
0.771 0.774 0.088 0.078 0.913 0.56 0.74 2.21
0.848 0.881 0.051 0.125 0.96 0.702 0.83 4.18
0.902 0.881 0.025 0.008* 0.983 0.807 0.89 4.18
0.772 0.723 0.085 0.071 0.915 0.563 0.74 1.81
0.613 0.585 0.198 0.134 0.806 0.332 0.57 1.2
0.808 0.742 0.063 0.431 0.936 0.625 0.78 1.94
Statistical analyses EH 0.771 OH 0.774 MP 0.092 P 0.645 PD 0.914 PE 0.561 PIC 0.74 TPI 2.21
EH: expected heterozygosity; OH: observed heterozygosity; MP: matching probability; P: p-value of the HWE exact test; PD: power of discrimination; PE: probability of exclusion; PIC: polymorphic information content; TPI: typical paternity index.
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Table 3 Numbers and percentages of observed microvariant alleles in Jordanian Chechens (n = 159). STR loci
Observed microvariant
Percentage (%)
D12S391 D19S433 D1S1656 D21S11 D2S441 FGA SE33 TH01
1/12 6/12 6/15 5/12 2/8 3/13 14/27 2/7
8.3 50 40 41.7 25 23.1 51.8 28.6
Table 6 The two most common observed alleles and their frequencies as well as the most common genotypic frequencies in Jordanian Chechens (n = 159).
Table 4 Phenotypes and allelic frequencies of the Amelogenin locus in Jordanian Chechens (n = 159). Allele
Genotype
Phenotype
Observed genotype
Genotype frequency
Allele frequency
Y X
X/Y X/X
Male Female
87 72
0.547 0.453
0.273 0.726
Table 5 Number of observed alleles and possible genotypes in Jordanian Chechens (n = 159). STR loci
Observed alleles
Possible genotypes n(n + 1)/2
CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vWA Total
6 6 12 8 7 11 12 15 12 5 12 8 6 7 7 8 13 27 7 6 8 203
21 21 78 36 28 66 78 120 78 15 78 36 21 28 28 36 91 378 28 21 36 3.037 × 1034
STR loci
Allele 1
Allele 2
Allele 1 frequency (p)
Allele 2 frequency (q)
Genotype frequency (2pq)
CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 FGA SE33 TH01 TPOX vWA
10 14 20 11 11 14 13 11 29 15 17 11 15 11 10 13 22 19 6 8 16,18
12 15 21,23 12 12 15 14 15 30 16 19 14 16 12 11 14 33 28.2 9.3 11 17
0.368 0.415 0.214 0.274 0.28 0.17 0.252 0.145 0.258 0.387 0.198 0.264 0.245 0.336 0.255 0.425 0.186 0.167 0.355 0.566 0.255
0.362 0.274 0.113 0.333 0.223 0.208 0.371 0.208 0.186 0.261 0.217 0.286 0.264 0.358 0.333 0.189 0.255 0.195 0.176 0.226 0.167
0.266 0.227 0.048 0.182 0.125 0.071 0.187 0.060 0.096 0.261 0.086 0.151 0.129 0.241 0.169 0.161 0.095 0.065 0.125 0.256 0.085
Table 7 Polymorphism Information Content (PIC) values in Jordanian Africans, Arabs, and Chechens. STR loci
Chechen Jordanians (n = 159)
Arab Jordanians (n = 159)
African Jordanians (n = 159)
CSF1PO D13S317 D16S539 D18S51 D21S11 D3S1358 D5S818 D7S820 D8S1179 FGA TH01 TPOX VWA Average
0.65 0.74 0.76 0.86 0.84 0.73 0.69 0.74 0.71 0.83 0.74 0.57 0.78 0.74
0.68 0.75 0.74 0.88 0.84 0.75 0.76 0.76 0.83 0.87 0.77 0.76 0.78 0.78
0.74 0.65 0.79 0.88 0.79 0.72 0.67 0.69 0.77 0.89 0.73 0.71 0.8 0.75
Table 8 Observed heterozygosity (OH) in Jordanian Africans, Arabs, and Chechens.
polymorphic and informative marker among Jordanian Chechens due to a marked imbalance in its allelic distribution. The most common allele among Jordanian Chechens was allele 8 of the TPOX marker, as it was detected in 57% of sampled individuals (n = 159 or 318 measured chromosomes). Table 6 illustrates the two most commonly observed alleles for each STR marker as well as their allelic and genotypic frequencies.
3.4. Forensic efficiency parameters 3.4.1. Matching probability (MP) The maximum and minimum MP values were calculated for the TPOX (0.198) and SE33 (0.025) markers, respectively, while the average MP value for all 21 investigated autosomal loci was 0.084. Despite having a higher number of alleles, TPOX had a poorer MP value than D22S1045, indicating that the former had a more balanced allelic distribution. The combined matching probability (CMP) was 5
STR loci
Chechen Jordanians (n = 159)
Arab Jordanians (n = 159)
African Jordanians (n = 159)
CSF1PO D13S317 D16S539 D18S51 D21S11 D3S1358 D5S818 D7S820 D8S1179 FGA TH01 TPOX VWA Average
0.704 0.774 0.824 0.918 0.862 0.748 0.736 0.774 0.73 0.881 0.723 0.585 0.742 0.77
0.695 0.736 0.776 0.749 0.64 0.779 0.689 0.76 0.735 0.824 0.749 0.623 0.802 0.74
0.811 0.695 0.768 0.874 0.842 0.653 0.737 0.726 0.8 0.947 0.779 0.768 0.905 0.79
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Table 9 Studies incorporating GlobalFiler in the STR analysis of different populations. Population
Sample size
STR with highest PD (%)
STR with lowest PD (%)
CMP
Chinese Kazakh (Zhang et al., 2016a) Chinese Uyghur (Zhang et al., 2016b) Emirati Arab (Ali Alhmoudi et al., 2015) Japanese (Fujii et al., 2015) Jordanian Chechen Korean (Park et al., 2016) Mongolian (Choi et al., 2017) Polish (Ossowski et al., 2017) Saudi Arabian (Alsafiah et al., 2017)
748 1962 519 1501 159 1000 267 600 500
SE33 SE33 SE33 SE33 SE33 SE33 SE33 SE33 SE33
TPOX TPOX TPOX TPOX TPOX TPOX TPOX TPOX TPOX
3.203 × 10−26 1.254 × 10−26 6.2468 × 10−27 1.84 × 10−25 1.06 × 10−24 2.937 × 10−25 1.139 × 10−24 1.8 × 10−26 1.42091 × 10−26
(99.32) (99.15) (99.3) (99.28) (98.3) (99.2) (99) (99.4) (99.3)
determined to be 1.06 × 10−24 or 1 in 1 × 1024 (septillion), which means that the probability that two randomly selected Jordanian Chechens will match at all of the 21 loci is impossible. Moreover, the CMP value for the most common DNA profile was calculated to be 2.43 × 19−19. Tables 2a and 2b details the CMP and MP values for all of the tested STR markers, while Table 6 shows the frequencies of the most common DNA profiles and their CMP values.
(79.87) (82.34) (85) (81.67) (80.6) (78.4) (79.8) (78.5) (84)
values for Jordanian Chechens obtained in the current study as well as previously published PIC values for Jordanian Arabs and Africans. In addition, Table 8 shows the observed heterozygosity (OH) values determined for Chechen Jordanians as well as those that were previously published for Jordanian Arabs and Africans. Apart from the TPOX locus in Chechens and the TPOX and D21S11 loci in Arab Jordanians, the vast majority of the 13 overlapping CODIS core STR markers were higher than 70%, meaning that they have a sufficiently high discriminating power and are suitable for use in human identification (Butler, 2005). Afro-Jordanians exhibited the highest OH (79%) followed by Chechens (77%) and Arab Jordanians (74%). With regard to non-Jordanians, the GlobalFiler™ PCR Amplification Kit has also been investigated in the Emirati (Ali Alhmoudi et al., 2015), Japanese (Fujii et al., 2015), Korean (Park et al., 2016), Mongolian (Choi et al., 2017), Polish (Ossowski et al., 2017), Saudi Arabian (Alsafiah et al., 2017), Chinese Kazakh (Zhang et al., 2016a), and Chinese Uyghur (Zhang et al., 2016b) populations. As Table 9 illustrates, our findings are similar to those found in other populations in terms of CMP and PD, among other forensic parameters. From these results, it can be concluded that the genetic efficiency of the 21 tested polymorphic autosomal STR markers is suitable for criminal and forensic investigations, paternity testing, and human identification studies for the Chechen subpopulation of Jordan.
3.4.2. Power of discrimination (PD) The maximum and minimum PD values for the investigated STR markers were found for the SE33 (0.983) and TPOX (0.806) loci, respectively. Additionally, the average PD value was found to be 0.920, and, apart from the TPOX locus, the PD values of the rest of the investigated STR loci were close to their average. The combined power of discrimination (CPD) for all the 21 investigated autosomal STR markers was calculated to be 0.9999997, which is high enough to allow the employment of these loci in several forensic applications. Tables 2a and 2b displays the calculated PD values for all 21 investigated autosomal STR loci. 3.4.3. Polymorphism information content (PIC) The PIC values for the 21 autosomal loci were found to be > 0.5 among Jordanian Chechens and had an average value of 0.759 (Tables 2a and 2b). Among Jordanian Chechens, the TPOX locus possessed the least PIC value (57%), while the SE33 locus exhibited the highest PIC value (89%). These PIC values indicate that the 21 investigated STR markers are highly polymorphic in the Jordanian-Chechen subpopulation.
Declaration of competing interest The authors have declared that no competing interest exists. Acknowledgements
3.4.4. Observed heterozygosity (OH) The maximum and minimum OH values were observed for the D18S51 (0.918) and TPOX (0.585) loci, respectively, and the average OH value was equal to 0.784. For both observed and expected heterozygosity, the average levels were above 78% (Tables 2a and 2b).
This study was funded by the Deanship of Research (RN: 244/2017), Jordan University of Science and Technology. Ethics committee approval and patient consent All procedures performed in this study involving human participants were in accordance with the ethical standards of the Institutional Review Board (IRB) at Jordan University of Science and Technology with ethical code number 18/107/2017. Written informed consent was obtained from all individual participants included in the study.
4. Discussion The present study is the first to investigate allelic frequencies and other forensic efficiency parameters of 21 autosomal STR markers in the Jordanian Chechen subpopulation. Few studies have carried out STR analysis in the Jordanian populations, and those that did employed different commercial STR kits in their investigations, making it difficult to directly compare their results with the current findings. However, the 13 CODIS core STR markers overlapped between the aforementioned studies on Arab and African Jordanians and the present study on Chechen Jordanians (Al-Eitan and Tubaishat, 2018; Yasin et al., 2005). Among the 13 overlapping STR markers, the D18S51 STR locus exhibited the highest PIC value in Chechen and Arab Jordanians, while the FGA STR locus exhibited the highest PIC value followed by D18S51 in African Jordanians. The TPOX locus had the least PIC value in Chechens, but the CSF and D13S317 loci exhibited the least PIC values in Arab and African Jordanians, respectively. Table 7 depicts the PIC
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