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Forensic parameters and mutation analysis of 23 short tandem repeat (PowerPlex® Fusion System) loci in Fujian Han Chinese population
Legal Medicine 37 (2019) 33–36
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Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Forensic parameters and mutation analysis of 23 short tandem repeat (PowerPlex® Fusion System) loci in Fujian Han Chinese population Beilei Zhanga,1, Zheng Lib,1, Kai Lib,1, Peng Chenb, , Feng Chenb,c, ⁎
T
⁎
a
Fujian Zhengtai Judicial Expertise Center, Xiamen, Fujian 361000, PR China Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, PR China c Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, PR China b
ABSTRACT
Kinship testing based on genetic markers has valuable practical applications. Short tandem repeat polymorphisms (STRPs) can have large number of alleles, and become the dominant marker for kinship identification. However, the high mutation rates affect the identification accuracy. Thus, accurate investigation of the mutation rate of STR loci in different populations is crucial for the reliability of phylogenetic relationships. In present study, forensic parameters and mutation rates (include 95% CI) of 23 short tandem repeats (STR) loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, D16S539, D18S51, D2S1338, CSF1PO, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045, PentaE, PentaD and DYS391) were investigated through PowerPlex® Fusion System in Fujian Han population. The high level of CDP (0.999999999999999999999999992) and CPE (0.999999993) indicated the panel was high efficiency in forensic DNA identification and paternity testing. In mutation analysis, 43 mutation cases were found through 54,124 parent-child meiotic transfers. The observed mutation rates ranged from 0 (D3S1358, D1S1656, D13S317, TH01, D19S433 and D22S1045) to 0.0025 (PentaE and FGA). The overall mutation rate across all loci was 0.0008 and the average mutation rate for the 23 loci was estimated to be 0.00078 per meiosis. The vast majority of mutations were single-step (88.4%) mutation and also include double-step (9.3%) and triple-step (2.3%) mutations. Paternal mutation rate was more common than maternal mutation rate with a ratio of 7.2:1. In addition, mutation rates indicated positive correlation (r = 0.633, p = 0.009) with the expected heterozygosity (He).
1. Introduction Short tandem repeats (STRs) are the most widely used genetic markers in forensic application with high polymorphism and heterozygosity [1,2]. However, STR have relatively high mutation rates due to their molecular architecture [3] comparing to other genetic markers such as indel [4] and microhaplotypes [5–7]. Mutations may occur between parent and offspring and cause incorrect interpretation in parentage testing [8–12]. Therefore, credible information of mutation rates of STR loci should be collected and taken into account in calculating cumulative paternity (CPI), especially in complex or deficient cases [13–15]. Fujian is a province on the southeast coast of China bordered by Zhejiang to the north, Jiangxi to the west and Guangdong to the South. Population in Fujian had reached nearly 37 million and Han is the most widely distributed population in Fujian province, making up about 98% of the total population [16]. Although numerous studies have reported the mutation rates of different STR loci in many Han Chinese populations, there are still necessary to establish larger population genetic researches [17–23]. PowerPlex® Fusion System was developed by Promega (Promega,
Madison, WI), consisting of 20 the Combined DNA Index System (CODIS) core loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, D16S539, D18S51, D2S1338, CSF1PO, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045) and 2 routinely tested loci (PentaE and PentaD). Amelogenin locus for sex discrimination and DYS391 were also included to prevent improper gender determination [24,25]. Here, we aimed to report the forensic parameters and mutation rates of the 23 STR loci in Chinese Fujian Han population. 2. Materials and methods 2.1. Sample selection This study was performed with the approval of the ethics committee, and all the families provided written informed consent. Ethical approval (approval number: 2018567) was obtained from Nanjing Medical University, and the procedures followed were in accordance with the human and ethical research principles of Nanjing Medical University. Totally, 1112 independent paternity cases with confirmed
Corresponding authors at: Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, PR China (P. Chen and F. Chen). E-mail addresses: [email protected] (P. Chen), [email protected] (F. Chen). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.legalmed.2019.01.005 Received 26 September 2018; Received in revised form 30 November 2018; Accepted 1 January 2019 Available online 02 January 2019 1344-6223/ © 2019 Elsevier B.V. All rights reserved.
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parentage were randomly selected from Fujian province. The details of our selected families were exhibited in Table 1. In other words, 2401 meiosis were studied at the 23 loci, resulting in 54,124 allele transfers in parent-child duos.
Table 1 Details of our selected families with confirmed parentage.
One-child Two-children Three-Children Four-children
Parents
Father
Mother
553 148 34 6
209 46 6 2
92 16 0 0
2.2. PCR amplification and STR typing Genomic DNA of each individual was extracted by using Chelex-100 [26]. The amplification of the 23 STR loci were performed by using PowerPlex® Fusion System (Promega, Madison, WI). The PCR products were separated and detected using capillary electrophoresis with an ABI 3130xl Genetic Analyzer (Thermo Fisher Scientific, MA, USA) and genotypes were generated using GeneMapper ID v3.2 (Thermo Fisher Scientific).
Parents: Children both have father and mother; Father: Children only have father; Mother: Children only have mother. Table 2 Forensic parameters of 23 STR loci. Locus
MP
DP
PIC
PE
TPI
D3S1358 D1S1656 D2S441 D10S1248 D13S317 PentaE D16S539 D18S51 D2S1338 CSF1PO PentaD TH01 vWA D21S11 D7S820 D5S818 TPOX DYS391 D8S1179 D12S391 D19S433 FGA D22S1045
0.1319 0.0446 0.0789 0.0958 0.0678 0.0146 0.0819 0.0370 0.0344 0.1117 0.0646 0.1475 0.0730 0.0563 0.0894 0.0764 0.2371 0.6467 0.0421 0.0433 0.0585 0.0364 0.0939
0.8681 0.9554 0.9211 0.9042 0.9322 0.9854 0.9181 0.9630 0.9656 0.8883 0.9354 0.8525 0.9270 0.9437 0.9106 0.9236 0.7629 0.3533 0.9579 0.9567 0.9415 0.9636 0.9061
0.6675 0.8160 0.7508 0.7209 0.7732 0.9054 0.7476 0.8395 0.8458 0.6988 0.7769 0.6322 0.7649 0.7939 0.7309 0.7573 0.5225 0.3117 0.8294 0.8243 0.7887 0.8421 0.7294
0.4781 0.6568 0.5568 0.5151 0.5933 0.8164 0.5740 0.7079 0.7019 0.4940 0.6336 0.3741 0.5951 0.6334 0.5407 0.6007 0.2710 – 0.6881 0.6900 0.6016 0.7059 0.5496
1.8601 2.9497 2.2404 2.0254 2.4617 5.5698 2.3404 3.4860 3.4144 1.9284 2.7528 1.4858 2.4739 2.7514 2.1533 2.5113 1.1992 0.5000 3.2582 3.2796 2.5177 3.4618 2.2009
2.3. Statistical analysis Forensic parameters (Matching probability, MP; discrimination power, DP; polymorphism information content, PIC; probability of exclusion, PE; the typical paternity index, TPI) were calculated by modified-PowerStats spreadsheet (Promega, Madison, WI, USA). And expected heterozygosity (He) was obtained using GenAlEx 6.593 (the Australian National University, Australia). The 95% confidence intervals (CI) for mutation rates were derived based on the binomial distribution and obtained via the website (http://statpages.org/confint. html). Spearman’s test was performed using SPSS 21 software. 3. Results and discussion 3.1. Forensic parameters of 23 STR loci On the basis of parents’ data, forensic parameters of the 23 STR loci in Fujian Han population was listed in Table 2. After Bonferroni correction (p = 0.002), no deviation from Hardy–Weinberg equilibrium (HWE) was observed for all 23 loci in our studied groups. MP were in range of 0.0146 (PentaE) to 0.6467 (DYS391) and the TPI ranged from 0.5000 (DYS391) to 5.5698 (PentaE). The lowest probability of
MP: Matching probability; DP: discrimination power; PIC: polymorphism information content; PE: probability of exclusion and TPI: the typical paternity index. Table 3 Mutation rate and 95% CI observed. Locus
D3S1358 D1S1656 D2S441 D10S1248 D13S317 PentaE D16S539 D18S51 D2S1338 CSF1PO PentaD TH01 vWA D21S11 D7S820 D5S818 TPOX D8S1179 D12S391 D19S433 FGA D22S1045 DYS391 Overall
Paternal
Maternal
U
Total
N.M
N.T
M.R
95% CI
N.M
N.T
M.R
95% CI
N.M
N.M
N.T
M.R
95% CI
0 0 1 1 0 5 1 2 2 2 0 0 5 3 1 1 1 1 5 0 4 0 1 36
1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 29,946
0.0000 0.0000 0.0008 0.0008 0.0000 0.0038 0.0008 0.0015 0.0015 0.0015 0.0000 0.0000 0.0038 0.0023 0.0008 0.0008 0.0008 0.0008 0.0038 0.0000 0.0031 0.0000 0.0008 0.0012
0.0000–0.0028 0.0000–0.0028 0.0000–0.0043 0.0000–0.0043 0.0000–0.0028 0.0012–0.0089 0.0000–0.0043 0.0002–0.0055 0.0002–0.0055 0.0002–0.0055 0.0000–0.0028 0.0000–0.0028 0.0012–0.0089 0.0005–0.0067 0.0000–0.0043 0.0000–0.0043 0.0000–0.0043 0.0000–0.0043 0.0012–0.0089 0.0000–0.0028 0.0008–0.0078 0.0000–0.0028 0.0000–0.0043 0.0008–0.0017
0 0 0 0 0 1 0 2 0 0 0 0 0 0 0 0 0 1 0 0 1 0 – 5
1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 – 24,178
0.0000 0.0000 0.0000 0.0000 0.0000 0.0009 0.0000 0.0018 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0009 0.0000 0.0000 0.0009 0.0000 – 0.0002
0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0051 0.0000–0.0034 0.0002–0.0066 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0051 0.0000–0.0034 0.0000–0.0034 0.0000–0.0051 0.0000–0.0034 – 0.0001–0.0005
0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 – 2
0 0 1 1 0 6 1 4 2 2 1 0 5 3 1 1 1 2 5 0 6 0 1 43
2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 1302 54,124
0.0000 0.0000 0.0004 0.0004 0.0000 0.0025 0.0004 0.0017 0.0008 0.0008 0.0004 0.0000 0.0021 0.0012 0.0004 0.0004 0.0004 0.0008 0.0021 0.0000 0.0025 0.0000 0.0008 0.0008
0.0000–0.0015 0.0000–0.0015 0.0000–0.0023 0.0000–0.0023 0.0000–0.0015 0.0009–0.0054 0.0000–0.0023 0.0005–0.0043 0.0001–0.0030 0.0001–0.0030 0.0000–0.0023 0.0000–0.0015 0.0007–0.0048 0.0003–0.0036 0.0001–0.0023 0.0000–0.0023 0.0000–0.0023 0.0001–0.0030 0.0007–0.0048 0.0000–0.0015 0.0009–0.0054 0.0000–0.0015 0.0000–0.0043 0.0006–0.0011
N.M: number of mutations, N.T: number of transmissions, M.R: mutation rate, U: mutation origin was unassigned. 34
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3.2. Mutation analysis
Table 4 Mutation rates of previous study in different regions of Chinese Han population. Loci
Fujian
Shanghai
Hebei
Guangdong
Guizhou
Yunnan
D3S1358 D1S1656 D2S441 D10S1248 D13S317 PentaE D16S539 D18S51 D2S1338 CSF1PO PentaD TH01 vWA D21S11 D7S820 D5S818 TPOX D8S1179 D12S391 D19S433 FGA D22S1045 DYS391 Number of paternity cases
0.00 0.00 0.40 0.40 0.00 2.50 0.40 1.70 0.80 0.80 0.40 0.00 2.10 1.20 0.40 0.40 0.40 0.80 2.10 0.00 2.50 0.00 0.80 1112
1.52 NA NA NA 1.68 3.20 0.61 2.89 1.07 2.59 0.61 0.15 1.52 1.98 0.30 1.07 0.30 2.89 1.68 0.91 4.57 NA NA 5846
0.34 NA NA NA 0.68 1.36 0.68 1.02 0.68 1.02 0.68 0.00 1.02 0.68 0.34 0.68 0.00 1.36 2.38 0.34 3.06 NA NA 2063
1.18 NA NA NA 0.94 2.75 0.71 2.20 0.86 1.18 0.86 0.16 1.33 1.41 1.10 1.41 0.08 1.73 2.43 0.94 2.59 NA NA 9626
1.80 1.30 0.40 0.40 0.90 1.80 0.90 0.90 0.40 0.40 0.40 NA 2.70 0.90 0.40 0.90 NA 0.40 1.30 0.40 1.30 NA NA 1647
0.91 0.76 NA NA 0.30 1.98 0.46 1.52 0.91 0.91 0.61 0.46 2.74 2.13 0.61 1.22 0.30 1.37 2.74 1.37 2.43 NA NA 4363
As shown in Table 3, we observed 43 mutations at 17 of the 23 loci. No mutation was found at six loci (D3S1358, D1S1656, D13S317, TH01, D19S433 and D22S1045) and the PentaE and FGA loci showed the highest mutation rate (0.0025). The overall mutation rate across all loci was 0.0008. Among these mutations, 38 single-step, 4 double-step and 1 triple-step mutations were observed. The finding that the vast majority of mutations were single-step changes (88.3%) was consistent with the stepwise mutation model (SMM) theory [29,30]. The ratio of average paternal mutations to maternal mutations was 7.2:1 indicating a higher mutation rate in paternal transmissions. The result was also consistent with previously researches [18,31] showing that the number of mutations of paternal origin considerably exceed the number of mutations of maternal origin. The phenomenon may be caused by different numbers and types of cell division in germ-cell genesis. Oogonia undergoes ∼22 cell divisions before meiosis starts and generate oocytes, while spermatogonia cells divide continuously by mitosis before they become sperm cells [3]. Thus, Men trend to undergo more divisions with the age increasing. For a 29-year-old man, one can assume ∼350 divisions as described by Vogel et al. [32]. Therefore, sperm cell from a father divides more often than oocytes which causes a significant higher mutation rate in paternal cases. As shown in Table 4, Our studied Fujian Han mutation rates were compared with previously reported Han populations in different geographical regions of China, including Shanghai Han [19], Hebei Han [20], Guangdong Han [21], Guizhou Han [22] and Yunnan Han [23]. The result revealed that the Han population’ mutation rate of each STR locus varied from one region to another. Previous study has confirmed that the length and structure of the repeat and the size of locus will affect the locus’ mutation rate [3]. Different alleles in a same locus could have different mutation rates due to their different repetitions. Therefore, when the alleles’ frequency differentially distributed in different regions, the mutation rate of a same locus could also be varied. Thus, more studies are still needed to illustrate the STR mutation rates in different regions and produce more accurate CPI. Previous study has indicated that STR loci’ heterozygosity has positive correlation to their mutation rate [33]. We also evaluated the correlation coefficient of each locus by suing the observed heterozygosity and mutation rate in the Fujian Han population. We found a
Mutation rates ×10−3; NA: data not available.
exclusion (PE) was found at TPOX locus (0.2710) and the highest PE was found at PentaE locus (0.8164) with cumulative PE (CPE) value of 0.999999993 for all loci. The cumulative DP (CDP) values were 0.999999999999999999999999992 with lowest DP value found at DYS391 (0.3533) and highest DP value found at PentaE locus (0.9854). The high level of CDP and CPE indicated that the kit was more efficient than many commercial kits, such as AmpFℓSTR® Profiler Plus™ [27] and PowerPlex® 21 kit [28]. PIC varied from 0.3117 (DYS391) to 0.9054 (PentaE) with a mean value of 0.7422. These forensic genetic parameters indicated these 23 STRs have a high forensic efficiency in forensic DNA identification and paternity testing among individuals from the Fujian population.
Fig. 1. The correlation analysis between logarithm of mutation rate and heterozygosity. 35
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linear correlation between the log-mutation rate and heterozygosity, with a linear correlation coefficient of r = 0.633 (p = 0.009) (Fig. 1). The result implied that STR loci with larger heterozygosity might have a higher mutation rate, which indicated that loci with higher heterozygosity may tend to carry more easily-mutation alleles. In another word, the higher the mutation rate of a locus, the easier it is to form new alleles, thus increasing the locus’s heterozygosity in a certain region. These loci could be more useful for discriminating individuals from different regions. Overall, during the present study, we investigated the mutation rate of PowerPlex® Fusion System’s 23 STR loci. The mutation rate ranged from 0 (D3S1358, D1S1656, D13S317, TH01, D19S433 and D22S1045) to 0.0025 (PentaE and FGA). Most mutations were single-step and paternal mutations were 7.2 times higher than that of the maternal mutations.
[10] [11] [12] [13] [14] [15] [16]
Acknowledgements
[17]
This work was supported by the National Natural Science Foundation of China, China (No. 81801879, No. 81570378 and No. 81772020), the Science and Technology of Jiangsu Province China, China (BK20170048), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China, China (18KJB340002), the Science and technology development fund of Nanjing Medical University, China (2017NJMUZD007) and Jiangsu Specially-Appointed Professor program.
[18] [19] [20]
Appendix A. Supplementary data
[21]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.legalmed.2019.01.005.
[22] [23]
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Forensic parameters and mutation analysis of 23 short tandem repeat (PowerPlex® Fusion System) loci in Fujian Han Chinese population Beilei Zhanga,1, Zheng Lib,1, Kai Lib,1, Peng Chenb, , Feng Chenb,c, ⁎
T
⁎
a
Fujian Zhengtai Judicial Expertise Center, Xiamen, Fujian 361000, PR China Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, PR China c Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, PR China b
ABSTRACT
Kinship testing based on genetic markers has valuable practical applications. Short tandem repeat polymorphisms (STRPs) can have large number of alleles, and become the dominant marker for kinship identification. However, the high mutation rates affect the identification accuracy. Thus, accurate investigation of the mutation rate of STR loci in different populations is crucial for the reliability of phylogenetic relationships. In present study, forensic parameters and mutation rates (include 95% CI) of 23 short tandem repeats (STR) loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, D16S539, D18S51, D2S1338, CSF1PO, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045, PentaE, PentaD and DYS391) were investigated through PowerPlex® Fusion System in Fujian Han population. The high level of CDP (0.999999999999999999999999992) and CPE (0.999999993) indicated the panel was high efficiency in forensic DNA identification and paternity testing. In mutation analysis, 43 mutation cases were found through 54,124 parent-child meiotic transfers. The observed mutation rates ranged from 0 (D3S1358, D1S1656, D13S317, TH01, D19S433 and D22S1045) to 0.0025 (PentaE and FGA). The overall mutation rate across all loci was 0.0008 and the average mutation rate for the 23 loci was estimated to be 0.00078 per meiosis. The vast majority of mutations were single-step (88.4%) mutation and also include double-step (9.3%) and triple-step (2.3%) mutations. Paternal mutation rate was more common than maternal mutation rate with a ratio of 7.2:1. In addition, mutation rates indicated positive correlation (r = 0.633, p = 0.009) with the expected heterozygosity (He).
1. Introduction Short tandem repeats (STRs) are the most widely used genetic markers in forensic application with high polymorphism and heterozygosity [1,2]. However, STR have relatively high mutation rates due to their molecular architecture [3] comparing to other genetic markers such as indel [4] and microhaplotypes [5–7]. Mutations may occur between parent and offspring and cause incorrect interpretation in parentage testing [8–12]. Therefore, credible information of mutation rates of STR loci should be collected and taken into account in calculating cumulative paternity (CPI), especially in complex or deficient cases [13–15]. Fujian is a province on the southeast coast of China bordered by Zhejiang to the north, Jiangxi to the west and Guangdong to the South. Population in Fujian had reached nearly 37 million and Han is the most widely distributed population in Fujian province, making up about 98% of the total population [16]. Although numerous studies have reported the mutation rates of different STR loci in many Han Chinese populations, there are still necessary to establish larger population genetic researches [17–23]. PowerPlex® Fusion System was developed by Promega (Promega,
Madison, WI), consisting of 20 the Combined DNA Index System (CODIS) core loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, D16S539, D18S51, D2S1338, CSF1PO, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045) and 2 routinely tested loci (PentaE and PentaD). Amelogenin locus for sex discrimination and DYS391 were also included to prevent improper gender determination [24,25]. Here, we aimed to report the forensic parameters and mutation rates of the 23 STR loci in Chinese Fujian Han population. 2. Materials and methods 2.1. Sample selection This study was performed with the approval of the ethics committee, and all the families provided written informed consent. Ethical approval (approval number: 2018567) was obtained from Nanjing Medical University, and the procedures followed were in accordance with the human and ethical research principles of Nanjing Medical University. Totally, 1112 independent paternity cases with confirmed
Corresponding authors at: Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, PR China (P. Chen and F. Chen). E-mail addresses: [email protected] (P. Chen), [email protected] (F. Chen). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.legalmed.2019.01.005 Received 26 September 2018; Received in revised form 30 November 2018; Accepted 1 January 2019 Available online 02 January 2019 1344-6223/ © 2019 Elsevier B.V. All rights reserved.
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parentage were randomly selected from Fujian province. The details of our selected families were exhibited in Table 1. In other words, 2401 meiosis were studied at the 23 loci, resulting in 54,124 allele transfers in parent-child duos.
Table 1 Details of our selected families with confirmed parentage.
One-child Two-children Three-Children Four-children
Parents
Father
Mother
553 148 34 6
209 46 6 2
92 16 0 0
2.2. PCR amplification and STR typing Genomic DNA of each individual was extracted by using Chelex-100 [26]. The amplification of the 23 STR loci were performed by using PowerPlex® Fusion System (Promega, Madison, WI). The PCR products were separated and detected using capillary electrophoresis with an ABI 3130xl Genetic Analyzer (Thermo Fisher Scientific, MA, USA) and genotypes were generated using GeneMapper ID v3.2 (Thermo Fisher Scientific).
Parents: Children both have father and mother; Father: Children only have father; Mother: Children only have mother. Table 2 Forensic parameters of 23 STR loci. Locus
MP
DP
PIC
PE
TPI
D3S1358 D1S1656 D2S441 D10S1248 D13S317 PentaE D16S539 D18S51 D2S1338 CSF1PO PentaD TH01 vWA D21S11 D7S820 D5S818 TPOX DYS391 D8S1179 D12S391 D19S433 FGA D22S1045
0.1319 0.0446 0.0789 0.0958 0.0678 0.0146 0.0819 0.0370 0.0344 0.1117 0.0646 0.1475 0.0730 0.0563 0.0894 0.0764 0.2371 0.6467 0.0421 0.0433 0.0585 0.0364 0.0939
0.8681 0.9554 0.9211 0.9042 0.9322 0.9854 0.9181 0.9630 0.9656 0.8883 0.9354 0.8525 0.9270 0.9437 0.9106 0.9236 0.7629 0.3533 0.9579 0.9567 0.9415 0.9636 0.9061
0.6675 0.8160 0.7508 0.7209 0.7732 0.9054 0.7476 0.8395 0.8458 0.6988 0.7769 0.6322 0.7649 0.7939 0.7309 0.7573 0.5225 0.3117 0.8294 0.8243 0.7887 0.8421 0.7294
0.4781 0.6568 0.5568 0.5151 0.5933 0.8164 0.5740 0.7079 0.7019 0.4940 0.6336 0.3741 0.5951 0.6334 0.5407 0.6007 0.2710 – 0.6881 0.6900 0.6016 0.7059 0.5496
1.8601 2.9497 2.2404 2.0254 2.4617 5.5698 2.3404 3.4860 3.4144 1.9284 2.7528 1.4858 2.4739 2.7514 2.1533 2.5113 1.1992 0.5000 3.2582 3.2796 2.5177 3.4618 2.2009
2.3. Statistical analysis Forensic parameters (Matching probability, MP; discrimination power, DP; polymorphism information content, PIC; probability of exclusion, PE; the typical paternity index, TPI) were calculated by modified-PowerStats spreadsheet (Promega, Madison, WI, USA). And expected heterozygosity (He) was obtained using GenAlEx 6.593 (the Australian National University, Australia). The 95% confidence intervals (CI) for mutation rates were derived based on the binomial distribution and obtained via the website (http://statpages.org/confint. html). Spearman’s test was performed using SPSS 21 software. 3. Results and discussion 3.1. Forensic parameters of 23 STR loci On the basis of parents’ data, forensic parameters of the 23 STR loci in Fujian Han population was listed in Table 2. After Bonferroni correction (p = 0.002), no deviation from Hardy–Weinberg equilibrium (HWE) was observed for all 23 loci in our studied groups. MP were in range of 0.0146 (PentaE) to 0.6467 (DYS391) and the TPI ranged from 0.5000 (DYS391) to 5.5698 (PentaE). The lowest probability of
MP: Matching probability; DP: discrimination power; PIC: polymorphism information content; PE: probability of exclusion and TPI: the typical paternity index. Table 3 Mutation rate and 95% CI observed. Locus
D3S1358 D1S1656 D2S441 D10S1248 D13S317 PentaE D16S539 D18S51 D2S1338 CSF1PO PentaD TH01 vWA D21S11 D7S820 D5S818 TPOX D8S1179 D12S391 D19S433 FGA D22S1045 DYS391 Overall
Paternal
Maternal
U
Total
N.M
N.T
M.R
95% CI
N.M
N.T
M.R
95% CI
N.M
N.M
N.T
M.R
95% CI
0 0 1 1 0 5 1 2 2 2 0 0 5 3 1 1 1 1 5 0 4 0 1 36
1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 1302 29,946
0.0000 0.0000 0.0008 0.0008 0.0000 0.0038 0.0008 0.0015 0.0015 0.0015 0.0000 0.0000 0.0038 0.0023 0.0008 0.0008 0.0008 0.0008 0.0038 0.0000 0.0031 0.0000 0.0008 0.0012
0.0000–0.0028 0.0000–0.0028 0.0000–0.0043 0.0000–0.0043 0.0000–0.0028 0.0012–0.0089 0.0000–0.0043 0.0002–0.0055 0.0002–0.0055 0.0002–0.0055 0.0000–0.0028 0.0000–0.0028 0.0012–0.0089 0.0005–0.0067 0.0000–0.0043 0.0000–0.0043 0.0000–0.0043 0.0000–0.0043 0.0012–0.0089 0.0000–0.0028 0.0008–0.0078 0.0000–0.0028 0.0000–0.0043 0.0008–0.0017
0 0 0 0 0 1 0 2 0 0 0 0 0 0 0 0 0 1 0 0 1 0 – 5
1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 1099 – 24,178
0.0000 0.0000 0.0000 0.0000 0.0000 0.0009 0.0000 0.0018 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0009 0.0000 0.0000 0.0009 0.0000 – 0.0002
0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0051 0.0000–0.0034 0.0002–0.0066 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0034 0.0000–0.0051 0.0000–0.0034 0.0000–0.0034 0.0000–0.0051 0.0000–0.0034 – 0.0001–0.0005
0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 – 2
0 0 1 1 0 6 1 4 2 2 1 0 5 3 1 1 1 2 5 0 6 0 1 43
2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 2401 1302 54,124
0.0000 0.0000 0.0004 0.0004 0.0000 0.0025 0.0004 0.0017 0.0008 0.0008 0.0004 0.0000 0.0021 0.0012 0.0004 0.0004 0.0004 0.0008 0.0021 0.0000 0.0025 0.0000 0.0008 0.0008
0.0000–0.0015 0.0000–0.0015 0.0000–0.0023 0.0000–0.0023 0.0000–0.0015 0.0009–0.0054 0.0000–0.0023 0.0005–0.0043 0.0001–0.0030 0.0001–0.0030 0.0000–0.0023 0.0000–0.0015 0.0007–0.0048 0.0003–0.0036 0.0001–0.0023 0.0000–0.0023 0.0000–0.0023 0.0001–0.0030 0.0007–0.0048 0.0000–0.0015 0.0009–0.0054 0.0000–0.0015 0.0000–0.0043 0.0006–0.0011
N.M: number of mutations, N.T: number of transmissions, M.R: mutation rate, U: mutation origin was unassigned. 34
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3.2. Mutation analysis
Table 4 Mutation rates of previous study in different regions of Chinese Han population. Loci
Fujian
Shanghai
Hebei
Guangdong
Guizhou
Yunnan
D3S1358 D1S1656 D2S441 D10S1248 D13S317 PentaE D16S539 D18S51 D2S1338 CSF1PO PentaD TH01 vWA D21S11 D7S820 D5S818 TPOX D8S1179 D12S391 D19S433 FGA D22S1045 DYS391 Number of paternity cases
0.00 0.00 0.40 0.40 0.00 2.50 0.40 1.70 0.80 0.80 0.40 0.00 2.10 1.20 0.40 0.40 0.40 0.80 2.10 0.00 2.50 0.00 0.80 1112
1.52 NA NA NA 1.68 3.20 0.61 2.89 1.07 2.59 0.61 0.15 1.52 1.98 0.30 1.07 0.30 2.89 1.68 0.91 4.57 NA NA 5846
0.34 NA NA NA 0.68 1.36 0.68 1.02 0.68 1.02 0.68 0.00 1.02 0.68 0.34 0.68 0.00 1.36 2.38 0.34 3.06 NA NA 2063
1.18 NA NA NA 0.94 2.75 0.71 2.20 0.86 1.18 0.86 0.16 1.33 1.41 1.10 1.41 0.08 1.73 2.43 0.94 2.59 NA NA 9626
1.80 1.30 0.40 0.40 0.90 1.80 0.90 0.90 0.40 0.40 0.40 NA 2.70 0.90 0.40 0.90 NA 0.40 1.30 0.40 1.30 NA NA 1647
0.91 0.76 NA NA 0.30 1.98 0.46 1.52 0.91 0.91 0.61 0.46 2.74 2.13 0.61 1.22 0.30 1.37 2.74 1.37 2.43 NA NA 4363
As shown in Table 3, we observed 43 mutations at 17 of the 23 loci. No mutation was found at six loci (D3S1358, D1S1656, D13S317, TH01, D19S433 and D22S1045) and the PentaE and FGA loci showed the highest mutation rate (0.0025). The overall mutation rate across all loci was 0.0008. Among these mutations, 38 single-step, 4 double-step and 1 triple-step mutations were observed. The finding that the vast majority of mutations were single-step changes (88.3%) was consistent with the stepwise mutation model (SMM) theory [29,30]. The ratio of average paternal mutations to maternal mutations was 7.2:1 indicating a higher mutation rate in paternal transmissions. The result was also consistent with previously researches [18,31] showing that the number of mutations of paternal origin considerably exceed the number of mutations of maternal origin. The phenomenon may be caused by different numbers and types of cell division in germ-cell genesis. Oogonia undergoes ∼22 cell divisions before meiosis starts and generate oocytes, while spermatogonia cells divide continuously by mitosis before they become sperm cells [3]. Thus, Men trend to undergo more divisions with the age increasing. For a 29-year-old man, one can assume ∼350 divisions as described by Vogel et al. [32]. Therefore, sperm cell from a father divides more often than oocytes which causes a significant higher mutation rate in paternal cases. As shown in Table 4, Our studied Fujian Han mutation rates were compared with previously reported Han populations in different geographical regions of China, including Shanghai Han [19], Hebei Han [20], Guangdong Han [21], Guizhou Han [22] and Yunnan Han [23]. The result revealed that the Han population’ mutation rate of each STR locus varied from one region to another. Previous study has confirmed that the length and structure of the repeat and the size of locus will affect the locus’ mutation rate [3]. Different alleles in a same locus could have different mutation rates due to their different repetitions. Therefore, when the alleles’ frequency differentially distributed in different regions, the mutation rate of a same locus could also be varied. Thus, more studies are still needed to illustrate the STR mutation rates in different regions and produce more accurate CPI. Previous study has indicated that STR loci’ heterozygosity has positive correlation to their mutation rate [33]. We also evaluated the correlation coefficient of each locus by suing the observed heterozygosity and mutation rate in the Fujian Han population. We found a
Mutation rates ×10−3; NA: data not available.
exclusion (PE) was found at TPOX locus (0.2710) and the highest PE was found at PentaE locus (0.8164) with cumulative PE (CPE) value of 0.999999993 for all loci. The cumulative DP (CDP) values were 0.999999999999999999999999992 with lowest DP value found at DYS391 (0.3533) and highest DP value found at PentaE locus (0.9854). The high level of CDP and CPE indicated that the kit was more efficient than many commercial kits, such as AmpFℓSTR® Profiler Plus™ [27] and PowerPlex® 21 kit [28]. PIC varied from 0.3117 (DYS391) to 0.9054 (PentaE) with a mean value of 0.7422. These forensic genetic parameters indicated these 23 STRs have a high forensic efficiency in forensic DNA identification and paternity testing among individuals from the Fujian population.
Fig. 1. The correlation analysis between logarithm of mutation rate and heterozygosity. 35
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linear correlation between the log-mutation rate and heterozygosity, with a linear correlation coefficient of r = 0.633 (p = 0.009) (Fig. 1). The result implied that STR loci with larger heterozygosity might have a higher mutation rate, which indicated that loci with higher heterozygosity may tend to carry more easily-mutation alleles. In another word, the higher the mutation rate of a locus, the easier it is to form new alleles, thus increasing the locus’s heterozygosity in a certain region. These loci could be more useful for discriminating individuals from different regions. Overall, during the present study, we investigated the mutation rate of PowerPlex® Fusion System’s 23 STR loci. The mutation rate ranged from 0 (D3S1358, D1S1656, D13S317, TH01, D19S433 and D22S1045) to 0.0025 (PentaE and FGA). Most mutations were single-step and paternal mutations were 7.2 times higher than that of the maternal mutations.
[10] [11] [12] [13] [14] [15] [16]
Acknowledgements
[17]
This work was supported by the National Natural Science Foundation of China, China (No. 81801879, No. 81570378 and No. 81772020), the Science and Technology of Jiangsu Province China, China (BK20170048), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China, China (18KJB340002), the Science and technology development fund of Nanjing Medical University, China (2017NJMUZD007) and Jiangsu Specially-Appointed Professor program.
[18] [19] [20]
Appendix A. Supplementary data
[21]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.legalmed.2019.01.005.
[22] [23]
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