Application of CPI cutoff value based on parentage testing of duos and trios typed by four autosomal kits.

In this study, we analyzed the application of four autosomal kits and the sensitivity of the combined paternity index (CPI) cutoff value (CPI≥10000) in parentage testing. First, 1442 real trios and 803 real duos were tested using the Goldeneye 25A kit. The Goldeneye 25A kit covers the autosomal short tandem repeat (STR) loci of the other three kits, so we calculated the CPI value of every case for the four kits. Second, three complex close relative kinship cases were also analyzed to evaluate the application of the CPI cutoff value. The CPI values of all trio cases were higher than 10000 using the four kits; the CPI values of all duo cases were higher than 10000 using the Goldeneye 25A kit; and the CPI values of a portion of the duo cases were lower than 10000 using the other three kits. In the three complex close relative cases, the alleged father or mother was not excluded using 40 autosomal STRs. Adding X chromosome short tandem repeats (X-STR) and samples of biological fathers or mothers, the conclusions were confirmed. The four kits were adequate to draw conclusions in the trio cases; the Goldeneye 25A Kit was adequate to draw conclusions in the duo cases; and the other three kits were not sufficient for a portion of the duo cases. The CPI cutoff value was sensitive for real trio and duo cases. In complex close relative kinship cases, high CPI values may result in false conclusions.

Introduction

In forensic laboratories, autosomal short tandem repeats (STRs) are used in forensic parentage testing [1]. At present, the commercial kits include 13 Combined DNA Index System (CODIS) STRs. The number of STRs in the kits determines the system efficiency. The combined power of exclusion and combined power of discrimination of kits should be above 0.9999 for duo and trio cases. For duo cases, the STR number was more than that of trio cases due to the lack of a biological mother or father [2]. STR locus mutation reduces the combined paternity index (CPI) value of the case and may lead to inconclusive results. It is necessary to increase the number of STR loci to obtain an adequate CPI value for the cases. High CPI values could also result in false conclusions when the STR gene type of two unrelated people matched [3]. X chromosome short tandem repeats (X-STR) and Y chromosome short tandem repeats (Y-STR) are inherited in a unique, non-Mendelian fashion. Y-STRs are passed down from father to son. A father’s X-STRs are passed down only to his daughter, and a mother’s X-STRs are inherited by her sons and daughters. X-STR and Y-STR analyses are important for supplementing autosomal STR kit results in forensic cases [4-6].

In Chinese forensic laboratories, the CPI cutoff value of autosomal STRs for parentage testing is provided. In duo and trio cases, the alleged father or mother is confirmed when the CPI is ≥10000. The results are inconclusive when the CPI is 0.00017]. The Goldeneye 25A kit includes 23 autosomal STRs. It covers all the STRs of Goldeneye 20A, AmpFlSTR SinoFiler and AmpFlSTR Identifiler kits. In the present study, we evaluated the application of 4 autosomal STR kits and the sensitivity of the CPI cutoff value (CPI ≥10000) based on routine parentage testing of duos and trios in our laboratory. At the same time, 3 complex close relative cases were also analyzed, which required the addition of other autosomal STRs and X-STRs to confirm the conclusions.

Materials and methods

Samples

In this study, 1442 real trio parentage testing cases, 803 real duo cases (including 412 motherless and 391 fatherless cases) and 3 complex close relative kinship cases were analyzed to evaluate the use of the CPI cutoff value. All the cases were typed using the Goldeneye 25A Kit in our laboratory. Three complex relative kinship cases were also tested, adding up to more autosomal SRTs and X-STRs for real conclusions. For all the cases, the parents or guardians of the children signed the informed consent forms with our laboratory. At the same time, the study was approved by the Ethics Committee of Jinan Central Hospital affiliated with Shandong University, China.

Goldeneye 25A kit includes 23 STRs (D2S441, TPOX, D22S1045, D7S820, D1S1656, Penta E, D10S1248, D8S1179, D5S818, D19S433, D16S539, CSF1PO, Penta D, D3S1358, vWA, D2S1338, D18S51, D6S1043, D13S317, TH01, D12S391, D21S11, FGA);

Goldeneye 20A kit includes 19 STRs (TPOX, D7S820, Penta E, D8S1179, D5S818, D19S433, D16S539, CSF1PO, Penta D, D3S1358, vWA, D2S1338, D18S51, D6S1043, D13S317, TH01, D12S391, D21S11, FGA);

AmpFlSTR Identifiler kit includes 15 STRs (TPOX, D7S820, D8S1179, D5S818, D19S433, D16S539, CSF1PO, D3S1358, vWA, D2S1338, D18S51, D13S317, TH01, D21S11, FGA);

AmpFlSTR SinoFiler kit includes 15 STRs (D7S820, D8S1179, D5S818, D19S433, D16S539, CSF1PO, D3S1358, vWA, D2S1338, D18S51, D6S1043, D13S317, D12S391, D21S11, FGA);

Goldeneye 22NC kit includes 4 STRs found in the Goldeneye 25A Kit (D3S1358, D2S441, D1S1656, D10S1248) and 17 other autosomal STRs (D4S2366, D6S477, GATA198B05, D15S659, D8S1132, D3S3045, D14S608, D17S1290, D3S1744, D18S535, D13S325, D7S1517, D10S1435, D11S2368, D19S253, D7S3048, D5S2500);

Goldeneye 17X kit includes 16 X-STRs (DXS6795, DXS9902, DXS8378, HPRTB, GATA165B12, DXS7132, DXS7424, DXS6807, DXS6803, GATA172D05, DXS6800, DXS10134, GATA31E08, DXS10159, DXS6789, DXS6810).

STR typing

DNA was extracted using Chelex-100 [8]. Goldeneye 25A, Goldeneye 22NC and Goldeneye 17X kits were used for PCR according to the manufacturer’s instructions. Autosomal STR and X-STR were typed by the ABI PRISM 3500 Genetic Analyzer. The data were analyzed using GeneMapper ID-X 1.3 software.

Statistical analysis

The CPI values of duo and trio cases for four kits were calculated according to the Specifications of Parentage Testing in China [7]; gene frequencies of 40 autosomal STRs from the Shandong Han population in China were used for CPI values of all cases [9, 10]. The CPI values of different groups (including trio, duo cases, and different kits) were compared using the Mann-Whitney U test; the sensitivity of the CPI cutoff value was compared using the chi-square test. The results were considered significant if P<0.05.

Results

Application of CPI cutoff value for parentage testing of trios

As shown in Table 1, the sensitivity of the CPI cutoff value (CPI≥10000) was high for trio cases. The CPI values of all the trio cases were higher than 10000 typed by the four autosomal kits, and every case had a confirmed conclusion. CPI values from high to low were the Goldeneye 25A kit> Goldeneye 20A kit>AmpFlSTR SinoFiler kit>AmpFlSTR Identifiler kit. There were significant differences between the four kits (P<0.05).

Table 1

Sensitivity of the CPI cutoff value for real trio and duo cases.

Cases	Scope	25A (23 STRs)		20A (19 STRs)		Sino (15 STRs)		ID (15 STRs)
		A	B	A	B	A	B	A	B
Trios	AF-C-M(n = 1442)	1442	0	1442	0	1442	0	1442	0
	AF-C (n = 1442)	1442	0	1416	26	1269	173	1101	341
Duos	AM-C (n = 1442)	1442	0	1425	17	1288	154	1142	300
	AF-C (n = 412)	412	0	407	5	363	49	322	90
	AM-C (n = 391)	391	0	389	2	339	52	296	95

25A: Goldeneye 25A Kit; 20A: Goldeneye 20A Kit; ID: AmpFlSTR Identifiler Kit;

Sino: AmpFlSTR SinoFiler Kit. AF: alleged father; AM: alleged mother; M: mother;

C: child. A: CPI≥10000; B: 0.0001

Application of CPI cutoff value for parentage testing of duos

As shown in Table 1, for every trio case, we regarded it as two real duo cases: a motherless case and a fatherless case. We found that the CPI values of all duo cases were higher than 10000 when typed by the Goldeneye 25A kit. The CPI value of a portion of the duo cases was 0.0001Goldeneye 20A kitkitkit. The CPI values and rate of inconclusive results were significantly different between the three kits (P<0.05). For the 412 motherless and 391 fatherless cases, the results were consistent with those of the duo cases separated from the trio cases. The CPI value and sensitivity of the CPI cutoff value were not significantly different between the motherless and fatherless cases (P<0.05).

Three complex close relative kinship trio cases

As shown in Table 2, in the first trio case, the child was a boy and the alleged father was the child’s uncle. Typed by the Goldeneye 25A kit, only 2 STR loci had no alleles from his uncle, with a CPI = 3.6787x102. After adding up to 40 STR loci using the Goldeneye 22NC kit, 5 STR loci had no alleles from his uncle, with a CPI = 0.8825. Without the mother’s information, as a duo case, only 3 STRs of the child had no alleles from his uncle, with a CPI = 3.5647x103. We could not exclude the possibility that his uncle was the biological father. The Y-STR kit was not used for the child and the child’s uncle, because the results of the Y-STR gene type were same for them. To obtain reliable conclusions, we added the STRs gene type of his biological father for comparison. The alleles at 40 STRs of the biological father matched that of the child (data not shown). The alleged fathers were excluded based on the results. In the second case, the child was a girl. The alleged mother was her aunt, who was typed using the Goldeneye 25A kit. As a trio case, only two STR loci had no alleles from her aunt, with a CPI = 5.2048 x106. Adding up to 40 STRs, 5 STRs had no alleles from her aunt, with a CPI = 2.3643 x104. As a duo case, all 40 STRs of child had alleles from her aunt, with a CPI = 1.1392 x1013. We added the X-STR kit test, and only one X-STR (DXS10134) had no alleles from her aunt (Table 3). Adding a sample from her biological mother for comparison, the child’s alleles at 40 autosomal STRs and 16 X-STRs came from her biological mother (data not shown). The alleged mother was excluded based on the results. In the third case, we did not exclude the possibility that his aunt was his biological mother based on 40 autosomal STR loci. The client could not provide a sample of the biological mother. The child was a boy, so we added the X-STRs test. We found that the alleles at three X-STR loci of the child did not come from his aunt. Therefore, we ruled out the possibility that his aunt was his biological mother (Tables 2 and 4).

Table 2

CPI values of three complex close relative kinship cases.

Cases	Scope	Tested by 25A	CPI Values	Adding 22NC test	CPI Values
1	AF-C-M	D6S1043,D22S1045	3.6787 x102	GATA198B05,D15S659, D14S608	0.8825
	AF-C	D6S1043	2.03447 x103	D15S659, D14S608	3.5647 x103
2	F-C-AM	TH01, D21S11	5.2048 x106	D8S1132, D17S1290, D18S535	2.3643 x104
	AM-C	0	6.4469 x106	0	1.1392 x1013
3	F-C-AM	D8S1179,D19S433	2.7814 x107	D3S3045,D14S608, D17S1290,D3S1744, D11S2368, D19S253	0.00043
	AM-C	D8S1179, D19S433	2.9800 x105	D14S608, D3S1744, D11S2368, D19S253	0.00012

F = father; M = mother; AF = alleged father; AM = alleged mother; C = child.

Table 3

X-STR gene type results of case 2.

X-STR	F	C	AM	X-STR	F	C	AM
DXS6795	13	11, 13	11, 11	DXS6803	11	11, 12	11.3, 12
DXS9902	10	10, 11	11, 12	GATA172D05	9	9, 10	6, 10
DXS8378	10	10, 11	10, 11	DXS6800	16	16, 21	16, 21
HPRTB	14	14, 14	12, 14	DXS10134	36	36, 37	33, 38
GATA165B12	10	10, 11	10, 11	GATA31E08	11	11, 12	11, 12
DXS7132	15	12, 15	12, 14	DXS10159	26	24, 26	24, 27
DXS7424	16	15, 16	15, 16	DXS6789	21	15, 21	15, 20
DXS6807	15	15, 15	14, 15	DXS6810	19	19, 19	18, 19

F = father; C = child; AM = alleged mother.

Table 4

X-STR gene type results of case 3.

X-STR	AM	C	X-STR	AM	C
DXS6795	11, 13	11	DXS6803	11, 12	12.3
DXS9902	10, 12	12	GATA172D05	8, 11	11
DXS8378	10, 12	12	DXS6800	16, 16	16
HPRTB	12, 13	12	DXS10134	34, 37	37
GATA165B12	9, 11	11	GATA31E08	10, 10	10
DXS7132	14, 16	14	DXS10159	24, 25	24
DXS7424	16, 16	13	DXS6789	16, 16	20
DXS6807	11, 11	11	DXS6810	18, 19	18

AM = alleged mother; C = child.

Discussion

Autosomal STR analysis is the primary method for parentage testing in forensic laboratories. The STR gene type of a child is based on his father and mother. In a real trio case, the paternity index (PI) of every STR locus can be calculated according to the STR gene type of the child’s mother and alleged father. In a real duo case, the PI of every STR locus can be calculated between two samples that share at least one allele. For one case, the CPI of all kit STR loci can be determined by multiplying the PI of every locus. The results of parentage testing are directly decided by CPI values. The conclusion of the probability of paternity can be determined from the CPI cutoff value set by the Specification of Parentage Testing in China [7].

In the present study, we calculated the CPI values of 1442 real trio cases for 4 kits, including the Goldeneye 25A, Goldeneye 20A, AmpFlSTR SinoFiler and AmpFlSTR Identifiler kit. All the CPI values of the trios were above 10000. The alleged father was the biological father without an STR mutation, and the CPI value was significantly different among the four kits (P<0.05). The four kits were sufficient to draw conclusions in the trio cases. In all the duo cases, including motherless and fatherless cases, typed by the Goldeneye 25A kit, all the CPI values of the duos were higher than 10000, but some of the CPI values were 0.0001Goldeneye 25A kit>Goldeneye 20A kit>AmpFlSTR SinoFiler kit>AmpFlSTR Identifiler kit. The AmpFlSTR SinoFiler Kit is based on the AmpFlSTR Identifiler kit. In the AmpFlSTR SinoFiler kit, D6S1043 and D12S39 with a high power of exclusion substitute for the THO1 and TPOX loci, so the system efficiency of the AmpFlSTR SinoFiler kit is higher than the AmpFlSTR Identifiler kit. This result was consistent with another study [11]. In the present study, the results showed that the four kits could meet the requirements of routine parentage testing of trios. The Goldeneye 25A kit was sufficient to draw conclusions in the duo cases, but the other three kits were insufficient for a portion of the duo cases. In order to resolve the duo cases with a low CPI value, we should add more autosomal STR loci to increase the CPI value.

For complex close relative cases, the alleged father usually was the brother of the biological father; the alleged mother was the mother’s sister. In the first case, the alleged father was the child’s uncle, and the CPI of the trio was 0.0001trio was higher than 10000, with 5 STR loci having no alleles from her aunt. Without a biological father, as an alleged mother and child case, the child’s alleles at 40 STRs came from the alleged mother, and the CPI value was 1.1392 X1013. Alleles at only one X-STR locus did not come from the alleged mother (Tables 2 and 3). Considering that alleles at 40 autosomal STRs and 16 X-STRs came from her biological mother (data not shown), we excluded the possibility that her aunt was his biological mother. In the third case, we did not exclude that his aunt was his mother when using 23 or 40 autosomal STRs, but we excluded his aunt using the X-STR gene type (Tables 2 and 4). In the second and third cases, two STR mutations were found after testing by the Goldeneye 25A kit, and the CPI>10000. If we did not know the expected results of the identification, it would result in a false conclusion that the alleged mother was the child’s biological mother with loci mutation. Therefore, evaluating simple CPI values of duo or trio cases may lead to false conclusions [2, 12]. Mutations of the STR loci are relatively common in forensic cases. They reduce the CPI value of the case, and affect the conclusion of parentage testing with a low CPI value. More autosomal STRs should be added to confirm the mutation [13-15]. One or two STR loci mismatches may be due to mutational events. Sometimes, we should consider the mutational events as error events. Considering the possibility that autosomal STR loci mutations may occur, it is necessary to increase the number of the required STR loci and supplement the samples of the triplet. In this way, the identification errors could be greatly decreased. In complex close relative cases, there is great similarity of STR gene type among the close relatives, because the alleged father or mother is in the child’s immediate family. In some cases, all the limited STR loci gene types of the child matched that of the alleged father or mother. In fact, they were not the child’s biological father or mother. Sometimes, high CPI values can lead to false conclusions [16]. In complex close relative cases, the genetic background of the case should be considered, and more autosomal STRs, X-STRs and Y-STRs should be used for further confirmation.

Conclusion

In summary, the four autosomal kits were adequate to draw conclusions in the trio cases, and the Goldeneye 25A kit was adequate to draw conclusions in the duo cases. The other three autosomal kits were not sufficient for satisfactory conclusions for all duo cases. For complex close relative kinship cases, more autosomal STR loci and other genetic markers are necessary. The CPI cutoff value (CPI≥10000) is satisfactory for all trio cases and most duo cases. In some complex close relative cases, high CPI values may result in false conclusions.

(XLS)

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9 Oct 2019

PONE-D-19-24597

The Title: Application of CPI Cutoff Value Based on Parentage Testing of Duos and Trios Typed by Four Autosomal Kits

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Reviewer #1: The manuscript entitled "Application of CPI Cutoff Value Based on Parentage Testing of Duos and Trios Typed by Four Autosomal Kits" by Hong-mei Gao provides quality results.

I also described some parts that could be improved where the sentences and paragraphs are even more difficult to follow.

In the paragraph "STR loci mutations affect the conclusions of parentage testing with a low CPI value, and more autosomal

STRs should be added to confirm the mutation" can you explain it's not clear written.

When you use abbreviations, for the first time in your manuscript, you have to give details about that.

I recommend that a native speaker of English review the manuscript to improve word choice, sentence structure, and grammar.

The conclusion need to clear and specific, with 3 short conclusion.

Thanks for the opportunity to read the manuscript.

Reviewer #2: The language and structure of the article are so unclear that the merit can't be assessed. Most of the sentences are overly long, complicated, as well as ungrammatical in some instances. Throughout the article the language is neither clear, nor concise. Typographical and spelling errors have also been encountered.

The article gives relations to three cases that are not included in the study groups described in the methods. Therefore part of the article rather looks as a case report (in this case sample sizes are not large enough to produce robust results).

Regarding the tables of this manuscript, they are not all placed directly after the paragrapgh where they were first cited (see Table 2).

The References formatting does not respect the style of the journal (does not list the first six authors followed by et.al.). Some of the title used as references are rather outdated (over 20 years old).

Laboratory protocols were not made fully availabe. Detailed protocols could enhance the reproductibility of the results.

In part the Discussions only repeat the results without interpreting them.

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25 Oct 2019

We are sorry for some flaws and incorrections appeared in this paper and appreciate the hard-work of your technical staff for revising. Here are the answers for the comments raised by the reviewers in the paper and we made changes in the paper accordingly.

Reviewer #1:

1. In the paragraph "STR loci mutations affect the conclusions of parentage testing with a low CPI value, and more autosomal STRs should be added to confirm the mutation" can you explain it's not clear written.

Answer: We explained it in discussion. “One or two STR loci mismatches may be due to mutational events. Sometimes, we should consider the mutational events as error events. Considering the possibility that autosomal STR loci mutations may occur, it is necessary to increase the number of the required STR loci and supplement the samples of the triplet. In this way, the identification errors could be greatly decreased.”

2. When you use abbreviations, for the first time in your manuscript, you have to give details about that.

Answer: we revised the abbreviations in the paper, including X-STRs and Y-STRs.

3. I recommend that a native speaker of English review the manuscript to improve word choice, sentence structure, and grammar.

Answer: The paper was revised by a native speaker of English (U.S.A).

4. The conclusion need to clear and specific, with 3 short conclusion.

Answer: We revised the conclusion according the suggestion of the reviewer.

Reviewer #2:

1. The language and structure of the article are so unclear that the merit can't be assessed. Most of the sentences are overly long, complicated, as well as ungrammatical in some instances. Throughout the article the language is neither clear, nor concise. Typographical and spelling errors have also been encountered.

Answer: The paper was revised by a native speaker of English (U.S.A).

2. The article gives relations to three cases that are not included in the study groups described in the methods. Therefore part of the article rather looks as a case report (in this case sample sizes are not large enough to produce robust results).

Answer:

(1) Three complex close relative kinship cases were analyzed to evaluate application of CPI Cutoff Value. At the same time, in order to explain the problem that high CPI value (CPI≥10000) will lead to false conclusions in a part of cases.

(2) In our present study, we only had 1442 real trio cases, 803 real duo cases and 3 complex close relative cases typed using Goldeneye 25A kit. We will study more cases in the future.

3. Regarding the tables of this manuscript, they are not all placed directly after the paragrapgh where they were first cited (see Table 2).

Answer: we revised it in the results and discussion.

4. The references formatting does not respect the style of the journal (does not list the first six authors followed by et.al.). Some of the title used as references are rather outdated (over 20 years old).

Answer: we revised the references formatting according to the style of the journal. One reference is over 20 years old (NO. 8), because DNA was extracted according to the method.

5. Laboratory protocols were not made fully availabe. Detailed protocols could enhance the reproductibility of the results.

Answer: We added the laboratory protocols in materials and methods (samples).

6. In part the discussions only repeat the results without interpreting them.

Answer: we amended the part of the discussion.

Submitted filename: Response to reviewers.doc

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31 Oct 2019

Application of CPI Cutoff Value Based on Parentage Testing of Duos and Trios Typed by Four Autosomal Kits

PONE-D-19-24597R1

Dear Dr. Zhang,

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Alexandru Rogobete, Ph.D., M.Sc., Clin Res

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Authors,

I read your paper carefully and it looks much better.

Thank you

BR,

Reviewers' comments:

5 Nov 2019

PONE-D-19-24597R1

Application of CPI Cutoff Value Based on Parentage Testing of Duos and Trios Typed by Four Autosomal Kits

Dear Dr. Zhang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alexandru Rogobete

Academic Editor

PLOS ONE