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Clin. Lab. 2018;64:XXX-XXX ©Copyright

ORIGINAL ARTICLE

A Single Step Mutation at D3S1358 Locus in a DNA Paternity Testing with 2 Alleged Fathers

Raluca Dumache 1, 3, Maria Puiu 1, Agneta M. Pusztai 1, Ramona Parvanescu 3, 4, Alexandra Enache 1, 2

1 Victor Babes University of Medicine and Pharmacy, Timisoara, Romania

2 Institute of Forensic Medicine, Timisoara, Romania 3 Laboratory of Forensic Genetics- Institute of Forensic Medicine, Timisoara, Romania

4 Doctoral School of Victor Babes University of Medicine and Pharmacy, Timisoara, Romania

SUMMARY Background: Genetic information is used very frequently in human identification in civil or judicial cases. Estab-lishing the kinship relationship between a child and his biological father involves many ethical facts. We describe a DNA paternity case with two alleged fathers and an inconsistency between alleged father-2 and the child at D3S1358 locus. Methods: As biological samples we used saliva collected from inside the cheek of each person using buccal swabs (Copan, Italy). We collected the biological samples from each of person after each person gave the consent. In or-der to find the concentration of salivary DNA, the DNA samples were quantified by 7500 ABI Real-time PCR us-ing the Quantifiler Human DNA kit (Applied Biosystems, USA). The next step was the amplification of the Sali-vary DNA samples by polymerase chain reaction (PCR). It was performed on a ProFlex PCR System (Applied Biosystems, USA) using the multiplex STR markers from the AmpFlSTR® Identifiler Plus Amplification Kit (Ap-plied Biosystems, USA). After amplification, the PCR products were run on capillary electrophoresis on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA). Results: AF-1 was excluded as biological father. The DNA profiles of AF-2 and the child had one mismatch at D3S1358 locus. Further, we amplified the Y-STR markers to confirm the mutation, obtaining a perfect match be-tween the 2 persons. Conclusions: In paternity testing, where one or two inconsistencies are present between the child and the alleged father on autosomal STR markers, the use of haploid markers X-STR or Y-STRs is needed for the confirmation or exclusion of paternity. (Clin. Lab. 2018;64:xx-xx. DOI: 10.7754/Clin.Lab.2018.180423) Correspondence: Raluca Dumache, MD, PhD, MSc Victor Babes University of Medicine and Pharmacy Timisoara Romania Email: raluca.dumache@umft.ro _________________________________________ Manuscript accepted April 17, 2018

KEY WORDS

paternity testing, mutation, locus, short tandem repeat (STR), Y-chromosomal STRs, alleged father (AF)

INTRODUCTION Nowadays, DNA analysis represents an important tool in forensic human identification. It is used in establish-ing the paternity/maternity relationship of a child. There are 2 known types of genetic markers used in human identification: First, the autosomal markers (short tan-dem repeats (STR)) and second, the uni-parental mark-ers, including the X-STR chromosome markers and the

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Y-STR chromosome markers [1]. The genetic informa-tion is transmitted in equal percentage (50%) from each of the biological parents to the offspring, based on Men-del’s laws of inheritance. Human identification for de-termining the genetic profile of the person uses the STR markers [2]. The uni-parental genetic markers (X and Y) are inherited through maternal lineage (X haplo-types) or paternal lineage (Y haplotypes). These uni-parental markers can be used in those cases of human identification where one of the alleged parents is not available for the DNA testing. The missing alleged par-ent can be replaced by a female or a male relative who has common ancestors on maternal or paternal lineage, if uni-parental markers are used [3-6]. When we talk about parentage DNA testing, in difficult cases the expert is dealing with one, two or three incon-sistencies between the child and the alleged father [7-9]. When the mismatches between the child and the alleged father are present on one or two loci, the expert has to consider the possibility of a paternal mutation [10-12]. Paternal mutations are five to six times more frequent compared to maternal ones due to a gain by insertion or a loss by deletion, of a single repeat unit during paternal meiosis [13-15]. Single step mutations are more fre-quent than the multistep mutational event [16,17]. In this article, we present a DNA paternity case with two men as alleged fathers and one mismatch between alleged father-2 and the child on the autosomal STR markers.

MATERIALS AND METHODS This year, our laboratory was requested to perform DNA parentage testing on a woman with her minor son and two men as alleged fathers. The study protocol was approved by the ethics committee of the Victor Babes University of Medicine and Pharmacy from Timisoara, Romania. It was conducted in accordance to the princi-ples of the Declaration of Helsinki. The biological sam-ples were collected after the parent’s of the child gave their consent for this procedure. After obtaining the in-formed consent from the mother and the two alleged fa-thers we collected the saliva samples by buccal swabs. The child being minor, 9 years old, did not have to give his consent for this DNA investigation and his mother consented on his behalf. For the saliva collection we used 3 buccal swabs (4N6 FLOQSwabs™, Copan, Italy), one for each person par-ticipating in the DNA testing, according to the internal procedure of the laboratory. The saliva samples were collected by swabbing from the inner cheek of each per-son. Extraction of the salivary DNA For the DNA isolation we used the Pure Link Genomic DNA kit (Invitrogen, USA), following the manufactur-er’s instructions.

Quantification of the salivary DNA samples Salivary DNA quantification was performed on a 7500 Real-Time PCR (Applied Biosystems, USA) using the Quantifiler® Human DNA Quantification Kit (Ap-plied Biosystems, USA). Thermal cycler conditions for the quantification were as follows: the sample volume was 25 µL containing 10.5 µL Quantifiler® Human Pri-mer Mix, 12.5 µL Quantifiler® PCR Reaction Mix and 2 µL of sample DNA, standard or control, following the manufacturer’s recommendations. Stage 1 consisted of 1 cycle at a temperature of 95°C for 10 minutes the sec-ond stage consisted of 40 cycles at 95°C for 15 seconds each, and a final hold for 1 minute at 60°C. Amplification of the autosomal STR markers The amplification reactions of the DNA samples were performed on a ProFlex PCR System (Applied Biosys-tems, USA). In this step, we used the AmpFlSTR® Iden-tifiler Plus Amplification Kit (Applied Biosystems, USA) [15]. Further, the PCR reactions of the salivary DNA samples were carried out in a total volume of 25 µL. The final volume contained 10 µL AmpFlSTR Identifiler Plus Master Mix, 5 µL AmpFlSTR Identifiler Plus Primer Set and 10 µL of salivary DNA reaction volume. The AmpFlSTR® Identifiler® Plus Amplifica-tion Kit contains 15 STR autosomal markers, as fol-lows: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, plus the gender gene Amelogenin. All these genetic markers are contained in a single reaction, known as multiplex. The conditions for the PCR amplification, were as follows: 11 minutes at 95C, followed by 28 cycles for 20 seconds at 94C, and 3 minutes at 59C, with a final extension for 10 minutes at 60C. Amplification of the Y-STR markers Because the child was a male, we amplified the Y-STR markers using the AmpFlSTR® Yfiler® PCR Amplifica-tion kit (Applied Biosystems, USA). The kit contains 17 Y-STR markers as follows: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385 a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438, and DYS448. The reaction volume contained: 9.2 µL of AmpFlSTR® PCR Reaction Mix; 5.0 µL of AmpFlSTR Yfiler® Prim-er Set and 0.8 µL of AmpliTaq Gold® DNA Polymer-ase. The amplification conditions were as follows: Initial in-cubation step (hold): 11 minutes at T = 95°C; 30 cycles as follows: denature: 1 minute at T = 94°C; anneal: 1 minute at T = 61°C; extend: 1 minute at T = 72°C. Final extension (hold): 80 minutes at T = 60°C and a final hold indefinite at T = 4°C. Detection of the STR products on capillary electro-phoresis The detection of the PCR was performed on a 3500 HID Genetic Analyzer (Applied Biosystems, USA).

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Table 1. Salivary DNA concentrations of the mother, child, AF-1 and AF-2.

Person Salivary DNA concentration/sample (ng/µL)

Mother 0.632

Child 0.345

Alleged father-1 1.81

Alleged father-2 1.87

Table 2. Genetic profiles of mother (M), child (C), and alleged father 1 (AF-1): the DNA markers of exclusion between the child and the alleged father-1 are marked.

Genetic DNA markers Genetic profile of the mother

Genetic profile of the child

Genetic profile of the alleged father-1

D8S1179 10; 11 11; 13 10; 13

D21S11 28; 32.2 28; 32.2 29; 29

D7S820 12; 13 8; 13 10; 11

CSF1PO 11; 12 10; 11 12; 12

D3S1358 15; 15 15; 15 18; 19

THO1 7; 8 7; 8 8; 8

D13S317 8; 10 10; 11 11; 12

D16S539 11; 11 11; 11 9; 11

D2S1338 17; 18 18; 20 17; 25

D19S433 14; 14 14; 15 12; 13

vWA 15; 17 14; 17 14; 16

TPOX 8; 8 8; 8 11; 11

D18S51 15; 15 13; 15 13; 14

D5S818 11; 12 11; 12 12; 13

FGA 20; 22 19; 22 24; 26

Amelogenin XX XY XY

In this case, following the manufacturer’s recommenda-tions, we used 1 µL of the amplified PCR product (DNA sample) and the allelic ladder (AL). They were added into the mix containing: 8.5 µL of Hi-Di Form-amide (Applied Biosystems, USA) and 0.5 µL of Gene Scan 600 LIZ (Applied Biosystems, USA). Gene Map-per ID-X Software version 1.4 (Applied Biosystems, USA) was used to analyze the obtained data. Detection of the Y-STR products on capillary elec-trophoresis For the capillary electrophoresis we followed the rec-ommendations of the manufacturer and used 0.5 µL GeneScan™ 600LIZ® Size Standard v2.0 (Applied Bio-systems, USA) and 8.5 µL Hi-Di ™ Formamide (Ap-plied Biosystems, USA). The final volume contained: 9 µL mixture of formamide and LIZ600 Size Standard

v2.0 and 1 µL of PCR product allelic ladder. We left the reaction plate 3 minutes at T = 95°C follow-ed by 3 minutes at T = -20°C.

RESULTS The statistical analysis of the probabilities of paternity in both cases, was calculated based on the Essen-Moller formula using the commercial software GenoProof - Parentage Examination (Qualitype, Germany). In the case of AF-1, we obtained a probability of paternity equal to zero, thus being excluded from paternity. AF-2 had a PP = 99.999 and a mismatch between him and the child at locus D3S1358. Because the child was a male, we amplified the Y-STR markers too.

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Table 3. The genetic profiles of the mother (M), child (C), and alleged father-2 (AF-2).

Genetic DNA markers Genetic profile of the mother Genetic profile of the

child Genetic profile of the

alleged father-2

D8S1179 10; 11 11; 13 13; 16

D21S11 28; 32.2 28; 32.2 28; 31

D7S820 12; 13 8; 13 8; 9

CSF1PO 11; 12 10; 11 10; 11

D3S1358 15; 15 15; 15 * * 16; 16

THO1 7; 8 7; 8 7; 9

D13S317 8; 10 10; 11 11; 11

D16S539 11; 11 11; 11 11; 11

D2S1338 17; 18 18; 20 18; 20

D19S433 14; 14 14; 15 12; 15

vWA 15; 17 14; 17 14; 18

TPOX 8; 8 8; 8 8; 9

D18S51 15; 15 13; 15 13; 17

D5S818 11; 12 11; 12 11; 12

FGA 20; 22 19; 22 19; 23

Amelogenin XX XY XY

Probability of paternity (PP)

99.999%

Combined paternity index (CPI)

12.303 x 106

The paternal alleles between the child and alleged father-2 (AF-2) are underlined, at locus D3S1358 the paternal alleles are * (AF-2: 16, child: 15). Table 4. Y-haplotypes of the child and AF-2.

Y-STR markers Y-haplotype of the child Y-haplotype of AF-2

DYS456 16 16

DYS389 I 13 13

DYS390 22 22

DYS389 II 29 29

DYS458 18 18

DYS19 15 15

DYS385 a/b 14; 14 14; 14

DYS393 14 14

DYS391 11 11

DYS439 12 12

DYS635 21 21

DYS392 11 11

Y GATA H4 12 12

DYS437 16 16

DYS438 10 10

DYS448 21 21

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Figure 1. Genetic profile of the mother.

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Figure 2. Genetic profile of the child.

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Figure 3. Genetic profile of AF-1.

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Figure 4. Genetic profile of AF-2.

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Figure 5. Y haplotype of the child and AF-2.

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DISCUSSION Genetic information represents the most important tool in establishing kinship relationships. Establishing the biological father of a child by DNA testing represents a legal act that takes place through the court. In Roman law there was a saying “mater semper certa est, pater semper incertus est” meaning that in establishing a kin-ship relationship the mother of the child is always con-sidered known. In DNA paternity testing, rare cases can occur in which there is an inconsistency in one or two loci between the child and the alleged father. These mutations occur dur-ing the process of meiosis when the cells are transferred from the father to the offspring [18-20]. In DNA pater-nity testing two situations are known: single step muta-tions and double step mutations. In such cases, if there is a mismatch between the alleged father and the child in one or two loci, the exclusion from paternity is con-sidered when the number of mismatches exceeds two. In such cases, additional autosomal markers need to be tested and, depending on the gender of the child haploid Y or X chromosome markers need to be analyzed [21-23]. In the presented case, on locus D3S1358 there is no match between the child and the AF-2. This mutational phenomenon represents a single step mutation which occurred from allele 16 in father to allele 15 in child by loss of a repeat unit through deletion. In this case, be-cause the child was a male we used the 17 Y-chromo-some STR markers. We obtained a complete match on all alleles between the alleged father and the child, con-firming the paternity of AF-2 to the child. Also, the fact that on the Y-haplotype we had a com-plete match for both males suggests that the inconsis-tency at D3S1358 locus was a mutational event.

CONCLUSION In cases of paternity or maternity with one or two mis-matches on the autosomal markers between the child and the alleged parent (mother, father), it is necessary to use supplementary haploid markers X-STR or Y-STR in addition to autosomal markers, depending on the gender of the child, to solve the inconclusive paternity/materni-ty case. Declaration of Interest: The authors declare they have no conflict of interest.

References: 1. Negi DS, Shrivastava P, Das SP. Biochemical characterization of

Molecular Markers for Human Genetic Identification in Paternity testing by DNA profiling. Asian Journal of Biomedical and Phar-maceutical Sciences 2014;4(37):52-6. http://www.alliedacade mies.org/articles/biochemical-characterization-of-molecular-mark ers-for-human-genetic-identification-in-paternity-testing-by-dna-profiling.pdf

2. Mantovani B. Tandem Repeats. Brenner’s Encyclopedia of Ge-netics 2nd Ed, Academic Press 2013 9-11 ISBN 9780123749840.

3. Morling N, Allen R, Carracedo A, et al. Paternity testing Com-mission of the International Society of Forensic Genetics: recom-mendations on genetic investigations in paternity cases. Forensic Sci Int 2002 Oct 9;129(3):148-57 (PMID: 12372685).

4. Santangelo R, Gonzalez-Andrade F, Borsting C, Torroni A, Pereira V, Morling N. Analysis of ancestry informative markers in three main ethnic groups from Ecuador supports a trihybrid origin of Ecuadorians. Forensic Sci Int Genet 2017;31:29-33 (PMID: 28826061).

5. Robino C, Lacerenza D, Aneli S, et al. Allele and haplotype di-versity of 12 X-STR in Sardinia. Forensic Sci Int Genet 2018: 33:e1-e3 (PMID: 29221994).

6. Kayser M, Sajantila A. Mutations at Y-STR loci: implications for paternity testing and forensic analysis. Forensic Sci Int 2001; 118:116-21 (PMID: 11311822).

7. Brinkmann B, Pfeiffer H, Schurenkamp M, Hohoff C. The evi-dential value of STRs. An analysis of exclusion cases. Int J Legal Med 2001;114:173-7 (PMID: 11296890).

8. Chakraborty R, Stivers DN. Paternity exclusion by DNA mark-ers: effects of paternal mutations. J Forensic Sci 1996;41:671-7 (PMID: 8754581).

9. Dauber EM, Kratzer A, Neuhuber F, et al. Germline mutations on STR-alleles include multi-step mutations as defined by sequenc-ing of repeat and flanking regions. Forensic Sci Int Genet 2012;6: 381-6 (PMID: 21873136).

10. Rothman ED, Neel JV, Hoppe FM. Assigning a probability of pa-ternity in apparent case of mutation. Am J Hum Genet 1980;33: 617-28 (PMID: 6942652).

11. O’Connor KL, Hill CR, Vallone PM, Butler JM. Linkage disequi-librium analysis of D12S391 and vWA in U.S. population and pa-ternity samples. Forensic Sci Int Genet 2011;5:538-40 (PMID: 21 067983).

12. Butler JM. Genetics and genomics of core short tandem repeat loci used in human identity testing. J Forensic Sci 2006;51(2): 253-65 (PMID: 16566758).

13. Junge A, Brinkmann B, Fimmers R, Madea B. Mutations or ex-clusions: an unusual case in paternity testing. Int J Legal Med 2006;120:360-3 (PMID: 16328425).

14. Muller M, Sibbing U, Hohoff C, Brinkmann B. Haplotype-assist-ed characterization of germline mutations at short tandem repeat loci. Int J Legal Med 2010;124:177-82 (PMID: 19904551).

15. Ellegren H. Microsatellite mutations in the germ line: implica-tions for evolutionary inference. Trends Genet 2000;16:551-8 (PMID: 11102705).

16. Dawid AP, Mortera J, Pascali VL. Non-fatherhood or mutation? A probabilistic approach to parental exclusion in paternity testing. Forensic Sci Int 2001;124:55-61 (PMID: 11741761).

DNA Mutation in Paternity Testing

Clin. Lab. 9/2018 11

17. Ellegren H. Heterogenous mutation process in human microsatel-lite DNA sequences. Nat Genet 2000;24:400-2 (PMID: 10742 106).

18. Yu K, Fung WK. Evaluation of parentage testing accuracy of child trafficking cases: Combining the exclusion probability and likelihood ratio approaches. Forensic Sci Int Genet 2018;24:81-7 (PMID: 29428891).

19. Rolf B, Keil W, Brinkmann B, Roewer L, Fimmers R. Paternity testing using Y-STR haplotypes: assigning a probability for pater-nity in cases of mutations. Int J Legal Med 2001;115:12-5 (PMID: 11599763).

20. Lindner I, von Wurmb-Scwark N, Meier P, Fimmers R, Büttner A. Usefulness of SNPs as supplementary markers in a paternity case with 3 genetic incompatibilities at autosomal and Y chromo-somal loci. Transfus Med Hemother 2014;41:117-21 (PMID: 248 47187).

21. Gjertson DW, Brenner CH, Baur MP, et al. ISFG: Recommenda-tions on biostatistics in paternity testing. Forensic Sci Int Genet 2007;1(3-4):223-1 (PMID: 190837660).

22. Gill P, Brenner C, Brinkmann B, et al. DNA Commission of the International Society of Forensic genetics: recommendations of forensic analysis using Y-chromosome STRs. Forensic Sci Int 2001;124:5-10 (PMID: 11741752).

23. Tilmar AO, Kling D, Butler JM, et al. DNA Commission of the International Society of Forensic Genetics (ISFG): Guidelines on the use of X-STRs in kinship analysis. Forensic Sci Int Genet 2017;29:269-75 (PMID: 28544956).