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Reporting paternity testing results when 2 exclusions are encountered
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Forensic Science International: Genetics Supplement Series 1 (2008) 492–493 www.elsevier.com/locate/FSIGSS
Research article
Reporting paternity testing results when 2 exclusions are encountered K.J.D. Balloch a,*, J. Marshall b, J. Clugston b, J.W. Gow a a
Centre for Forensic Investigation, Charles Oakley Laboratories, Glasgow Caledonian University, Cowcaddens Road, Glasgow, Lanarkshire G4 0BA, United Kingdom b Crucial Genetics, University Department of Neurology, Southern General Hospital, Govan Road, Glasgow G51 4TF, United Kingdom Received 21 August 2007; accepted 10 October 2007
Abstract A married couple whose commercial paternity test was reported as negative requested a second opinion. The disputed result was based on two apparent mismatches between the tested male (TM) and child at D8S1179 and CSF1PO (PowerPlex 161). Our laboratory repeated the test using PowerPlex 161, FFFL System1 and SGM Plus1 kits (total 21 loci). Apparent exclusions at D8S1179 and CSF1PO were observed. The Ychromosome haplotypes of the TM and male child matched (PowerPlex Y1 kit). From the 13 matching loci, PI > 10 million. The reproducibility of the two exclusions, high PI, matching Y-chromosome haplotypes and the absence of further mismatches with the additional six loci indicate that it is possible that the exclusions were due to mutations at the D8S1179 and CSF1PO loci in the putative father. It is possible that the alleles observed at CSF1PO: TM (11,12), Child (10,11), mother (11,12) arose as a result of a mutation from either the mother or father. The laboratory that carried out the initial paternity test were incorrect in reporting this case as a negative paternity based on two mismatches without considering the mutation rates for the discrepant loci. When mutation rates were considered the value of the PI was > 10 million and the probability of paternity > 99.9999%. The conclusions of the initial test also failed to consider the possibility that only one mismatch may exist between the TM and child. We were unable to rule out the possible involvement of a close male relative of the TM due to lack of further information and the questionable ethics of raising this issue directly with clients. This case highlights the necessity for caution and awareness of the literature and current standards when interpreting paternity testing data. # 2008 Published by Elsevier Ireland Ltd. Keywords: Paternity; Mutations; Ethics
1. Introduction A married couple commissioned our laboratory to repeat a paternity test that had already been performed by another, unrelated, commercial DNA paternity testing company. The report issued by the first laboratory concluded that the tested male (TM) was excluded from the paternity of the male child based on mismatches at the D8S1179 and CSF1PO loci. It is standard practice in our laboratory to exclude paternity only when a minimum of three mismatches have been observed, which is in agreement with Di Lonardo et al. [1]. The possibility of mutations must be taken into account for cases where 1 or 2 mismatches are observed [2,3]. The laboratory that carried out the initial paternity test did not address the possibility of mutational events in their report despite
* Corresponding author. Tel.: +44 141 331 3000; fax: +44 141 331 3208. E-mail address: [email protected] (K.J.D. Balloch). 1875-1768/$ – see front matter # 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigss.2007.10.096
existing literature on methods for the analysis and interpretation of the effects of apparent mutations on paternity testing conclusions [1–4]. 2. Materials and methods 2.1. STR profiling DNA samples were extracted from buccal swabs using the DNA IQ extraction kit (Promega). The samples were profiled using 3 separate autosomal STR marker kits: Promega PowerPlex 161, Promega Geneprint FFFL1 kit and the Applied Biosystems SGM Plus1 kit. The combination of the 3 kits above gave a total of 21 autosomal STR loci. The Promega 12 PowerPlex Y1 kit was used to obtain 12 STR locus Y-chromosome haplotypes for the TM and male child. Products were analysed using the ABI Prism 3101 Genetic Analyser.
K.J.D. Balloch et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 492–493 Table 1 Genotypes of TM, child and mother at the discrepant loci Locus
TM
Child
Mother
Mutation rate
PI
CSF1PO D8S1179
11, 12 13, 15
10, 11 12, 14
11, 12 12, 13
0.16% 0.13%
0.186 0.072
PI is the paternity index calculated when the mutation rate was incorporated.
2.2. Statistical analysis Paternity indices (PI) were calculated using the Familias 1.81 software package [5]. Mutation frequencies of 0.16% for the CSF1PO locus and 0.13% for the D8S1179 locus were used in the Familias PI calculations [5]. 3. Results DNA typing of the TM, child and mother was successful for all of the autosomal STR loci included in the 3 amplification kits used. The apparent mismatches between father and child at D8S1179 and CSF1PO were reproduced using PowerPlex161 (Table 1). No additional mismatches were observed when the additional 6 autosomal STR loci from the FFFL1 and SGM Plus1 kits were compared. Incorporating the mutation rates for D8S1179 and CSF1PO into the Familias PI calculation gave a PI for all 15 PowerPlex 16 loci that was > 10 million, with probability of paternity > 99.99999%, excluding the involvement of a close male relative of the TM. The Y-chromosome haplotypes of the TM and male child matched at all of the 12 Y-chromosome STR loci tested, indicating that they are likely to share the same paternal line. The presence of null alleles was considered to be unlikely as the trio were all heterozygous at D8S1179 and CSF1PO. 4. Discussion The laboratory that performed the initial paternity test for this trio was incorrect in their conclusion that the TM could be excluded as the biological father of the child on the basis of discrepant alleles at the CSF1PO and D8S1179 loci [1,6]. Incorporating the mutation rates for these loci resulted in high values for the combined PI and probability of paternity, strongly supporting the hypothesis that the TM is the biological father of the child [2,6]. The first laboratory should have also considered the hypothesis that the discrepant alleles at CSF1PO could have arisen as a result of a mutation from the mother, leaving just one mismatch between TM and child at D8S1179.
493
The third hypothesis to be considered is that a close male relative from the same paternal line of descent as the TM is the true biological father. Our laboratory reported a similar instance of a paternity test with two mismatches where it was subsequently revealed that the brother of the TM was suspected as the possible father [4]. There were no mismatches observed when the TM’s brother was tested against the child and the TM was excluded as the father. In the present case, we were not provided with information on whether a close male relative of the TM was suspected of involvement. It is standard practice in our laboratory to include the statement ‘. . .excluding the involvement of a close male relative. . .’ when reporting paternity testing results. It could be argued that our results would be more conclusive if we were able to ask our clients whether any close relatives of the TM could be the true father. However, the potential harm that such a discussion could cause to the family concerned was deemed to outweigh the benefit of producing a more conclusive result by ruling out close male relatives of the TM as potential fathers. Therefore, the decision was made to report that the TM could not be excluded as the biological father but that our findings were inconclusive pending further analysis.
5. Conclusion This case highlights the problem of interpreting testing results when there is a lack of case information and the need for awareness of current literature within paternity testing laboratories. Caution and sensitivity should be exercised when interpreting and reporting the results of complex paternity cases.
Acknowledgement We would like to thank Max Hamilton of Crucial Genetics for his assistance in the preparation of this article.
References [1] Di Lonardo, et al. Int. Congr. Ser. 1261 (2004) 488–490. [2] C. Brenner, Mutations in paternity, http://dna-view.com (accessed August 14, 2007). [3] J. Butler, Forensic DNA Typing, Elsevier, USA, 2005, p. 141. [4] W. Goodwin, et al. Int. Congr. Ser. 1261 (2004) 460–462. [5] P. Mostad, T. Egeland, http://www.math.chalmers.se/(mostad/familias/ (accessed August 6, 2007). [6] C. Brenner, Multiple mutations, covert mutations, and false exclusions in paternity casework, http://dna-view.com (accessed August 14, 2007).
Forensic Science International: Genetics Supplement Series 1 (2008) 492–493 www.elsevier.com/locate/FSIGSS
Research article
Reporting paternity testing results when 2 exclusions are encountered K.J.D. Balloch a,*, J. Marshall b, J. Clugston b, J.W. Gow a a
Centre for Forensic Investigation, Charles Oakley Laboratories, Glasgow Caledonian University, Cowcaddens Road, Glasgow, Lanarkshire G4 0BA, United Kingdom b Crucial Genetics, University Department of Neurology, Southern General Hospital, Govan Road, Glasgow G51 4TF, United Kingdom Received 21 August 2007; accepted 10 October 2007
Abstract A married couple whose commercial paternity test was reported as negative requested a second opinion. The disputed result was based on two apparent mismatches between the tested male (TM) and child at D8S1179 and CSF1PO (PowerPlex 161). Our laboratory repeated the test using PowerPlex 161, FFFL System1 and SGM Plus1 kits (total 21 loci). Apparent exclusions at D8S1179 and CSF1PO were observed. The Ychromosome haplotypes of the TM and male child matched (PowerPlex Y1 kit). From the 13 matching loci, PI > 10 million. The reproducibility of the two exclusions, high PI, matching Y-chromosome haplotypes and the absence of further mismatches with the additional six loci indicate that it is possible that the exclusions were due to mutations at the D8S1179 and CSF1PO loci in the putative father. It is possible that the alleles observed at CSF1PO: TM (11,12), Child (10,11), mother (11,12) arose as a result of a mutation from either the mother or father. The laboratory that carried out the initial paternity test were incorrect in reporting this case as a negative paternity based on two mismatches without considering the mutation rates for the discrepant loci. When mutation rates were considered the value of the PI was > 10 million and the probability of paternity > 99.9999%. The conclusions of the initial test also failed to consider the possibility that only one mismatch may exist between the TM and child. We were unable to rule out the possible involvement of a close male relative of the TM due to lack of further information and the questionable ethics of raising this issue directly with clients. This case highlights the necessity for caution and awareness of the literature and current standards when interpreting paternity testing data. # 2008 Published by Elsevier Ireland Ltd. Keywords: Paternity; Mutations; Ethics
1. Introduction A married couple commissioned our laboratory to repeat a paternity test that had already been performed by another, unrelated, commercial DNA paternity testing company. The report issued by the first laboratory concluded that the tested male (TM) was excluded from the paternity of the male child based on mismatches at the D8S1179 and CSF1PO loci. It is standard practice in our laboratory to exclude paternity only when a minimum of three mismatches have been observed, which is in agreement with Di Lonardo et al. [1]. The possibility of mutations must be taken into account for cases where 1 or 2 mismatches are observed [2,3]. The laboratory that carried out the initial paternity test did not address the possibility of mutational events in their report despite
* Corresponding author. Tel.: +44 141 331 3000; fax: +44 141 331 3208. E-mail address: [email protected] (K.J.D. Balloch). 1875-1768/$ – see front matter # 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigss.2007.10.096
existing literature on methods for the analysis and interpretation of the effects of apparent mutations on paternity testing conclusions [1–4]. 2. Materials and methods 2.1. STR profiling DNA samples were extracted from buccal swabs using the DNA IQ extraction kit (Promega). The samples were profiled using 3 separate autosomal STR marker kits: Promega PowerPlex 161, Promega Geneprint FFFL1 kit and the Applied Biosystems SGM Plus1 kit. The combination of the 3 kits above gave a total of 21 autosomal STR loci. The Promega 12 PowerPlex Y1 kit was used to obtain 12 STR locus Y-chromosome haplotypes for the TM and male child. Products were analysed using the ABI Prism 3101 Genetic Analyser.
K.J.D. Balloch et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 492–493 Table 1 Genotypes of TM, child and mother at the discrepant loci Locus
TM
Child
Mother
Mutation rate
PI
CSF1PO D8S1179
11, 12 13, 15
10, 11 12, 14
11, 12 12, 13
0.16% 0.13%
0.186 0.072
PI is the paternity index calculated when the mutation rate was incorporated.
2.2. Statistical analysis Paternity indices (PI) were calculated using the Familias 1.81 software package [5]. Mutation frequencies of 0.16% for the CSF1PO locus and 0.13% for the D8S1179 locus were used in the Familias PI calculations [5]. 3. Results DNA typing of the TM, child and mother was successful for all of the autosomal STR loci included in the 3 amplification kits used. The apparent mismatches between father and child at D8S1179 and CSF1PO were reproduced using PowerPlex161 (Table 1). No additional mismatches were observed when the additional 6 autosomal STR loci from the FFFL1 and SGM Plus1 kits were compared. Incorporating the mutation rates for D8S1179 and CSF1PO into the Familias PI calculation gave a PI for all 15 PowerPlex 16 loci that was > 10 million, with probability of paternity > 99.99999%, excluding the involvement of a close male relative of the TM. The Y-chromosome haplotypes of the TM and male child matched at all of the 12 Y-chromosome STR loci tested, indicating that they are likely to share the same paternal line. The presence of null alleles was considered to be unlikely as the trio were all heterozygous at D8S1179 and CSF1PO. 4. Discussion The laboratory that performed the initial paternity test for this trio was incorrect in their conclusion that the TM could be excluded as the biological father of the child on the basis of discrepant alleles at the CSF1PO and D8S1179 loci [1,6]. Incorporating the mutation rates for these loci resulted in high values for the combined PI and probability of paternity, strongly supporting the hypothesis that the TM is the biological father of the child [2,6]. The first laboratory should have also considered the hypothesis that the discrepant alleles at CSF1PO could have arisen as a result of a mutation from the mother, leaving just one mismatch between TM and child at D8S1179.
493
The third hypothesis to be considered is that a close male relative from the same paternal line of descent as the TM is the true biological father. Our laboratory reported a similar instance of a paternity test with two mismatches where it was subsequently revealed that the brother of the TM was suspected as the possible father [4]. There were no mismatches observed when the TM’s brother was tested against the child and the TM was excluded as the father. In the present case, we were not provided with information on whether a close male relative of the TM was suspected of involvement. It is standard practice in our laboratory to include the statement ‘. . .excluding the involvement of a close male relative. . .’ when reporting paternity testing results. It could be argued that our results would be more conclusive if we were able to ask our clients whether any close relatives of the TM could be the true father. However, the potential harm that such a discussion could cause to the family concerned was deemed to outweigh the benefit of producing a more conclusive result by ruling out close male relatives of the TM as potential fathers. Therefore, the decision was made to report that the TM could not be excluded as the biological father but that our findings were inconclusive pending further analysis.
5. Conclusion This case highlights the problem of interpreting testing results when there is a lack of case information and the need for awareness of current literature within paternity testing laboratories. Caution and sensitivity should be exercised when interpreting and reporting the results of complex paternity cases.
Acknowledgement We would like to thank Max Hamilton of Crucial Genetics for his assistance in the preparation of this article.
References [1] Di Lonardo, et al. Int. Congr. Ser. 1261 (2004) 488–490. [2] C. Brenner, Mutations in paternity, http://dna-view.com (accessed August 14, 2007). [3] J. Butler, Forensic DNA Typing, Elsevier, USA, 2005, p. 141. [4] W. Goodwin, et al. Int. Congr. Ser. 1261 (2004) 460–462. [5] P. Mostad, T. Egeland, http://www.math.chalmers.se/(mostad/familias/ (accessed August 6, 2007). [6] C. Brenner, Multiple mutations, covert mutations, and false exclusions in paternity casework, http://dna-view.com (accessed August 14, 2007).