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Presence of two mutations between father and child in two cases of paternity testing
International Congress Series 1239 (2003) S15 – S18
Presence of two mutations between father and child in two cases of paternity testing C. Brandt-Casadevall a,*, M. Gene´ b, E. Pique´ b, N. Borrego b, C. Gehrig a, N. Dimo-Simonin a, P. Mangin a a
Institut Universitaire de Me´decine Le´gale (IUML), Rue du Bugnon 21, 1005, Lausanne, Switzerland b Departament de Medicina Legal, Universitat de Barcelona, Barcelona, Spain
Abstract We report two cases of paternity testing presenting two inconsistencies between father and child in the same two STR-loci from a total of 16 STR and several VNTR loci examined. D 2003 Published by Elsevier B.V. Keywords: STR-mutations; Double paternal inconsistency; STR-multiplex kits
1. Introduction The presence of mutations and especially step mutations (loss or win of a tandem repeat) in STR-loci is well-known and relatively frequent [1]. The mutation rate for each STR-locus, used in paternity testing or in individual identification, is reported by laboratories that carry out this kind of analyses [2– 5]. We report here two cases of paternity testing presenting two different mutations from a total of 16 STR-loci and several VNTR loci examined.
2. Material and methods The two observed cases came from routine paternity cases analysed in two laboratories (Institut Universitaire de Me´decine Le´gale de Lausanne and Departament de Medicina
* Corresponding author. Tel.: +41-21-314-70-70; fax: +41-21-314-70-90. E-mail address: [email protected] (C. Brandt-Casadevall). 0531-5131/ D 2003 Published by Elsevier B.V. doi:10.1016/S0531-5131(03)01052-5
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Legal de l’Universitat de Barcelona). Furthermore, the identity control pointed out that the mother and the alleged father were not related. Concerning the first case, the STR profiles were performed using three triplex (THO1CSF1PO-TPOX, D3S1358-D19S433-D18S51, D5S818-D13S317-D7S820), one duplex (D16S539-D21S11) and three single determinations for the loci VWA, ACTBP2 and D8S1179. The phenol-chloroform extracted DNA samples were amplified with hot start method (Taq gold) using fluorescein-labelled primers. The PCR products were analysed by 6% denaturing polyacrylamide gel electrophoresis with the automated monochrome fluorescent sequencer ALF (Pharmacia). The VNTR AmpFLP loci D17S5 (YNZ22), D1S80 and 3VApoB were amplified and detected on a submarine agarose gel electrophoresis stained with ethidium bromide. Furthermore, RFLP-Southern profiles were also performed with the probes MS43a, MS31, MS621 and YNH24. In addition, PCR amplification and genotyping were performed using two multiplex kits SGM Plus (Applied Biosystems = AB) and the PowerPlex16 (Promega). The STR loci were separated and detected using the AB-ABI 310 Genetic Analyzer and GeneScan and GenoTyper softwares. The second case was tested using the multiplex kits Cofiler, Profiler Plus, SGM Plus (AB) and the PowerPlex16 (Promega) kit. The ACTBP2 locus was typed in a single reaction followed by detection on an ABI 310 Genetic Analyzer. Additional testing was performed with HLA-DQA1 and Polymarker kits (Perkin-Elmer). RFLP profiles were also analysed with the probes MS43a, MS31, MS621, YNH24 and MS1.
3. Results For case no. 1, all results are consistent with the alleged father being the biological father of the child with the exception of the loci VWA (mother 16– 17/child 15– 16/alleged father 17) and D5S818 (mother 11/child 11 –12/alleged father 11– 13). For case no. 2, all results are also consistent with the alleged father being the biological father of the child, with the exception of the loci D5S818 (mother 11 –12/ child 11– 13 and alleged father 12) and VWA (mother 14/child 18/alleged father 14– 19). In this case, a mismatch was also observed between the mother and the child at the locus VWA when typing with AB kits Profiler Plus and SGM Plus. Interestingly, the mother showed a 14– 18 phenotype for the VWA locus when typing with the Powerplex16 (Promega) kit.
4. Discussion and conclusions The two cases were examined with more than 16 PCR-based loci and 4 RFLP-Southern probes and the probability of paternity for every one was greater than 99.999% (including the mutations in the calculation). It is interesting to notice that in both cases, the mutations were present at the same two loci (VWA and D5S818). Furthermore, in the first case, a double-step mutation, which is quite rare, was observed at the VWA locus between father and child.
C. Brandt-Casadevall et al. / International Congress Series 1239 (2003) S15–S18
S17
Genetic new mutations observed so far are either insertions or deletions of one repeat, rarely two or more [1]. The mutation rate for the different loci used in the forensic analysis (paternity testing or individuals identification) is reported by manufacturers of the Multiplex kits. On the other hand, the mutation rates reported in the literature are linked to the chance of the laboratories to detect them. A number of studies have shown that some loci as VWA, ACTBP2 and FGA display a higher mutation rate [2– 5]. However, as already reported, the mutation rate can be higher for some alleles for a given locus [6]. Another kind of mutation can be observed in case of a null allele caused by a deletion or primer site mutation [7]. In this respect, some authors have mentioned that one individual was homozygous for a locus using a multiplex kit whereas the same individual was heterozygous for the same locus using another multiplex kit [8,9]. In parentage testing, the presence of one single mutation is currently observed but two mutations on two different chromosomes seem to be rare [10]. Despite the fact that the observed mutation rate for each locus is relatively low, the values are very different between laboratories. Thomson et al. [11] reported a surprisingly high mutation rate for the locus D16S539 (1.3 10 2) whereas no mutation for the same locus was reported by others. Furthermore, Thomson et al. calculated that two mutations would occur in 1 of 15,000 cases. The double mutation (father/child) global rate observed in the two laboratories concerned shows a value of about 0.7% of all paternal meiosis (N = 294). Concerning our results, and despite the fact that when we give a probability of paternity we also take account the mutations [12], we require a minimum of three independent exclusions before concluding that a putative father is not the biological father of the child.
Acknowledgements The authors wish to thank Mrs C. Besancßon, A. Grini, S. Delessert, Mr C. Simili and C. Folly for the technical assistance.
References [1] C. Schlo¨tterer, Microsatellites, in: A.R. Hoelzel (Ed.), Molecular Genetic Analysis of populations. A Practical Approach, IRL Press, Oxford, 1998, pp. 252 – 255. [2] B. Brinkmann, A. Mo¨ller, P. Wiegand, Structure of new mutations in 2 STR systems, Int. J. Leg. Med. 107 (1995) 201 – 203. [3] B. Brinkmann, M. Klintschar, F. Neuhuber, J. Hu¨hne, B. Rolf, Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat, Am. J. Hum. Genet. 62 (1998) 1408 – 1415. [4] C.M. Hsu, N.E. Huang, L.C. Tsai, L.G. Kao, C.H. Chao, A. Linacre, J.C.I. Lee, Identification of victims of the 1998 Taoyuan Airbus crash accident using DNA analysis, Int. J. Leg. Med. 113 (1999) 43 – 46. [5] E.M. Dauber, B. Glock, D.W.M. Schwartz, W.R. Mayr, Mutational events at human micro- and minisatellite loci: mutation rates and new STR-alleles, in: G.F. Sensabaugh, P.J. Lincoln, B. Olaisen (Eds.), Progress in Forensic Genetics, vol. 8, Elsevier, Amsterdam, 2000, pp. 21 – 23. [6] C.J. Fre´geau, B. Leclair, K. Bowen, J. Elliott, S. Borys, R. Fourney, The Swissair 111 disaster: short tandem repeat mutations observed, in: G.F. Sensabaugh, P.J. Lincoln, B. Olaisen (Eds.), Progress in Forensic Genetics, vol. 8, Elsevier, Amsterdam, 2000, pp. 40 – 43.
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[7] J.M. Pemberton, J. Slate, D.R. Bancroft, J.A. Barrett, Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies, Mol. Ecol. 4 (1995) 249 – 252. [8] C. Alves, A. Amorim, L. Gusmao, VWA str Genotyping: inconsistency between Perkin Elmer’s profiler plusk kit and Promega’s GenePrintk CTTv, in: G.F. Sensabaugh, P.J. Lincoln, B. Olaisen (Eds.), Progress in Forensic Genetics, vol. 8, Elsevier, Amsterdam, 2000, pp. 467 – 469. [9] R.W. Allen, J. Pritchard, Phenotypic Differences at the HUMvWA locus amplified with different STR kits, J. Forensic Sci. 45 (2000) 1166 – 1167. [10] P.R. Gunn, K. Trueman, P. Stapleton, D.B. Klarkowski, DNA analysis in disputed parentage: the occurrence of two apparently false exclusions of paternity, both at short tandem repeat (STR) loci, in the one child, Electrophoresis 18 (1997) 1650 – 1652. [11] J.A. Thomson, V. Pilotti, P. Stevens, K.L. Ayres, P.G. Debenham, Validation of short tandem repeat analysis for the investigation of cases of disputed paternity, Forensic Sci. Int. 100 (1999) 1 – 16. [12] B. Brinkmann, H. Pfeiffer, M. Schu¨renkamp, C. Hohoff, The evidential value of STRs. An analysis of exclusion cases, Int. J. Leg. Med. 114 (2001) 173 – 177.
Presence of two mutations between father and child in two cases of paternity testing C. Brandt-Casadevall a,*, M. Gene´ b, E. Pique´ b, N. Borrego b, C. Gehrig a, N. Dimo-Simonin a, P. Mangin a a
Institut Universitaire de Me´decine Le´gale (IUML), Rue du Bugnon 21, 1005, Lausanne, Switzerland b Departament de Medicina Legal, Universitat de Barcelona, Barcelona, Spain
Abstract We report two cases of paternity testing presenting two inconsistencies between father and child in the same two STR-loci from a total of 16 STR and several VNTR loci examined. D 2003 Published by Elsevier B.V. Keywords: STR-mutations; Double paternal inconsistency; STR-multiplex kits
1. Introduction The presence of mutations and especially step mutations (loss or win of a tandem repeat) in STR-loci is well-known and relatively frequent [1]. The mutation rate for each STR-locus, used in paternity testing or in individual identification, is reported by laboratories that carry out this kind of analyses [2– 5]. We report here two cases of paternity testing presenting two different mutations from a total of 16 STR-loci and several VNTR loci examined.
2. Material and methods The two observed cases came from routine paternity cases analysed in two laboratories (Institut Universitaire de Me´decine Le´gale de Lausanne and Departament de Medicina
* Corresponding author. Tel.: +41-21-314-70-70; fax: +41-21-314-70-90. E-mail address: [email protected] (C. Brandt-Casadevall). 0531-5131/ D 2003 Published by Elsevier B.V. doi:10.1016/S0531-5131(03)01052-5
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Legal de l’Universitat de Barcelona). Furthermore, the identity control pointed out that the mother and the alleged father were not related. Concerning the first case, the STR profiles were performed using three triplex (THO1CSF1PO-TPOX, D3S1358-D19S433-D18S51, D5S818-D13S317-D7S820), one duplex (D16S539-D21S11) and three single determinations for the loci VWA, ACTBP2 and D8S1179. The phenol-chloroform extracted DNA samples were amplified with hot start method (Taq gold) using fluorescein-labelled primers. The PCR products were analysed by 6% denaturing polyacrylamide gel electrophoresis with the automated monochrome fluorescent sequencer ALF (Pharmacia). The VNTR AmpFLP loci D17S5 (YNZ22), D1S80 and 3VApoB were amplified and detected on a submarine agarose gel electrophoresis stained with ethidium bromide. Furthermore, RFLP-Southern profiles were also performed with the probes MS43a, MS31, MS621 and YNH24. In addition, PCR amplification and genotyping were performed using two multiplex kits SGM Plus (Applied Biosystems = AB) and the PowerPlex16 (Promega). The STR loci were separated and detected using the AB-ABI 310 Genetic Analyzer and GeneScan and GenoTyper softwares. The second case was tested using the multiplex kits Cofiler, Profiler Plus, SGM Plus (AB) and the PowerPlex16 (Promega) kit. The ACTBP2 locus was typed in a single reaction followed by detection on an ABI 310 Genetic Analyzer. Additional testing was performed with HLA-DQA1 and Polymarker kits (Perkin-Elmer). RFLP profiles were also analysed with the probes MS43a, MS31, MS621, YNH24 and MS1.
3. Results For case no. 1, all results are consistent with the alleged father being the biological father of the child with the exception of the loci VWA (mother 16– 17/child 15– 16/alleged father 17) and D5S818 (mother 11/child 11 –12/alleged father 11– 13). For case no. 2, all results are also consistent with the alleged father being the biological father of the child, with the exception of the loci D5S818 (mother 11 –12/ child 11– 13 and alleged father 12) and VWA (mother 14/child 18/alleged father 14– 19). In this case, a mismatch was also observed between the mother and the child at the locus VWA when typing with AB kits Profiler Plus and SGM Plus. Interestingly, the mother showed a 14– 18 phenotype for the VWA locus when typing with the Powerplex16 (Promega) kit.
4. Discussion and conclusions The two cases were examined with more than 16 PCR-based loci and 4 RFLP-Southern probes and the probability of paternity for every one was greater than 99.999% (including the mutations in the calculation). It is interesting to notice that in both cases, the mutations were present at the same two loci (VWA and D5S818). Furthermore, in the first case, a double-step mutation, which is quite rare, was observed at the VWA locus between father and child.
C. Brandt-Casadevall et al. / International Congress Series 1239 (2003) S15–S18
S17
Genetic new mutations observed so far are either insertions or deletions of one repeat, rarely two or more [1]. The mutation rate for the different loci used in the forensic analysis (paternity testing or individuals identification) is reported by manufacturers of the Multiplex kits. On the other hand, the mutation rates reported in the literature are linked to the chance of the laboratories to detect them. A number of studies have shown that some loci as VWA, ACTBP2 and FGA display a higher mutation rate [2– 5]. However, as already reported, the mutation rate can be higher for some alleles for a given locus [6]. Another kind of mutation can be observed in case of a null allele caused by a deletion or primer site mutation [7]. In this respect, some authors have mentioned that one individual was homozygous for a locus using a multiplex kit whereas the same individual was heterozygous for the same locus using another multiplex kit [8,9]. In parentage testing, the presence of one single mutation is currently observed but two mutations on two different chromosomes seem to be rare [10]. Despite the fact that the observed mutation rate for each locus is relatively low, the values are very different between laboratories. Thomson et al. [11] reported a surprisingly high mutation rate for the locus D16S539 (1.3 10 2) whereas no mutation for the same locus was reported by others. Furthermore, Thomson et al. calculated that two mutations would occur in 1 of 15,000 cases. The double mutation (father/child) global rate observed in the two laboratories concerned shows a value of about 0.7% of all paternal meiosis (N = 294). Concerning our results, and despite the fact that when we give a probability of paternity we also take account the mutations [12], we require a minimum of three independent exclusions before concluding that a putative father is not the biological father of the child.
Acknowledgements The authors wish to thank Mrs C. Besancßon, A. Grini, S. Delessert, Mr C. Simili and C. Folly for the technical assistance.
References [1] C. Schlo¨tterer, Microsatellites, in: A.R. Hoelzel (Ed.), Molecular Genetic Analysis of populations. A Practical Approach, IRL Press, Oxford, 1998, pp. 252 – 255. [2] B. Brinkmann, A. Mo¨ller, P. Wiegand, Structure of new mutations in 2 STR systems, Int. J. Leg. Med. 107 (1995) 201 – 203. [3] B. Brinkmann, M. Klintschar, F. Neuhuber, J. Hu¨hne, B. Rolf, Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat, Am. J. Hum. Genet. 62 (1998) 1408 – 1415. [4] C.M. Hsu, N.E. Huang, L.C. Tsai, L.G. Kao, C.H. Chao, A. Linacre, J.C.I. Lee, Identification of victims of the 1998 Taoyuan Airbus crash accident using DNA analysis, Int. J. Leg. Med. 113 (1999) 43 – 46. [5] E.M. Dauber, B. Glock, D.W.M. Schwartz, W.R. Mayr, Mutational events at human micro- and minisatellite loci: mutation rates and new STR-alleles, in: G.F. Sensabaugh, P.J. Lincoln, B. Olaisen (Eds.), Progress in Forensic Genetics, vol. 8, Elsevier, Amsterdam, 2000, pp. 21 – 23. [6] C.J. Fre´geau, B. Leclair, K. Bowen, J. Elliott, S. Borys, R. Fourney, The Swissair 111 disaster: short tandem repeat mutations observed, in: G.F. Sensabaugh, P.J. Lincoln, B. Olaisen (Eds.), Progress in Forensic Genetics, vol. 8, Elsevier, Amsterdam, 2000, pp. 40 – 43.
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[7] J.M. Pemberton, J. Slate, D.R. Bancroft, J.A. Barrett, Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies, Mol. Ecol. 4 (1995) 249 – 252. [8] C. Alves, A. Amorim, L. Gusmao, VWA str Genotyping: inconsistency between Perkin Elmer’s profiler plusk kit and Promega’s GenePrintk CTTv, in: G.F. Sensabaugh, P.J. Lincoln, B. Olaisen (Eds.), Progress in Forensic Genetics, vol. 8, Elsevier, Amsterdam, 2000, pp. 467 – 469. [9] R.W. Allen, J. Pritchard, Phenotypic Differences at the HUMvWA locus amplified with different STR kits, J. Forensic Sci. 45 (2000) 1166 – 1167. [10] P.R. Gunn, K. Trueman, P. Stapleton, D.B. Klarkowski, DNA analysis in disputed parentage: the occurrence of two apparently false exclusions of paternity, both at short tandem repeat (STR) loci, in the one child, Electrophoresis 18 (1997) 1650 – 1652. [11] J.A. Thomson, V. Pilotti, P. Stevens, K.L. Ayres, P.G. Debenham, Validation of short tandem repeat analysis for the investigation of cases of disputed paternity, Forensic Sci. Int. 100 (1999) 1 – 16. [12] B. Brinkmann, H. Pfeiffer, M. Schu¨renkamp, C. Hohoff, The evidential value of STRs. An analysis of exclusion cases, Int. J. Leg. Med. 114 (2001) 173 – 177.