Pii: s0379-0738(01)00494-7

Forensic Science International 123 (2001) 239±242 A STR mutation in a heteropaternal twin case Helena Geadaa,b,*, Teresa Ribeirob, Rui M. Britob, Rosa Espinheirab, Burkhard Rolfc, Carsten Hohoff d, Bernd Brinkmannd aDepartment of Legal Medicine, Faculty of Medicine, University of Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal bForensic Genetic and Biology Laboratory, National Institute of Legal Medicine-Lisbon, Rua Manuel Bento de Sousa 3, 1150-219 Lisbon, Portugal cInstitute of Legal Medicine, University of MuÈnchen, Frauenlobstraûe 7A, D-80337 MuÈnchen, Germany dInstitute of Legal Medicine, University of MuÈnster, Von-Esmarch-Str. 62, D-48149 MuÈnster, Germany Received 10 September 2000; received in revised form 1 March 2001; accepted 6 April 2001 A heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the ®rst report on a STR mutation in a double paternity case where both biological fathers were indisputably identi®ed. The STR systems enable the resolution ofthese complex genetic relationships even in a case where a mutation in one STR locus was encountered. # 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Paternity investigation; Heteropaternal twins; Minisatellites; Short tandem repeats; ACTBP2 mutation Dizygous twins may arise by fertilisation of two ova at the same (superfecundation) or at different menstrual cycles (superfoetation). Sexual intercourse of a woman during a Genomic DNA was extracted from blood stains by the polyovulatory period with at least two men may thus lead to Chelex method [9]. Polymarker and HLA-DQA1 were superfecundation with the resulting twins having two dif- detected by reverse dot-blot (Perkin-Elmer), YNH24 and MS 43A were analysed using the RFLP technique with Paternity investigation ofmale twins was performed as probes, the PCR-based STR loci were analysed by auto- requested by a Portuguese Court. In the ®rst stage, only one mated ¯uorescent detection (ALF DNA Sequencer, Phar- alleged father (Af1) was investigated and the results were macia and ABI Prism 310 DNA Sequencer, Perkin-Elmer/ somewhat puzzlingÐAf1 seemed to match only one child Applied Biosystems). References for the DNA systems (Ch1) while several genetic inconsistencies existed to the studied are given in Table 1 [10±24].
other child (Ch2). Relative to child 1 there also existed two possible genetic inconsistenciesÐa second-order genetic inconsistency in Duffy (data not shown) and a possible ®rst order genetic inconsistency in ACTBP2 (Table 1). The Sequencing ofall ACTBP2 alleles was performed using second alleged father (Af2) was then studied. Biostatistical the Taq Cycle sequencing kit and an ABI Prism 373A DNA evaluation ®nally lead to two matching fathers, i.e. Af1 Sequencer (Perkin-Elmer/Applied Biosystems).
matching Ch1 and Af2 matching Ch2, elucidating a rare case ofheteropaternal twins arisen by superfecundation [1±8].
* Corresponding author. Tel.: ‡351-21-8811800; The DNA typing was performed with respect to classical E-mail address: hgeada@mail.telepac.pt (H. Geada).
polymarker loci, minisatellites and microsatellites. Due to 0379-0738/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 9 - 0 7 3 8 ( 0 1 ) 0 0 4 9 4 - 7 Paternity investigation results and paternity index valuesa a Paternity indices are based on calculations using the preceding systems above the respective index value and including all preceding calculations.
H. Geada et al. / Forensic Science International 123 (2001) 239±242 ACTBP2 allele sequences from mother, alleged father 1 and the dizygotic twins their high discrimination power short tandem repeats are haplotypes; URL:http://ystr.charite.de), which does not today's method of choice for forensic individualisation include DXYS156, we have very conservatively assumed a frequency of 1% for this haplotype.
The genotypes ofthe loci analysed for the mother, the two Inclusion ofthe Y-STR loci strongly supported paternity alleged fathers and the twins are given in Table 1.
over non-paternity (paternity index ˆ 1,249,999,999 and paternity probability ˆ 99.99999992%, based on the assumption ofa 0.5 a priori probability). Therefore, it is beyond reasonable doubt that Af1 is the biological father of While Af1 showed 14 genetic inconsistencies towards Ch1. Also, a paternal one-step mutation at the ACTBP2 Ch2, leading to a Ch2 paternity exclusion, only a single locus was proven, which resulted in a tetrameric repeat unit genetic inconsistency to Ch1 at the ACTBP2 locus was loss in an uninterrupted regular allele (Table 2).
observed (Table 1). We have therefore analysed this case assuming a mutation at this locus [26].
In addition to the length polymorphism based on the number ofrepeats, an enormous number ofsequence var- While Af2 showed 10 genetic inconsistencies towards iants has been observed at the ACTBP2 locus [18,19]. We Ch1 leading to a Ch1 paternity exclusion, there was no have sequenced all the ACTBP2 alleles and observed a genetic inconsistencies relative to Ch2 (Table 1) and the ®nal regular 5H-¯anking region (Table 2), except for ACTBP2 paternity probability reaches 99.999991% (paternity allele 13.2 ofthe mother, representing an AG loss, which has index ˆ 11,111,110, based on the assumption ofa 0.5 a been previously found by Rolf et al. [19].
priori probability). Therefore, it can be concluded beyond Alleles 19 (Ch1) and 20 (Af1) both show simple repeat reasonable doubt that Af2 is the biological father of Ch2.
structures, respectively, with 19 and 20 AAAG repeats in the This is the ®rst case ofa STR mutation in heteropaternal repetitive region and no variation in the ¯anking region.
twins where both fathers were intensively studied by PCR- There is only one repeat unit difference between these two based DNA polymorphisms. Microsatellites as performed by alleles. Losses or gains ofsingle repeat units are observed in most forensic laboratories have been found to enable the most microsatellite mutations events [27,28]. The mutation resolution ofcomplex genetic relationships even in double rate ofthe ACTBP2 locus was determined to be 7 Â 10À3 paternal twin cases where mutations can be encountered.
[26], which is very high compared to other loci (e.g. mutation rate ofTH01 is 4 Â 10À5 [29]). Our biostatistical calculation for the inclusion of the mutation [16,30] at the ACTBP2 locus made, indeed, use ofa mutation rate of7 Â 10À3.
Excluding the ACTBP2 locus from the statistical analy- The authors wish to thank Profs A. Carracedo and M.S.
sis, a high paternity probability was observed (Table 1), RodrõÂguez-Calvo from the Institute of Legal Medicine, while the inclusion ofthe ACTBP2 locus led to a lower Santiago de Compostela, for the YNH24 and MS43A study paternity probability value. We therefore analysed, in this and also Dr. M.C. Vide from the National Institute of Legal special case, additional autosomal and gonosomal STRs Medicine-Coimbra, for the APOAI1 study.
Concerning Y-chromosomal loci, six Y-loci and the XY locus DXYS156 were statistically evaluated using their haplotype frequencies. Since the haplotype present in Af1 [1] P.I. Terasaki, D. Gjertson, D. Bernoco, S. Perdue, M.R.
has been observed twice in the Caucasian Y-STR database Mickey, J. Bond, Twins with two different fathers identi®ed (based on a European population sample of4115 minimal by HLA, N. Engl. J. Med. 299 (1978) 590±592.
H. Geada et al. / Forensic Science International 123 (2001) 239±242 [2] F.A. Bsat, M.A.F. Seoud, Superfetation secondary to ovula- single locus probes, in: C. Rittner, P.M. Schneider (Eds.), tion induction with clomiphene citrate: a case report, Fertil.
Advances in Forensic Haemogenetics, Vol. 4, Springer, [3] R.S. Verma, S. Luke, P. Dhawan, Twins with different fathers, [18] A. MoÈller, M. SchuÈrenkamp, B. Brinkman, Evaluation ofan ACTBP2 ladder of26 sequenced alleles, Int. J. Leg. Med. 108 [4] R.E. Wenk, T. Houtz, M. Brooks, F.A. Chiafari, How frequent is heteropaternal superfecundantion? Acta Genet. Med.
[19] B. Rolf, M. SchuÈrenkamp, B. Brinkman, Sequence poly- morphism at the tetranucleotide repeat ofthe human beta- [5] W.H. James, The incidence ofsuperfecundation and ofdouble actin related pseudogene H-Beta-Actin (ACTBP2) locus, Int.
paternity in the general population, Acta Genet. Med.
[20] P. Wiegand, H.R. Schneider, M. SchuÈrenkamp, M. Kleiber, B.
[6] H.L. Lu, C.X. Wang, F.Q. Wu, J.J. Li, Paternity identi®cation Brinkmann, Tetranucleotide STR system D8S1132: sequen- in twins with different fathers, J. Forensic Sci. 39 (1994) cing data and population genetic comparisons, Int. J. Leg.
[7] E. Girela, L.A. Lorente, J.C. Alvarez, M.D. Rodrigo, M.
[21] M.V. Lareu, S. Barral, A. Salas, C. Pestoni, A. Carracedo, Lorente, E. Villanueva, Indisputable double paternity in Sequence variation ofa hypervariable short tandem repeat dizygous twins, Fertil. Steril. 67 (1997) 1159±1161.
at the D1S1656 locus, Int. J. Leg. Med. 111 (1998) 244± [8] E. Ambach, W. Parson, C. Brezinka, Superfecundation and dual paternity in a twin pregnancy ending with placental [22] S.M.M. Santos, B. Budowle, J.B. Smerick, K.M. Keys, T.R.
abruption, J. Forensic Sci. 45 (2000) 181±183.
Moretti, Portuguese population data on the six short tandem [9] J. Singer-Sam, R.L. Tanguay, A.D. Riggs, Use ofChelex to repeat loci Ð CSF1PO, TPOX, TH01, D3S1358, VWA and improve the PCR signal from a small number of cells, FGA, Forensic Sci. Int. 83 (1996) 229±235.
[23] M. Kayser, A. Caglia, D. Corach, N. Fretwell, C. Gehrig, [10] R.M. Brito, T. Ribeiro, R. Espinheira, H. Geada, South G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M. Hidding, Portuguese population data on the loci HLA-DQA1, LDLR, K. Honda, M. Jobling, M. Krawczak, K. Leim, S. Meuser, GYPA, HBGG, D7S8 and Gc, J. Forensic Sci. 43 (1998) E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G.
Penacino, A. Perez-Lezaun, A. Piccinini, M. Prinz, C.
[11] H. Geada, R. Espinheira, R. Ribeiro, L. Reys, Population Schmitt, P.M. Schneider, R. Szibor, J. Teifel-Greding, G.
genetics ofD1S80, HUMvWA31 and HUMF13A1 from Weichhold, P. de Knijff, L. Roewer, Evaluation of Portugal and Goa (India), in: A. Carracedo, B. Brinkmann, W.
Y-chromosomal STRs: a multicenter study, Int. J. Leg. Med.
BaÈr (Eds.), Advances in Forensic Haemogenetics, Vol. 6, Springer, Berlin, 1996, pp. 465±467.
[24] P. de Knijff, M. Kayser, A. Caglia, D. Corach, N. Fretwell, C.
[12] R. Espinheira, H. Geada, T. Ribeiro, L. Reys, STR analysis: Gehrig, G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M.
HUMTH01 and HUMFES/FPS for forensic application, in: A.
Hidding, K. Honda, M. Jobling, M. Krawczak, K. Leim, S.
Carracedo, B. Brinkmann, W. BaÈr (Eds.), Advances in Meuser, E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G.
Forensic Haemogenetics, Vol. 6, Springer, Berlin, 1996, Penacino, A. Perez-Lezaun, A. Piccinini, M. Prinz, C.
Schmitt, P.M. Schneider, R. Szibor, J. Teifel-Greding, G.
[13] T. Ribeiro, R.M. Brito, R. Espinheira, H. Geada, Duplex STR Weichhold, L. Roewer, Chromosome Y microsatellites: analysis ofD19S253 and D18S51 in a Portuguese population, population genetic and evolutionary aspects, Int. J. Leg.
in: B. Olaisen, B. Brinkmann, P. Lincoln (Eds.), Progress in Forensic Genetics 7, Elsevier, Amsterdam, 1998, pp. 285± [25] C. Hohoff, B. Brinkmann, Human identity testing with PCR- based systems, Mol. Biotechnol. 13 (1999) 123±136.
[14] T. Ribeiro, R.M. Brito, R. Espinheira, H. Geada, New alleles [26] B. Brinkmann, M. Klintschar, F. Neuhuber, J. HuÈhne, B. Rolf, ofD12S391 STR locus in a Portuguese population, in: B.
Mutation rate in human microsatellites: in¯uence ofthe Olaisen, B. Brinkmann, P. Lincoln (Eds.), Progress in structure and length ofthe tandem repeat, Am. J. Hum. Genet.
Forensic Genetics 7, Elsevier, Amsterdam, 1998, pp. 275± [27] W. Amos, S.J. Sawcer, R.W. Feakes, D.C. Rubinsztein, [15] H. Geada, R.M. Brito, T. Ribeiro, R. Espinheira, Portuguese Microsatellites show mutational bias and heterozygote population and paternity investigation studies with a mega- instability, Nat. Genet. 13 (1996) 390±391.
plex PCRÐthe AmpFlSTR Pro®ler Plus, Forensic Sci. Int.
[28] J.L. Weber, C. Wong, Mutation ofhuman short tandem repeats, Hum. Mol. Genet. 8 (1993) 1123±1128.
[16] J. Henke, R. Fimmers, M.P. Baur, L. Henke, DNA- [29] R. Chakraborty, N. Stivers, Paternity exclusion by DNA minisatellite mutations: recent investigations concerning markers. Effects of paternal mutations, J. Forensic Sci. 41 distribution and impact on parentage testing, Int. J. Leg.
[30] B. Brinkmann, A. MoÈller, P. Wiegand, Structure ofnew [17] E. Valverde, C. Cabrero, A. Diez, A. Carracedo, T. BorraÂs, mutations in 2 STR systems, Int. J. Leg. Med. 107 (1995) Allele frequency in the population of Spain using several

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