High levels of mitochondrial DNA heteroplasmy in single hair roots ...

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1164 T. Grzybowski et al. Electrophoresis 2003, 24, 1159–1165 plasmic positions revealed after nested PCR. This mechanism was previously considered by other authors reporting mtDNA heteroplasmy data [10, 16]. There are three reasons for which enzyme errors might contribute to some of the nested PCR results. The first reason is the lack of reproducibility of results for samples No. 1, 10, 12 and 13 in which the group III mutations occurred (Table 2). The second reason is that specific conditions of nested PCR favor the occurrence of PCR replication errors. One cannot exclude these mechanisms where errors introduced by low-fidelity Taq polymerase in the first round of PCR and normally undetectable by direct sequencing, are eventually made visible due to the subsequent amplification for additional 32 cycles. One must also take into consideration the length of the first D-loop amplicon (1333 bp), which may not represent the most abundant size class of mtDNA available for amplification from the hair extracts. As it was demonstrated earlier, small amounts of template DNA, longer amplicons, additional amplification cycles and high enzyme concentrations may contribute to the increase of the error rate of a conventional PCR [45–47]. The third reason is that group III mutations do not respect phylogenetic hierarchy of the data (see Table 2). Moreover, Table 3 shows that the spectrum of group III mutations is biased toward transitions T–C, one of the most frequent errors made by the wild-type Taq polymerase I [48]. Finally, the majority of T–C transitions occurred on the 5’-GTand 5’-CTcontext, which may influence the rate of substitutions made by wild-type and mutants of Taq DNA polymerase [47–49] (Table 3). It must be stressed, however, that misincorporation during PCR is not proposed as a general explanation of all rare heteroplasmic events. This mechanism may contribute to a limited number of observations. On the other hand, heteroplasmy at rare positions does occur, as demonstrated by this study and other reports. In the face Table 3. Spectra of three groups of mutations revealed in the HV1 sequences from hair and the effect of 5’-sequence context in T–C transitions Transitions Group I Group II Group III T–C 2 4 7 C–T 3 5 2 G–A 1 0 1 A–G 1 1 1 % of T–C out of 28.6 40.0 63.6 all transitions 5’-Sequence context in T–C transitions CT 0 2 2 GT 2 2 3 AT 0 0 2 of these uncertainties, the exclusion of nested PCR from the techniques employed in forensic casework would be a more conservative approach. The interpretation of sequence data obtained by direct PCR (i.e., lower number of cycles) appears to be more straightforward and leads to unambiguous conclusions even in the presence of heteroplasmy. 4 Concluding remarks While the general conclusions of the previous study on highly variable levels of heteroplasmy in hair are still warranted, this reanalysis strongly emphasizes a different ability of current protocols to reveal mixed sequences. The uniquely sensitive nested PCR technique leads to the detection of an additional variability, which may be difficult to interpret or in some instances, may appear to be an artifact. At the same time, this reinvestigation into the widely discussed data shows that phylogenetic analysis may be of a considerable value in a posteriori evaluation of authenticity and plausibility of the newly obtained mtDNA sequences. Phylogenetic rules, which are similar to those recently demonstrated as the effective tools in a quality control of mtDNA population data, can also be helpful in interpreting the difficult forensic cases in which heteroplasmy is encountered. Supplementary information. The electropherograms illustrating the differences between the detection of heteroplasmic positions with nested and direct PCR strategies are available online, http://www.zms-bydgoszcz. w.pl. This site also includes control region sequences found in nuclear- and mtDNA-enriched fractions of the semen samples. We would like to thank in particular Dr. Miroslava V. Derenko who has offered her valuable critical comments and constructive suggestions during the numerous discussions we have had together. Received October 26, 2002 5 References [1] Richards, M., Côrte-Real, H., Forster, P., Macaulay, V., Wilkinson-Herbots, H., Demaine, A., Papiha, S., Hedges, R., Bandelt, H.-J., Am. J. Hum. Genet. 1996, 59, 185–203. [2] Richards, M. B., Macaulay, V. A., Bandelt, H.-J., Sykes, B. C., Ann. Hum. Genet. 1998, 62, 241–260. [3] Richards, M., Macaulay, V., Hickey, E., Vega, E., Sykes, B., Guida, V., Rengo, C., Sellito, D., Cruciani, F., Kivisild, T., Villems, R., Thomas, M., Rychkov, S., Rychkov, O., Rychkov, Y., Gölge, M., Dimitrov, D., Hill, E., Bradley, D., Romano, V., Calì, F., Vona, G., Demaine, A., Papiha, S., Triantaphyllidis, C., Stefanescu, G., Hatina, J., Belledi, M., Di Rienzo, A.,
Electrophoresis 2003, 24, 1159–1165 Reanalysis of mitochondrial DNA heteroplasmy 1165 Novelletto, A., Oppenheim, A., Norby, S., Al-Zaheri, N., Santachiara-Benerecetti, S., Scozzari, R., Torroni, A., Bandelt, H.-J., Am. J. Hum. Genet. 2000, 67, 1251–1276. [4] Röhl, A., Brinkmann, B., Forster, L., Forster, P., Int. J. Legal Med. 2001, 115, 29–39. [5] Holland, M. M., Parsons T. J., Forensic Sci. Rev. 1999, 11, 21–50. [6] Comas, D., Pääbo, S., Bertranpetit, J., Genome Res. 1995, 5, 89–90. [7] Jazin, E., Cavelier, L., Eriksson, I., Oreland, L., Gyllensten, U., Proc. Natl. Acad. Sci. USA 1996, 93, 12382–12387. [8] Wilson, M. R., Polanskey, D., Replogle, J., DiZinno, J. A., Budowle, B., Hum. Genet. 1997, 100, 167–171. [9] Hühne, J., Pfeiffer, H., Brinkmann, B., Int. J. Legal Med. 1998, 112, 27–30. [10] Calloway, C. D., Reynolds, R. L., Herrin Jr., G. L., Anderson W. W., Am. J. Hum. Genet. 2000, 66, 1384–1397. [11] Sullivan, K. M., Alliston-Greiner, F. I. A., Archampong, R., Piercy, G., Tully, P., Gill, P., Lloyd-Davies, C., Proceedings from the Seventh International Symposium on Human Identification 1996, Promega Corporation, Madison, WI, USA 1996, pp. 126–130. [12] Bendall, K. E., Macaulay, V. A., Sykes, B. C., Am. J. Hum. Genet. 1997, 61, 1303–1308. [13] Salas, A., Lareu, M. V., Carracedo, A., Int. J. Legal Med. 2001, 114, 186–190. [14] Alonso, A., Salas, A., Albarrán, C., Arroyo, E., Castro, A., Crespillo, M., di Lonardo, A. M., Lareu, M. V., Cubría, C. L., Soto, M. L., Lorente, J. A., Semper, M. M., Palacio, A., Paredes, M., Pereira, L., Lezaun, A. P., Brito, J. P., Sala, A., Vide, M. C., Whittle, M., Yunis, J. J., Gómez, J., Forensic Sci. Int. 2002, 125, 1–7. [15] Stoneking, M., Am. J. Hum. Genet. 2000, 67, 1029–1032. [16] Tully, L. A., Parsons, T. J., Steighner, R. J., Holland, M. M., Marino, M. A., Prenger, V. L., Am. J. Hum. Genet. 2000, 67, 432–443. [17] Bendall, K. E., Macaulay, V. A., Baker, J. R., Sykes, B. C., Am. J. Hum. Genet. 1996, 59, 1276–1287. [18] Sigurgardóttir, S., Helgason, A., Gulcher, J. R., Stefansson, K., Donnelly, P., Am. J. Hum. Genet. 2000, 66, 1599–1609. [19] Liu, M. R., Pan, K. F., Li, Z. F., Wang, Y., Deng, D. J., Zhang, L., Lu, Y. Y., World J. Gastroenterol. 2002, 8, 426–430. [20] Tully, G., Lareu, M., Electrophoresis 2001, 22, 180. [21] Budowle, B., Allard, M. W., Wilson, M. R., Forensic Sci. Int. 2002, 126, 30–33. [22] Bandelt, H.-J., Lahermo, P., Richards, M., Macaulay, V., Int. J. Legal Med. 2001, 115, 64–69. [23] Bandelt, H.-J., Quintana-Murci, L., Salas, A., Macaulay, V., Am. J. Hum. Genet. 2002, 71, 1150–1160. [24] Grzybowski, T., Electrophoresis 2000, 21, 548–553. [25] Sullivan, K. M., Hopgood, R., Lang, B., Gill, P., Electrophoresis 1991, 12, 17–21. [26] Hopgood, R., Sullivan, K. M., Gill, P., BioTechniques 1992, 13, 82–92. [27] Sullivan, K. M., Hopgood, R., Gill, P., Int. J. Legal Med. 1992, 105, 83–86. [28] Carracedo, A., D’Aloja, E., Dupuy, B., Jangblad, A., Karjalainen, M., Lambert, C., Parson, W., Pfeiffer, H., Pfizinger, H., Sabatier, M., Syndercombe Court, D., Vide, C., Forensic Sci. Int. 1998, 97, 165–170. [29] Budowle, B., Allard, M. W., Wilson, M., Forensic Sci. Int. 2002, 130, 68–70. [30] D’Eustachio, P., Forensic Sci. Int. 2002, 130, 63–67. [31] Wilson, M. R., Polanskey, D., Butler, J., DiZinno, J. A., Reprogle, J., Budowle, B., BioTechniques 1995, 18, 662– 669. [32] Wilson, M. R., DiZinno, J. A., Polanskey, D., Reprogle, J., Budowle, B., Int. J. Legal Med. 1995, 108, 68–74. [33] Melton, T., Nelson, K., Croat. Med. J. 2001, 42, 298–303. [34] Macaulay, V., Richards, M., Hickey, E., Vega, E., Cruciani, F., Guida, V., Scozzari, R., Bonné-Tamir, Sykes, B., Torroni, A., Am. J. Hum. Genet. 1999, 64, 232–249. [35] Malyarchuk, B. A., Grzybowski, T., Derenko, M. V., Czarny, J., Wozniak, M., Miścicka-Śliwka, D., Ann. Hum. Genet. 2002, 66, 261–283. [36] Malyarchuk, B. A., Rogozin, I. B., Berikov, V. B., Derenko, M. V., Hum. Genet. 2002, 111, 46–53. [37] Zischler, H., Geisert, H., von Haeseler, A., Pääbo, S., Nature 1995, 378, 489–492. [38] Sajantila, A., Lahermo, P., Anttinen, T., Lukka, M., Sistonen, P., Savontaus, M. L., Aula, P., Beckman, L., Tranebjaerg, L., Gedde-Dahl, T., Issel-Tarver, L., DiRienzo, A., Pääbo, S., Genome Res. 1995, 5, 42–52. [39] Lutz, S., Weisser, H.-J., Heizmann, J., Pollak, S., Int. J. Legal Med. 1998, 111, 67–77. [40] Linch, C. A., Whiting, D. A., Holland, M. M., J. Forensic Sci. 2001, 46, 844–853. [41] Grzybowski, T., Electrophoresis 2001, 22, 181–182. [42] Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R., Young, I. G., Nature 1981, 290, 457–465. [43] Woischnik, M., Moraes, C. T., Genome Res. 2002, 12, 885– 893. [44] Morgan, M. A., Parsons, T. J., Holland, M. M., Proceedings from the Eight International Symposium on Human Identification 1997, Promega Corporation, Madison, WI, USA 1998, p. 128. [45] Keohavong, P., Thilly, W. G., Proc. Natl. Acad. Sci. USA 1989, 86, 9253–9257. [46] Eckert, K. A., Kunkel, T. A., PCR Methods Appl. 1991, 1, 17–24. [47] Tosaka, A., Ogawa, M., Yoshida, S., Suzuki, M., J. Biol. Chem. 2001, 276, 27562–27567. [48] Suzuki, M., Yoshida, S., Adman, E. T., Blank, A., Loeb, L. A., J. Biol. Chem. 2000, 275, 32728–32735. [49] Yoshida, K., Tosaka, A., Kamiya, H., Murate, T., Kasai, H., Nimura, Y., Ogawa, M., Yoshida, S., Suzuki, M., Nucleic Acids Res. 2001, 29, 4206–4214. [50] Malyarchuk, B. A., Derenko, M. V., Russ. J. Genet. 2001, 37, 823–832.
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