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Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy)
Forensic Science International 135 (2003) 48–52
Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy) C. Binia, S. Ceccardia, D. Luisellib, G. Ferria, S. Pelottia, C. Colalongoa, M. Falconia, G. Pappalardoa,* a
Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, via Irnerio 49, 40126 Bologna, Italy b Department of Biology, Section of Anthropology, University of Bologna, via Selmi 3, Bologna, Italy Received 3 March 2003; received in revised form 15 April 2003; accepted 15 April 2003
Abstract Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI–HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mitochondrial DNA; Hypervariable regions (HVRs); Sequencing; Polymorphism
1. Introduction The outstanding features of mtDNA have been thoroughly described in previous reports [1,2]. Sequence analysis of the hypervariable regions (HVRs) located in the non-coding D-loop is used mainly for forensic and anthropological applications [3–6]. Most sequence variations are located in two segments, HVI and HVII, but Lutz et al. [7] recently described a third hypervariable region within the control region of mtDNA, called HVIII. The aims of this study were to determine the mtDNA polymorphism of these three HVRs in an Italian sample and to estimate the single or cumulative discrimination power for forensic applications.
2. Materials and methods DNA from blood samples of 100 maternally unrelated individuals from Bologna (Italy) was extracted by the salting out method [8]. PCR was performed in a 25 ml reaction * Corresponding author. Tel.: þ39-051-243250; fax: þ39-051-248896. E-mail address: [email protected] (G. Pappalardo).
volume using Biometra Trio-thermo block (Biometra, Germany) with primers L15971-H16414 for HVI, L00015H00389 for HVII, according to the Technical Booklet on Mitochondrial DNA Sequencing of PE Applied Biosystem, USA, and L182-H619 for HVIII according to Lutz et al. [7]. Each reaction contained 50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, 50 mM of each dNTP, 0.25 mM of each primer and 1.25 U Taq DNA polymerase (PE Applied Biosystem, USA). Thirty cycles of 1 min at 94 8C, 30 s at 54 8C, 1 min at 72 8C with a pre-denaturation step of 1 min at 94 8C and 7 min of final extension at 72 8C were performed. PCR products were purified by Centrisep (PE Applied Biosystem, USA) and sequenced using BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystem, USA) according to the manufacturer’s manual and with the above-mentioned primers. Sequencing of both strands was performed to reduce ambiguities in sequence determination, as recommended by the DNA Commission of the International Society for Forensic Genetics [9]. Sequences were aligned and compared with the reference sequence using the Sequence Navigator computer program (Applied Biosystem, Sequence Navigator version 1.0.1). The population diversity indices were estimated by Arlequin software ver. 2.000 [10]. The probability (P) of two randomly selected individuals from a population having
0379-0738/$ – see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0379-0738(03)00167-1
C. Bini et al. / Forensic Science International 135 (2003) 48–52
49
Table 1 Comparison of diversity indices in 100 individuals from Bologna (Italy)
Haplotype diversity Mean number of pairwise differences Random match probability (P) Nucleotide diversity Observed substitutions Observed insertion/deletion Polymorphic sites Haplotypes
HVI
HVII
HVIII
HVI þ HVII
0.967 0.012 4.533 2.248 0.042 0.070 0.038 67 1 64 66
0.965 0.008 3.418 1.762 0.044 0.092 0.052 35 3 36 52
0.590 0.056 1.342 0.051 0.415 0.074 0.051 9 9 18 15
0.992 0.004 0.995 0.003 7.951 3.727 9.293 4.305 0.017 0.015 0.078 0.040 0.078 0.040 102 111 4 13 100 118 85 88
P identical mtDNA types is P ¼ x2 , where x is the frequency of each mtDNA haplotype [11].
HVI þ HVII þ HVIII
The values of the statistical parameters (Table 1) are in agreement with the range of European populations [12]. In Table 2 are presented all the sequences with the polymorphic positions: sequence classification into haplogroups is based on HVRI and position 73 of HVRII according to nomenclature of Macaulay et al. [13]. As reported by Salas et al. [14], the position 73 is slightly unstable and haplogroup H classification sometimes can result ambiguous; this is the
3. Results and discussion The HVI, HVII and HVIII sequences of the mtDNA control region were determined in 100 Italians from Bologna.
Table 2 Haplotypes and variant positions from the Cambridge Reference Sequence (CRS) Sample
HG
HVI
HVII
HVIII
bo56 bo11 bo94 bo76 bo26 bo27 bo19 bo20 bo30 bo9 bo80 bo22 bo1 bo61 bo35 bo64 bo2 bo69 bo98 bo13 bo44 bo12 bo39 bo96 bo3 bo15 bo18 bo25 bo54 bo58 bo62
H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H
093, 368 093 093 129 145, 309, 356 192 235, 311, 327 256, 352 265 C 287, 311 288 292, 319 293, 311 297 299 299 304 304 304 311 311 362 362 362 CRS CRS CRS CRS CRS CRS CRS
263, 263, 263, 152, 263, 152, 073, 263, 195, 152, 263, 263, 263, 152, 150, 263, 263, 263, 195, 263, 263, 073, 239, 239, 263, 263, 263, 263, 263, 263, 263,
CRS CRS CRS CRS CRS CRS CRS CRS CRS 523.1 A, 523.2 C CRS 523 D, 524 D CRS 523 D, 524 D CRS CRS 456 456, 506 456 CRS 523 D, 524 D 477 CRS CRS 523 D, 524 D CRS CRS CRS CRS CRS CRS
315.1 C 309.1 C, 315.1 C 315.1 C 263, 309.1 C, 315.1 C 315.1 C 263, 315.1 C 146, 150, 152, 195, 241, 263, 315.1 C 309.1 C, 315.1 C 263, 315.1 C, 335 263, 315.1 C 309.1 G, 309.2 C, 315.1 C 315.1 C 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 152, 263, 315.1 C 309.1 C, 315.1 C 315.1 C 309.1 C, 315.1 C 263, 309.1 C, 309.2 C, 315.1 C 315.1 C 309.1 C, 309.2 C, 315.1 C 239, 263, 309.1 C, 309.2 C, 315.1 C 263, 309.1 C, 309.2 C, 315.1 C 263, 315.1 C, 318 315.1 C 309.1 C, 315.1 C 309.1 C, 315.1 C 309.1 C, 315.1 C 279, 309.1 C, 309.2 C, 315.1 C 309.1 C, 315.1 C 309.1 C, 315.1 C
50
C. Bini et al. / Forensic Science International 135 (2003) 48–52
Table 2 (Continued ) Sample
HG
HVI
HVII
HVIII
bo71 bo73 bo74 bo75 bo82 bo86 bo92 bo99 bo100 bo97 bo57
H H H H H H/U* H H H HV* I
CRS CRS CRS CRS CRS CRS CRS CRS CRS CRS 129, 223, 391
263, 152, 263, 263, 263, 073, 152, 263, 263, 072, 073,
315.1 C 263, 315.1 C 315.1 C 315.1 C 309.1 C, 315.1 C 263, 315.1 C 263, 315.1 C 315.1 C 315.1 C 195, 263, 309.1 C, 309.2 C, 315.1 C 152, 199, 204, 207, 250, 263, 309.1 C, 315.1 C
bo5 bo59 bo24 bo43 bo77 bo14 bo23 bo41 bo48 bo72 bo63
J* J* J* J* J* J* J* J* J* K K
063, 069, 069, 069, 069, 069, 069, 069, 069, 093, 093,
073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073,
228, 182, 228, 185, 185, 185, 152, 185, 152, 150, 146,
bo45
K
093, 224, 311
073, 263, 315.1 C
bo49
K
093, 224, 311
073, 146, 195, 263, 315.1 C
bo6 bo60 bo84
K K K
172, 224, 311, 357, 362 129, 224, 311 224, 245, 311
073, 263, 309.1 C, 315.1 C 073, 150, 152, 199, 263, 309.1 C, 315.1 C 073, 263, 309.1 C, 315.1 C
bo33 bo36 bo8 bo70
K K K K
224, 224, 224, 224,
270, 311 270, 311 311 311
073, 073, 073, 073,
146, 146, 152, 146,
263, 263, 263, 195,
bo85 bo88 bo66 bo38 bo16 bo46
K K N1b N1b N1b T*
094, 263, 152, 152, 152, 195,
263, 309.1 C, 315.1 C 315.1 C 195, 263, 315.1 C 263, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 309.2 C, 315.1 C
T*
bo83
T*
bo34 bo91 bo81 bo40 bo79 bo47 bo89 bo28 bo87 bo95
T* T* T* T1 T1 T2 T2 T2 T2 T2
311 311 145, 176 G, 222, 390 176 G, 223, 284, 390 176 G, 223, 390 182 C, 183 C, 189, 294, 298 182 C, 183 C, 189, 294, 298 182 C, 183 C, 189, 294, 298 294, 296 294 270, 292, 294, 296, 304 163, 186, 189, 261, 294 163, 186, 189, 294 147, 294, 296, 297, 304 265, 294, 296, 304 294, 296, 304 294, 296, 304 294, 296, 344
073, 073, 073, 073, 073, 073,
bo50
224, 224, 126, 145, 145, 126, 296, 126, 296, 126, 296, 126, 126, 126, 126, 126, 126, 126, 126, 126, 126,
CRS CRS 477 CRS CRS CRS 477 CRS CRS CRS 573.1 C, 573.2 C, 573.3 C 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 497 497, 523.1 A, 523.2 C, 597 497, 523.1 A, 523.2 C 497, 523.1 A, 523.2 C 497 497 497, 573.1 C, 573.2 C, 573.3 C CRS CRS 497 523.1 A, 523.2 C, 523.3 A, 523.4 C CRS 497 CRS 593 523 D, 524 D CRS
069, 126, 126, 126, 126, 126, 126 126 126 129, 224,
126, 348 145, 222, 261, 274 176 185, 189 222 240
189, 224, 311 227, 239, 311
263, 263, 263, 188, 228, 188, 185, 188, 185, 190, 195,
295, 295, 295, 263, 263, 222, 188, 228, 188, 263, 263,
309.1 C, 315.1 C 309.1 C, 315.1 C 315.1 C 295, 309.1 C, 315.1 C 295, 315.1 C 228, 263, 295, 315.1 C 228, 263, 295, 315.1 C 263, 295, 309.1 C, 315.1 C 228, 263, 295, 315.1 C 309.1 C, 315.1 C 315.1 C
315.1 C 315.1 C 315.1 C 263, 309.1 C, 315.1 C
073, 195, 263, 309.1 C, 309.2 C, 315.1 C
CRS
073, 146, 263, 315.1 C
CRS
073, 073, 073, 073, 073, 073, 073, 073, 073, 073,
CRS CRS CRS CRS CRS CRS CRS CRS CRS CRS
200, 263, 263, 152, 150, 152, 152, 195, 263, 263,
263, 309.1 C, 315.1 C 309.1 C, 315.1 C 315.1 C, 334 263, 309.1 C, 315.1 C, 384 199, 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 200, 263, 309.1 C, 315.1 C 263, 315.1 C 315.1 C 309.1 C, 315.1 C
C. Bini et al. / Forensic Science International 135 (2003) 48–52
51
Table 2 (Continued ) Sample
HG
HVI
HVII
bo51 bo53 bo21 bo68 bo65 bo93 bo7 bo29 bo37 bo42 bo10 bo4 bo55 bo78 bo90 bo32 bo17 bo31 bo67 bo52
T5 T5 T5 T5 U* U* U* U2 U3 U4 U5 U5 U5 U7 V V V W W X
126, 153, 292, 294, 296 126, 153, 294, 296, 327 A 126, 153, 294 126, 153, 294 162 192, 256, 270, 291 292, 311 051, 129 C, 183 C, 189, 362 343, 390 300, 311, 356, 362 183 C, 193.1 C, 270 189, 192, 270, 311, 336 256, 270, 293 318 T 153, 298 189, 222, 298 223, 298 145, 223, 260, 292 223, 292 189, 223, 278, 344
073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 072, 072, 072, 073, 073, 073,
150, 150, 150, 150, 263, 263, 263, 152, 150, 195, 150, 150, 263, 152, 093, 263, 263, 189, 189, 153,
HVIII 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 309.1 C, 309.2 C, 315.1 C 315.1 C 295 A, 309.1 C, 315.1 C 217, 263, 309.1 C, 315.1 C 263, 315.1 C 263, 310 263, 309.1 C, 315.1 C 263, 315.1 C 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 315.1 C 315.1 C 195, 204, 207, 263, 315.1 C 194, 199, 204, 207, 263, 309.1 C, 315.1 C 195, 225, 226, 263, 315.1 C
CRS CRS CRS CRS CRS CRS CRS 508 CRS 499, 523 D, 524 D CRS CRS CRS 523 D, 524 D CRS CRS CRS 513 C 523 D, 524 D CRS
HG: haplogroup names; 0.1, 0.2, 0.3 and 0.4: insertions; D: deletions; nucleotide positions correspond to transition; transversions are specified.
reason, why we classify haplotype ‘‘bo86’’ as H/U and haplotypes ‘‘bo19’’ and ‘‘bo12’’ (despite carrying 073 variant) as haplogroup H. Screening the CA-repeat of the HVIII region at positions 514–523, we found 87 sequences with (CA)5, 8 with (CA)51, 4 with (CA)5þ1 and 1 with (CA)5þ2 repeats; these frequencies are similar to those reported by Lutz et al. [7]. Besides, among our 100 individuals, 2/20 identical sequences in HVI and HVII (same variants at position 263 and 315.1) showed a different pattern in HVIII region: a point mutation at 477 and two deletions at 523/524, respectively. The value of this further differentiation is significant even if lower compared to that obtained (13/68) in the wider Magdeburg database comprising 904 sequences [15]. Therefore, we suggest that also sequence polymorphisms of the HVIII region (corresponding to positions 438–574), may be useful in forensic field, especially when nuclear DNA cannot be investigated (e.g. matrilineal analysis, degraded and keratinised tissues). Acknowledgements We kindly thank Antonio Torroni (University of Pavia) for criticism and suggestions. References [1] G.S. Michaels, W.W. Hauswirth, P.J. Laipis, Mitochondrial DNA copy number in bovine oocytes and somatic cells, Dev. Biol. 94 (1982) 246–251.
[2] E.D. Robin, R. Wong, Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells, J. Cell Physiol. 136 (1988) 507–513. [3] M.R. Wilson, J.A. Di Zinno, D. Polansky, J. Repogle, B. Budowle, Validation of mitochondrial DNA sequencing for forensic casework analysis, Int. J. Legal Med. 108 (1995) 68–74. [4] W. Parson, T.J. Parson, R. Scheithauer, M.M. Holland, Population data for 101 Austrian Caucasian mitochondrial DNA D-loop sequences: application of mtDNA sequence analysis to a forensic case, Int. J. Legal Med. 111 (1998) 124–132. [5] B. Budowle, M.R. Wilson, J.A. Di Zinno, C. Stauffer, M.A. Fasano, M.H. Holland, K.L. Monson, Mitochondrial DNA regions HVI and HVII population data, Forensic Sci. Int. 103 (1999) 23–35. [6] A. Torroni, M. Richards, V. Macaulay, P. Foster, R. Villems, S. Norby, M.L. Sayontaus, K. Huoponen, R. Scozzari, H.J. Bandelt, MtDNA haplogroups and frequency patterns in Europe, Am. J. Hum. Genet. 66 (2000) 1173–1177. [7] S. Lutz, H.J. Weisser, J. Heizmann, S. Pollak, Location and frequency of polymorphic positions in the mtDNA control region of individuals from Germany, Int. J. Legal Med. 111 (1998) 67–77 [Erratum of Int. J. Legal Med. 112 (1999) 145–150]. [8] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells, Nucl. Acids Res. 16 (1988) 1215. [9] A. Carracedo, W. Ba¨ r, B. Brinkmann, B. Budowle, P. Gill, M. Holland, P.J. Lincoln, W. Mayr, N. Morling, B. Olaisen, P.M. Schneider, G. Tully, M. Wilson, DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85. [10] S. Schneider, D. Roessli, L. Excoffier, Arlequin ver. 2.000: A Software for Population Genetics Data Analysis, Genetic and
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Biometry Laboratory, University of Geneva, Switzerland, 2000. [11] M. Stoneking, D. Hedgecock, R.G. Higuchi, L. Vigilant, H.A. Erlich, Populations variation of human mtDNA control region sequences detected by enzymatic amplification and sequencespecific oligonucleotide probes, Am. J. Hum. Genet. 48 (1981) 370–382. [12] A. Salas, M.V. Lareu, P. Sa´ nchez-Diz, F. Calafell, A. Carracedo, MtDNA hypervariable region II (HVII) sequences in human evolution studies: impact of mutation rate heterogeneity, Progress Forensic Genet. 8 (2000) 329–331. [13] V. Macaulay, M. Richards, E. Hickey, E. Vega, F. Cruciani, V. Guida, R. Scozzari, B. Bonne-Tamir, B. Sykes, A. Torroni,
The emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLPs, Am. J. Hum. Genet. 64 (1999) 232–249. [14] A. Salas, D. Comas, M.V. Lareu, J. Bertranpetit, A. Carracedo, mtDNA analysis of the Galician population: a genetic edge of European variation, Eur. J. Hum. Genet. 6 (1998) 365–375. [15] S. Lutz, H. Wittig, H.J. Weisser, J. Heizmann, A. Junge, N. Dimo-Simonin, W. Parson, J. Edelmann, K. Anslinger, S. Jung, C. Augustin, Is it possible to differentiate mtDNA by means of HVIII in samples that cannot be distinguished by sequencing the HVI and HVII regions? Forensic Sci. Int. 113 (2000) 97–100.
Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy) C. Binia, S. Ceccardia, D. Luisellib, G. Ferria, S. Pelottia, C. Colalongoa, M. Falconia, G. Pappalardoa,* a
Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, via Irnerio 49, 40126 Bologna, Italy b Department of Biology, Section of Anthropology, University of Bologna, via Selmi 3, Bologna, Italy Received 3 March 2003; received in revised form 15 April 2003; accepted 15 April 2003
Abstract Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI–HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mitochondrial DNA; Hypervariable regions (HVRs); Sequencing; Polymorphism
1. Introduction The outstanding features of mtDNA have been thoroughly described in previous reports [1,2]. Sequence analysis of the hypervariable regions (HVRs) located in the non-coding D-loop is used mainly for forensic and anthropological applications [3–6]. Most sequence variations are located in two segments, HVI and HVII, but Lutz et al. [7] recently described a third hypervariable region within the control region of mtDNA, called HVIII. The aims of this study were to determine the mtDNA polymorphism of these three HVRs in an Italian sample and to estimate the single or cumulative discrimination power for forensic applications.
2. Materials and methods DNA from blood samples of 100 maternally unrelated individuals from Bologna (Italy) was extracted by the salting out method [8]. PCR was performed in a 25 ml reaction * Corresponding author. Tel.: þ39-051-243250; fax: þ39-051-248896. E-mail address: [email protected] (G. Pappalardo).
volume using Biometra Trio-thermo block (Biometra, Germany) with primers L15971-H16414 for HVI, L00015H00389 for HVII, according to the Technical Booklet on Mitochondrial DNA Sequencing of PE Applied Biosystem, USA, and L182-H619 for HVIII according to Lutz et al. [7]. Each reaction contained 50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, 50 mM of each dNTP, 0.25 mM of each primer and 1.25 U Taq DNA polymerase (PE Applied Biosystem, USA). Thirty cycles of 1 min at 94 8C, 30 s at 54 8C, 1 min at 72 8C with a pre-denaturation step of 1 min at 94 8C and 7 min of final extension at 72 8C were performed. PCR products were purified by Centrisep (PE Applied Biosystem, USA) and sequenced using BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystem, USA) according to the manufacturer’s manual and with the above-mentioned primers. Sequencing of both strands was performed to reduce ambiguities in sequence determination, as recommended by the DNA Commission of the International Society for Forensic Genetics [9]. Sequences were aligned and compared with the reference sequence using the Sequence Navigator computer program (Applied Biosystem, Sequence Navigator version 1.0.1). The population diversity indices were estimated by Arlequin software ver. 2.000 [10]. The probability (P) of two randomly selected individuals from a population having
0379-0738/$ – see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0379-0738(03)00167-1
C. Bini et al. / Forensic Science International 135 (2003) 48–52
49
Table 1 Comparison of diversity indices in 100 individuals from Bologna (Italy)
Haplotype diversity Mean number of pairwise differences Random match probability (P) Nucleotide diversity Observed substitutions Observed insertion/deletion Polymorphic sites Haplotypes
HVI
HVII
HVIII
HVI þ HVII
0.967 0.012 4.533 2.248 0.042 0.070 0.038 67 1 64 66
0.965 0.008 3.418 1.762 0.044 0.092 0.052 35 3 36 52
0.590 0.056 1.342 0.051 0.415 0.074 0.051 9 9 18 15
0.992 0.004 0.995 0.003 7.951 3.727 9.293 4.305 0.017 0.015 0.078 0.040 0.078 0.040 102 111 4 13 100 118 85 88
P identical mtDNA types is P ¼ x2 , where x is the frequency of each mtDNA haplotype [11].
HVI þ HVII þ HVIII
The values of the statistical parameters (Table 1) are in agreement with the range of European populations [12]. In Table 2 are presented all the sequences with the polymorphic positions: sequence classification into haplogroups is based on HVRI and position 73 of HVRII according to nomenclature of Macaulay et al. [13]. As reported by Salas et al. [14], the position 73 is slightly unstable and haplogroup H classification sometimes can result ambiguous; this is the
3. Results and discussion The HVI, HVII and HVIII sequences of the mtDNA control region were determined in 100 Italians from Bologna.
Table 2 Haplotypes and variant positions from the Cambridge Reference Sequence (CRS) Sample
HG
HVI
HVII
HVIII
bo56 bo11 bo94 bo76 bo26 bo27 bo19 bo20 bo30 bo9 bo80 bo22 bo1 bo61 bo35 bo64 bo2 bo69 bo98 bo13 bo44 bo12 bo39 bo96 bo3 bo15 bo18 bo25 bo54 bo58 bo62
H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H
093, 368 093 093 129 145, 309, 356 192 235, 311, 327 256, 352 265 C 287, 311 288 292, 319 293, 311 297 299 299 304 304 304 311 311 362 362 362 CRS CRS CRS CRS CRS CRS CRS
263, 263, 263, 152, 263, 152, 073, 263, 195, 152, 263, 263, 263, 152, 150, 263, 263, 263, 195, 263, 263, 073, 239, 239, 263, 263, 263, 263, 263, 263, 263,
CRS CRS CRS CRS CRS CRS CRS CRS CRS 523.1 A, 523.2 C CRS 523 D, 524 D CRS 523 D, 524 D CRS CRS 456 456, 506 456 CRS 523 D, 524 D 477 CRS CRS 523 D, 524 D CRS CRS CRS CRS CRS CRS
315.1 C 309.1 C, 315.1 C 315.1 C 263, 309.1 C, 315.1 C 315.1 C 263, 315.1 C 146, 150, 152, 195, 241, 263, 315.1 C 309.1 C, 315.1 C 263, 315.1 C, 335 263, 315.1 C 309.1 G, 309.2 C, 315.1 C 315.1 C 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 152, 263, 315.1 C 309.1 C, 315.1 C 315.1 C 309.1 C, 315.1 C 263, 309.1 C, 309.2 C, 315.1 C 315.1 C 309.1 C, 309.2 C, 315.1 C 239, 263, 309.1 C, 309.2 C, 315.1 C 263, 309.1 C, 309.2 C, 315.1 C 263, 315.1 C, 318 315.1 C 309.1 C, 315.1 C 309.1 C, 315.1 C 309.1 C, 315.1 C 279, 309.1 C, 309.2 C, 315.1 C 309.1 C, 315.1 C 309.1 C, 315.1 C
50
C. Bini et al. / Forensic Science International 135 (2003) 48–52
Table 2 (Continued ) Sample
HG
HVI
HVII
HVIII
bo71 bo73 bo74 bo75 bo82 bo86 bo92 bo99 bo100 bo97 bo57
H H H H H H/U* H H H HV* I
CRS CRS CRS CRS CRS CRS CRS CRS CRS CRS 129, 223, 391
263, 152, 263, 263, 263, 073, 152, 263, 263, 072, 073,
315.1 C 263, 315.1 C 315.1 C 315.1 C 309.1 C, 315.1 C 263, 315.1 C 263, 315.1 C 315.1 C 315.1 C 195, 263, 309.1 C, 309.2 C, 315.1 C 152, 199, 204, 207, 250, 263, 309.1 C, 315.1 C
bo5 bo59 bo24 bo43 bo77 bo14 bo23 bo41 bo48 bo72 bo63
J* J* J* J* J* J* J* J* J* K K
063, 069, 069, 069, 069, 069, 069, 069, 069, 093, 093,
073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073,
228, 182, 228, 185, 185, 185, 152, 185, 152, 150, 146,
bo45
K
093, 224, 311
073, 263, 315.1 C
bo49
K
093, 224, 311
073, 146, 195, 263, 315.1 C
bo6 bo60 bo84
K K K
172, 224, 311, 357, 362 129, 224, 311 224, 245, 311
073, 263, 309.1 C, 315.1 C 073, 150, 152, 199, 263, 309.1 C, 315.1 C 073, 263, 309.1 C, 315.1 C
bo33 bo36 bo8 bo70
K K K K
224, 224, 224, 224,
270, 311 270, 311 311 311
073, 073, 073, 073,
146, 146, 152, 146,
263, 263, 263, 195,
bo85 bo88 bo66 bo38 bo16 bo46
K K N1b N1b N1b T*
094, 263, 152, 152, 152, 195,
263, 309.1 C, 315.1 C 315.1 C 195, 263, 315.1 C 263, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 309.2 C, 315.1 C
T*
bo83
T*
bo34 bo91 bo81 bo40 bo79 bo47 bo89 bo28 bo87 bo95
T* T* T* T1 T1 T2 T2 T2 T2 T2
311 311 145, 176 G, 222, 390 176 G, 223, 284, 390 176 G, 223, 390 182 C, 183 C, 189, 294, 298 182 C, 183 C, 189, 294, 298 182 C, 183 C, 189, 294, 298 294, 296 294 270, 292, 294, 296, 304 163, 186, 189, 261, 294 163, 186, 189, 294 147, 294, 296, 297, 304 265, 294, 296, 304 294, 296, 304 294, 296, 304 294, 296, 344
073, 073, 073, 073, 073, 073,
bo50
224, 224, 126, 145, 145, 126, 296, 126, 296, 126, 296, 126, 126, 126, 126, 126, 126, 126, 126, 126, 126,
CRS CRS 477 CRS CRS CRS 477 CRS CRS CRS 573.1 C, 573.2 C, 573.3 C 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 462, 489 497 497, 523.1 A, 523.2 C, 597 497, 523.1 A, 523.2 C 497, 523.1 A, 523.2 C 497 497 497, 573.1 C, 573.2 C, 573.3 C CRS CRS 497 523.1 A, 523.2 C, 523.3 A, 523.4 C CRS 497 CRS 593 523 D, 524 D CRS
069, 126, 126, 126, 126, 126, 126 126 126 129, 224,
126, 348 145, 222, 261, 274 176 185, 189 222 240
189, 224, 311 227, 239, 311
263, 263, 263, 188, 228, 188, 185, 188, 185, 190, 195,
295, 295, 295, 263, 263, 222, 188, 228, 188, 263, 263,
309.1 C, 315.1 C 309.1 C, 315.1 C 315.1 C 295, 309.1 C, 315.1 C 295, 315.1 C 228, 263, 295, 315.1 C 228, 263, 295, 315.1 C 263, 295, 309.1 C, 315.1 C 228, 263, 295, 315.1 C 309.1 C, 315.1 C 315.1 C
315.1 C 315.1 C 315.1 C 263, 309.1 C, 315.1 C
073, 195, 263, 309.1 C, 309.2 C, 315.1 C
CRS
073, 146, 263, 315.1 C
CRS
073, 073, 073, 073, 073, 073, 073, 073, 073, 073,
CRS CRS CRS CRS CRS CRS CRS CRS CRS CRS
200, 263, 263, 152, 150, 152, 152, 195, 263, 263,
263, 309.1 C, 315.1 C 309.1 C, 315.1 C 315.1 C, 334 263, 309.1 C, 315.1 C, 384 199, 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 200, 263, 309.1 C, 315.1 C 263, 315.1 C 315.1 C 309.1 C, 315.1 C
C. Bini et al. / Forensic Science International 135 (2003) 48–52
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Table 2 (Continued ) Sample
HG
HVI
HVII
bo51 bo53 bo21 bo68 bo65 bo93 bo7 bo29 bo37 bo42 bo10 bo4 bo55 bo78 bo90 bo32 bo17 bo31 bo67 bo52
T5 T5 T5 T5 U* U* U* U2 U3 U4 U5 U5 U5 U7 V V V W W X
126, 153, 292, 294, 296 126, 153, 294, 296, 327 A 126, 153, 294 126, 153, 294 162 192, 256, 270, 291 292, 311 051, 129 C, 183 C, 189, 362 343, 390 300, 311, 356, 362 183 C, 193.1 C, 270 189, 192, 270, 311, 336 256, 270, 293 318 T 153, 298 189, 222, 298 223, 298 145, 223, 260, 292 223, 292 189, 223, 278, 344
073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 073, 072, 072, 072, 073, 073, 073,
150, 150, 150, 150, 263, 263, 263, 152, 150, 195, 150, 150, 263, 152, 093, 263, 263, 189, 189, 153,
HVIII 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 309.1 C, 309.2 C, 315.1 C 315.1 C 295 A, 309.1 C, 315.1 C 217, 263, 309.1 C, 315.1 C 263, 315.1 C 263, 310 263, 309.1 C, 315.1 C 263, 315.1 C 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 263, 309.1 C, 315.1 C 315.1 C 315.1 C 195, 204, 207, 263, 315.1 C 194, 199, 204, 207, 263, 309.1 C, 315.1 C 195, 225, 226, 263, 315.1 C
CRS CRS CRS CRS CRS CRS CRS 508 CRS 499, 523 D, 524 D CRS CRS CRS 523 D, 524 D CRS CRS CRS 513 C 523 D, 524 D CRS
HG: haplogroup names; 0.1, 0.2, 0.3 and 0.4: insertions; D: deletions; nucleotide positions correspond to transition; transversions are specified.
reason, why we classify haplotype ‘‘bo86’’ as H/U and haplotypes ‘‘bo19’’ and ‘‘bo12’’ (despite carrying 073 variant) as haplogroup H. Screening the CA-repeat of the HVIII region at positions 514–523, we found 87 sequences with (CA)5, 8 with (CA)51, 4 with (CA)5þ1 and 1 with (CA)5þ2 repeats; these frequencies are similar to those reported by Lutz et al. [7]. Besides, among our 100 individuals, 2/20 identical sequences in HVI and HVII (same variants at position 263 and 315.1) showed a different pattern in HVIII region: a point mutation at 477 and two deletions at 523/524, respectively. The value of this further differentiation is significant even if lower compared to that obtained (13/68) in the wider Magdeburg database comprising 904 sequences [15]. Therefore, we suggest that also sequence polymorphisms of the HVIII region (corresponding to positions 438–574), may be useful in forensic field, especially when nuclear DNA cannot be investigated (e.g. matrilineal analysis, degraded and keratinised tissues). Acknowledgements We kindly thank Antonio Torroni (University of Pavia) for criticism and suggestions. References [1] G.S. Michaels, W.W. Hauswirth, P.J. Laipis, Mitochondrial DNA copy number in bovine oocytes and somatic cells, Dev. Biol. 94 (1982) 246–251.
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