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Evaluating the forensic informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale
Forensic Science International 159 (2006) 43–50 www.elsevier.com/locate/forsciint
Evaluating the forensic informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale Luı´sa Pereira a,*, Martin Richards b, Ana Goios a,c, Antonio Alonso d, Cristina Albarra´n d, Oscar Garcia e, Doron M. Behar f, Mukaddes Go¨lge g, Jirˇi Hatina h, Lihadh Al-Gazali i, Daniel G. Bradley j, Vincent Macaulay k, Anto´nio Amorim a,c a
Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), R. Dr. Roberto Frias s/n, 4200-465 Porto, Portugal b Schools of Biology and Computing, University of Leeds, Leeds, UK c Faculdade de Cieˆncias da Universidade do Porto, Porto, Portugal d Instituto de Toxicologı´a, Seccio´n de Biologia, Madrid, Spain e Area de Laboratorio Ertzaintza, Gobierno Vasco, Bilbao, Spain f Bruce Rappaport Faculty of Medicine and Research Institute, Technion and Rambam Medical Center, Haifa, Israel g Department of Physiology, University of Kiel, Kiel, Germany h Charles University, Medical Faculty in Pilsen, Institute of Biology, Pilsen, Czech Republic i Department of Paediatrics, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates j Smurfit Institute of Genetics, Trinity College, Dublin, Ireland k Department of Statistics, University of Glasgow, Glasgow, UK Received 30 March 2005; received in revised form 17 June 2005; accepted 18 June 2005 Available online 1 August 2005
Abstract The impact of phylogeographic information on mtDNA forensics has been limited to the quality control of published sequences and databases. In this work we use the information already available on Eurasian mtDNA phylogeography to guide the choice of coding-region SNPs for haplogroup H. This sub-typing is particularly important in forensics since, even when sequencing both HVRI and HVRII, the discriminating power is low in some Eurasian populations. We show that a small set (eight) of coding-region SNPs resolves a substantial proportion of the identical haplotypes, as defined by control-region variation alone. Moreover, this SNP set, while substantially increasing the discriminating efficiency in most Eurasian populations by roughly equal amounts, discloses population-specific profiles. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human mtDNA; Haplogroup H; Haplotype diversity; HVRI and HVRII; Coding-region SNPs; Eurasia
1. Introduction
* Corresponding author. Tel.: +351 22 5570700; fax: +351 22 5570799. E-mail address: [email protected] (L. Pereira).
The advent of new methodological strategies for SNP typing, allied to the publication of complete mtDNA sequence population data, has given birth to a new phase in the application of mtDNA to forensic casework, characterized by the incorporation of coding-region information.
0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.06.008
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Searches for the most informative coding-region polymorphisms are currently in progress, either by randomly screening some mtDNA genes in specific population samples [1], or by performing complete mtDNA sequencing on some of the more common HVRI/HVRII haplotypes [2,3]. This latter strategy has led to the identification of several coding-region polymorphisms, requiring a set of eight multiplex panels. Up to now, only the first one of these panels has been adapted to the SNaPshot methodology [4]. Other multiplex assays for haplogroup classification have been developed (also based on SNaPshot), suitable not only for forensic but also for anthropological purposes: Brandstatter et al. [5] designed an assay of 16 SNPs for the characterization of western Eurasian haplogroups; Quinta´ns et al. [6] combined 17 SNPs in two multiplexes, the first including SNPs that define common European haplogroups, and the second for polymorphisms defining sub-haplogroups within haplogroup H. It is widely agreed that this strategy, at least in forensics, will not replace HVRI/HVRII sequencing. It can be used, nonetheless, to increase the discrimination power in cases of a HVRI/HVRII haplotype match. It remains to be determined to what extent this gain of information due to coding-region typing holds throughout different west Eurasian populations. The best candidate for this evaluation is haplogroup H due to (1) its poor phylogenetic resolution in HVRI, and (2) its being by far the most common west Eurasian haplogroup, accounting for 40–50% of the mtDNA pool in most of Europe, and 20–30% in the Near East and the Caucasus region [7–9]. Recently, using polymorphism information derived from the growing complete mtDNA sequence database, we sequenced 1580-bp of targeted coding-region segments of the mtDNA genome in 649 individuals harbouring mtDNA haplogroup H from populations throughout Europe, the Caucasus and the Near East [10]. This screening included, besides the haplogroup H diagnostic polymorphism 7028C, all of the seven SNPs (3010A, 4769A, 6776C, 3992T, 4336C, 3915A and 4793G, defining, respectively, sub-haplogroups H1–H7) combined in multiplex 2 by Quinta´ns et al. [6], and the polymorphism 4745G that defines the additional sub-haplogroup H13 [8]. This enhanced genealogical resolution clearly showed that sub-clades of haplogroup H have highly distinctive geographical distributions throughout Eurasia. This improvement in phylogeographic resolution could provide information relevant to forensics, namely to guide the choice of SNPs to be typed. To test this hypothesis, we have evaluated the potential of sub-typing these eight coding-region polymorphisms to increase the haplotype discrimination power inside haplogroup H in populations throughout west Eurasia. Framed in a more practical forensic context, we have also analysed, for the same purpose, a subset of these population samples for which combined HVRI/HVRII information, along with the above-mentioned SNPs, was available. This is an attempt to extend the use of
phylogeographic approaches to mtDNA forensics, which have been largely limited up to now to the quality control of published sequences and databases [11].
2. Materials and methods 2.1. Samples and sequencing We dissected haplogroup H variation in 649 samples from 20 populations from Europe, the Caucasus and the Near East (see Supplementary material), previously analysed only for HVRI sequence variation and some haplogroup-diagnostic RFLPs. The populations were grouped as: SW (Portugal [12], Spain-Madrid [10] and Basque Country [10]), NW (France [7], Ireland [7] and Norway [7]), Med (Italy [7], Sardinia [7] and Crete [7]), NE (Finland [13], Russia [7] and Chuvash [7]), CSE (Poland [7], Czech Republic [7], Romania [7] and Bulgaria [7]), AJ (Ashkenazi Jews [14]), CA (Caucasus [7]) and NRE (Palestine [7], Kurd [7] and Gulf states [7]). We sequenced four mtDNA coding-region segments encompassing the principal diagnostic positions in haplogroup H samples: 3001–3360, 3661–4050, 4281–4820, and 6761–7050 (a total of 1580 bp) according to the numbering of the reference sequence (CRS) [15]. The new sequences generated for this work have been deposited in GenBank, accession nos. AY776364–AY778959. We also included 31 complete sequences from Finland [13], available in GenBank, accession nos. AY339402–AY339432. In Supplementary material, we give tables of the diversity in these coding regions, together with diversity in HVRI and, in some cases, also in HVRII. For amplifying and sequencing the four coding-region segments, the following four sets of primers were used: L2978 50 -GTC CAT ATC AAC AAT AGG GT-30 and H3361 50 -CGT TCG GTA AGC ATT AGG AA-30 ; L3640 50 -TCT AGC CAC CTC TAG CCT AG-30 and H4051 50 -TAG AGT TCA GGG GAG AGT GC-30 ; L4264 50 -CAT TCC CCC TCA AAC CTA AG-30 and H4821 50 -AGA GGG GTG CCT TGG GTA AC-30 ; L6740 50 -TGG TCT GAG CTA TGA TAT CA-30 and H7051 50 -GAT GGC AAATAC AGC TCC TA-30 . The temperature profiles for the PCR were: 95 8C for 10 s, 64 8C for 30 s, and 72 8C for 30 s, for 35 cycles, for the third pair of primers, and the same, except with 58 8C as annealing temperature, for the others. We carried out automated sequencing in an ABI 3100, using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit (AB Applied Biosystems). 2.2. Genetic analysis Control-region positions considered for analysis were between nucleotide positions 16024–16365 for HVRI, and 73–340 for HVRII. The hypervariable positions 16182 and 16183 in HVRI and the indels at positions 309 and 315 in HVRII were not considered in the analysis [16]. Assignment
L. Pereira et al. / Forensic Science International 159 (2006) 43–50
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Fig. 1. mtDNA haplotype frequencies and number of haplotypes observed in the total sample as defined by HVRI alone or with the information provided by eight coding-region SNPs. For comparison, HVRI data were plotted above the x-axis and HVRI + coding region below it.
to sub-haplogroups within haplogroup H based on codingregion polymorphism was as follows: H1 was defined by 3010A; H2 by 4769A; H3 by 6776C; H4 by 3992T; H5a by 4336C; H6 by 3915A; H7 by 4793G; H13 by 4745G. Regarding H5/H5a, we follow the nomenclature of [9],
which updates the earlier H5 of [6]; H5 now refers to the sub-group defined by the control-region substitution 16304C, which seems to have occurred before the one to 4336C. We refer to the paraphyletic collection of H mtDNAs outside these eight main sub-clades as H*.
Fig. 2. The effect of SNP sub-typing on discriminating the most common mtDNA HVRI haplogroup H haplotypes. At the top of each chart, the identification of haplotypes: CRS and the defining polymorphic positions. Total haplotype frequencies are shown in black bars and their subdivision after typing the eight coding-region polymorphisms in grey.
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Diversity measures were estimated using the Arlequin 2.0 software [17], and we constructed the reduced-median network [18] using the program ‘‘Network’’ (Shareware Phylogenetic Network Software, version 4).
3. Results and discussion 3.1. Phylogeography of the major sub-clades within haplogroup H A strategy involving the sequencing of coding region 1580-bp in a large number of samples is obviously not suitable for forensic purposes. Nevertheless, this sequencing effort confirmed that at such a wide geographical scale, the seven major clades defined previously (and already adapted to a SNaPshot assay by Quinta´ns et al. [6]), plus a further sub-haplogroup defined by 4745G (recently labelled as H13 by Achilli et al. [8]), are the main Eurasian H sub-haplogroups. Further polymorphisms included in the haplogroup H network of Herrnstadt et al. [19], at positions 3277, 3333, 3666, 3591, 4310 and 6869, can be disregarded as being insufficiently informative for H sub-classification in Eurasia. Loogva¨li et al. [9] have checked another branch of
the Herrnstadt et al. [19] network, the one defined by substitutions 8448C and 13759A, showing that it is also a minor sub-haplogroup. Most of the European populations sampled display an overall haplogroup H frequency of 40–50%, with frequencies decreasing towards the south-east, reaching 20% in the Near East and Caucasus, and <10% in the Gulf. However, genealogical dissection into sub-clades points to a mosaic sub-structure, with different sub-clades displaying different geographical patterns. H1 and H3 (amounting to around half of the haplogroup H samples in our codingregion database) comprise 65% of haplogroup H lineages in Iberia, 46% in the north-west, 27% in central and eastern Europeans, and 5–15% in the Near East/Caucasus, and are absent from the Gulf. The frequency of H5a appears to be highest on the central European plain; it occurs at low levels across Europe but is absent from the Caucasus and the Near East. H2 and H6 are both common in Eastern Europe and the Caucasus, and are not found in the Near Eastern sample. The less common sub-clades H4, H7 and H13 occur in both Europe and the Near East; H13 also present in the Caucasus. The paraphyletic cluster H* predominates in the Near East, its range at least partly the mirror-image of that for H1 and H3, but it is most common in East–Central
Fig. 3. The effect of SNP sub-typing on discriminating the most common haplogroup H HVRI haplotypes in two Eurasian regions: southwest (SW) and central–south-east (CSE) Europe. Samples included in each region are described in Supplementary material. On the top of each chart, the relevant HVRI haplotype is shown. The HVRI haplotype frequencies are shown by black bars and the frequencies after subdivision by the eight coding-region polymorphisms in grey.
L. Pereira et al. / Forensic Science International 159 (2006) 43–50
Europe and the Balkans, and is frequent in Atlantic Europe as well. 3.2. The gain of information by haplogroup H subcharacterization The sub-characterization of the eight coding-region polymorphisms reduces the frequency of the most abundant haplotype from 36% to 17% (Fig. 1), increasing the number of haplotypes observed once from 121 to 162, and increasing the haplotype diversity from 0.8638 0.0129 to 0.9545 0.0049. Specifying by haplotype, Fig. 2 represents the six most frequent H haplotypes in HVRI, and how they can be resolved by typing the eight coding-region polymorphisms. (a) Although the CRS haplotype (in HVRI) is partly resolved, there still remain three frequent haplotypes: 3010A with a frequency of 17.3%, 9.3% within H*, and 6776C with 5.9%. (b) The second most frequent HVRI subclade, H5, is defined by 16304C (4.9%), which includes all of the 4336C (3.4%) and a fraction (1.5%) of the H* lineages. (c) The next most frequent HVRI haplotype is defined by 16189C (3.3%); half of which is associated with polymorphism 3010A (1.8%; defining sub-clades H1b and H1f according to [9]), and the rest split between the sub-haplogroups defined by 6776C, 3992T, 4793G and the paragroup H*. (d) The substitution 16362C is present in all the individuals
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bearing polymorphism 3915A, but it was found also on the background 3010A and in H*. (e) Haplotype 16129A (2.7%) is mainly observed on the background of 3010A (1.9%), but appears in a few instances with 4769A or 6776C and also in H*. (f) Finally, the haplotype 16311C (1.9%) is mainly within H* (0.9%) and probably corresponds largely to the sub-haplogroup H11a defined by Loogva¨li et al. [9], members of which bear this polymorphism in association with the coding-region polymorphisms 8448C and 13759A. Since the frequencies of these coding-region defined subclades of haplogroup H vary across the continent, the picture of the main HVRI haplotypes also varies to some extent between the geographic regions. For instance, we can consider the two population groups for which we have a particularly large sample, located quite distantly in the continent (Fig. 3), Iberia (SW) and central/south-eastern Europe (CSE). There are higher frequencies of HVRI haplotypes CRS and 16129A in SW than in CSE (the latter is in fact absent in CSE), with the converse being true for the remaining haplotypes; the frequency of the H* cluster is higher in CSE. Some private lineages are also observed. In SW, an HVRI haplotype defined by 16126C (1.3%) appears on the backgrounds 6776C (0.3%) and H* (1.0%); another defined by 16320T (1.3%), on the backgrounds 3010A (0.3%) and H* (1.0%); another defined by 16291T (2.6%; appearing only in the Basque Country), linked to 4769A. For CSE, there were two private lineages, one defined by
Fig. 4. Reduced-median network of SW European mtDNA lineages displaying 16129A and/or 3010A 4733C. The sequence indicated by an asterisk bears the polymorphisms 3010A–16129A (in the coding regions and HVRI analysed here).
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Fig. 5. The effect of typing eight coding-region SNPs (in addition to HVRI) on the number and diversity of haplogroup H sequences in various west Eurasian populations. Ratios to the values calculated from HVRI alone are graphed. Population groups as defined in Section 2.
16092C–16293G–16311C (2.7%) and the other by 16051G– 16312G (also 2.7%), both in H*. As we sequenced coding-region stretches totalling 1580 bp, we detected further polymorphisms observed so far only in Iberia, which increase resolution of the relatively common HVRI haplotype defined by 16129A in this geographic region (Fig. 4). Those were the transitions at np 3849A, 4659A and 4733C, appearing in the background of 3010A; 3849A was previously described in two individuals from haplogroup U2 and 4733C in one haplogroup L3e1 individual [19]. On the right side of the network (Fig. 4), we observe four individuals who, most probably, have reverted at position 16129 (which is rather fast-evolving) [9]. Despite these instances, the increase both in number of different haplotypes and in haplotype diversity, due to the characterization of the eight coding-region polymorphisms, in relation to HVRI diversity, is broadly similar in all the population groups (Fig. 5), a fact which is particularly convenient for forensic applications.
It is worth detailing the gain of information obtained from HVRII and coding-region SNPs in the case of the HVRI-defined CRS haplotype, due to its high frequency (47.4% of the H haplotypes), which is split into the following most frequent sub-haplotypes: (a) 263G 315.1/3010A (13.9%); (b) 150T 263G 315.1/3010A (3.1%); (c) 152C 263G 315.1/3010A (4.1%); (d) 263G 315.1/6776C (8.3%); (e) 152C 263G 315.1/6776C (2.1%); (f) 263G 315.1/H* (3.6%). For the rest of the haplotypes, the splits defined by HVRII and coding-region SNPs sub-typing reveal 18 haplotypes with frequencies between 1.0% and 2.1% (i.e. shared between two and four individuals), and 83 unique haplotypes. It is also noteworthy that the information gain is not equally apportioned between the number of different haplotypes and haplotype diversity, the increase of the former being far more impressive than the latter. This fact is, however, not attributable to any artefact of the typing strategy employed. Indeed the new haplotypes are usually observed at low frequencies (reflecting the strongly star-like
3.3. The gain of information due to HVRII sequencing and coding-region SNPs In Fig. 6, we compare the gain of information provided by HVRII and/or the eight coding-region polymorphisms in the Portuguese sample, the only one for which the corresponding information is available. Both HVRII and the coding-region variation considerably increase the subcharacterization of H haplotypes. However, if we take into consideration that there are 23 polymorphic sites in HVRII compared with only eight coding-region polymorphisms (a ratio of 2.9), the proportion of the gain of information in both number of haplotypes and haplotype diversity due to HVRII is only roughly half that provided by the coding-region polymorphisms. Nevertheless, in absolute numbers, HVRII is more informative for haplotype discrimination.
Fig. 6. Increase in the discriminating power among mtDNA haplogroup H Portuguese samples, by typing successively HVRII and a set of coding-region SNPs in addition to HVRI sequence.
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phylogeny of haplogroup H) and therefore haplotype diversity changes very little.
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Peter and Anna Katharina Forster, Ariella Oppenheim, Sergei and Oksana Rychkov, and Gheorghe Stefanescu for samples and some DNA extractions.
4. Conclusions Appendix A. Supplementary data The publication of complete mtDNA sequences has led to many population studies at a large geographic scale, focused on the detailed characterization of certain haplogroups. This was the case for haplogroups L2 [20], V [21] and, more recently, H [8–10]. These data are particularly useful when developing coding-region based multiplexes for forensics with the aim of increasing the haplotype discrimination and forecasting their likely success rate across populations. Out of the previously described assays for H sub-characterization, the one developed by Quinta´ns et al. [6] is the most sensitive. The one developed by Vallone et al. [4], corresponding to the first out of the eight multiplexes indicated by Coble et al. [3] misses the informative polymorphism 6776C. We confirmed that the haplotype CRS/ 263G 315.1/6776C can reach a high frequency in western Eurasia (8.3% in the Portuguese sample). Moreover, the basal frequency of the haplotype CRS/263G 315.1 is also much higher (26.8%) than the one (7.1%) detected in the database used by Coble et al. [3]. Another important outcome of our analysis is related to the issue of population heterogeneity. It could happen, for instance, that a SNP typing assay, while increasing substantially the discrimination among a specific population, would be poorly informative in another one. Fortunately for the forensic field, that is not the case, at least within west Eurasia. The SNPs described here provide roughly the same gain, in terms both of haplotype numbers and haplotype diversity, in distinct populations (Fig. 3), and additionally reveal quite different sub-typing profiles. This can be of potential value for inferring the population or region of origin of DNA samples, as for Y-SNPs [22], as the phylogenetic dissection of mtDNA haplogroups is revealing gradients previously hidden, on the Eurasian scale [8–10]. We conclude that it is possible to increase considerably the discrimination power of current mtDNA typing, with the addition of a moderate number of coding-region SNPs (which can be carried out in a single PCR multiplex reaction, as in [6]), at a large geographic scale.
Acknowledgments This work was partially supported by a research grant to AG (SFRH/BD/16518/2004) from Fundac¸a˜o para a Cieˆncia e a Tecnologia and IPATIMUP by Programa Operacional Cieˆncia, Tecnologia e Inovac¸a˜o (POCTI), Quadro Comunita´rio de Apoio III. This study is included in the Project POCTI/ANT/45139/2002 financed by Fundac¸a˜o para a Cieˆncia e a Tecnologia (Eixo 2, Medida 2.3 do POCTI, QCA III). We thank Pierre-Marie Danze, Dimitar Dimitrov,
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.forsciint.2005.06.008.
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Evaluating the forensic informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale Luı´sa Pereira a,*, Martin Richards b, Ana Goios a,c, Antonio Alonso d, Cristina Albarra´n d, Oscar Garcia e, Doron M. Behar f, Mukaddes Go¨lge g, Jirˇi Hatina h, Lihadh Al-Gazali i, Daniel G. Bradley j, Vincent Macaulay k, Anto´nio Amorim a,c a
Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), R. Dr. Roberto Frias s/n, 4200-465 Porto, Portugal b Schools of Biology and Computing, University of Leeds, Leeds, UK c Faculdade de Cieˆncias da Universidade do Porto, Porto, Portugal d Instituto de Toxicologı´a, Seccio´n de Biologia, Madrid, Spain e Area de Laboratorio Ertzaintza, Gobierno Vasco, Bilbao, Spain f Bruce Rappaport Faculty of Medicine and Research Institute, Technion and Rambam Medical Center, Haifa, Israel g Department of Physiology, University of Kiel, Kiel, Germany h Charles University, Medical Faculty in Pilsen, Institute of Biology, Pilsen, Czech Republic i Department of Paediatrics, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates j Smurfit Institute of Genetics, Trinity College, Dublin, Ireland k Department of Statistics, University of Glasgow, Glasgow, UK Received 30 March 2005; received in revised form 17 June 2005; accepted 18 June 2005 Available online 1 August 2005
Abstract The impact of phylogeographic information on mtDNA forensics has been limited to the quality control of published sequences and databases. In this work we use the information already available on Eurasian mtDNA phylogeography to guide the choice of coding-region SNPs for haplogroup H. This sub-typing is particularly important in forensics since, even when sequencing both HVRI and HVRII, the discriminating power is low in some Eurasian populations. We show that a small set (eight) of coding-region SNPs resolves a substantial proportion of the identical haplotypes, as defined by control-region variation alone. Moreover, this SNP set, while substantially increasing the discriminating efficiency in most Eurasian populations by roughly equal amounts, discloses population-specific profiles. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human mtDNA; Haplogroup H; Haplotype diversity; HVRI and HVRII; Coding-region SNPs; Eurasia
1. Introduction
* Corresponding author. Tel.: +351 22 5570700; fax: +351 22 5570799. E-mail address: [email protected] (L. Pereira).
The advent of new methodological strategies for SNP typing, allied to the publication of complete mtDNA sequence population data, has given birth to a new phase in the application of mtDNA to forensic casework, characterized by the incorporation of coding-region information.
0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.06.008
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Searches for the most informative coding-region polymorphisms are currently in progress, either by randomly screening some mtDNA genes in specific population samples [1], or by performing complete mtDNA sequencing on some of the more common HVRI/HVRII haplotypes [2,3]. This latter strategy has led to the identification of several coding-region polymorphisms, requiring a set of eight multiplex panels. Up to now, only the first one of these panels has been adapted to the SNaPshot methodology [4]. Other multiplex assays for haplogroup classification have been developed (also based on SNaPshot), suitable not only for forensic but also for anthropological purposes: Brandstatter et al. [5] designed an assay of 16 SNPs for the characterization of western Eurasian haplogroups; Quinta´ns et al. [6] combined 17 SNPs in two multiplexes, the first including SNPs that define common European haplogroups, and the second for polymorphisms defining sub-haplogroups within haplogroup H. It is widely agreed that this strategy, at least in forensics, will not replace HVRI/HVRII sequencing. It can be used, nonetheless, to increase the discrimination power in cases of a HVRI/HVRII haplotype match. It remains to be determined to what extent this gain of information due to coding-region typing holds throughout different west Eurasian populations. The best candidate for this evaluation is haplogroup H due to (1) its poor phylogenetic resolution in HVRI, and (2) its being by far the most common west Eurasian haplogroup, accounting for 40–50% of the mtDNA pool in most of Europe, and 20–30% in the Near East and the Caucasus region [7–9]. Recently, using polymorphism information derived from the growing complete mtDNA sequence database, we sequenced 1580-bp of targeted coding-region segments of the mtDNA genome in 649 individuals harbouring mtDNA haplogroup H from populations throughout Europe, the Caucasus and the Near East [10]. This screening included, besides the haplogroup H diagnostic polymorphism 7028C, all of the seven SNPs (3010A, 4769A, 6776C, 3992T, 4336C, 3915A and 4793G, defining, respectively, sub-haplogroups H1–H7) combined in multiplex 2 by Quinta´ns et al. [6], and the polymorphism 4745G that defines the additional sub-haplogroup H13 [8]. This enhanced genealogical resolution clearly showed that sub-clades of haplogroup H have highly distinctive geographical distributions throughout Eurasia. This improvement in phylogeographic resolution could provide information relevant to forensics, namely to guide the choice of SNPs to be typed. To test this hypothesis, we have evaluated the potential of sub-typing these eight coding-region polymorphisms to increase the haplotype discrimination power inside haplogroup H in populations throughout west Eurasia. Framed in a more practical forensic context, we have also analysed, for the same purpose, a subset of these population samples for which combined HVRI/HVRII information, along with the above-mentioned SNPs, was available. This is an attempt to extend the use of
phylogeographic approaches to mtDNA forensics, which have been largely limited up to now to the quality control of published sequences and databases [11].
2. Materials and methods 2.1. Samples and sequencing We dissected haplogroup H variation in 649 samples from 20 populations from Europe, the Caucasus and the Near East (see Supplementary material), previously analysed only for HVRI sequence variation and some haplogroup-diagnostic RFLPs. The populations were grouped as: SW (Portugal [12], Spain-Madrid [10] and Basque Country [10]), NW (France [7], Ireland [7] and Norway [7]), Med (Italy [7], Sardinia [7] and Crete [7]), NE (Finland [13], Russia [7] and Chuvash [7]), CSE (Poland [7], Czech Republic [7], Romania [7] and Bulgaria [7]), AJ (Ashkenazi Jews [14]), CA (Caucasus [7]) and NRE (Palestine [7], Kurd [7] and Gulf states [7]). We sequenced four mtDNA coding-region segments encompassing the principal diagnostic positions in haplogroup H samples: 3001–3360, 3661–4050, 4281–4820, and 6761–7050 (a total of 1580 bp) according to the numbering of the reference sequence (CRS) [15]. The new sequences generated for this work have been deposited in GenBank, accession nos. AY776364–AY778959. We also included 31 complete sequences from Finland [13], available in GenBank, accession nos. AY339402–AY339432. In Supplementary material, we give tables of the diversity in these coding regions, together with diversity in HVRI and, in some cases, also in HVRII. For amplifying and sequencing the four coding-region segments, the following four sets of primers were used: L2978 50 -GTC CAT ATC AAC AAT AGG GT-30 and H3361 50 -CGT TCG GTA AGC ATT AGG AA-30 ; L3640 50 -TCT AGC CAC CTC TAG CCT AG-30 and H4051 50 -TAG AGT TCA GGG GAG AGT GC-30 ; L4264 50 -CAT TCC CCC TCA AAC CTA AG-30 and H4821 50 -AGA GGG GTG CCT TGG GTA AC-30 ; L6740 50 -TGG TCT GAG CTA TGA TAT CA-30 and H7051 50 -GAT GGC AAATAC AGC TCC TA-30 . The temperature profiles for the PCR were: 95 8C for 10 s, 64 8C for 30 s, and 72 8C for 30 s, for 35 cycles, for the third pair of primers, and the same, except with 58 8C as annealing temperature, for the others. We carried out automated sequencing in an ABI 3100, using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit (AB Applied Biosystems). 2.2. Genetic analysis Control-region positions considered for analysis were between nucleotide positions 16024–16365 for HVRI, and 73–340 for HVRII. The hypervariable positions 16182 and 16183 in HVRI and the indels at positions 309 and 315 in HVRII were not considered in the analysis [16]. Assignment
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Fig. 1. mtDNA haplotype frequencies and number of haplotypes observed in the total sample as defined by HVRI alone or with the information provided by eight coding-region SNPs. For comparison, HVRI data were plotted above the x-axis and HVRI + coding region below it.
to sub-haplogroups within haplogroup H based on codingregion polymorphism was as follows: H1 was defined by 3010A; H2 by 4769A; H3 by 6776C; H4 by 3992T; H5a by 4336C; H6 by 3915A; H7 by 4793G; H13 by 4745G. Regarding H5/H5a, we follow the nomenclature of [9],
which updates the earlier H5 of [6]; H5 now refers to the sub-group defined by the control-region substitution 16304C, which seems to have occurred before the one to 4336C. We refer to the paraphyletic collection of H mtDNAs outside these eight main sub-clades as H*.
Fig. 2. The effect of SNP sub-typing on discriminating the most common mtDNA HVRI haplogroup H haplotypes. At the top of each chart, the identification of haplotypes: CRS and the defining polymorphic positions. Total haplotype frequencies are shown in black bars and their subdivision after typing the eight coding-region polymorphisms in grey.
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Diversity measures were estimated using the Arlequin 2.0 software [17], and we constructed the reduced-median network [18] using the program ‘‘Network’’ (Shareware Phylogenetic Network Software, version 4).
3. Results and discussion 3.1. Phylogeography of the major sub-clades within haplogroup H A strategy involving the sequencing of coding region 1580-bp in a large number of samples is obviously not suitable for forensic purposes. Nevertheless, this sequencing effort confirmed that at such a wide geographical scale, the seven major clades defined previously (and already adapted to a SNaPshot assay by Quinta´ns et al. [6]), plus a further sub-haplogroup defined by 4745G (recently labelled as H13 by Achilli et al. [8]), are the main Eurasian H sub-haplogroups. Further polymorphisms included in the haplogroup H network of Herrnstadt et al. [19], at positions 3277, 3333, 3666, 3591, 4310 and 6869, can be disregarded as being insufficiently informative for H sub-classification in Eurasia. Loogva¨li et al. [9] have checked another branch of
the Herrnstadt et al. [19] network, the one defined by substitutions 8448C and 13759A, showing that it is also a minor sub-haplogroup. Most of the European populations sampled display an overall haplogroup H frequency of 40–50%, with frequencies decreasing towards the south-east, reaching 20% in the Near East and Caucasus, and <10% in the Gulf. However, genealogical dissection into sub-clades points to a mosaic sub-structure, with different sub-clades displaying different geographical patterns. H1 and H3 (amounting to around half of the haplogroup H samples in our codingregion database) comprise 65% of haplogroup H lineages in Iberia, 46% in the north-west, 27% in central and eastern Europeans, and 5–15% in the Near East/Caucasus, and are absent from the Gulf. The frequency of H5a appears to be highest on the central European plain; it occurs at low levels across Europe but is absent from the Caucasus and the Near East. H2 and H6 are both common in Eastern Europe and the Caucasus, and are not found in the Near Eastern sample. The less common sub-clades H4, H7 and H13 occur in both Europe and the Near East; H13 also present in the Caucasus. The paraphyletic cluster H* predominates in the Near East, its range at least partly the mirror-image of that for H1 and H3, but it is most common in East–Central
Fig. 3. The effect of SNP sub-typing on discriminating the most common haplogroup H HVRI haplotypes in two Eurasian regions: southwest (SW) and central–south-east (CSE) Europe. Samples included in each region are described in Supplementary material. On the top of each chart, the relevant HVRI haplotype is shown. The HVRI haplotype frequencies are shown by black bars and the frequencies after subdivision by the eight coding-region polymorphisms in grey.
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Europe and the Balkans, and is frequent in Atlantic Europe as well. 3.2. The gain of information by haplogroup H subcharacterization The sub-characterization of the eight coding-region polymorphisms reduces the frequency of the most abundant haplotype from 36% to 17% (Fig. 1), increasing the number of haplotypes observed once from 121 to 162, and increasing the haplotype diversity from 0.8638 0.0129 to 0.9545 0.0049. Specifying by haplotype, Fig. 2 represents the six most frequent H haplotypes in HVRI, and how they can be resolved by typing the eight coding-region polymorphisms. (a) Although the CRS haplotype (in HVRI) is partly resolved, there still remain three frequent haplotypes: 3010A with a frequency of 17.3%, 9.3% within H*, and 6776C with 5.9%. (b) The second most frequent HVRI subclade, H5, is defined by 16304C (4.9%), which includes all of the 4336C (3.4%) and a fraction (1.5%) of the H* lineages. (c) The next most frequent HVRI haplotype is defined by 16189C (3.3%); half of which is associated with polymorphism 3010A (1.8%; defining sub-clades H1b and H1f according to [9]), and the rest split between the sub-haplogroups defined by 6776C, 3992T, 4793G and the paragroup H*. (d) The substitution 16362C is present in all the individuals
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bearing polymorphism 3915A, but it was found also on the background 3010A and in H*. (e) Haplotype 16129A (2.7%) is mainly observed on the background of 3010A (1.9%), but appears in a few instances with 4769A or 6776C and also in H*. (f) Finally, the haplotype 16311C (1.9%) is mainly within H* (0.9%) and probably corresponds largely to the sub-haplogroup H11a defined by Loogva¨li et al. [9], members of which bear this polymorphism in association with the coding-region polymorphisms 8448C and 13759A. Since the frequencies of these coding-region defined subclades of haplogroup H vary across the continent, the picture of the main HVRI haplotypes also varies to some extent between the geographic regions. For instance, we can consider the two population groups for which we have a particularly large sample, located quite distantly in the continent (Fig. 3), Iberia (SW) and central/south-eastern Europe (CSE). There are higher frequencies of HVRI haplotypes CRS and 16129A in SW than in CSE (the latter is in fact absent in CSE), with the converse being true for the remaining haplotypes; the frequency of the H* cluster is higher in CSE. Some private lineages are also observed. In SW, an HVRI haplotype defined by 16126C (1.3%) appears on the backgrounds 6776C (0.3%) and H* (1.0%); another defined by 16320T (1.3%), on the backgrounds 3010A (0.3%) and H* (1.0%); another defined by 16291T (2.6%; appearing only in the Basque Country), linked to 4769A. For CSE, there were two private lineages, one defined by
Fig. 4. Reduced-median network of SW European mtDNA lineages displaying 16129A and/or 3010A 4733C. The sequence indicated by an asterisk bears the polymorphisms 3010A–16129A (in the coding regions and HVRI analysed here).
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Fig. 5. The effect of typing eight coding-region SNPs (in addition to HVRI) on the number and diversity of haplogroup H sequences in various west Eurasian populations. Ratios to the values calculated from HVRI alone are graphed. Population groups as defined in Section 2.
16092C–16293G–16311C (2.7%) and the other by 16051G– 16312G (also 2.7%), both in H*. As we sequenced coding-region stretches totalling 1580 bp, we detected further polymorphisms observed so far only in Iberia, which increase resolution of the relatively common HVRI haplotype defined by 16129A in this geographic region (Fig. 4). Those were the transitions at np 3849A, 4659A and 4733C, appearing in the background of 3010A; 3849A was previously described in two individuals from haplogroup U2 and 4733C in one haplogroup L3e1 individual [19]. On the right side of the network (Fig. 4), we observe four individuals who, most probably, have reverted at position 16129 (which is rather fast-evolving) [9]. Despite these instances, the increase both in number of different haplotypes and in haplotype diversity, due to the characterization of the eight coding-region polymorphisms, in relation to HVRI diversity, is broadly similar in all the population groups (Fig. 5), a fact which is particularly convenient for forensic applications.
It is worth detailing the gain of information obtained from HVRII and coding-region SNPs in the case of the HVRI-defined CRS haplotype, due to its high frequency (47.4% of the H haplotypes), which is split into the following most frequent sub-haplotypes: (a) 263G 315.1/3010A (13.9%); (b) 150T 263G 315.1/3010A (3.1%); (c) 152C 263G 315.1/3010A (4.1%); (d) 263G 315.1/6776C (8.3%); (e) 152C 263G 315.1/6776C (2.1%); (f) 263G 315.1/H* (3.6%). For the rest of the haplotypes, the splits defined by HVRII and coding-region SNPs sub-typing reveal 18 haplotypes with frequencies between 1.0% and 2.1% (i.e. shared between two and four individuals), and 83 unique haplotypes. It is also noteworthy that the information gain is not equally apportioned between the number of different haplotypes and haplotype diversity, the increase of the former being far more impressive than the latter. This fact is, however, not attributable to any artefact of the typing strategy employed. Indeed the new haplotypes are usually observed at low frequencies (reflecting the strongly star-like
3.3. The gain of information due to HVRII sequencing and coding-region SNPs In Fig. 6, we compare the gain of information provided by HVRII and/or the eight coding-region polymorphisms in the Portuguese sample, the only one for which the corresponding information is available. Both HVRII and the coding-region variation considerably increase the subcharacterization of H haplotypes. However, if we take into consideration that there are 23 polymorphic sites in HVRII compared with only eight coding-region polymorphisms (a ratio of 2.9), the proportion of the gain of information in both number of haplotypes and haplotype diversity due to HVRII is only roughly half that provided by the coding-region polymorphisms. Nevertheless, in absolute numbers, HVRII is more informative for haplotype discrimination.
Fig. 6. Increase in the discriminating power among mtDNA haplogroup H Portuguese samples, by typing successively HVRII and a set of coding-region SNPs in addition to HVRI sequence.
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phylogeny of haplogroup H) and therefore haplotype diversity changes very little.
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Peter and Anna Katharina Forster, Ariella Oppenheim, Sergei and Oksana Rychkov, and Gheorghe Stefanescu for samples and some DNA extractions.
4. Conclusions Appendix A. Supplementary data The publication of complete mtDNA sequences has led to many population studies at a large geographic scale, focused on the detailed characterization of certain haplogroups. This was the case for haplogroups L2 [20], V [21] and, more recently, H [8–10]. These data are particularly useful when developing coding-region based multiplexes for forensics with the aim of increasing the haplotype discrimination and forecasting their likely success rate across populations. Out of the previously described assays for H sub-characterization, the one developed by Quinta´ns et al. [6] is the most sensitive. The one developed by Vallone et al. [4], corresponding to the first out of the eight multiplexes indicated by Coble et al. [3] misses the informative polymorphism 6776C. We confirmed that the haplotype CRS/ 263G 315.1/6776C can reach a high frequency in western Eurasia (8.3% in the Portuguese sample). Moreover, the basal frequency of the haplotype CRS/263G 315.1 is also much higher (26.8%) than the one (7.1%) detected in the database used by Coble et al. [3]. Another important outcome of our analysis is related to the issue of population heterogeneity. It could happen, for instance, that a SNP typing assay, while increasing substantially the discrimination among a specific population, would be poorly informative in another one. Fortunately for the forensic field, that is not the case, at least within west Eurasia. The SNPs described here provide roughly the same gain, in terms both of haplotype numbers and haplotype diversity, in distinct populations (Fig. 3), and additionally reveal quite different sub-typing profiles. This can be of potential value for inferring the population or region of origin of DNA samples, as for Y-SNPs [22], as the phylogenetic dissection of mtDNA haplogroups is revealing gradients previously hidden, on the Eurasian scale [8–10]. We conclude that it is possible to increase considerably the discrimination power of current mtDNA typing, with the addition of a moderate number of coding-region SNPs (which can be carried out in a single PCR multiplex reaction, as in [6]), at a large geographic scale.
Acknowledgments This work was partially supported by a research grant to AG (SFRH/BD/16518/2004) from Fundac¸a˜o para a Cieˆncia e a Tecnologia and IPATIMUP by Programa Operacional Cieˆncia, Tecnologia e Inovac¸a˜o (POCTI), Quadro Comunita´rio de Apoio III. This study is included in the Project POCTI/ANT/45139/2002 financed by Fundac¸a˜o para a Cieˆncia e a Tecnologia (Eixo 2, Medida 2.3 do POCTI, QCA III). We thank Pierre-Marie Danze, Dimitar Dimitrov,
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.forsciint.2005.06.008.
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