Multiplex mtDNA coding region SNP assays for molecular dissection of haplogroups U/K and J/T

Mitochondrial DNA (mtDNA) U/K and J/T are sister haplogroups within the superhaplogroup R. They are both common in Europe, with a combined overall frequency similar to the one reported for H, the most common European haplogroup (40–50%). In this study, we selected 159 Italian subjects, already ascribed to U/K and J/T by RFLP typing, and assigned each mtDNA to specific clades/subclades by investigating at least one diagnostic coding region SNP. For each sister haplogroup, one multiplex PCR and one SNaPshot minisequencing reaction were set up targeting 16 U/K and 7 J/T coding region SNPs. Each mtDNA sample was clearly assigned to a specific subclade, which could be further subdivided into several minor sub-branches according to peculiar HVS I/II motifs.Such a molecular dissection of haplogroups U/K and J/T could be extremely useful to reduce the overall analysis time and labor intensive sequencing procedures in high volume forensic casework, for example when it is important to rapidly exclude samples in order to restrict the number of suspects.     

Complex interactions of the Eastern and Western Slavic populations with other European groups as revealed by mitochondrial DNA analysis

Mitochondrial DNA sequence variation was examined by the control region sequencing (HVS I and HVS II) and RFLP analysis of haplogroup-diagnostic coding region sites in 570 individuals from four regional populations of Poles and two Russian groups from northwestern part of the country. Additionally, sequences of complete mitochondrial genomes representing K1a1b1a subclade in Polish and Polish Roma populations have been determined. Haplogroup frequency patterns revealed in Poles and Russians are similar to those characteristic of other Europeans. However, there are several features of Slavic mtDNA pools seen on the level of regional populations which are helpful in the understanding of complex interactions of the Eastern and Western Slavic populations with other European groups. One of the most important is the presence of subhaplogroups U5b1b1, D5, Z1 and U8a with simultaneous scarcity of haplogroup K in populations of northwestern Russia suggesting the participation of Finno-Ugrian tribes in the formation of mtDNA pools of Russians from this region. The results of genetic structure analyses suggest that Russians from Velikii Novgorod area (northwestern Russia) and Poles from Suwalszczyzna (northeastern Poland) differ from all remaining Polish and Russian samples. Simultaneously, northwestern Russians and northeastern Poles bear some similarities to Baltic (Latvians) and Finno-Ugrian groups (Estonians) of northeastern Europe, especially on the level of U5 haplogroup frequencies. The occurrence of K1a1b1a subcluster in Poles and Polish Roma is one of the first direct proofs of the presence of Ashkenazi-specific mtDNA lineages in non-Jewish European populations.     

Mitochondrial DNA analysis of Swedish population samples

As a contribution to the geographic coverage of EMPOP, currently the best available forensic mitochondrial DNA (mtDNA) database, a total of 299 Swedish individuals were analysed by sequencing of the first and second hypervariable regions of the mtDNA genome. In this sample set, a total of 179 different haplotypes were detected. The genetic diversity was estimated to be 0.9895 (±0.0023), and the random match probability was 1.39 %. The most abundant haplogroups were HV (including its subhaplogroups H and V) with a frequency of 46.5 %, followed by haplogroup U (including its subhaplogroup K) at 27.8 %, haplogroup T at 10.0 % and haplogroup J at 7.0 %, a distribution that is consistent with previous observations in other European populations.     

Characterization of human control region sequences for Spanish individuals in a forensic mtDNA data set

Population data on the hypervariable regions of the mitochondrial DNA (mtDNA) genome are used to convey the relative rarity of mtDNA profiles obtained from evidence samples and of profiles used to identify missing persons. In this study, mtDNA profiles of Spanish individuals (n=312) were analyzed to describe haplogroup distributions and to determine relevant single nucleotide polymorphisms (SNPs) of those haplogroups. All nine common European haplogroups were observed in the sample, and these were divided into subgroups when possible. Haplogroup H was the most common haplogroup. The haplogroups U, J, T, and V were the next most frequent groups, each occurring at a frequency of 6.4% or greater. In addition, African and Asian sequences were present though rare in the samples. The data were compared with and found to be similar to other published data sets. There were 109 SNPs observed in the data set, including 10 positions not previously reported. The most variable sites are consistent with other studies.     

Homogeneity in mitochondrial DNA control region sequences in Swedish subpopulations

In order to promote mitochondrial DNA (mtDNA) testing in Sweden we have typed 296 Swedish males, which will serve as a Swedish mtDNA frequency database. The tested males were taken from seven geographically different regions representing the contemporary Swedish population. The complete mtDNA control region was typed and the Swedish population was shown to have high haplotype diversity with a random match probability of 0.5%. Almost 47% of the tested samples belonged to haplogroup H and further haplogroup comparison with worldwide populations clustered the Swedish mtDNA data together with other European populations. AMOVA analysis of the seven Swedish subregions displayed no significant maternal substructure in Sweden (F _ST = 0.002). Our conclusion from this study is that the typed Swedish individuals serve as good representatives for a Swedish forensic mtDNA database. Some caution should, however, be taken for individuals from the northernmost part of Sweden (provinces of Norrbotten and Lapland) due to specific demographic conditions. Furthermore, our analysis of a small sample set of a Swedish Saami population confirmed earlier findings that the Swedish Saami population is an outlier among European populations.     

A rapid mtDNA assay of 22 SNPs in one multiplex reaction increases the power of forensic testing in European Caucasians

We have developed a multiplex mitochondrial (mtDNA) assay of 21 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable region 1 (HVR1) and hypervariable region 2 (HVR2) that can be amplified in a single reverse touchdown polymerase chain reaction. Single base extension using the SNaPshot technique is also carried out as one multiplex. Besides the nine major European haplogroups (i.e. H, I, J, K, T, U, V, W, and X), 16 additional subclades (i.e. N1, X2, X2b, U2′-4/7′-9′, J/T, J1, J1c, HV, H1, H1a1, H1c, H3, H4, H6a, H7a H10) can be detected and classified into a phylogenetic mtDNA tree. By analyzing 130 Caucasoid samples from Germany, 36 different haplotypes were found resulting in a power of discrimination of 93.2%. Although 49% of all samples belonged to superhaplogroup H, the most common haplotype, i.e., haplogroup-specific SNPs plus haplogroup unspecific SNPs, had a frequency of only 18%. This assay is applicable for high-throughput mtDNA analysis and forensic mass screening. It will give additional information to the common control region sequencing of HVR1 and HVR2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: Application of mtDNA sequence analysis to a forensic case

The sequence of the two hypervariable segments of the mitochondrial DNA (mtDNA) control region was generated for 101 random Austrian Caucasians. A total of 86 different mtDNA sequences was observed, where 11 sequences were shared by more than 1 individual, 7 sequences were shared by 2 individuals and 4 sequences were shared by 3 individuals. One of the four most common mtDNA sequences in Austrians is also the most common sequence in both U.S. and British Caucasians, found in approximately 3.0% of Austrians, 4.0% of British, and 3.9% of U.S. Caucasians. Of the remaining three common Austrian sequences, one was not observed in either U.S. or British Caucasians. However, three British Caucasians exhibited a similar sequence type. Therefore, this particular cluster of sequence polymorphisms may represent a common “European” mtDNA sequence type. In general, Austrian Caucasians show little deviation from other Caucasian databases of European descent. Finally, mtDNA sequence analysis was applied to a forensic case, where hairs found at a crime scene matched the control hairs from the suspect. Received: 30 June 1997 / Received in revised form: 12 November 1997     

Mitochondrial DNA variation is associated with elite athletic status in the Polish population

There is mounting evidence that genetic factors located in mitochondrial and nuclear genomes influence sport performance. Certain mitochondrial haplogroups and polymorphisms were associated with the status of elite athlete, especially in endurance performance. The aim of our study was to assess whether selected mitochondrial DNA (mtDNA) and nuclear DNA variants are associated with elite athlete performance in a group of 395 elite Polish athletes (213 endurance athletes and 182 power athletes) and 413 sedentary controls. Our major finding was that the mtDNA haplogroup H and HV cluster influence endurance performance at the Olympic/World Class level of performance (P = 0.018 and P = 0.0185, respectively). We showed that two polymorphisms located in the mtDNA control region were associated with achieving the elite performance level either in the total athlete's group as compared with controls (m.16362C, 3.8% vs 9.2%, respectively, P = 0.0025, odds ratio = 0.39, 95% confidence interval: 0.21–0.72), or in the endurance athletes as compared with controls (m.16080G, 2.35% vs 0%, respectively, P = 0.004). Our results indicate that mtDNA variability affects the endurance capacity rather than the power one. We also propose that mtDNA haplogroups and subhaplogroups, as well as individual mtDNA polymorphisms favoring endurance performance, could be population‐specific, reflecting complex cross‐talk between nuclear and mitochondrial genomes.     

HVI and HVII mitochondrial DNA data in Apaches and Navajos

Most mtDNA studies on Native Americans have concentrated on hypervariable region I (HVI) sequence data. Mitochondrial DNA haplotype data from hypervariable regions I and II (HVI and HVII) have been compiled from Apaches (N=180) and Navajos (N=146). The inclusion of HVII data increases the amount of information that can be obtained from low diversity population groups. Less mtDNA variation was observed in the Apaches and Navajos than in major population groups. The majority of the mtDNA sequences were observed more than once; only 17.8% (32/180) of the Apache sequences and 25.8% of the Navajo sequences were observed once. Most of the haplotypes in Apaches and Navajos fall into the A and B haplogroups. Although a limited number of haplogroups were observed, both sample populations exhibit sufficient variation for forensic mtDNA typing. Genetic diversity was 0.930 in the Apache sample and 0.963 in the Navajo sample. The random match probability was 7.48% in the Apache sample and 4.40% in the Navajo sample. The average number of nucleotide differences between individuals in a database is 9.0 in the Navajo sample and 7.7 in the Apache sample. The data demonstrate that mtDNA sequencing can be informative in forensic cases where Native American population data are used.     

Hungarian mtDNA population databases from Budapest and the Baranya county Roma

To facilitate forensic mtDNA testing in Hungary, we have generated control region databases for two Hungarian populations: 211 individuals were sampled from the urban Budapest population and 208 individuals were sampled from a Romani (“gypsy”) population in Baranya county. Sequences were generated using a highly redundant approach to minimize potential database errors. The Budapest population had high sequence diversity with 180 lineages, 183 polymorphic positions, and a random match probability of 1%. In contrast, the Romani population exhibited low sequence diversity, with only 56 lineages, 109 segregating sites, and a random match probability of 8.8%. The mtDNA haplogroup compositions of the two populations were also distinct, with the large proportion of haplogroup M samples (35%) in the Roma the most obvious difference between the two populations. These factors highlight the importance of considering population structure when generating reference databases for forensic testing purposes. Comparisons between our Romani population sample and other published data indicate the need for heightened caution when sampling and using mtDNA databases of small endogamous populations. The Romani populations that we compared showed significant departures from genetic uniformity. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Amerindian mitochondrial DNA haplogroups predominate in the population of Argentina: towards a first nationwide forensic mitochondrial DNA sequence database

The study presents South American mitochondrial DNA (mtDNA) data from selected north (N = 98), central (N = 193) and south (N = 47) Argentinean populations. Sequence analysis of the complete mtDNA control region (CR, 16024–576) resulted in 288 unique haplotypes ignoring C-insertions around positions 16193, 309, and 573; the additional analysis of coding region single nucleotide polymorphisms enabled a fine classification of the described lineages. The Amerindian haplogroups were most frequent in the north and south representing more than 60% of the sequences. A slightly different situation was observed in central Argentina where the Amerindian haplogroups represented less than 50%, and the European contribution was more relevant. Particular clades of the Amerindian subhaplogroups turned out to be nearly region-specific. A minor contribution of African lineages was observed throughout the country. This comprehensive admixture of worldwide mtDNA lineages and the regional specificity of certain clades in the Argentinean population underscore the necessity of carefully selecting regional samples in order to develop a nationwide mtDNA database for forensic and anthropological purposes. The mtDNA sequencing and analysis were performed under EMPOP guidelines in order to attain high quality for the mtDNA database.     

Helena,the hidden beauty: Resolving the most common West Eurasian mtDNA control region haplotype by massively parallel sequencing an Italian population sample

The analysis of mitochondrial (mt)DNA is a powerful tool in forensic genetics when nuclear markers fail to give results or maternal relatedness is investigated. The mtDNA control region (CR) contains highly condensed variation and is therefore routinely typed. Some samples exhibit an identical haplotype in this restricted range. Thus, they convey only weak evidence in forensic queries and limited phylogenetic information. However, a CR match does not imply that also the mtDNA coding regions are identical or samples belong to the same phylogenetic lineage. This is especially the case for the most frequent West Eurasian CR haplotype 263G 315.1C 16519C, which is observed in various clades within haplogroup H and occurs at a frequency of 3–4% in many European populations.In this study, we investigated the power of massively parallel complete mtGenome sequencing in 29 Italian samples displaying the most common West Eurasian CR haplotype – and found an unexpected high diversity. Twenty-eight different haplotypes falling into 19 described sub-clades of haplogroup H were revealed in the samples with identical CR sequences. This study demonstrates the benefit of complete mtGenome sequencing for forensic applications to enforce maximum discrimination, more comprehensive heteroplasmy detection, as well as highest phylogenetic resolution.     

Confirmation of Y haplogroup tree topologies with newly suggested Y-SNPs for the C2, O2b and O3a subhaplogroups

Y chromosome single nucleotide polymorphisms (Y-SNPs) are useful markers for reconstructing male lineages through hierarchically arranged allelic sets known as haplogroups, and are thereby widely used in the fields such as human evolution, anthropology and forensic genetics. The Y haplogroup tree was recently revised with newly suggested Y-SNP markers for designation of several subgroups of haplogroups C2, O2b and O3a, which are predominant in Koreans. Therefore, herein we analyzed these newly suggested Y-SNPs in 545 unrelated Korean males who belong to the haplogroups C2, O2b or O3a, and investigated the reconstructed topology of the Y haplogroup tree. We were able to confirm that markers L1373, Z1338/JST002613-27, Z1300, CTS2657, Z8440 and F845 define the C2 subhaplogroups, C2b, C2e, C2e1, C2e1a, C2e1b and C2e2, respectively, and that markers F3356, L682, F11, F238/F449 and F444 define the O subhaplogroups O2b1, O2b1b, O3a1c1, O3a1c2 and O3a2c1c, respectively. Among six C2 subhaplogroups (C2b, C2e, C2e1*, C2e1a, C2e1b and C2e2), the C2e haplogroup and its subhaplogroups were found to be predominant, and among the four O2b subhaplogroups (O2b*, O2b1*, O2b1a and O2b1b), O2b1b was most frequently observed. Among the O3a subhaplogroups, O3a2c1 was predominant and it was further divided into the subhaplogroups O3a2c1a and O3a2c1c with a newly suggested marker. However, the JST002613-27 marker, which had been known to define the haplogroup C2f, was found to be an ancestral marker of the C2e haplogroup, as is the Z1338 marker. Also, the M312 marker for the O2b1 haplogroup designation was replaced by F3356, because all of the O2b1 haplotypes showed a nucleotide change at F3356, but not at M312. In addition, the F238 marker was always observed to be phylogenetically equivalent to F449, while both of the markers were assigned to the O3a1c2 haplogroup. The confirmed phylogenetic tree of this study with the newly suggested Y-SNPs could be valuable for anthropological and forensic investigations of East Asians including Koreans.     

Mitochondrial diversity in Amerindian Kichwa and Mestizo populations from Ecuador

This study presents mitochondrial DNA (mtDNA) data from 107 unrelated individuals from two of the major ethnic groups in Ecuador: Amerindian Kichwas (n = 65) and Mestizos (n = 42). We characterized the diversity of the matrilineal lineages of these Ecuadorian groups by analyzing the entire mtDNA control region. Different patterns of diversity were observed in the two groups as result of the unique historical and demographic events which have occurred in each population. Higher genetic diversity values were obtained for the Mestizo group than for the Amerindian group. Interestingly, only Native American lineages were detected in the two population samples, but with differences in the haplogroup distribution: Kichwa (A, 49%; B, 3%; C, 8%; and D, 40%) and Mestizo (A, 33%; B, 33%; C, 10%; and D, 24%). Analysis of the complete mtDNA control region proved to be useful to increase the discrimination power between individuals who showed common haplotypes in HVSI and HVSII segments; and added valuable information to the phylogenetic interpretation of mtDNA haplotypes.     

Mitochondrial DNA analysis of a Viking age mass grave in Sweden

In 1998, a Viking Age mass grave was discovered and excavated at St. Laurence´s churchyard in Sigtuna, Sweden. The excavated bones underwent osteoarchaeological analysis and were assigned to at least 19 individuals. Eleven skeletons showed sharp force trauma from bladed weapons. Mass graves are an unusual finding from this time period, making the burial context extraordinary. To investigate a possible maternal kinship among the individuals, bones and teeth from the skeletal remains were selected for mitochondrial DNA (mtDNA) analysis. Sanger sequencing of short stretches of the hypervariable segments I and II (HVS-I and HVS-II) was performed. A subset of the samples was also analysed by massively parallel sequencing analysis (MPS) of the entire mtDNA genome using the Precision ID mtDNA Whole Genome Panel. A total of 15 unique and three shared mtDNA profiles were obtained. Based on a combination of genetic and archaeological data, we conclude that a minimum of 20 individuals was buried in the mass grave. The majority of the individuals were not maternally related. However, two possible pairs of siblings or mother-child relationships were identified. All individuals were assigned to West Eurasian haplogroups, with a predominance of haplogroup H. Although the remains showed an advanced level of DNA degradation, the combined use of Sanger sequencing and MPS with the Precision ID mtDNA Whole Genome Panel revealed at least partial mtDNA data for all samples.     

Mitochondrial DNA control region data reveal high prevalence of Native American lineages in Jujuy province,NW Argentina

Mitochondrial control region (16024-576) sequences were generated from 180 individuals of four population nuclei from the province of Jujuy (NW Argentina), located at different altitudes above sea level. The frequency at which a randomly selected mtDNA profile would be expected to occur in the general population (random match probability) was estimated at 0.011, indicating a relatively high diversity. Analysis of the haplogroup distribution revealed that Native American lineages A2 (13.9%), B (56.7%), C1 (17.8%), D1 (8.9%) and D4h3a (1.1%) accounted for more than 98% of the total mtDNA haplogroup diversity in the sample examined. We detected a certain degree of genetic heterogeneity between two subpopulations located at different points along the altitudinal gradient (Valles and Puna), suggesting that altitude above sea level cannot be ruled out as a factor promoting divergences in mtDNA haplogroup frequencies, since altitude is closely associated with human living conditions, and consequently, with low demographic sizes and the occurrence of genetic drift processes in human communities. In all, mitochondrial DNA database obtained for Jujuy province strongly points to the need for creating local mtDNA databases, to avoid bias in forensic estimations caused by genetic substructuring of the populations.     

Italian mitochondrial DNA database: results of a collaborative exercise and proficiency testing

This work is a review of a collaborative exercise on mtDNA analysis undertaken by the Italian working group (Ge.F.I.). A total of 593 samples from 11 forensic genetic laboratories were subjected to hypervariable region (HVS-I/HVS-II) sequence analysis. The raw lane data were sent to MtDNA Population Database (EMPOP) for an independent evaluation. For the inclusion of data for the Italian database, quality assurance procedures were applied to the control region profiles. Only eight laboratories with a final population sample of 395 subjects passed the quality conformance test. Control region haplogroup (hg) assignments were confirmed by restriction fragment length polymorphism (RFLP) typing of the most common European hg-diagnostic sites. A total of 306 unique haplotypes derived from the combined analysis of control and coding region polymorphisms were found; the most common haplotype--CRS, 263, 309.1C, 315.1C/ not7025 AluI--was shared by 20 subjects. The majority of mtDNAs detected in the Italian population fell into the most common west Eurasian hgs: R0a (0.76%), HV (4.81%), H (38.99%), HV0 (3.55%), J (7.85%), T (13.42%), U (11.65%), K (10.13%), I (1.52%), X (2.78%), and W (1.01%).     

Mitochondrial DNA sequences for 118 individuals from northeastern Spain

A population database was generated from 118 unrelated Caucasoid individuals living in Spain. Sequence polymorphisms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II (HVRI and HVRII) were determined using the polymerase chain reaction (PCR) and direct sequencing. A total of 102 different sequences were found as defined by 105 variable positions. The most common sequence occurred six times, and this sequence is also the most frequent in other European populations such as Austria, Germany and Britain. The mean pair-wise difference for the two HVR regions taken together was 7.74. The study revealed that transitions made up the majority of the variations (88%), whereas we observed a significantly lower frequency of transversions (8%). Also one individual in this study was observed with two positions of heteroplasmy at nucleotides 150 (C/T) and 153 (G/A). A statistical estimate of the results for this population showed a genetic diversity of 0.99. The probability of two random individuals showing identical mtDNA haplotypes is 1.3%. In order to use the mtDNA analysis in forensic casework, we consider that it is of crucial importance to know the frequency of the different sequences of mtDNA, and this data base study could be a useful tool to statistically evaluate the results. Received: 12 July 1999 / Accepted: 11 April 2000     

Mitochondrial DNA control region variation in an autochthonous Basque population sample from the Basque Country

Mitochondrial control region (16024-576) sequences were generated from 106 samples from autochthonous Basques from the Autonomous Community of the Basque Country. It is especially important to generate mtDNA databases from isolated populations in order to maximize the power of discrimination of this molecular marker. It also represents a useful approach to carry out a more accurate haplogroup classification. This is the first database report of complete control region sequences in an autochthonous Basque population sample. Strict selection criteria of autochthonous individuals, automation of laboratory processing and independent reviews of the raw electropherograms ensure the high quality of these sequences and their utility as reference population data of the autochthonous Basque population.     

Recent progress in mitochondrial DNA analysis

In this review, we describe the current state of knowledge of mitochondrial genetics of East Asian populations and its application to forensic science. Recent advances in mitochondrial DNA (mtDNA) phylogeny have identified haplogroup-specific single nucleotide polymorphisms (SNPs) and the control region motifs of haplogroups. By analyzing haplogroup-specific SNPs, we can rapidly and accurately connect the mtDNA under study to the relevant haplogroup. Haplogroups are fairly continent- and/or region-specific; therefore, we can infer the ethnic background of that mtDNA. In addition, errors in hypervariable region sequences can be detected by means of haplogroup motif analysis.