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     

Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population

The forensic application of mitochondrial DNA (mtDNA) typing requires large and regionally well-defined databases. To expand the database for forensic identification purposes in Slovenia, the mtDNA control region sequences of the hypervariable regions HVI and HVII were determined in a population of 129 maternally unrelated Slovenians, using a fluorescent-based capillary electrophoresis sequencing method. A total of 111 different haplotypes resulting from 124 polymorphic positions (80 polymorphic positions in HVI and 44 in HVII) were found. Of these, 101 mtDNA types were unique, 6 haplotypes were shared by 2 individuals, 1 haplotype by 3 individuals, 2 haplotypes by 4 individuals, and the most common haplotype was found in 5 individuals. The most frequent haplotypes in the Slovenian population ,263(G), 315.1(C) and 263(G), 309.1(C), 315.1(C) are also the most common in other European populations. The data support the concept that these haplotypes may represent a common European mtDNA sequence types. The sequence poymorphisms were compared to the databases of west Austria and central Italy and the HVI and HVII sequence matching probabilities within and between populations were calculated. It is 1.1–4.5 times more likely to find a sequence match in a random pair of Slovenians than in a random Slovenian-Italian pair and in a random Slovenian-Austrian pair. The length heteroplasmy in the homopolymeric C-stretch regions located at nucleotide positions 16184–16193 in HVI and at positions 303–315 in HVII was observed in 17% and 8% of individuals, respectively. A statistical estimate of the results for this population showed the random match probability and the genetic diversity of 1.16% and 0.996, respectively.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00414-003-0394-3     

Sequence diversity of the uniparentally transmitted portions of the genome in the resident population of Catalonia

Genomic reference databases of residing populations are available in different countries and regions. Since they represent the whole genetic diversity of a geographical region, they have wide applications, from biomedical studies to forensic identifications. Uniparentally transmitted portions of the genome specifically are highly suitable for kinship analyses, mixed DNA cases and geographical ancestry inferences. We have sampled 808 individuals currently residing in Catalonia within the GCAT cohort, from which we have generated 808 high-quality whole mitochondrial DNA (mtDNA) genomes and 399 sequences of the male-specific part of the Y chromosome (MSY). We observe higher genetic diversity than in classical population genetics datasets. We test the robustness of whole sequences for unequivocal identifications, and we found that they have higher resolution than mitochondrial control region and Y chromosome short tandem repeats (Y-STRs), and that most of the variants they present are at low frequencies, increasing the discrimination capacity between individuals. These results confirm the forensic applicability of whole uniparental sequences and provide one of the largest high-quality reference datasets ever published.     

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.     

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     

Sequence polymorphisms of mtDNA HV1, HV2, and HV3 regions in the Malay population of Peninsular Malaysia

The uniparentally inherited mitochondrial DNA (mtDNA) is in the limelight for the past two decades, in studies relating to demographic history of mankind and in forensic kinship testing. In this study, human mtDNA hypervariable segments 1, 2, and 3 (HV1, HV2, and HV3) were analyzed in 248 unrelated Malay individuals in Peninsular Malaysia. Combined analyses of HV1, HV2, and HV3 revealed a total of 180 mtDNA haplotypes with 149 unique haplotypes and 31 haplotypes occurring in more than one individual. The genetic diversity was estimated to be 99.47%, and the probability of any two individuals sharing the same mtDNA haplotype was 0.93%. The most frequent mtDNA haplotype (73, 146, 150, 195, 263, 315.1C, 16140, 16182C, 16183C, 16189, 16217, 16274, and 16335) was shared by 11 (4.44%) individuals. The nucleotide diversity and mean of pair-wise differences were found to be 0.036063 ± 0.020101 and 12.544022 ± 6.230486, respectively.     

Sequence polymorphism of mitochondrial DNA in Japanese individuals from Gifu Prefecture

Sequence polymorphisms of the hypervariable region HV1 in mitochondrial DNA (mtDNA) were analyzed in a sample of 137 unrelated Japanese individuals living in Gifu Prefecture (central region of Japan) using polymerase chain reaction amplification and direct sequencing. Eighty-two different haplotypes resulting from 81 variable sites were found in the mtDNA HV1 region between positions 16061 and 16450. The most frequent haplotype (16223T, 16362C) was shared by ten individuals. The genetic diversity and the genetic identity were 0.985 and 0.022, respectively. The C-stretch region located around position 16189 was observed in 23.4% of this population sample. Sequence heteroplasmy at the position 16103 (A/G) was found in one individual.     

Investigation of mtDNA control region sequences in an Egyptian population sample

The sequences of mitochondrial DNA (mtDNA) control region were investigated in 101 unrelated individuals living in the northern region of Nile delta (Gharbia, N = 55 and Kafrelsheikh, N = 46). DNA was extracted from blood stained filter papers or buccal swabs. HV1, HV2 and HV3 were PCR amplified and sequenced; the resulted sequences were aligned and compared with revised Cambridge sequence (rCRS). The results revealed presence of total 93 different haplotypes, 86 of them are unique and 7 are shared haplotypes, the most common haplotype, was observed with a frequency, 2.97% of population sample. High mtDNA diversity was observed with genetic diversity and power of discrimination, 0.9982 and 0.9883, respectively. In this dataset the west Eurasian haplogroups predominated over the African haplogroups. The results would be useful for forensic examinations and human genetic studies.     

Location and frequency of polymorphic positions in the mtDNA control region of individuals from Germany

In order to identify polymorphic positions and to determine their frequency in the human mitochondrial D-loop containing region, the mitochondrial DNA (mtDNA) control region of 200 unrelated individuals from Germany were amplified and directly sequenced. Sequence comparison led to the identification of 190 mitochondrial lineages as defined by 202 variable positions. The most frequently occurring lineage comprised 5 individuals, whereas 186 types of D-loop sequences were observed in only one individual. Of the sequences studied 7% are not unique but show at least one counterpart with an identical haplotype. The majority (61%) of the control regions investigated showed between four and eight nucleotide positions deviating from the reference sequence. The maximum number of deviations observed in a single control region was 18. The majority of the variable positions in the D-loop region (88%) are located within three hypervariable regions. Sequence variations are caused by nucleotide substitutions, insertions or deletions. As compared to insertions and deletions, nucleotide substitutions make up the vast majority of the mutations (90%). We have predominantly found transitions (75%) and a significantly lower frequency of transversions (15%) whereas insertions (6%) as well as deletions (4%) are rather rare. Upon sequencing the mitochondrial control region from 200 German Caucasians the genetic diversity was estimated at 0.99. The probability of two randomly selected individuals from a population having identical mtDNA types is 0.6%. Received: 21 May 1997 / Received in revised form: 9 September 1997     

Haplotype distribution in a forensic full mtDNA genome database of admixed Southern Brazilians and its association with self-declared ancestry and pigmentation traits

Background:The advent of massively parallel sequencing (MPS) applications focused on the generation of forensic-quality full mitochondrial genome sequences led to a popularization of the technique on a global scale. However, the lack of forensic-graded population databases has refrained a wider adoption of full genome sequences as the industry standard, despite its better discrimination capacity of individual maternal lineages.Purpose:This work describes a forensic-oriented full mtDNA genome database comprised of 480 samples from a Southern Brazilian population.Methods:A collection of mitochondrial sequences were obtained from low-pass, full genome DNA sequencing results. The complete sample set was evaluated regarding haplotype composition and distribution. Summary statistics and forensic parameters were calculated and are presented for the database, with detailed information concerning the impact of removing genetic information in the form of specific variants or increasingly larger genomic regions. Interpopulational analysis comparing haplotypical diversity in Brazilian and 26 worldwide populations was also performed. The association between mitochondrial genetic variability and phenotypic diversity was also evaluated in populations, with self-declared ancestry and three distinct phenotypic pigmentation traits (eyes, skin and hair colors) as parameters.Results:The presented database can be used to evaluate mitochondrial-related genetic evidence, providing LR values of up to 20,465 for unobserved haplotypes. Haplotype distribution in Southern Brazil seems to be different than the remaining of the country, with a larger contribution of maternal lines with European origin. Despite association can be found between lighter and darker phenotypes or self-declared ancestry and haplotype distribution, prediction models cannot be reliably proposed due to the admixed nature of the Brazilian population.Conclusions:The proposed database provides a basis for statistical calculation and frequency estimation of full mitochondrial genomes, and can be part of an integrated, representative, national database comprising most of the genetic diversity of maternal lineages in the country.     

A new database of mitochondrial DNA hypervariable regions I and II sequences from 162 Japanese individuals

A database of mitochondrial DNA (mtDNA) hypervariable region 1 (HV1) and region 2 (HV2) sequences of the mtDNA control region was established from 162 unrelated Japanese individuals. The random match probability and the genetic diversity for this database were 0.96% and 0.997, respectively. Length heteroplasmy in the C-stretch regions located around position 16189 in HV1 and 310 in HV2 was observed in 37% and 38% of the samples, respectively. A strategy using internal sequencing primers was devised to obtain confirmed sequences in these length heteroplasmic individuals. This database, combined with other mtDNA sequence databases from the Japanese population, will permit the significance of mtDNA match results to be properly reported in mtDNA typing casework in Japan.Received: 10 August 2000 / Accepted: 7 January 2001An erratum to this article can be found at     

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.     

A view of the maternal inheritance of Espírito Santo populations: The contrast between the admixed and Pomeranian descent groups

In addition to its valuable utility in forensic investigations, mitochondrial DNA (mtDNA) analysis is a reliable tool to uncover the origins of admixed populations, such as Brazilians. The state of Espírito Santo (ES), similar to other coastal Brazilian states, has a population shaped by 3 main ancestral roots: Amerindian, African and European. Among the latter, the Pomeranian descendants stand out due to the preservation of the traditional aspects of their culture, especially the Pomeranian language. Despite the genetic data already available, there is no mtDNA database that adequately reflects the diversity, the geographic distribution, and the origins of the maternal lineages from ES. To increase the knowledge of maternal ancestry and to investigate the population’s genetic stratification, a total of 291 samples were collected in the 4 macroregions (Metropolitan, South, Central and North) of ES and in the Pomeranian communities. Complete control region data were produced for the general (N=214) and Pomeranian (N=77) groups. Regarding the general population, the high values of haplotype diversity (H=99.9%) and pairwise differences (MNPD=16.9) found are in agreement with those reported for other populations in the southeast region of the country. Regarding maternal inheritance, the ES populations stood out due to the predominance of European haplogroups (49.5%), although the North macroregion had a higher African ancestry (47.1%). Among the Pomeranians, the lowest MNPD value (11.2) and the high percentage of shared haplotypes (15%) were indicative of founder events. The F_ST analysis showed that the Pomeranians (98.7% of European lineages) are genetically isolated from the other admixed populations in Brazil. This study demonstrated that the ES state contains singularities regarding the intrapopulational and interpopulational diversity of mtDNA. Even after 5 centuries of interethnic admixture, the present-day population of Espírito Santo harbors genetic marks that trace back to the historical aspects of its formation.     

Subtyping mtDNA haplogroup H by SNaPshot minisequencing and its application in forensic individual identification

Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1–H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs (∼70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis.     

Mitochondrial DNA heteroplasmy in the emerging field of massively parallel sequencing

Long an important and useful tool in forensic genetic investigations, mitochondrial DNA (mtDNA) typing continues to mature. Research in the last few years has demonstrated both that data from the entire molecule will have practical benefits in forensic DNA casework, and that massively parallel sequencing (MPS) methods will make full mitochondrial genome (mtGenome) sequencing of forensic specimens feasible and cost-effective. A spate of recent studies has employed these new technologies to assess intraindividual mtDNA variation. However, in several instances, contamination and other sources of mixed mtDNA data have been erroneously identified as heteroplasmy. Well vetted mtGenome datasets based on both Sanger and MPS sequences have found authentic point heteroplasmy in approximately 25% of individuals when minor component detection thresholds are in the range of 10–20%, along with positional distribution patterns in the coding region that differ from patterns of point heteroplasmy in the well-studied control region. A few recent studies that examined very low-level heteroplasmy are concordant with these observations when the data are examined at a common level of resolution. In this review we provide an overview of considerations related to the use of MPS technologies to detect mtDNA heteroplasmy. In addition, we examine published reports on point heteroplasmy to characterize features of the data that will assist in the evaluation of future mtGenome data developed by any typing method.     

Expanding the forensic German mitochondrial DNA control region database: genetic diversity as a function of sample size and microgeography

Mitochondrial DNA control region sequences were determined in 109 unrelated German Caucasoid individuals from north west Germany for both hypervariable regions 1 (HV1) and 2 (HV2) and 100 polymorphic nucleotide positions (nps) were found, 63 in HV1 and 37 in HV2. A total of 100 different mtDNA lineages was revealed, of which 7 were shared by 2 individuals and 1 by 3 individuals. The probability of drawing a HV1 sequence match within the north west Germans or within published sets of south Germans and west Austrians is similar (within a factor of 2) to drawing a sequence match between any two of these three population samples. Furthermore, HV1 sequences of 700 male inhabitants of one village in Lower Saxony were generated and these showed a nearly linear increase of the number of different haplotypes with increasing number of individuals, demonstrating that the commonly used haplotype diversity measure (Nei 1987) for population samples tends to underestimate mtDNA diversity in the actual population. Received: 14 January 1999 / Accepted: 15 February 1999     

Population and forensic genetic analyses of mitochondrial DNA control region variation from six major provinces in the Korean population

We generated complete mitochondrial DNA (mtDNA) control region sequences from 704 unrelated individuals residing in six major provinces in Korea. In addition to our earlier survey of the distribution of mtDNA haplogroup variation, a total of 560 different haplotypes characterized by 271 polymorphic sites were identified, of which 473 haplotypes were unique. The gene diversity and random match probability were 0.9989 and 0.0025, respectively. According to the pairwise comparison of the 704 control region sequences, the mean number of pairwise differences between individuals was 13.47 ± 6.06. Based on the result of mtDNA control region sequences, pairwise F_ST genetic distances revealed genetic homogeneity of the Korean provinces on a peninsular level, except in samples from Jeju Island. This result indicates there may be a need to formulate a local mtDNA database for Jeju Island, to avoid bias in forensic parameter estimates caused by genetic heterogeneity of the population. Thus, the present data may help not only in personal identification but also in determining maternal lineages to provide an expanded and reliable Korean mtDNA database. These data will be available on the EMPOP database via accession number EMP00661.     

mtDNA sequence diversity of Hazara ethnic group from Pakistan

The present study was undertaken to investigate mitochondrial DNA (mtDNA) control region sequences of Hazaras from Pakistan, so as to generate mtDNA reference database for forensic casework in Pakistan and to analyze phylogenetic relationship of this particular ethnic group with geographically proximal populations. Complete mtDNA control region (nt 16024-576) sequences were generated through Sanger Sequencing for 319 Hazara individuals from Quetta, Baluchistan. The population sample set showed a total of 189 distinct haplotypes, belonging mainly to West Eurasian (51.72%), East & Southeast Asian (29.78%) and South Asian (18.50%) haplogroups. Compared with other populations from Pakistan, the Hazara population had a relatively high haplotype diversity (0.9945) and a lower random match probability (0.0085). The dataset has been incorporated into EMPOP database under accession number EMP00680. The data herein comprises the largest, and likely most thoroughly examined, control region mtDNA dataset from Hazaras of Pakistan.     

Haplotype diversity in mitochondrial DNA hypervariable region in a population of southeastern Brazil

Brazilian population derives from Native Amerindians, Europeans, and Africans. Southeastern Brazil is the most populous region of the country. The present study intended to characterize the maternal genetic ancestry of 290 individuals from southeastern (Brazil) population. Thus, we made the sequencing of the three hypervariable regions (HV1, HV2, and HV3) of the mitochondrial DNA (mtDNA). The statistical analyses were made using Arlequin software, and the median-joining haplotype networks were generated using Network software. The analysis of three hypervariable regios showed 230 (79.3 %) unique haplotypes and the most common haplotype was “263G” carried by 12 (4.1 %) individuals. The strikingly high variability generated by intense gene flow is mirrored in a high sequence diversity (0.9966?±?0.0010), and the probability of two random individuals showing identical mtDNA haplotypes were 0.0068. The analysis of haplogroup distribution revealed that 36.9 % (n?=?107) presented Amerindian haplogroups, 35.2 % (n?=?102) presented African haplogroups, 27.6 % (n?=?80) presented European haplogroups, and one (0.3 %) individual presented East Asian haplogroup, evidencing that the southeastern population is extremely heterogeneous and the coexistence of matrilineal lineages with three different phylogeographic origins. The genetic diversity found in the mtDNA control region in the southeastern Brazilian population reinforces the importance of increased national database in order to be important and informative in forensic cases.     

Mitochondrial DNA control region variation in a population sample from Hong Kong,China

Entire mitochondrial control region sequences were generated from 377 unrelated individuals from urban Hong Kong. In line with other control region datasets from China, the sample from Hong Kong exhibited significant genetic diversity that was reflected in a random match probability of 0.19% and a mean pairwise difference of 13.14. A total of 305 haplotypes were identified, of which 262 were unique. These sequences will be made publicly available to serve as forensic mtDNA reference data for China.