Polymorphism of the mitochondrial DNA control region in Italians

Mitochondrial DNA sequences of the hypervariable regions HVI and HVII were analysed in 83 Caucasians living in central Italy to expand the database for forensic identification purposes, and 75 different haplotypes resulting from 62 polymorphic positions in HVI and 44 in HVII were observed. The most frequent haplotype (263G, 309.1C, 315.1C) was shared by 7 individuals, 2 haplotypes were shared by 2 individuals, and 72 were unique. The genetic diversity was found to be 0.99 and the random match probability 1.9%. A condition of sequence heteroplasmy was found in only one case at nt 16311, whereas a length heteroplasmy was found in the homopolymeric stretch of cytosines 303–315. Our results indicate that in direct sequencing beyond the poly-cytosine stretch, the overlap is due to length heteroplasmy, whereas the blurred signal occurs when the stretch is composed of more than 10 cytosines. Received: 14 February 2000 / Accepted: 23 May 2000     

Sequence polymorphisms of the mitochondrial DNA control region and phylogenetic analysis of mtDNA lineages in the Japanese population

Sequence polymorphisms of the hypervariable mitochondrial DNA (mtDNA) regions HVI and HVII, and coding region polymorphisms were investigated in 211 unrelated individuals from the Japanese population. Sequence comparison of the HVI and HVII regions led to the identification of 169 mitochondrial haplotypes defined by 147 variable positions. Among them 145 types were observed in only 1 individual; the other 24 types were shared by 2 or more individuals. The gene diversity was estimated at 0.9961, and the probability of two randomly selected individuals from the population having identical mtDNA types was 0.86%. We also established phylogenetic haplogroups in the Japanese population based on the coding and control region polymorphisms and compared the haplotypes with those in other Japanese, Korean and Chinese populations. As a result, three new subhaplogroups, G4a, G4b, and N9b, and several haplotypes specific for the Japanese and Korean populations were identified. The present database can be used not only for personal identification but also as an aid for geographic or phenotype (race) estimation in forensic casework in Japan.Electronic Supplementary Material Supplementary material is available for this article if you access the article at     

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.     

Mitochondrial DNA population data of HV1 and HV2 sequences from Japanese individuals

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were determined for 1204 unrelated Japanese individuals. A total of 741 different mtDNA haplotypes were found, 157 of which were seen in multiple individuals. Twenty-seven of these individuals showed point heteroplasmy. The most frequent haplotype (16223T-16362C-73G-263G-315.1C) was found in 31 individuals and the second most frequent haplotypes (16129A-16223T- 16362C-73G-152C-263G-309.1C-315.1C) was found in 24 individuals. The haplotypes diversity and random match probability were calculated to be 0.9969 and 0.0040, respectively.     

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 the mitochondrial DNA hypervariable regions I and II in 205 Singapore Malays

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were analyzed in 205 unrelated ethnic Malays residing in Singapore as an initial effort to generate a database for forensic identification purposes. Sequence polymorphism was detected using PCR and direct sequencing analysis. A total of 152 haplotypes was found containing 152 polymorphisms. Out of the 152 haplotypes, 115 were observed only once and 37 types were seen in multiple individuals. The most common haplotype (16223T, 16295T, 16362C, 73G, 146C, 199C, 263G, and 315.1C) was shared by 7 (3.41%) individuals, two haplotypes were shared by 4 individuals, seven haplotypes were shared by 3 individuals, and 27 haplotypes by 2 individuals. Haplotype diversity and random match probability were estimated to be 0.9961% and 0.87%, respectively.     

Identification of single nucleotide polymorphisms within the mtDNA genome of the domestic dog to discriminate individuals with common HVI haplotypes

We sequenced the entire ～16 kb canine mitochondrial genome (mtGenome) of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. We then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. Haplotype frequencies in our data set were highly correlated with previous ones (e.g., F(ST)=0.02, r=0.90), suggesting the total data set reasonably reflected the broader dog population. A total of 128 HVI haplotypes was represented. The 10 most common HVI haplotypes (n=184 dogs) represented 53.3% of the sample. We identified a total 71 SNPs in the mtGenomes (external to the control region) that resolved the 10 most common HVI haplotypes into 63 mtGenome subhaplotypes. The random match probability of the dataset based solely on the HVI sequence was 4%, whereas the random match probability of the mtGenome subhaplotypes was <1%. Thus, the panel of 71 SNPs identified in this study represents a useful forensic tool to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).     

Mitochondrial DNA sequence analysis of a native Bolivian population

Mitochondrial DNA analysis is very useful for the interpretation of the history of human migration and to estimate the frequency of a haplotype in the forensic context. From a human settlement perspective, La Paz area is greatly interesting since the first planned city of the region is located there. Samples from 110 individuals from La Paz were studied analysing the polymorphisms in the D-loop, hypervariable region I (HVI) and hypervariable region II (HVII) in order to verify the genetic diversity. The aim of this study was to start the creation of a population database in order to obtain the genetic interpopulation variability and classify haplotypes into characteristic haplogroups of South America. A total of 97 different haplotypes were identified, 90 being unique, expressed by 122 polymorphic nucleotide positions. Nucleotide and sequence diversity were estimated to be 0.015 ± 0.0075 and 0.996, respectively. Haplogroup distribution in the samples was 57.27% B4, 19.09% C1, 10.00% A2, 3.64% D1, 2.73% D4h3, 1.82% H, and 0.91% for each of the haplogroups A4, B4c1a, CZ, D4J, M7a and M8/N9b. The rate of length heteroplasmy was 36.36% in HVI and 52.73% in HVII. Phylogenetic analysis reveals proximity to the Korean, Chilean aboriginal, Japanese and Australian populations. The estimated genetic variability of the studied population was high, suggesting an early settlement.     

Forensic evaluation of mtDNA in a population from south west Switzerland

The polymorphism of the two hypervariable segments (HVI and HVII) of the control region of mtDNA was analyzed in a population of 154 unrelated individuals from south west Switzerland using a fluorescent based capillary electrophoresis sequencing method. In our population data of 154 random individuals, 137 mtDNA types were observed. Of these, 124 sequences were observed only in one individual whereas 10 sequences were observed in 2 individuals, 2 sequences in 3 individuals and 1 sequence in 4 individuals. The probability of two unrelated individuals having the same sequence was 0.84%. The results were compared with four other Caucasian populations. Furthermore, the usefulness of the mtDNA sequencing was tested, for exclusion and inclusion, in 18 forensic cases including 69 evidence samples and 44 reference samples. Despite the fact that 55% of the evidence samples yielded a negative result for the nuclear DNA with the human dot quantitation system, the success rate of the mtDNA sequencing was 71.0%. This validation study proves the great usefulness and sensitivity of the mtDNA sequencing technique using nested PCR and fluorescent capillary electrophoresis.     

Forensic mitochondrial coding region analysis for increased discrimination using pyrosequencing technology

Analysis of mitochondrial DNA (mtDNA) is very useful when nuclear DNA analysis fails due to degradation or insufficient amounts of DNA in forensic analysis. However, mtDNA analysis has a lower discrimination power compared to what can be obtained by nuclear DNA (nDNA) analysis, potentially resulting in multiple individuals showing identical mtDNA types in the HVI/HVII region. In this study, the increase in discrimination by analysis of mitochondrial coding regions has been evaluated for identical or similar HVI/HVII sequences. A pyrosequencing-based system for coding region analysis, comprising 17 pyrosequencing reactions performed on 15 PCR fragments, was utilised. This assay was evaluated in 135 samples, resulting in an average read length of 81 nucleotides in the pyrosequencing analysis. In the sample set, a total of 52 coding region SNPs were identified, of which 18 were singletons. In a group of 60 samples with 0 or 1 control region difference from the revised Cambridge reference sequence (rCRS), only 12 samples could not be resolved by at least two differences using the pyrosequencing assay. Thus, the use of this pyrosequencing-based coding region assay has the potential to substantially increase the discriminatory power of mtDNA analysis.     

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.     

Sequence polymorphism of the mitochondrial DNA control region in the population of Vojvodina Province,Serbia

In order to generate and establish the database for forensic identification purposes in Vojvodina Province (Serbia), the sequence of the hypervariable regions 1 (HV1) and 2 (HV2) of the mtDNA control region were determined in a population of 104 unrelated individuals from Vojvodina Province, using a fluorescent-based capillary electrophoresis sequencing method. A total of 93 different haplotypes were found, of these 83 mtDNA types were unique, nine haplotypes were shared by two individuals and one haplotype by three individuals. The variation of mtDNA HV1 and HV2 regions was confined to 116 nucleotide positions, of which 72 were observed in the HV1 and 44 in the HV2. A statistical estimate of the results for this population showed the genetic diversity of 0.9977 and the random match probability of 1.18%. Haplogroup H was the most common haplogroup (43.3%). Haplogroups observed at intermediate levels included clusters U (13.5%), T (10.6%), J (8.6%) and W (5.8%).     

Predicting sampling saturation of mtDNA haplotypes: an application to an enlarged Portuguese database

An enlarged mtDNA database (n=549) for the Portuguese population, comprising HVRI and HVRII regions is reported. This database was used to test the effect of sample size on the estimation of relevant parameters such as haplotype diversity, number of different haplotypes, nucleotide diversity and number of polymorphic positions. Simulations were performed generating sets of random subsamples of variable sizes (n=50, 100, 200, 300 and 400). The results show that while haplotype and nucleotide diversities do not vary significantly with sample size, the numbers of haplotypes and polymorphic positions rise continuously inside the tested interval. These trends are interpretable by the evolution of the proportions of sequences that are found once or twice, which drop dramatically as sample size increases, with the corresponding rise in the frequency of those encountered 3 times or more. The generated data were also used to extrapolate saturation curves for the referred parameters. When considering for instance the number of haplotypes, it is shown that a sample size of 1,000 individuals is required for practical saturation (defined as the point where a sample size increase of 100 individuals corresponds to an increment in the diversity measure below 5%). For HVRII the same level is reached at n=900 and n=1,300 is needed when both regions are analysed simultaneously. Consequently, we can infer that currently used sample sizes are still rather inadequate for both anthropological and forensic purposes.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00414-003-0424-1     

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.     

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.     

Sequence polymorphisms of mtDNA HV1, HV2 and HV3 regions in eight population groups living in Taiwan

Analysis of human mitochondrial DNA (mtDNA) polymorphisms in the D-loop region has become a useful tool in forensic casework and matrilineal origin research. In this study, the mtDNA D-loop region including hypervariable region 1 (HV1), hypervariable region 2 (HV2), segment between HV1 and HV2 (7S DNA spanned region), and extended hypervariable region 3 (HV3ex) was sequenced in 539 unrelated individuals from eight population groups living in Taiwan. Combined analyses of the complete D-loop revealed a total of 383 haplotypes with 319 unique haplotypes. The probability of any two individuals sharing the same mtDNA haplotype decreased as the combination of control region segments extended and reached 0.48% with the combination of a complete D-loop region. Sequence variants in HV3ex can further discriminate the haplotypes in some population groups. Phylogenetic haplogroups of these subjects were analyzed. The multidimensional scaling plots of these population groups, constructed based on sequence of the complete D-loop, demonstrated a clear matrilineal genetic substructure in this area. In conclusion, this database of mtDNA complete D-loop sequence including HV3 can serve as a reference for forensic identification. Sequence polymorphisms of the D-loop located outside the HV1 and HV2 may be helpful in further haplogroup characterization.     

Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4°C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month period. Electronic supplementary material Supplementary material is available for this article at .     

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     

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.     

The results of an mtDNA study of 1200 inhabitants of a German village in comparison to other Caucasian databases and its relevance for forensic casework

Mitochondrial DNA control region sequences were determined in 1200 male volunteers from one village area of Lower Saxony for the hypervariable region 1 (HV1). The 154 variable positions found resulted in 460 different haplotypes with a haplotype diversity value of 0.98165. The number of different haplotypes showed a nearly linear increase with the number of individuals typed. The haplotype diversity approached saturation level at a value of approximately 0.981 after typing 400 individuals. Furthermore, the number of different haplotypes and the haplotype diversity were calculated for four short amplicons of HV1 in order to establish the most variable section with a high efficiency for forensic casework. Received: 29 January 2000 / Accepted: 24 April 2000