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     

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     

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     

Mitochondrial DNA in human hair shafts – existence of intra-individual differences?

The sequences of the hypervariable region 1 (HV1) of the mitochondrial DNA control region from multiple hair shafts from 10 unrelated individuals were compared to determine the frequency of differences in hairs from one individual. The extraction method described herein showed an average success rate of 67% for all 150 hair shafts tested in HV1. The mtDNA sequences from the hair shafts matched the sequences from the corresponding blood and saliva samples taken from the same donor and no evidence of heteroplasmy was found. The results emphasize the reliability of DNA extraction and mtDNA typing from human hair shafts for forensic purposes. Received: 16 July 1998 / Received in revised form: 31 August 1998     

Mitochondrial DNA typing from human axillary, pubic and head hair shafts – success rates and sequence comparisons

The analysis of mitochondrial DNA (mtDNA) from shed hairs has gained high importance in forensic casework since telogen hairs are one of the most common types of evidence left at the crime scene. In this systematic study of hair shafts from 20 individuals, the correlation of mtDNA recovery with hair morphology (length, diameter, volume, colour), with sex, and with body localisation (head, armpit, pubis) was investigated. The highest average success rate of hypervariable region 1 (HV 1) sequencing was found in head hair shafts (75%) followed by pubic (66%) and axillary hair shafts (52%). No statistically significant correlation between morphological parameters or sex and the success rate of sequencing was found. MtDNA sequences of buccal cells, head, pubic and axillary hair shafts did not show intraindividual differences. Heteroplasmic base positions were observed neither in the hair shafts nor in control samples of buccal cells. Received: 18 January 1999 / Accepted: 15 February 1999     

MtDNA control region and RFLP data for Sicily and France

The forensic application of mtDNA typing requires large databases which are regionally well defined. To further this aim, we have typed mtDNA in a sample of 111 French and 106 Sicilians. The French were typed for both hypervariable segments (HVR1 and HVR2) of the mtDNA control region, whereas the Sicilians were only typed for HVR1, but in addition for the coding region RFLP markers for mtDNA groups H, I, J, K, L, M, T, U, V and X. In both samples, the predominant sequence type by far was the Cambridge reference sequence. Comparing HVR1 sequences, we found that the French sample was twice as diverse as the Sicilian sample as measured by sequence matches. A further set of sequence match comparisons including the French, Sicilian, and the published British mtDNA samples, demonstrate that sequence matching probabilities within samples differ by less than a factor of 2 from the matching probabilities between samples. Received: 10 February 2000 / Accepted: 13 May 2000     

Aerobic capacity of Polish soldiers

Performances on 1.5-mile and 12-minute runs were used to compare aerobic fitness in groups of Polish, Austrian, and U.S. male military members. The Polish soldiers achieved better times than either the Americans or the Austrians, and both Polish and Austrians logged better times than the Americans, a possible indication of better fitness. Non-smokers performed better than smokers. Since coronary heart disease (CHD) is the leading non-accidental cause of death in the U.S. Armed Forces, higher levels of physical fitness may decrease the incidence of CHD in the U.S. military. A healthy lifestyle and motivation seem to have positive effects on physical fitness.     

The application of mitochondrial DNA typing to the study of white Caucasian genetic identification

Summary Mitochondrial DNA (mtDNA) from 100 unrelated British White Caucasians was extracted, amplified and directly sequenced. Sequences of approximately 800 nucleotides were obtained from 2 hypervariable segments within the non-coding region of the mitochondrial genome. A total of 91 different sequences were observed with an average nucleotide diversity of 1.1%. The most diverse pair of sequences differed at 3.6% of their nucleotide (nt) sites. Comparison to a consensus reference sequence showed that each region was polymorphic to a similar extent. Different methods of genetic analysis were used to examine the variation in each region, including pairwise comparisons, which demonstrated that although the data did not fit a Poisson distribution, the fit was closer to a Negative Binomial distribution.     

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     

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.     

An annotated mtDNA database

We have compiled a database of mitochondrial DNA (mtDNA) control region, hypervariable regions 1 (HVR1) and 2 (HVR2) sequences of a total of 14,138 individuals compiled from 103 mtDNA publications before 1 January 2000, 13 data sets published in 2000 and 2001 and 2 unpublished data sets of Iraqi Kurds and Indians from Kerala. By contacting the authors and by other means, we have confirmed and corrected sequence errors, eliminated duplications and harmonised the sequence format. These changes affected all but 26 of the 116 publications. Furthermore, we have implemented a geographic information system (“mtradius”) which searches for closest matches to a given mtDNA control region sequence and displays them on a geographic map. A potential application is to estimate a chance matching probability when a forensic stain and a suspect have an identical mtDNA sequence: we suggest that the geographic area with the highest frequency of closely related mtDNA sequence types may be used to define a reference population to give the suspect the maximum benefit of doubt in accordance with the ceiling principle. Received: 14 August 2000 / Accepted: 22 December 2000     

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%).     

Population genetics of ten STR loci (AmpFℓSTR SGM plus) in Austria

A population study on the ten short tandem repeat (STR) loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA was performed on 204 unrelated Austrian Caucasians. The DNA was amplified by multiplex PCR using the AmpFℓSTR SGM plus kit. All loci met Hardy-Weinberg expectations. The combined power of exclusion for the ten STR loci was 0.999976. The results show that these loci are very useful for forensic purposes. Received: 28 February 2000 / Accepted: 22 May 2000     

Variability of mitochondrial DNA in a population sample from Iceland

Over the past decade investigations of human mitochondrial DNA (mtDNA) have considerably contributed to our knowledge about human evolution and migration. The genome of the Icelandic population is of special interest since Iceland has been genetically isolated for centuries. The sequence of the hypervariable regions HVS-I and HVS-II of the mtDNA control region was generated for 100 Icelandic individuals. A total of 75 different mtDNA sequences were observed, of which 19 sequences were shared by more than one individual, 16 sequences were shared by two individuals and two sequences were shared by three individuals; the most frequent haplotype (16129 A, 16239 T, 00263 G and 00315.1 C) was found six times. Both the genetic diversity (0.9925+/-0.0031) and the average number of pairwise nucleotide differences (7.371) were comparable with most of the other European populations. However, we found a smaller number of distinct mitochondrial lineages, suggesting that founder effects and genetic drift may have exerted a visible influence on the Icelandic genetic diversity. We compared these data with 1400 other European sequences from the D-Loop-BASE database. The paper discusses the evolutionary relationship between Icelandic and Central European mtDNA under due consideration of the historical context. Finally, our study has been aimed at increasing the number of mtDNA sequences available throughout the world and contributing to human genome investigations.     

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     

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     

Isolation and characterization of human DNA from mosquitoes (Culicidae)

Human DNA was prepared from mosquitoes (Culicidae) which were collected in a room shared by four human individuals. Several insects did not contain human blood and DNA preparation from them was not successful. However, high molecular weight human genomic DNA could be isolated from four insects. HLA-DQα and D1S80 analysis showed that the blood from one insect was a mixture from two persons, whereas the others contained blood from single individuals. Human DNA isolated 26 h after ingestion was still suitable for typing. These results showed that DNA isolated from mosquitoes is qualitatively and quantitatively sufficient for DNA typing and could be helpful to identify individuals involved in certain cases of body violence or captivity. Received: 3 November 1997 / Received in revised form: 21 July 1998     

Detection of the age-dependent 4977 bp deletion of mitochondrial DNA

In recent years a number of mitochondrial DNA (mtDNA) deletions have been detected in various tissues from individuals over 20 years of age. It has been postulated that these deletions are associated with natural aging. In order to determine whether a correlation exists between age and the amount of deleted 4977 bp mtDNA, we used two PCR reactions to study total DNA (nuclear and mitochondrial DNA) extracted from skeletal muscle (m. iliopsoas) obtained at autopsy from 93 individuals representing a wide age spectrum (range: 3 months–97 years). The primer pair L15/H15 was used to amplify a 533 bp fragment of intact mtDNA to determine the percentage of total DNA. A second PCR with the primer pair L35/H35 was then employed to amplify a 667 bp fragment of the deleted mtDNA. The amount of template DNA necessary to amplify the specific fragments of deleted mtDNA was found to decrease with age. Whereas no 4977 bp deletion could be detected in subjects under 20 years of age even with 1000 ng of total DNA, in individuals aged 21 to 30 years 1000 ng total DNA were sufficient. Only 1 ng total DNA was needed in all individuals over 70. Our results show that the 4977 bp deletion can be a useful marker of natural aging in human subjects. Received: 24 September 1996 / Received in revised form: 24 April 1997     

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.     

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.