The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives

This paper presents an overview of the organisation and the results of the collaborative exercises (CE) of the European DNA Profiling (EDNAP) Group's mitochondrial DNA population database project (EMPOP). The aim of the collaborative exercises was to determine whether uniformity of mtDNA sequencing results could be achieved among different laboratories. These were asked to sequence either the complete mtDNA control region or the two hypervariable regions HVI (16024-16365) and HVII (73-340) from DNA extracts, buccal swabs or bloodstains, proceeding in accordance with the protocol and strategies used in each individual laboratory. The results of the collaborative exercises were employed to identify possible sources of errors that could arise during the analysis and interpretation of mtDNA profiles. These findings were taken as a basis to tentatively make suitable arrangements for the construction of a high quality mtDNA database. One hundred fifty mtDNA profiles were submitted to the evaluating laboratory, and disaccording profiles were classified into four groups corresponding to the source of error: clerical errors, sample mix-ups, contaminations and discrepancies with respect to the mtDNA nomenclature. Overall, 14 disaccording haplotypes (16 individual errors) were observed. The errors included 10 clerical errors, 3 interpretation problems, 2 cases of sample mix-up and 1 case of point heteroplasmic mixture, where the 2 sequencing reactions brought inconsistent base calls. This corresponds to an error rate of 10.7% in a virtual mtDNA database consisting of the collaborative exercise results. However, this estimate is still conservative compared to conclusions drawn by authors of meanwhile numerous publications critically reviewing published mtDNA population databases. Our results and earlier published concerns strongly emphasize the need for appropriate safety regulations when mtDNA profiles are compiled for database purposes in order to accomplish the high standard required for mtDNA databases that are used in the forensic context.     

Mitochondrial DNA regions HVI and HVII population data.

Data from 1393 unrelated individuals have been compiled from eight population groups: African Americans, Africans (Sierra Leone), U.S. Caucasians, Austrians, French, Hispanics, Japanese, and Asian Americans. The majority of the mtDNA sequences were observed only once within each population group (i.e., ranging from a low of 60.3% (35/58) of the Asian American sequences to a high of 85.3% (93/109) of the French sequences). Genetic diversity ranged from 0.990 in the African sample to 0.998 in African Americans. Random match probability ranged from 2.50% in the Asian American sample to 0.52% in U.S. Caucasians. The average number of nucleotide differences between individuals in a database is greatest for the African American and African samples (14.1 and 13.1, respectively), and the least variable are the Caucasians (ranging from 7.2 to 8.4). Substitutions are the predominate polymorphism, and at least 92% of the substitutions are transitions. The most prevalent transversions are As substituted for Cs and Cs substituted for As. For most population groups these transversions occurred predominately in the HVI region; however, the African, African American, and Hispanic samples also demonstrated a large portion of their C to A and A to C transversions in the HVII region (at sites 186 and/or 189). Most insertions occur in the HVII region at sites 309.1 and 315.1, within a stretch of C's. Insertions of an additional C are common in all population groups. The sequence data were converted to SSO mtDNA types and compared with population data on Caucasians, Africans, Asians, Japanese, and Mexicans described by Stoneking et al. [M. Stoneking, D. Hedgecock, R.G. Higuchi, L. Vigilant, H.A. Erlich, Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes, Am. J. Hum. Genet. 48 (1991) 370-382] using an R x C contingency table test. Differences between major population groups (i.e., between African, Caucasian, and Asian) are quite evident, and similar ethnic population groups carried similar SSO polymorphism frequencies. There were only a few SSO types that showed significant differences between subpopulation groups. The SSO data alone can not be used to describe the population genetics with complete sequence data. However, the results of the SSO comparisons are similar to other analyses, and differences in sequence data in regions HVI and HVII are greater between major population groups than between subgroups.     

Finnish mitochondrial DNA HVS-I and HVS-II population data

We have analyzed the two hypervariable regions HVS-I and HVS-II of 200 Finnish male individuals for forensic purposes. The distribution of the haplotypes within Finland was determined by the geographical knowledge of the donors' maternal ancestors. In our population sample, we identified 135 different mtDNA haplotypes. Different mtDNA sequences were further divided to haplogroups using the EMPOP software. The most common haplogroups were H (40.0%) and U (27.5%). Subgroup U5b, which contains earlier described "Saami motif", consisted majority (65.5%) of the sample in the U haplogroup. Analysis of the mtDNA sequence hypervariable regions I and II showed that the mtDNA diversity within the Finnish population sample was comparable to other European populations and uniformly distributed. This is contrary to the Y-STR "minimal haplotype" diversity, which in Finland is lower than in any of the other European populations studied so far.     

Mitochondrial DNA in the Central European population. Human identification with the help of the forensic mt-DNA D-loop-base database

Sequencing of mtDNA is an advanced method for the individualisation of traces. Disadvantages of this method are expensive and time-consuming analysis and evaluation procedures as well as the necessary stock of population-genetic data which is still insufficient. Central European institutes of forensic medicine from Germany, Austria, and Switzerland have been working together since the beginning of 1998 to establish a mtDNA database. The aim is to build up a large stock of forensically established data and provide population-genetic data for frequency investigations, which will serve as a basis for expert opinions and scientific research. Good data quality is ensured by using original sequences only. Ring tests, which have been conducted to enhance analytical reliability, revealed a high correspondence rate of the analytical results obtained by the individual member institutes. Today 1410 sequences are available for comparison, of which 1285 sequences in the HV1 and HV2 regions cover the full ranges from 16051 to 16365 and from 73 to 340 (according to Anderson). The major part is formed by Central European sequences comprising 1256 data sets from Germany, Austria, and Switzerland. Today the database contains sequences from a total of 12 European, six African and three Asian countries including 100 sequences from Japan. This paper is aimed at discussing the individualisation potentials of mtDNA as well as the possibilities and limits of ethnic differentiation by means of pairwise sequence differences on the basis of the data stock available.     

Mitochondrial DNA population data of HVS-I and HVS-II sequences from a northeast German sample

Mitochondrial DNA sequences of the control region's two hypervariable regions HVS-I and HVS-II were determined for 213 unrelated west Eurasian individuals from northeast Germany (Mecklenburg). A total of 174 different mtDNA haplotypes were found, 25 of which were shared by more than 1 individual. The most frequent haplotypes were 263G-309.1C-315.1C, found in seven individuals, 263G-309.1C-309.2C-315.1C, found in six individuals and 263G-315.1C, found in five individuals. These sequences are also the most common haplotypes in other published European data sets. The sequence polymorphisms consisting of 150 polymorphic nucleotide positions were compared with other European databases. The genetic diversity and random match probability were calculated. Our results corroborate certain features which are characteristic for west Eurasian mtDNA population samples.     

Forensic genetic analysis of mitochondrial DNA hypervariable region I/II sequences: an expanded Korean population database

We have analyzed variation of the mitochondrial DNA (mtDNA) hypervariable segments I and II (HVS-I and HVS-II) in 185 randomly chosen individuals from Korea to provide an expanded and reliable Korean database. Combined sequence comparison of HVS-I and HVS-II led to the identification of 167 different haplotypes characterized by 154 variable sites. One hundred and fifty-one of the haplotypes were individual-specific, 14 were found in two individuals and 2 were found in three individuals. A pairwise comparison of the 185 HVS-I/II sequences found an average of 10.11 +/- 4.63 differences between individuals. The random match probability and gene diversity for the combined hypervariable regions were estimated at 0.66% and 0.9988, respectively. Analyzing the expanded database including three previously reported data sets and the present data using haplogroup-based comparisons and comparison with closely related sequences allowed errors to be detected and eliminated, thus considerably improving data quality. Sample division comparisons based on PhiST genetic distance measures revealed no significant population differentiation in the distribution of mtDNA sequence variations between the present data set and a database in The Scientific Working Group on DNA Analysis Methods (SWGDAM), but did indicate differences from other sets of data. Based on the results of mtDNA profiles, almost all of the mtDNA types studied here could be classified into subsets of haplogroups common in east Asia, and show that the Koreans possess lineages from both the southern and the northern haplogroup complexes of east Asian populations. The new data, combined with other mtDNA sequences, demonstrate how useful comparison with closely related mtDNA sequences can be for improving database quality, as well as providing haplotype information for forensic and population genetic analyses in the Korean population.     

Mitochondrial DNA control region population data from Macedonia

Mitochondrial DNA sequences of the entire control region were analyzed in 200 unrelated individuals from Macedonia. A total of 163 different haplotypes were found as determined by 177 polymorphic sites. The probability of a random match was calculated as 1:121 (0.83%). The basic phylogenetic structure of the Macedonian population as derived from its haplogroup distribution is in agreement with other West-Eurasian populations. Upon publication, the population data are going to be available in the EMPOP database (www.empop.org) [W. Parson, A. Dür, EMPOP—a forensic mtDNA database, FSI:Genetics 1 (2) (2007) 88–92; W. Parson, A. Brandstätter, A. Alonso, N. Brandt, B. Brinkmann, A. Carracedo, et al., The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives, Forensic Sci. Int. 139 (2–3) (2004) 215–226.].     

Polymorphic sites in human mitochondrial DNA control region sequences: population data and maternal inheritance

Sequence analysis of the mitochondrial DNA (mtDNA) control region is of central importance for forensic identity testing as well as for studies of human evolution. Here we report the sequencing data of the hypervariable regions I and II from 50 unrelated individuals from a western German population (Rhine area). In regions I and II, 52 and 26 sites of sequence polymorphism, respectively, were noted. Nucleotide substitution rather than insertion/deletion was the majority of variation. The distribution showed a large bias towards transitional changes than transversional changes. Furthermore we investigated uniparental inheritance in seven CEPH families each family with 7–9 maternal descendants. Most maternal relatives shared identical mtDNA sequences. Additionally sequences were compared for father:child pairs and as expected no evidence for paternal transmission of mtDNA was observed. The high variability of mtDNA control region sequences permits utility in forensic identity investigations. The data also indicate that the neomutation rate seems to be very low from one generation to the other.     

Mitochondrial DNA control region polymorphism in the population of Alagoas state, north-eastern Brazil

The sequences of the two hypervariable (HV) segments of the mitochondrial DNA (mtDNA) control region were determined in 167 randomly selected, unrelated individuals living in the state of Alagoas, north-eastern Brazil. One hundred and forty-five different haplotypes, associated with 139 variable positions, were determined. More than 95% of the mtDNA sequences could be allocated to specific mtDNA haplogroups according to the mutational motifs. Length heteroplasmy in the C-stretch HV1 and HV2 regions was observed in 22 and 11%, respectively, of the population sample. The genetic diversity was estimated to be 0.9975 and the probability of two random individuals presenting identical mtDNA haplotypes was 0.0084. The most frequent haplotype was shared by six individuals. All sequences showed high-quality values and phantom mutations were not detected. The diversity revealed in the mitochondrial control region indicates the importance of this locus for forensic casework and population studies within Alagoas, Brazil.     

Characterization of human control region sequences of the African American SWGDAM forensic mtDNA data set

The scientific working group on DNA analysis Methods (SWGDAM) mitochondrial DNA (mtDNA) population data set is used to infer the relative rarity of control region mtDNA profiles obtained from evidence samples and of profiles used for identification of missing persons. In this study, the African American haplogroup patterns in the SWGDAM data were analyzed in a phylogenetic context to determine relevant single nucleotide polymorphisms (SNPs) and to describe haplogroup distributions for Africans observed in these data sets. Over 200 SNPs (n=217) were observed in the African American data set (n=1148). These SNPs ranged from having 1-39 changes in the phylogenetic tree, with sites 152 and 16519 being the most variable. On average there were 5.8 changes for a character on the tree. The most variable sites (with 19 or more changes each) observed included 16093, 16129, 16189, 16311, 16362, 16519, 146, 150, 152, 189, and 195. These rapidly changing sites are consistent with other published analyses. Only 34 SNPs are needed to identify all clusters containing 10 or more individuals in the African American data set. The results show that the African American SWGDAM mtDNA data set contains variation consistent with that described in continental African populations. Thirteen of the 18 haplogroups previously observed in African populations were observed and include: L1a, L1b, L1c, L2a, L2b, L2c, L3b, L3d, L3e1, L3e2, L3e3, L3e4 and L3f. Haplogroup L2a is the most commonly observed cluster (18.8%) in the African American data set. The next most common haplogroups in the African American data set include the clusters L1c (11.0%), L1b (9.1%), L3e2 (9.0%) and L3b (8.1%). Approximately 8% of the haplogroups observed within African Americans were common in European Caucasians or East Asians; these were H (n=32), J (n=4), K (n=5), T (n=2), U5 (n=6), U6 (n=9 also known from North Africa), A (n=12), B (n=7), C (n=4), and M (n=16), respectively. The European Caucasian and East Asian haplogroups are expected due to admixture between individuals with recent ancestry in Western Eurasia and sub-Saharan Africa. The genetic characterization of these relevant data sets is fully consistent with other published mtDNA genetic variation. The sequence diversity observed in this data set makes it a valuable tool for forensic applications.     

Population data for 8 X-chromosome STR loci in a population sample from Northern Italy and from the Sardinia island

To establish a database for DXS8378, HPRTB, DXS7423, DXS7132, DXS10134, DXS10074, DXS10101, DXS10135 loci in an Italian population sample from Brescia (Northern Italy) and from the Sardinia island, 131 unrelated individuals were typed. DNA was amplified in a multiplex reaction with subsequent automatic detection using capillary electrophoresis. The obtained data give a contribution to the definition of Italian population STR allele frequencies for the eight analysed loci.     

Belgian canine population and purebred study for forensics by improved mitochondrial DNA sequencing

In canine population studies for forensics, the mitochondrial DNA is profiled by sequencing the two hyper variable regions, HV1 and HV2 of the control region.In a first effort to create a Belgian population database some samples showed partially poor sequence quality. We demonstrated that a nuclear pseudogene was co-amplified with the mtDNA control region. Using a new combination of primers this adverse result was no longer observed and sequencing quality was improved. All former samples with poor sequence data were reanalyzed. Furthermore, the forensic canine population study was extended to 208 breed and mixed dogs. In total, 58 haplotypes were identified, resulting in an exclusion capacity of 0.92. The profile distribution of the Belgian population sample was not significantly different from those observed in population studies of three other countries.In addition to the total population study 107 Belgian registered pedigree dogs of six breeds were profiled. Per breed, the obtained haplotypes were supplemented with those from population and purebred studies. The combined data revealed that some haplotypes were more or less prominent present in particular dog breeds. The statistically significant differences in haplotype distribution between breeds and population sample can have consequences on mtDNA databasing and matching probabilities in forensics.     

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.     

Mitochondrial diversity of a northeast German population sample

Mitochondrial DNA sequences of the hypervariable regions HV I and HV II were analyzed in 300 unrelated individuals born and living in the northeast corner of Germany (Western Pomerania) to generate a database for forensic identification purposes in this region. Sequence polymorphism were detected using PCR and direct sequencing analysis. A total of 242 different haplotypes were found as determined by 147 variable positions. The most frequent haplotype (263G, 315.1C) was found in 10 individuals and is also the most common sequence in Europe. Three other haplotypes were shared by 5 individuals, 2 sequences by 4, 8 haplotypes by 3, 15 sequences by 2 persons, and 213 sequences were unique. The genetic diversity was estimated to be 0.99 and the probability of two random individuals showing identical mitochondrial DNA (mtDNA) haplotypes is 0.6%. A comparison with other studies from Germany showed only little differences in the distribution of haplogroups. Nevertheless, one frequent haplotype in northeast Germany (five unrelated individuals) could only rarely be found in other German and European regions. Our results may indicate that despite a high admixture proportion in the German population some regions could demonstrate certain characteristic features.     

Detection of sequence variation in the HVII region of the human mitochondrial genome in 689 individuals using immobilized sequence-specific oligonucleotide probes

We have developed a rapid, immobilized probe-based assay for the detection of sequence variation in the hypervariable segment II (HVII) of the mitochondrial DNA (mtDNA) control region. Using a panel of 17 sequence-specific oligonucleotide (SSO) probes immobilized on nylon membrane strips, we typed 689 individuals from four population groups. The genetic diversity value for each population was calculated from the frequency data, and the frequencies of distinct "mitotypes" in each group were determined. We performed DNA sequence analysis of 129 samples to characterize the sequences associated with "blanks" (absence of probe signals) and weak probe signals. Out of 689 samples, we observed five heteroplasmic samples (excluding the variable C-stretch beginning at position 303) using the immobilized SSO probe panel. The SSO probe strips were used for the analysis of shed hairs and bloodstains from several criminal cases in Sweden, one of which is described here. We conclude that this mtDNA typing system is useful for human identification and significantly decreases casework turnaround time.     

Is it possible to differentiate mtDNA by means of HVIII in samples that cannot be distinguished by sequencing the HVI and HVII regions?

The mitochondrial control region includes three so-called hypervariable (HV) regions, in which the polymorphic positions show a particularly high frequency. According to a population study of 200 unrelated individuals from Germany, HVI (positions 16,024-16,365, according to Anderson) showed 88 variable positions in a total length of 342 bp (26%) and HVII (positions 73-340) displayed 65 mutable sites in 268 bp (24%). HVIII (positions 438-574) exhibited a slightly lower variability, with 25 polymorphic sites within 137 bp (18%), but contrasted clearly with the background, which showed variability rates of only 7% (positions 16,366-16,569, 1-72) and 3% (positions 341-437), respectively. At present, the displacement (D)-loop database in Magdeburg comprises 904 sequences of the mitochondrial HVI region and HVII region from Germans, Austrians and Swiss. By means of this material, the extent to which the mtDNA sequences that do not differ in the HVI and HVII regions can be differentiated by additionally sequencing HVIII was investigated.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.     

A call for mtDNA data quality control in forensic science

There is increasing evidence that many of the mitochondrial DNA (mtDNA) databases published in the fields of forensic science and molecular anthropology are flawed. An a posteriori phylogenetic analysis of the sequences could help to eliminate most of the errors and thus greatly improve data quality. However, previously published caveats and recommendations along these lines were not yet picked up by all researchers. Here we call for stringent quality control of mtDNA data by haplogroup-directed database comparisons. We take some problematic databases of East Asian mtDNAs, published in the Journal of Forensic Sciences and Forensic Science International, as examples to demonstrate the process of pinpointing obvious errors. Our results show that data sets are not only notoriously plagued by base shifts and artificial recombination but also by lab-specific phantom mutations, especially in the second hypervariable region (HVR-II).     

mtDNAprofiler: A Web Application for the Nomenclature and Comparison of Human Mitochondrial DNA Sequences,

Mitochondrial DNA (mtDNA) is a valuable tool in the fields of forensic, population, and medical genetics. However, recording and comparing mtDNA control region or entire genome sequences would be difficult if researchers are not familiar with mtDNA nomenclature conventions. Therefore, mtDNAprofiler, a Web application, was designed for the analysis and comparison of mtDNA sequences in a string format or as a list of mtDNA single‐nucleotide polymorphisms (mtSNPs). mtDNAprofiler which comprises four mtDNA sequence‐analysis tools (mtDNA nomenclature, mtDNA assembly, mtSNP conversion, and mtSNP concordance‐check) supports not only the accurate analysis of mtDNA sequences via an automated nomenclature function, but also consistent management of mtSNP data via direct comparison and validity‐check functions. Since mtDNAprofiler consists of four tools that are associated with key steps of mtDNA sequence analysis, mtDNAprofiler will be helpful for researchers working with mtDNA. mtDNAprofiler is freely available at http://mtprofiler.yonsei.ac.kr .     

Brazilian population data on the polymerase chain reaction-based loci LDLR,GYPA, HBGG,D7S8, and Gc

Gene and genotype frequencies in relation to the low density lipoprotein receptor (LDLR), glycophorin A (GYPA), hemoglobin G gammaglobin (HBGG), D7S8, and group specific component (Gc) loci were determined in a sample of 344 unrelated individuals (250 whites and 94 mulattoes) living in the city of S?o Paulo, Brazil. DNA was extracted from 5 mL of peripheral blood obtained from each of the 344 volunteers by the salting-out procedure. Polymerase chain reaction and reverse dot-blot analysis were performed with the Amplitype PM PCR Amplification and Typing Kit (Polymarker Multiplex; Applied Biosystems, Foster City, CA) under conditions recommended by the manufacturer. Estimated allele frequencies in the white sample were in the usual range of that of other United States and European population groups. In any case, genotype distributions for these loci did not deviate significantly from Hardy-Weinberg equilibrium proportions. Only 1 marginally significant (0.01 < P < 0.05) association, between loci HBGG and Gc, was detected in our mulatto sample out of a total of 20 possible pairwise comparisons of the 5 loci for both data sets. Allele frequencies were significantly different (P < 0.001) at the HBGG and Gc loci when the white and mulatto samples were compared. Biologic relationship exclusion probabilities (test powers) were calculated for the data. A Brazilian database has thus been established for the loci LDLR, GYPA, HBGG, D7S8, and Gc, 5 polymerase chain reaction-based loci systems that have been shown to be a useful tool for biologic relationship identification and exclusion.