[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org).
[Show abstract][Hide abstract] ABSTRACT: A voluntary collaborative exercise aiming at the mitochondrial analysis of canine biological samples was carried out in 2006-2008 by the Non-Human Forensic Genetics Commission of the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Genetics (ISFG). The participating laboratories were asked to sequence two dog samples (one bloodstain and one hair sample) for the mitochondrial D-loop region comprised between positions 15,372 and 16,083 using suggested primers and PCR conditions, and to compare their results against a reference sequence. Twenty-one participating laboratories reported a total of 67.5% concordant results, 15% non-concordant results, and 17.5% no results. The hair sample analysis presented more difficulty to the participants than the bloodstain analysis, with a high percentage (29%) failing to obtain a result. The high level of participation showed the interest of the community in the analysis of dog forensic samples but the results reveal that crucial methodological issues need to be addressed and further training is required in order to respond proficiently to the demands of forensic casework.
[Show abstract][Hide abstract] ABSTRACT: Allele frequencies and forensic parameters for six miniSTR autosomal loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639 and D17S1290) were obtained from a sample of 205 unrelated Basque autochthous individuals using the recently designed hexaplex amplification and typing system, “Midi-6”. No significant deviations from Hardy–Weinberg expectations were found. Due to the small PCR products (<200bp), the use of these miniSTRs can increase the probability that a degraded sample can be typed. Additionally, these systems can be used in routine paternity analyses where more markers are needed to increase the power of exclusion or in complex paternity cases (e.g. involving closely related individuals).
Full-text · Article · Oct 2007 · Forensic Science International
[Show abstract][Hide abstract] ABSTRACT: Allele frequencies and forensic parameters for six miniSTR autosomal loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) were obtained from a sample of 264 unrelated individuals from Spain. No significant deviations from Hardy-Weinberg expectations were found. Due to the small PCR products (<125 bp), the use of these non-CODIS (NC) miniSTRs can increase the probability that a degraded sample can be typed. Additionally, these systems can be used in routine paternity analyses where more markers are needed to increase the power of exclusion or in complex paternity cases (e.g. involving closely related individuals).
Full-text · Article · Jul 2007 · Forensic science international
[Show abstract][Hide abstract] ABSTRACT: The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.
Full-text · Article · Jun 2007 · Forensic science international
[Show abstract][Hide abstract] ABSTRACT: We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.
[Show abstract][Hide abstract] ABSTRACT: The mitochondrial DNA (mtDNA) working group of the GEP-ISFG carried out an inter-laboratory exercise consisting of the study of mixture stains (saliva/semen and blood/semen) in order to investigate the behaviour of these common forensic samples when analysing their mtDNA using standard sequencing methodology. All labs extracted the DNA by preferential lysis and amplified and sequenced the first hypervariable region I (HVS-I). The results showed high consensus between labs for the first fraction of the lysis but not for the second one. We also observed differences between mixtures prepared from different donors and different body fluids. The present study has important consequences for the analysis and interpretation of mtDNA mixtures.
Full-text · Article · Apr 2006 · International Congress Series
[Show abstract][Hide abstract] ABSTRACT: DNA typing was requested to investigate a presumptive cancer diagnosis error by confirming whether benign and cancerous prostatic tissue in the same presurgical haematoxylin and eosin stained slide belonged to the same person. After independent histological re-examination of the slide by a pathologist, manual slide dissection was used to guarantee independent and high recovery DNA isolation from each tissue section, avoiding carryover and background contamination. Nuclear DNA quantification performed by real time polymerase chain reaction (PCR) revealed the absence of human DNA for short tandem repeat (STR) typing. Mitochondrial DNA was only obtained by performing PCR of very short fragments ( approximately 100 bp), indicating high DNA degradation. Different low frequency hypervariable region I haplotypes were obtained from each tissue section (normal tissue section haplotype: 16224C, 16234T, 16311C, 16356C; cancer tissue section haplotype: 16256T, 16270T, 16293G). Only the normal tissue section haplotype matched that obtained from the patient's blood sample, indicating that the cancer tissue section originated from an unknown patient. These results supported the hypothesis of sample mix up during block processing or slide preparation by a carryover mechanism. Mitochondrial genetic typing is recommended to exclude the possibility of carryover artefacts when low DNA content and high degradation compromise conventional STR typing.
Full-text · Article · Feb 2005 · Journal of Clinical Pathology
[Show abstract][Hide abstract] ABSTRACT: Information on complete human mitochondrial DNA has revealed polymorphisms located in the coding region, which are useful for a more secure mtDNA haplogroup subdivision. We sequenced 1580 bp in the coding region informative for subclassification of haplogroup H, the most poorly resolved European haplogroup when studying only control-region variation. This revealed nine coding polymorphisms (at positions 3010, 3915, 3992, 4024, 4336, 4745, 4769, 4793 and 6776, besides the diagnostic position 7028) that allowed subclassification of around 80% of the 306 Iberian H samples in eight subhaplogroups. Frequencies for these haplogroups vary widely, with half of the samples sharing the substitution at position 3010 (but some structure is already apparent within this
subhaplogroup). The HVRI diversity suggested the following age estimates for some subhaplogroups: 3010: 13,400F3000 years; 6776: 8600F2800 years; 4336: 4700F2700 years; 3915: 17,300F9100 years. Those ages indicate that these H subhaplogroups may be informative for different time scales of European demographic history.
Full-text · Article · Apr 2004 · International Congress Series
[Show abstract][Hide abstract] ABSTRACT: The results of some forensic validation studies on the Y-PLEXk 6 Kit (Reliagene Technologies) are presented. The evaluation of specificity (including imbalanced male/female mixtures, DNA from microorganisms) and sensitivity (28–32 cycles) allowed us to conclude that the Y-Plex 6 Kit system is a sensitive and reproducible Y-STR multiplex system. However, some nonspecific PCR products amplified from female DNA were observed for the yellow channel.
Full-text · Article · Jan 2003 · International Congress Series
[Show abstract][Hide abstract] ABSTRACT: In this study, we have analysed the nucleotide sequence variation of the 12S rRNA mitochondrial gene (648–1601 bp) from five different populations (Spanish Caucasian, Autochthonous from the Basque Country, Chinese, New Guinea Highlander and Africans of Benin) by full sequencing of two overlapping PCR fragments using d-rhodamine cycle sequencing coupled with an ABI377 sequencer. Preliminary data indicate different patterns of sequence variation between Africans and non-Africans. Africans are much more polymorphic than non-African populations, which have only a very restricted subset of haplotypes. Furthermore, the greater part of Africans analysed showed two specific nucleotide substitutions (769G-A and 1018G-A) that were not observed in non-African individuals. In conclusion, the mtDNA 12SRNA gene in combination with other systems could be an interesting ethnic marker that could help to differentiate between African and non-African maternal lineages.
Full-text · Article · Jan 2003 · International Congress Series
[Show abstract][Hide abstract] ABSTRACT: Population data were generated for four tetrameric short tandem repeat loci systems (D8S1179, D16S539, D18S51 and D21S11) for a Spanish Caucasian population sample (n = 218-219 individuals) using PCR. All loci were highly polymorphic, met Hardy-Weinberg expectations and the results demonstrated the assumption of independence of the loci analysed. The allele frequency data can be used in identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Spanish population.
Full-text · Article · Feb 1999 · Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin
[Show abstract][Hide abstract] ABSTRACT: Allele and genotype frequencies for four tetrameric short tandem repeat loci were determined in a Spanish population sample (N=193-225) using PCR. All loci met Hardy-Weinberg expectations and the results demonstrated the assumption of independence of the loci analysed. The allele frequency data can be used in identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Spanish population.
Full-text · Article · Feb 1999 · Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin
[Show abstract][Hide abstract] ABSTRACT: Blood samples from 202-208 unrelated Basque Country autochthonous individuals were amplified, typed and their allele frequencies were determined. Results demonstrate the assumption of independence within and between the loci analyzed. Therefore, a Basque population database can be used in identity testing to estimate the frequency of a multiple PCR-based locus DNA profile.
Full-text · Article · Feb 1998 · Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin