M.F. Pinheiro

The GHEP–EMPOP collaboration on mtDNA population data—A new resource for forensic casework

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).

Forensic analysis of dog (Canis lupus familiaris) mitochondrial DNA sequences: an inter-laboratory study of the GEP-ISFG working group.

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.

Results of the GEP-ISFG collaborative study on two Y-STRs tetraplexes: GEPY I (DYS461, GATA C4, DYS437 and DYS438) and GEPY II (DYS460, GATA A10, GATA H4 and DYS439)

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)n, but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)2(TGTA)2(TCTA)n. The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems.

New clues to the evolutionary history of the main European paternal lineage M269: dissection of the Y-SNP S116 in Atlantic Europe and Iberia.

The dissection of S116 in more than 1500 individuals from Atlantic Europe and the Iberian Peninsula has provided important clues about the controversial evolutionary history of M269. First, the results do not point to an origin of M269 in the Franco–Cantabrian refuge, owing to the lack of sublineage diversity within M269, which supports the new theories proposing its origin in Eastern Europe. Second, S116 shows frequency peaks and spatial distribution that differ from those previously proposed, indicating an origin farther west, and it also shows a high frequency in the Atlantic coastline. Third, an outstanding frequency of the DF27 sublineage has been found in Iberia, with a restricted distribution pattern inside this peninsula and a frequency maximum in the area of the Franco–Cantabrian refuge. This entire panorama indicates an old arrival of M269 into Western Europe, because it has generated at least two episodes of expansion in the Franco–Cantabrian area. This study demonstrates the importance of continuing the dissection of the M269 lineage in different European populations because the discovery and study of new sublineages can adjust or even completely revise the theories about European peopling, as has been the case for the place of origin of M269.

Analysis of the R1b-DF27 haplogroup shows that a large fraction of Iberian Y-chromosome lineages originated recently in situ

Haplogroup R1b-M269 comprises most Western European Y chromosomes; of its main branches, R1b-DF27 is by far the least known, and it appears to be highly prevalent only in Iberia. We have genotyped 1072 R1b-DF27 chromosomes for six additional SNPs and 17 Y-STRs in population samples from Spain, Portugal and France in order to further characterize this lineage and, in particular, to ascertain the time and place where it originated, as well as its subsequent dynamics. We found that R1b-DF27 is present in frequencies ~40% in Iberian populations and up to 70% in Basques, but it drops quickly to 6–20% in France. Overall, the age of R1b-DF27 is estimated at ~4,200 years ago, at the transition between the Neolithic and the Bronze Age, when the Y chromosome landscape of W Europe was thoroughly remodeled. In spite of its high frequency in Basques, Y-STR internal diversity of R1b-DF27 is lower there, and results in more recent age estimates; NE Iberia is the most likely place of origin of DF27. Subhaplogroup frequencies within R1b-DF27 are geographically structured, and show domains that are reminiscent of the pre-Roman Celtic/Iberian division, or of the medieval Christian kingdoms.

Analysis of Penta D and Penta E STR loci in a Northern Portuguese population

Abstract Allele and genotype frequency data for two STR loci, Penta D and Penta E, were obtained from a sample of 291 unrelated inhabitants from Northern Portugal, with a commercial PCR-based typing kit. No significant deviations from Hardy–Weinberg equilibrium were found. There was no evidence for correlation between alleles of the two loci. Appropriate statistical evaluations revealed that these systems have a high forensic efficiency and can be very useful for personal identification among the Northern Portuguese population.