E.F. Carvalho

A GEP-ISFG collaborative study on the optimization of an X-STR decaplex: data on 15 Iberian and Latin American populations

In a collaborative work carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG), a polymerase chain reaction multiplex was optimized in order to type ten X-chromosome short tandem repeats (STRs) in a single reaction, including: DXS8378, DXS9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7133, GATA172D05, GATA31E08, and DXS7423. Using this X-decaplex, each 17 of the participating laboratories typed a population sample of approximately 200 unrelated individuals (100 males and 100 females). In this work, we report the allele frequencies for the ten X-STRs in 15 samples from Argentina (Buenos Aires, Córdoba, Río Negro, Entre Ríos, and Misiones), Brazil (São Paulo, Rio de Janeiro, Paraná, and Mato Grosso do Sul), Colombia (Antioquia), Costa Rica, Portugal (Northern and Central regions), and Spain (Galicia and Cantabria). Gene diversities were calculated for the ten markers in each population and all values were above 56%. The average diversity per locus varied between 66%, for DXS7133, and 82%, for DXS6809. For this set of STRs, a high discrimination power was obtained in all populations, both in males (≥1 in 5 × 105) and females (≥1 in 3 × 109), as well as high mean exclusion chance in father/daughter duos (≥99.953%) and in father/mother/daughter trios (≥99.999%). Genetic distance analysis showed no significant differences between northern and central Portugal or between the two Spanish samples from Galicia and Cantabria. Inside Brazil, significant differences were found between Rio de Janeiro and the other three populations, as well as between São Paulo and Paraná. For the five Argentinean samples, significant distances were only observed when comparing Misiones with Entre Ríos and with Río Negro, the only two samples that do not differ significantly from Costa Rica. Antioquia differed from all other samples, except the one from Río Negro.

Stannous chloride mediates single strand breaks in plasmid DNA through reactive oxygen species formation.

Stannous ion (Sn) has been employed in nuclear medicine and in food industry. We described that Stannous Chloride (SnCl2) inactivation effect in Escherichia coli is mediated by a Fenton-like reaction. The effect of SnCl2 was studied through: (i) the alteration of plasmid topology in neutral and acidic pH by gel electrophoresis; and (ii) the transformation efficiency of an wild type E. coli strain. Treatment of plasmid DNA pUC 9.1 with SnCl2, at pH 7.4, results in DNA single-strand breaks (SSB), in a dose-dependent manner. Addition of sodium benzoate partly inhibited the DNA damage, while EDTA completely abolishes DNA-SSB. Furthermore, the ability of the plasmid to transform E. coli was reduced. At pH 1.3, SnCl2 exerts a protective effect on plasmid against HCI depurination. Our results suggest the generation of ROS, such as *OH by a Fenton-like reaction, close to the site of the lesions due to a possible complexation of stannous ion to DNA.

Revisiting the Genetic Ancestry of Brazilians Using Autosomal AIM-Indels

There are many different studies that contribute to the global picture of the ethnic heterogeneity in Brazilian populations. These studies use different types of genetic markers and are focused on the comparison of populations at different levels. In some of them, each geographical region is treated as a single homogeneous population, whereas other studies create different subdivisions: political (e.g., pooling populations by State), demographic (e.g., urban and rural), or ethnic (e.g., culture, self-declaration, or skin colour). In this study, we performed an enhanced reassessment of the genetic ancestry of ~ 1,300 Brazilians characterised for 46 autosomal Ancestry Informative Markers (AIMs). In addition, 798 individuals from twelve Brazilian populations representing the five geographical macro-regions of Brazil were newly genotyped, including a Native American community and a rural Amazonian community. Following an increasing North to South gradient, European ancestry was the most prevalent in all urban populations (with values up to 74%). The populations in the North consisted of a significant proportion of Native American ancestry that was about two times higher than the African contribution. Conversely, in the Northeast, Center-West and Southeast, African ancestry was the second most prevalent. At an intrapopulation level, all urban populations were highly admixed, and most of the variation in ancestry proportions was observed between individuals within each population rather than among population. Nevertheless, individuals with a high proportion of Native American ancestry are only found in the samples from Terena and Santa Isabel. Our results allowed us to further refine the genetic landscape of Brazilians while establishing the basis for the effective application of an autosomal AIM panel in forensic casework and clinical association studies within the highly admixed Brazilian populations.

Continent-Wide Decoupling of Y-Chromosomal Genetic Variation from Language and Geography in Native South Americans

Numerous studies of human populations in Europe and Asia have revealed a concordance between their extant genetic structure and the prevailing regional pattern of geography and language. For native South Americans, however, such evidence has been lacking so far. Therefore, we examined the relationship between Y-chromosomal genotype on the one hand, and male geographic origin and linguistic affiliation on the other, in the largest study of South American natives to date in terms of sampled individuals and populations. A total of 1,011 individuals, representing 50 tribal populations from 81 settlements, were genotyped for up to 17 short tandem repeat (STR) markers and 16 single nucleotide polymorphisms (Y-SNPs), the latter resolving phylogenetic lineages Q and C. Virtually no structure became apparent for the extant Y-chromosomal genetic variation of South American males that could sensibly be related to their inter-tribal geographic and linguistic relationships. This continent-wide decoupling is consistent with a rapid peopling of the continent followed by long periods of isolation in small groups. Furthermore, for the first time, we identified a distinct geographical cluster of Y-SNP lineages C-M217 (C3*) in South America. Such haplotypes are virtually absent from North and Central America, but occur at high frequency in Asia. Together with the locally confined Y-STR autocorrelation observed in our study as a whole, the available data therefore suggest a late introduction of C3* into South America no more than 6,000 years ago, perhaps via coastal or trans-Pacific routes. Extensive simulations revealed that the observed lack of haplogroup C3* among extant North and Central American natives is only compatible with low levels of migration between the ancestor populations of C3* carriers and non-carriers. In summary, our data highlight the fact that a pronounced correlation between genetic and geographic/cultural structure can only be expected under very specific conditions, most of which are likely not to have been met by the ancestors of native South Americans.

Population and mutation analysis of 17 Y-STR loci from Rio de Janeiro (Brazil)

The 17 Y chromosome STR loci DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460, DYS461, GATA C4, GATA H4 and GATA A10 were analyzed in a male sample of 126 unrelated individuals from Rio de Janeiro. No shared haplotypes were observed, demonstrating the usefulness and informative power of these Y-STRs in male lineage identification in Rio de Janeiro. Pairwise haplotype analysis showed no significant differences in the comparison of Rio de Janeiro with Iberian samples from different regions of Portugal and Spain, as well as with other Caucasian samples from South America, namely Costa Rica, Buenos Aires (Argentina) and São Paulo (Brazil). The same set of Y-STRs was also typed in 119 father/son pairs and among 2,023 allele transfers, 8 mutations were observed with an overall mutation rate of 0.003955±0.001396 per locus/meiosis across the 17 loci. Except in one case, all mutations were single step. For DYS438 a four-step mutation was found which has never been reported before, where allele 10 mutated to allele 6.

Population and segregation data on 17 Y-STRs: results of a GEP-ISFG collaborative study

A collaborative work was carried out by the Spanish and Portuguese International Society for Forensic Genetics Working Group in order to extend the existing data on Y-short tandem repeat (STR) mutations at the 17 Y chromosome STR loci included in the AmpFlSTR YFiler kit (Applied Biosystems): DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATA H4.1. In a sample of 701 father/son pairs, 26 mutations were observed among 11,917 allele transfers across the 17 loci. After summing previously reported mutation data with our sample, mutation rates varied between 4.25 × 10−4 (95% CI 0.05 × 10−3–1.53 × 10−3) at DYS438 and 6.36 × 10−3 (95% CI 2.75 × 10−3–12.49 × 10−3) at DYS458. All mutations were single step, and mutations in the same father/son pair were found twice.

Analysis of genetic ancestry in the admixed Brazilian population from Rio de Janeiro using 46 autosomal ancestry-informative indel markers.

The Brazilian population is highly heterogeneous as a result of five centuries of inter-ethnic mating between native Amerindians, European colonizers and Africans arrived during slavery. This study aimed to assess the proportions of inter-ethnic admixture in the Brazilian population of Rio de Janeiro using autosomal Ancestry-Informative Markers (AIMs). The autosomal data were also compared to the results expected from uniparental genetic markers. A total of 413 individuals were genotyped for 46 AIM-Indels and ancestry estimates were then assessed using HGDP-CEPH samples as ancestral reference. Individuals from Rio de Janeiro presented highly diverse admixture patterns. The global admixture estimates showed a predominantly European ancestry, above 55%, followed by African and Amerindian contributions. A separate self-declared Afro-descendant group also included in this study revealed an increased African ancestry, from ∼30% to ∼50%. The inter-ethnic admixture landscape of Rio de Janeiro captured by autosomal AIM-Indels is in agreement with historical records and similar to that expected from uniparental mtDNA and Y-chromosome information. The AIM-Indel panel proved to be a rapid strategy to estimate autosomal genetic ancestry at individual and population levels in Rio de Janeiro, which is useful in population genetics and in case-control association studies.

Sub-Saharan Africa descendents in Rio de Janeiro (Brazil): population and mutational data for 12 Y-STR loci.

A male sample of 135 African descendents from the Rio de Janeiro population were typed for the 12 Y-chromosome short tandem repeat (STR) loci included in the PowerPlex Y System. A high haplotype diversity was observed (0.9971), with 91% of haplotypes being unique, demonstrating the usefulness and informative power of this Y-STR set in male lineage identification. Samples with shared haplotypes were additionally typed with the Yfiler kit, which includes five extra markers. The haplotype diversity when using the 17-Yfiler loci increased to (0.9998) with 97% unique haplotypes. The same set of Y-STRs was also typed in 135 father/son pairs and three single-step mutations were observed: one at DYS19 and two at DYS385. Genetic distance analysis showed highly significant differences in all pairwise comparisons between this sample of African descendents and the general population from Rio de Janeiro, as well as with Iberian and African samples from Portugal, Mozambique, Angola and Equatorial Guinea. Comparisons with samples from other regions in Brazil showed that heterogeneity does exist, indicating that a Y-haplotype database for the whole country should take into account the population sub-structure. Moreover, a strong European influence was detected, and thus, a Y-chromosome STR profile proves a rather poor indicator for the ethnic origin of an individual in Rio de Janeiro.

Indel markers: Genetic diversity of 38 polymorphisms in Brazilian populations and application in a paternity investigation with post mortem material

Aiming to evaluate the usefulness of 38 non-coding bi-allelic autosomal indels in genetic identification and kinship testing, three Brazilian population samples were studied: two from Rio de Janeiro (including a sample of individuals with self-declared African ancestry) and one Native American population of Terena from Mato Grosso do Sul. Based on the observed allele frequencies, parameters of forensic relevance were calculated. The combined power of discrimination of the 38 indels was high in all studied groups (PD≥0.9999999999997), although slightly lower in Native Americans. Genetic distance analysis showed significant differences between the allele frequencies in the Rio de Janeiro population and those previously reported for Europeans, Africans and Asians explained by its intermediate position between Europeans and Africans. As expected, the Terena sample was significantly different from all the other populations: Brazilians from Rio de Janeiro general population and with self-declared African ancestry, Europeans, Africans and East Asians. Finally, the performance of the 38-indel multiplex assay was tested in post-mortem material with positive results, supporting the use of short amplicon bi-allelic markers as an additional tool to STR analysis when DNA molecules are degraded.

A New Cryptic Species of South American Freshwater Pufferfish of the Genus Colomesus (Tetraodontidae), Based on Both Morphology and DNA Data

The Tetraodontidae are an Acantomorpha fish family with circumglobal distribution composed of 189 species grouped in 19 genera, occurring in seas, estuaries, and rivers between the tropical and temperate regions. Of these, the genus Colomesus is confined to South America, with what have been up to now considered only two species. C. asellus is spread over the entire Amazon, Tocantins-Araguaia drainages, and coastal environments from the Amazon mouth to Venezuela, and is the only freshwater puffers on that continent. C. psittacus is found in coastal marine and brackish water environments from Cuba to the northern coast of South America as far south as to Sergipe in Brazil. In the present contribution we used morphological data along with molecular systematics techniques to investigate the phylogeny and phylogeography of the freshwater pufferfishes of the genus Colomesus. The molecular part is based on a cytochrome C oxidase subunit I dataset constructed from both previously published and newly determined sequences, obtained from specimens collected from three distinct localities in South America. Our results from both molecular and morphological approaches enable us to identify and describe a new Colomesus species from the Tocantins River. We also discuss aspects of the historical biogeography and phylogeography of the South American freshwater pufferfishes, suggesting that it could be more recent than previously expected.