Population structure and forensic genetic analyses in Guarani and Kaingang Amerindian populations from Brazil.

When DNA profile comparisons between a crime scene trace and a reference sample generate correspondence, the match probability has to be estimated, so that evaluation of the strength of the forensic DNA evidence can be made. The random match probability estimations require information on allele frequencies and an adjustment factor, referred to as theta (θ) or Fst, a co-ancestry correction factor for subpopulation effects. The θ value has been standardized for urban and isolated populations, but inconsistencies have been reported when it is specifically calculated for smaller and isolated populations, including Amerindian populations. Notably, attempts to characterize forensic markers of these minor populations have been extensively limited and more conservative estimates of the correction factor may be generated for each of them. Therefore, we estimate allele frequencies of 21 autosomal STR markers used for forensic testing and calculated relevant forensic parameters for the set. In addition, we featured the possible structure of five Brazilian Amerindian populations that have been genetically isolated for centuries so we could obtain the appropriate θ value for them. The sample consisted of 319 individuals: (1) 121 Kaingang, from two communities: Ivaí (KIV=61) and Rio das Cobras (KRC=60); and (2) 198 Guaranis from three communities: Mbya from Rio das Cobras (GRC=51), Guarani Ñandeva (GND=71) and Guarani Kaiowá (GKW=76). Between Guarani populations low (Rst=0.0402, p < 10-4) to high (Rst=0.1557, p < 10-5) differentiation was found. Regarding Guarani and Kaingang populations, intermediate (Rst=0.0590, p < 10-5) to high (Rst=0.1604, p < 10-5) differentiation was found. The two Kaingang populations showed very low differentiation between them (Rst=0.0017, p = 0.27), which justifies the union of both genetic data for forensic databases and calculations. The combined power of discrimination (PD) and the combined power of exclusion (PE) were calculated for each population, demonstrating the usefulness of this set of markers in forensic and kinship analysis regarding these populations. Considering the demographic heterogeneity of Amerindian populations in general, the Fst mean value (0.03) was evaluated regarding 43 different indigenous populations from the Americas, including Guaranis and Kaingangs. This result confirms the adequacy of the standardized θ value for the forensic random match probability estimations involving Amerindian populations.

Genetic data for 26 autosomal STR markers from Brazilian population.

The allelic frequency distributions and statistical forensic parameters of 26 mini short tandem repeat (mini-STR) loci in a sample of 1575 unrelated individuals from five different Brazilian regions were obtained. All the analyzed loci showed great diversity and were highly informative. The results were compared with those of the US Caucasian, African American, and Hispanic population studies. This study aimed to contribute to forensic analysis for human identification and inference of the evidential value in familial bond tests.

Genetic profile characterization of ten X-STRs in a sample from Paraná, Brazil.

This work reports the allele frequencies for ten X-STRs (DXS8378, DXS7132, DXS9898, DXS6809, DXS9902, DXS6789, DXS7133, DXS7423, GATA172D05, GATA31E08) in a sample of 800 individuals from Paraná, Brazil. No deviations from the Hardy-Weinberg equilibrium were observed. Linkage disequilibrium analysis did not reveal association between the X-STRs. High overall power of discrimination was obtained for female and male samples, and high probability of exclusion was observed in father/mother/daughter trios and father/daughter duos. Genetic comparisons revealed significant differences between Paraná and other Brazilian populations.

Recombinant antigen production for assays of intradermoreaction for diagnosis and surveillance of tuberculosis.

The goal of the present work was to develop reagents with potential for tuberculosis diagnosis. Genetic sequences of Mycobacterium tuberculosis secretion antigens were amplified by PCR, cloned into the Gateway(®) system, and expressed in Escherichia coli. The recombinant M. tuberculosis proteins were purified by metal affinity chromatography and preparative gel SDS-PAGE electrophoresis followed by electroelution and removal of endotoxins using Triton X-114. In total, seven recombinant proteins were obtained (ESAT-6, CFP10, TB10.3, TB10.4, MTSP11, MPT70, and MPT83). Delayed hypersensitivity reactions (DHR) was evaluated in Cavia porcellus and compared to the response using a standard purified protein derivative (PPD). All seven recombinant proteins produced a positive induration reaction in an intradermal test in guinea pigs previously sensitized with M. tuberculosis. When applied together, at a concentration of each recombinant protein 0.04 mg/mL, the intradermoreaction in C. porcellus was significantly higher than that obtained by standard PPD (p-value=0.00386).

Molecular characterization of Mycobacterium tuberculosis isolated in the State of Parana in southern Brazil.

Sequence IS6110 has been successfully used throughout the world for characterizing the Mycobacterium tuberculosis lineages. The aim of this study was to obtain data about circulating strains of M. tuberculosis in patients from the State of Parana in southern Brazil. Sixty-two clinical specimens obtained from sputum, bronchial aspirate, biopsy and urine from 62 patients clinically diagnosed with tuberculosis and admitted to the SUS-Brazil - The Brazilian Centralized Health Service System - were genotyped by the mixed-linker PCR DNA fingerprinting technique. The analysis demonstrated that the number of copies of the IS6110 sequence per isolates varied from four to 13 bands, with an average number of 8.5. From this, 93% of the isolates presented multiple copies. Isolates with no copies of the IS6110 element were not observed. The genetic analysis by UPGMA grouped the 62 isolates by similarity into three different groups: the first group contained two strains, the second was composed of 23, and the third, a more heterogeneous group, contained 37 isolates. Only two isolates (3.2%) formed a cluster; in other words, they presented a pattern of polymorphism with similarity above 95%. Such findings suggest that in the State of Parana, illness predominantly develops through reactivation of the latent infection as opposed to exogenous transmission. The methodology used (mixed-linker PCR DNA fingerprinting) allowed for 93.5% differentiation of the isolates tested, and proved to be a powerful tool for differentiation in the molecular genotyping of M. tuberculosis.

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 (> or =1 in 5 x 10(5)) and females (> or =1 in 3 x 10(9)), as well as high mean exclusion chance in father/daughter duos (> or =99.953%) and in father/mother/daughter trios (> or =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.

The 2000-2001 GEP-ISFG Collaborative Exercise on mtDNA: assessing the cause of unsuccessful mtDNA PCR amplification of hair shaft samples.

We report the results of Spanish and Portuguese working group (GEP) of International Society of Forensic Genetics (ISFG) Collaborative Exercise 2001-2002 on mitochondrial DNA (mtDNA) analysis. 64 laboratories from Spain, Portugal and several Latin-American countries participated in this quality control exercise. Five samples were sent to the participating laboratories, four blood stains (M1-M4) and a sample (M5) consisting of two hair shaft fragments. M4 was non-human (Felis catus) in origin; therefore, the capacity of the labs to identify the biological source of this sample was an integral part of the exercise. Some labs detected the non-human origin of M4 by carrying out immuno-diffussion techniques using antihuman serum, whereas others identified the specific animal origin by testing the sample against a set of animal antibodies or by means of the analysis of mtDNA regions (Cyt-b, 12S, and 16S genes). The results of the other three human blood stains (M1-M3) improved in relation to the last Collaborative Exercises but those related to hairs yielded a low rate of success which clearly contrasts with previous results. As a consequence of this, some labs performed additional analysis showing that the origin of this low efficiency was not the presence of inhibitors, but the low quantity of DNA present in these specific hair samples and the degradation. As a general conclusion the results emphasize the need of external proficiency testing as part of the accreditation procedure for the labs performing mtDNA analysis in forensic casework.