RESUMO
With the advent of Next-Generation Sequencing technology, sequencing of short tandem repeats (STRs) allows for a more detailed analysis when compared to size-based fragment methods (capillary electrophoresis-CE). The implementation of high-throughput sequencing can help uncover deeper genetic diversities of different populations. Subjects from the South region of Brazil present a particular and more homogeneous ancestry background when compared to other regions of the country. Both autosomal and Y- STRs have been analyzed in these individuals; however, all analyses published to date encompass data from CE-based fragment analysis. In this study, a genetic analysis of 59 individuals from Southern Brazil was performed on STR sequences. Forensically relevant STRs were PCR-enriched using a prototype of the PowerSeq™ AUTO/Y system (Promega Corp.). Next-generation sequencing was performed on an Illumina MiSeq instrument. The raw data (FASTQ files) were processed using a custom designed sequence processing tool, Altius. Isoalleles, which are sequence-based allelic variants that do not differ in length, were observed in nine autosomal and in six Y- STRs from the core global forensic marker set. The number of distinctive alleles based on sequence was higher when compared to those based on length, 37.3% higher in autosomal STRs and 13.8% higher in Y-STRs. The most polymorphic autosomal locus was D12S391, which presented 38 different sequence-based alleles. Among the loci in the Y chromosome, DYS389II presented the highest number of isoalleles. In comparison to CE analysis, Observed and Expected Heterozygosity, Polymorphic Information Content (PIC) and Genetic Diversity also presented higher values when the alleles were analyzed based on their sequence. For autosomal loci, Polymorphic Information Content (PIC) was 2.6% higher for sequence-based data. Diversity was 9.3% and 6.5% higher for autosomal and Y markers, respectively. In the analysis of the repeat structures for the STR loci, a new allele variant was found for allele 18 in the vWA locus. The STR flanking regions were also further investigated and sixteen variations were observed at nine autosomal STR loci and one Y-STR locus. The results obtained in this study demonstrate the importance of genetic analysis based on sequencing and highlight the diversity of the South Brazilian population when characterized by STR sequencing.
Assuntos
Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Análise de Sequência de DNA , Brasil , Cromossomos Humanos Y , Impressões Digitais de DNA , Feminino , Frequência do Gene , Variação Genética , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
Determining the type and origin of body fluids in a forensic investigation can provide important assistance in reconstructing crime scenes. A set of epigenetic markers, ZC3H12D, BCAS4 and cg06379435, have been developed to produce unique and specific patterns of DNA methylation that can be used to identify semen, saliva, and blood, respectively. To ensure the efficacy of these markers, developmental validation studies were performed to determine the conditions and limitations of this new tool for forensic analysis. DNA was extracted from human samples and bisulfite modified using commercial bisulfite modification kits. Specific primers were used to amplify the region of interest and the methylation profile of the CpG sites were determined by pyrosequencing. The percent methylation values at each CpG site were determined in multiple samples and averaged for each tissue type. The versatility of these new markers is presented by showing the results of validation studies on sensitivity, human specificity, stability and mixture resolution. When testing the markers using different organisms, we did obtain positive results for certain non-human primate samples, however, all other tested species were negative. The lowest concentration consistently detected varied from 0.1 to 10ng, depending on the locus, indicating the importance of primer design and sequence in the assay. The method also proved to be effective when inhibitors were present in the samples or when samples were degraded by heat. Simulated case- samples were also tested. In the case of mixtures of different cell types, the overall methylation values varied in a consistent and predictable manner when multiple cell types were present in the same sample. Overall, the validation studies demonstrate the robustness and effectiveness of this new tool for body fluid identification.
Assuntos
Análise Química do Sangue , Metilação de DNA , Marcadores Genéticos , Saliva/química , Sêmen/química , Animais , Ilhas de CpG/genética , Primers do DNA , Epigenômica , Genética Forense/métodos , Humanos , Masculino , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodos , Especificidade da EspécieRESUMO
Neutrality investigations of markers with forensic use are important to see if a phenotypic trait is being expressed in relation to the alleles of the marker. MiniSTR marker D22S1045 (locus 22q12.3) is localized near the breakpoint region of the EWS gene (22q12.2), which leads to the development of Ewing's Sarcoma. Analyzing allele frequencies and linkage disequilibrium in Ewing's sarcoma patients and non-affected populations, we found that the marker mD22S1045 was neutral when related to Ewing's Sarcoma.
Assuntos
Cromossomos Humanos 21-22 e Y/genética , Genética Forense/métodos , Desequilíbrio de Ligação/genética , Sarcoma de Ewing/genética , Alelos , Estudos de Casos e Controles , Impressões Digitais de DNA/métodos , Marcadores Genéticos , Humanos , Repetições de Microssatélites/genética , FenótipoRESUMO
The Ewing Sarcoma is an important tumor of bone and soft tissue. The SNPs Arg72Pro of TP53 and T309G of MDM2 have been associated with many cancer types and have been differently distributed among populations worldwide. Based on a case-control design, this study aimed to assess the role of these SNPs in 24 Ewing Sarcoma patients, compared to 91 control individuals. DNA samples were extracted from blood and genotyped for both SNPs by PCR-RFLP and confirmed by DNA sequencing. The results showed an association between the G allele of the T309G and Ewing Sarcoma (P=0.02). Comparing to the TT carriers, the risk of G allele carriers was 3.35 (95% CI=1.22-9.21) with P=0.02. At the genotypic level, an association of the TT genotype with the control group (P=0.03) was found. Comparing to the TT genotype, the risk of TG and GG was 2.97 (95% CI=1.03-8.58) with P=0.04 and 5.00 (95% CI=1.23-20.34) with P=0.02, respectively. No associations regarding the Arg72Pro SNP were found. Considering that the T309G has been associated with several types of cancer, including sarcomas, our results indicate that this SNP may also be important to Ewing Sarcoma predisposition.
Assuntos
Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Sarcoma de Ewing/genética , Proteína Supressora de Tumor p53/genética , Substituição de Aminoácidos/genética , Brasil , Estudos de Casos e Controles , Estudos de Associação Genética , Genótipo , Humanos , Modelos Logísticos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNARESUMO
Erythrina velutina Willd., Fabaceae, is a medicinal plant that can be found in the tropics and subtropics, including in the semi-arid northeastern Brazil. It is commonly used in folk medicine to treat anxiety, agitation and insomnia. E. velutina has been known to present analgesic, anti-inflammatory and antibacterial activities, however, it is unknown if this plant present a protective effect on DNA. We assessed the antigenotoxic effect of E. velutina against the genotoxic effects induced by MMS in the root meristem cells of Allium cepa. Three concentrations of the aqueous extract (100, 200 and 400 mg/L) of this medicinal plant were used in three different types of treatment (pre-, post- and simultaneous). The effects of the extracts on the root meristem cells of A. cepa were analyzed at both macroscopic and microscopic levels. Protective effects were observed at higher concentrations in pre-treatment and in simultaneous treatment. The results suggest that E. velutina may present antigenotoxic properties and demonstrate its chemopreventive potential.
RESUMO
A task-force to resolve 26 pending forensic caseworks was carried out. We tested four different protocols to extract DNA from molar and pre-molar teeth from 26 cadavers with post-mortem intervals from 2 months to 12 years. We compared the amount of DNA and DNA profiles with the time elapsed between death and laboratory procedures. Molar or pre-molar teeth were removed from the corpses, cleaned, and DNA was extracted using 2 or 12h of incubation on lysis buffer and filtered using concentration column or precipitated with isopropanol. DNA profiles were obtained using PowerPlex16™ System PCR Amplification Kit, AmpFlSTR(®) Yfiler™ and/or mtDNA sequencing. Complete DNA profiles comparison and statistical evaluation allowed unambiguous identification of the 26 victims. No significant differences were observed in the amount of DNA obtained with the distinct incubation times. The use of concentration column resulted in an increased amount of DNA when compared to isopropanol. However, the lower concentration of DNA obtained with isopropanol seemed to have been compensated by the higher purity. No significant differences in the number of amplified loci were found. A non-significant tendency was found between the amount of total DNA recovered and the time elapsed between death and laboratory procedures. The increase of post-mortem time did not interfere in the analysed autosomal loci. In conclusion, molar and pre-molar teeth were shown to be good candidates to obtain satisfactory DNA profiles, suggesting the high potential of tooth samples as source for DNA typing independently of the decomposed corpse's time or laboratory procedures.
Assuntos
Cadáver , Impressões Digitais de DNA/métodos , DNA/isolamento & purificação , Odontologia Legal/métodos , Dente Molar/química , Análise de Variância , Feminino , Loci Gênicos , Humanos , Masculino , Mudanças Depois da MorteRESUMO
We aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p=0.02; Chi Square Test). About 300 bp from the rs4820804 SNP lies a palindromic hexamer (5'-GCTAGC-3') and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility.
Assuntos
Neoplasias Ósseas/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Adolescente , Adulto , Sequência de Bases , Estudos de Casos e Controles , Criança , Pré-Escolar , DNA de Neoplasias/genética , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
The effects of the decoction of Erythrina velutina Willd., Fabaceae, were investigated using the root meristem cells of Allium cepa L., Amaryllidaceae. Ten concentrations of the aqueous extract (0.125 to 1.25 percent) of this medicinal plant were analyzed at both macroscopic and microscopic levels. All concentrations showed root growth inhibition after 96 h treatment. Although there were no significant differences between the mitotic indexes of any concentration and the control, there were changes in the frequencies of cell stages at three different concentrations. Additionally, the presence of five different cells abnormalities was recorded: chromosome bridging, lagging chromosomes, chromosome fragments, disturbed metaphase and disturbed anaphase. These results suggest inhibitory and genotoxic activity of the decoction of E. velutina on Allium cepa.