Avaliação da qualidade do DNA extraído de saliva humana armazenada e sua aplicabilidade na identificação em Odontologia Legal

PURPOSE: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. METHODS: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at -20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. RESULTS: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. CONCLUSION: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.OBJETIVO: Este trabalho objetivou avaliar a qualidade do DNA obtido de saliva humana armazenada e sua aplicabilidade da identificação de pessoas. METODOLOGIA: Analisaram-se amostras salivares de n=20 sujeitos da pesquisa, coletadas nas formas de saliva in natura e de swab bucal, sendo armazenadas a 20ºC. Após 7 dias, o DNA foi extraído das 40 amostras de saliva e submetido à PCR e à eletroforese. Após 180 dias repetiu-se a técnica nas 20 amostras de swab. RESULTADOS: Os resultados da primeira etapa indicaram que o DNA foi extraído com sucesso em 97,5% das reações, e, analisando-se separadamente, em 95% de saliva in natura e em 100% da saliva do swab, não havendo diferenças estatisticamente significantes entre as duas formas de saliva. Na segunda fase, o resultado foi positivo para as 20 amostras analisadas (100%). Posteriormente, para analisar a qualidade do DNA obtido da saliva humana, o gene SIX3-2 foi testado nas 20 amostras de swab bucal e foi feita a digestão do produto da PCR com a enzima de restrição MbO1 para avaliar polimorfismo do gene ADRA-2 obtendo-se resultados positivos para a maioria das amostras. CONCLUSÃO: Concluiu-se que a quantidade e a qualidade do DNA advindo de saliva e as técnicas empregadas estão adequadas à análise forense do DNA

Validation of a physical anthropology methodology using mandibles for gender estimation in a Brazilian population

Validation studies of physical anthropology methods in the different population groups are extremely important, especially in cases in which the population variations may cause problems in the identification of a native individual by the application of norms developed for different communities. OBJECTIVE: This study aimed to estimate the gender of skeletons by application of the method of Oliveira, et al. (1995), previously used in a population sample from Northeast Brazil. MATERIAL AND METHODS: The accuracy of this method was assessed for a population from Southeast Brazil and validated by statistical tests. The method used two mandibular measurements, namely the bigonial distance and the mandibular ramus height. The sample was composed of 66 skulls and the method was applied by two examiners. The results were statistically analyzed by the paired t test, logistic discriminant analysis and logistic regression. RESULTS: The results demonstrated that the application of the method of Oliveira, et al. (1995) in this population achieved very different outcomes between genders, with 100% for females and only 11% for males, which may be explained by ethnic differences. However, statistical adjustment of measurement data for the population analyzed allowed accuracy of 76.47% for males and 78.13% for females, with the creation of a new discriminant formula. CONCLUSION: It was concluded that methods involving physical anthropology present high rate of accuracy for human identification, easy application, low cost and simplicity; however, the methodologies must be validated for the different populations due to differences in ethnic patterns, which are directly related to the phenotypic aspects. In this specific case, the method of Oliveira, et al. (1995) presented good accuracy and may be used for gender estimation in Brazil in two geographic regions, namely Northeast and Southeast; however, for other regions of the country (North, Central West and South), previous methodological adjustment is recommended as demonstrated in this study

Genetic screening for IRF6 and GRHL3 in Brazilians with non-syndromic cleft lip/palate / Triagem genética de IRF6 e GRHL3 em brasileiros com fissura lábio/palato não sindrômica

Cleft lip with or without cleft palate (CL/P) is the most frequent craniofacial anomaly. Advances in molecular and quantitative analysis suggests that the etiology is multifactorial of nonsyndromic CL/P (NSCL/P), and provide new opportunities to identify genes and gene-environment interactions relevant to the etiology of this common and representative birth defect. The present study aimed at detecting genetic variants in IRF6 and GRHL3 genes and susceptibility to NSCL/P in West Central and Northern Brazilian populations. We analyzed a set of 80 individuals with NSCL/P from Associação de Combate as Deformidades Faciais, recruited from Midwest and Northern Brazil. We performed Multiplex Ligation-dependent Probe Amplification (P304-B1-IRF6/GRHL3 (Lot B1-0116)) and PCR analysis for confirmation. In the MPLA study exon 4 of GRHL3, show possible alteration. Therefore, we performed a PCR validation of these alterations. The results showed no alteration on these genes (IRF6 and GRHL3) corroborating with previous studies. To our knowledge, this study of both genes is the first in these specific areas of Brazil, analyzing individuals with NSCL/P. Studies have identified a missense variant in the gene grainyhead-like-3 (GRHL3) in cleft palate individuals. The contribution of these genetic variants to NSCL/P susceptibility should be further investigated in different populations and cohorts. Thus, the underlying genetic causes of NSCL/P remain largely unknown

Sonic hedgehog (SHH) mutation in patients within the spectrum of holoprosencephaly

Holoprosencephaly (HPE) is a malformation sequence where the cerebral hemispheres fail to separate into distinct left and right halves. It can be associated with midline structural anomalies of the central nervous system and/or face. SHH is the major gene implicated in HPE and it plays a critical role in early forebrain and central nervous system development. SHH is expressed in the human embryo in the notochord, the floorplate of the neural tube, and the posterior limb buds. In the present Study we performed mutational analysis of the entire coding region of the SHH gene in 37 Unrelated individuals with the HPE spectrum. Three different variants were found throughout the extent of the gene. No genotype-phenotype correlation is evident based oil the type or position of the mutations. This study confirms the great genetic heterogeneity of the disease and the difficulty to establish genotype-phenotype correlations. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Validation of a physical anthropology methodology using mandibles for gender estimation in a Brazilian population

Validation studies of physical anthropology methods in the different population groups are extremely important, especially in cases in which the population variations may cause problems in the identification of a native individual by the application of norms developed for different communities. OBJECTIVE: This study aimed to estimate the gender of skeletons by application of the method of Oliveira, et al. (1995), previously used in a population sample from Northeast Brazil. MATERIAL AND METHODS: The accuracy of this method was assessed for a population from Southeast Brazil and validated by statistical tests. The method used two mandibular measurements, namely the bigonial distance and the mandibular ramus height. The sample was composed of 66 skulls and the method was applied by two examiners. The results were statistically analyzed by the paired t test, logistic discriminant analysis and logistic regression. RESULTS: The results demonstrated that the application of the method of Oliveira, et al. (1995) in this population achieved very different outcomes between genders, with 100% for females and only 11% for males, which may be explained by ethnic differences. However, statistical adjustment of measurement data for the population analyzed allowed accuracy of 76.47% for males and 78.13% for females, with the creation of a new discriminant formula. CONCLUSION: It was concluded that methods involving physical anthropology present high rate of accuracy for human identification, easy application, low cost and simplicity; however, the methodologies must be validated for the different populations due to differences in ethnic patterns, which are directly related to the phenotypic aspects. In this specific case, the method of Oliveira, et al. (1995) presented good accuracy and may be used for gender estimation in Brazil in two geographic regions, namely Northeast and Southeast; however, for other regions of the country (North, Central West and South), previous methodological adjustment is recommended as demonstrated in this study

Human identification analysis using PCR from the root portion of dental elements under different conditions of temperature and exposure time

Introduction and objective: The main exogenous factors limiting the retrieval of information from human remains are fire and accidents involving high temperatures. Teeth, due to their relatively high degree of chemical and physical resistance, offer the possibility for the recovery of genetic material, becoming important in forensic cases. With the aim to contribute to a standardization of the protocols employed in DNA extraction and analysis, it was evaluated the integrity of DNA recovered from dental roots submitted to high temperatures, simulating what happens to burnt people. Material and methods: Extractions of genomic DNA were made from the dental root after exposure to high temperatures (600ºC, 800ºC and 1000ºC), during 10, 30 and 60 minutes. Results and conclusion: After molecular analysis through PCR technique, it was verified that DNA amplification of the samples was not possible at any of the periods and temperatures analyzed