Histopathological characterization and effective methodologies for DNA analysis in exhumed bones for human identification

• Grant number: 12/24918-4
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2013
• End date: May 31, 2016
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Edna Sadayo Miazato Iwamura
• Grantee: Rafael Dias Astolphi
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

In Brazil, the practice of forensic identification of bodies has singular importance because of the high numbers of missing person’s records. Besides the investigation of crimes related to the elucidation, skeletal remains have been used as a source of DNA to determine, with virtually absolute reliability, paternity or filiation, human identification in natural disasters, wars and conflicts and mass graves, DNA has high chemical stability even after a long period of time and this molecule is present on all nucleated cells in the human body, which facilitates obtaining it. However, it is known that many factors present in the location where the material is found, such as heat, humidity and the presence of inhibitors in polymerase chain reaction (PCR), influence both the quality and the quantity of DNA used in subsequent analyzes. Moreover, the resulting samples of remains represent one of the most difficult sources to obtain DNA to be employed in the processes of human identification. This study aims to evaluate: 1) the microscopic changes in bones exhumed, 2) extraction protocol which is more efficient in these DNA samples, 3) the complete genetic profiles of all samples, 4) which sample, or tooth fragment Femoral provides more complete results in relation to genetic markers for human identification, 5) The technique of laser microdissection is a solution in the recovery of DNA in bone samples very poor and many inhibitors; 6) the correlation between the microscopic characteristics of bones exhumed with its molecular results to evaluate the potential of histopathologic analysis in the process of sorting these samples, 7) the histopathology of skeletons buried with the bones found on the ground. PATIENTS: Will be sectioned and transferred by the Centre of Forensic Medicine (CEMEL) Department of Pathology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (FMRP-USP) cortico-medullary fragments of compact bone of the femoral diaphysis of 100 skeletons exhumed coming Bom Pastor Cemetery in Ribeirão Preto. Also will be transferred by these same individuals, teeth. Methodologies will be used for histopathological and molecular analysis: 1) the blocks will be cut in automatic microtome stained by hematoxylin and eosin and periodic acid-Schiff and Silver Methenamine, and immunohistochemistry. 2) kits for DNA extraction and quantification (Applied Biosystems), will be used and the laser microdissection technique will be used on samples where the complete genetic profile is not obtained. Amplifications are carried out in PCR System 9700 thermal cycler (Applied Biosystems) employing the STR from multiplex system Powerplex ESX 17 ® System (Promega). The electrophoresis is performed on the ABI 3130 capillary sequencer Genetic Analyser ® (Applied Biosystems). Ethical aspects: the work will be developed under the authorization of the board of the Economic Development Company of Ribeirão Preto (CODERP), the entity responsible administratively by Bom Pastor Cemetery. There will be no disclosure or exposure of personal information exhumed, and ensured confidentiality of the data, because only the project participants will have access to them. We hope to determine histopathological changes in the bones exhumed and define what is the best method for obtaining complete genetic profiles in these samples, which is essential to aid forensic specialists to adopt a effective flow in laboratory procedures. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

MELLO, RAFAEL BARRIOS; REGIS SILVA, MARIA REGINA; SEIXAS ALVES, MARIA TERESA; EVISON, MARTIN PAUL; GUIMARAES, MARCO AURELIO; FRANCISCO, RAFAELLA ARRABACA; ASTOLPHI, RAFAEL DIAS; MIAZATO IWAMURA, EDNA SADAYO. Tissue Microarray Analysis Applied to Bone Diagenesis. SCIENTIFIC REPORTS, v. 7, JAN 4 2017. (12/24918-4, 12/12344-3, 13/17901-0)