Advancing Molecular Diagnostics in Sudden Unexplained Deaths

As submitted by the proposer: The concept of molecular autopsies has been proposed for more than a decade. However, a systematic molecular evaluation of Sudden Unexplained Death (SUD) cases, for which the causes of death remain undetermined after comprehensive forensic investigation, complete autopsy, and various ancillary laboratory tests (e.g. toxicology and microbiology tests), has not been feasible until the advent of next-generation sequencing (NGS) technology. This powerful molecular diagnostic tool is being used in our NIJ FY2011 Basic Research Grant project (2011-DN-BX-K535), which aimed to evaluate 114 genes in a large SUD cohort. Our basic research results have shown that over 40% of SUD cases are expected to have identifiable molecular defects either causing or contributing to the death when comprehensive molecular testing is performed. This is a significant improvement over the current 10 to 15% positive rate obtained by testing only six major cardiac channelopathy genes using the traditional Sanger Sequencing method. Through this grant application, we aim to directly translate the first-hand knowledge gained from our basic research into applied research via the following two objectives: 1) validate a molecular testing panel specifically targeting underlying molecular defects in SUD, using samples routinely collected at New York City Office of Chief Medical Examiner (NYC OCME), such as RNALater® fixed tissue and blood spot card samples; and 2) validate the SUD-specific molecular test panel for commonly collected, yet technically challenging, postmortem samples, including formalin-fixed (FF), formalin-fixed paraffin-embedded (FFPE), embalmed, and decomposed/degraded tissue samples. The focus of this grant project is to measure the analytical performance of the testing method (e.g. sensitivity, specificity, reproducibility, and sample input range) and to evaluate a feasible bioinformatic pipeline for high throughput NGS data analysis and genetic variant reporting. This project will be carried out within the Molecular Genetics Laboratory at NYC OCME, a College of American Pathologists (CAP)-accredited laboratory, using existing laboratory equipment and by residing scientists who possess a wealth of knowledge and skill, which were further enhanced through their direct involvement in the NIJ FY2011 Basic Research Grant project. As a result of this project, the quality of SUD investigation in NYC OCME will be drastically improved and provide answers to families and to the criminal justice system in cases for which no such answers previously existed. Ultimately, by disseminating our work through scientific presentations/publications and providing professional training to forensic pathologists/medicolegal investigators, we expect to help improve SUD investigation throughout the United States. ca/ncf