title: Postmortem genetic testing for conventional autopsy-negative sudden unexplained death: an evaluation of different DNA extraction protocols and the feasibility of mutational analysis from archival paraffin-embedded heart tissue.
abstract: One third of autopsy-negative sudden unexplained deaths (SUDs) can be attributed to a cardiac channelopathy. Typically, paraffin-embedded tissue (PET) is the only source of DNA available for genetic analyses. We examined different DNA extraction procedures, involving 2 deparaffinization methods, 2 digestion methods, 4 laboratory-based purification methods, and 5 commercial kits. Mutational analysis involving 25 RYR2 exons was performed on PET DNA from 35 SUD cases to evaluate the feasibility of using PET DNA for genetic testing. With the best PET-DNA extraction method, an average of only two thirds of the region of interest could be evaluated. Although we initially identified 5 missense mutations in 5 of 35 SUD cases, repeated analysis failed to confirm these mutations. DNA from PET should be considered error prone and unreliable in comprehensive surveillance of SUD-associated genes. Given these shortcomings, the standard autopsy for SUD should include archiving EDTA-preserved blood or frozen tissue to facilitate postmortem genetic testing.
authors.affiliation: Department of Medico-diagnostic Sciences and Special Therapies, University of Padua Medical School, Padua, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Carturan
authors.forename: David J
authors.initials: DJ
authors.lastname: Tester
authors.forename: Brian C
authors.initials: BC
authors.lastname: Brost
authors.forename: Cristina
authors.initials: C
authors.lastname: Basso
authors.forename: Gaetano
authors.initials: G
authors.lastname: Thiene
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ackerman
mesh_terms: D001344:Autopsy; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification*; D004252:DNA Mutational Analysis; D003645:Death, Sudden / Q000209:etiology* / Q000473:pathology; D016757:Death, Sudden, Cardiac / Q000209:etiology* / Q000473:pathology; D003933:Diagnosis; D005069:Evaluation Studies as Topic; D005557:Formaldehyde; D005821:Genetic Techniques; D006321:Heart; D006801:Humans; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics; D016707:Tissue Fixation
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D005557:Formaldehyde; D004247:DNA