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.

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.

[1]  James H. Davis.  Sudden death in the young. , 2009, EMS magazine.

[2]  D. Tester,et al.  The role of molecular autopsy in unexplained sudden cardiac death , 2006, Current opinion in cardiology.

[3]  Michael J Ackerman,et al.  Spectrum and prevalence of cardiac ryanodine receptor (RyR2) mutations in a cohort of unrelated patients referred explicitly for long QT syndrome genetic testing. , 2005, Heart rhythm.

[4]  Wojciech Zareba,et al.  Sudden cardiac death without structural heart disease: Update on the long QT and brugada syndromes , 2005, Current cardiology reports.

[5]  G. Danieli,et al.  Juvenile sudden death in a family with polymorphic ventricular arrhythmias caused by a novel RyR2 gene mutation: evidence of specific morphological substrates. , 2005, Human pathology.

[6]  M. Ackerman,et al.  Cardiac causes of sudden unexpected death in children and their relationship to seizures and syncope: genetic testing for cardiac electropathies. , 2005, Seminars in pediatric neurology.

[7]  J. Halgunset,et al.  Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. , 2005, The Journal of molecular diagnostics : JMD.

[8]  D. Tester,et al.  Targeted mutational analysis of the RyR2-encoded cardiac ryanodine receptor in sudden unexplained death: a molecular autopsy of 49 medical examiner/coroner's cases. , 2004, Mayo Clinic proceedings.

[9]  D. Corrado,et al.  Cardiomyopathies: is it time for a molecular classification? , 2004, European heart journal.

[10]  S. Chugh,et al.  Postmortem molecular screening in unexplained sudden death. , 2004, Journal of the American College of Cardiology.

[11]  M. Ackerman,et al.  Cardiac channelopathies: it's in the genes , 2004, Nature Medicine.

[12]  E. Wever,et al.  Sudden death in patients without structural heart disease. , 2004, Journal of the American College of Cardiology.

[13]  Michael J Ackerman,et al.  Ethnic differences in cardiac potassium channel variants: implications for genetic susceptibility to sudden cardiac death and genetic testing for congenital long QT syndrome. , 2003, Mayo Clinic proceedings.

[14]  E. Behr,et al.  Cardiological assessment of first-degree relatives in sudden arrhythmic death syndrome , 2003, The Lancet.

[15]  B. Aguilera,et al.  Sudden unexplained death among persons 1-35 years old. , 2003, Forensic science international.

[16]  A. Giuliano,et al.  Molecular lymphatic mapping of sentinel lymph nodes , 2002 .

[17]  M. Durigon,et al.  DNA genotyping of unbuffered formalin fixed paraffin embedded tissues. , 2002, Forensic science international.

[18]  A. von Haeseler,et al.  DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. , 2001, Nucleic acids research.

[19]  J. Towbin,et al.  Postmortem molecular analysis of SCN5A defects in sudden infant death syndrome. , 2001, JAMA.

[20]  D. Tester,et al.  Molecular Autopsy of Sudden Unexplained Death in the Young , 2001, The American journal of forensic medicine and pathology.

[21]  G Thiene,et al.  Postmortem diagnosis in sudden cardiac death victims: macroscopic, microscopic and molecular findings , 2001, Cardiovascular research.

[22]  D. Corrado,et al.  Sudden cardiac death in young people with apparently normal heart. , 2001, Cardiovascular research.

[23]  C. Walker,et al.  Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC). , 2001, Journal of biochemical and biophysical methods.

[24]  S. Chugh,et al.  Sudden cardiac death with apparently normal heart. , 2000, Circulation.

[25]  S. Priori,et al.  Brugada syndrome and sudden cardiac death in children , 2000, The Lancet.

[26]  M. Correia.  [Molecular diagnosis of the inherited long-QT syndrome in a woman who died after near-drowning]. , 2000, Revista portuguesa de cardiologia : orgao oficial da Sociedade Portuguesa de Cardiologia = Portuguese journal of cardiology : an official journal of the Portuguese Society of Cardiology.

[27]  F. Pontén,et al.  A high frequency of sequence alterations is due to formalin fixation of archival specimens. , 1999, The American journal of pathology.

[28]  F O Mueller,et al.  Sudden death in young competitive athletes. Clinical, demographic, and pathological profiles. , 1996, JAMA.

[29]  R. Liberthson,et al.  Sudden death from cardiac causes in children and young adults. , 1996, The New England journal of medicine.

[30]  S. Mitchell,et al.  Microwave-based DNA extraction from paraffin-embedded tissue for PCR amplification. , 1995, BioTechniques.

[31]  C. Wheeler,et al.  Sample preparation and PCR amplification from paraffin-embedded tissues. , 1994, PCR methods and applications.

[32]  R. Salomon,et al.  Rapid Detection and Species Identification of Mycobacteria in Paraffin‐Embedded Tissues by Polymerase Chain Reaction , 1992, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[33]  T. Greiner,et al.  An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication. , 1991, BioTechniques.

[34]  D. Shibata,et al.  Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction , 1988, The Journal of experimental medicine.