Molecular Autopsy for Sudden Unexpected Death Victims

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1 Molecular Autopsy for Sudden Unexpected Death Victims DEII Barcelona 2018 Jeppe Dyrberg Andersen Assistant Professor Department of Forensic Medicine, University of Copenhagen

2 19/11/ Forensic autopsy and molecular autopsy Autopsy: Medical examination of a dead body to determine the cause of death Natural Accident Homicide Suicide Undetermined Toxicology Microscopy (histopathology) Bacteriology Molecular autopsy: Postmortem genetic testing to help establish the cause of death Genetic testing of relatives

3 Sudden unexpected death Unexpected natural death of an apparently healthy individual Astori 31yrs Pheidippides 40yrs

4 Sudden unexpected death Unexpected natural death of an apparently healthy individual Sudden Cardiac Death (SCD) Structural ABNORMAL heart Structural NORMAL heart Hypertrophic cardiomyopathy (HCM) Long QT syndrome (LQTS) Arrhythmogenic right ventricular Short QT syndrome (SQTS) cardiomyopathy (ARVC) Brugada syndrome (BrS) Dilated cardiomyopathy (DCM) Catecholaminergic polymorphic ventricular Myocarditis tachycardia (CPVT) One third of presumed Sudden Cardiac Death remain unexplained after autopsy Molecular investigation

5 Aim of study Investigate if molecular autopsy using massively parallel sequencing (MPS) could improve the diagnostic outcome of sudden unexpected death victims Genetic heterogeneous Supplement to the forensic autopsy and contribute to finding the cause of death

6 19/11/ How molecular autopsy started in Copenhagen ( ) Autopsies <50yrs n=829 Unnatural causes n=486 Natural causes n=343 Suspected cardiac aetiology n=210 Suspected inherited SCD n=142 Non-inherited n=46 Structural n=20 SUD n=61 SUDI n=9 Unspecific n=52 Low quality DNA n=22

7 Molecular autopsy using massively parallel sequencing (MPS) Suspected inherited SCD n=142 Structural n=20 SUD n=61 SUDI n=9 Unspecific n=52 MiSeq/FGx NextSeq 25,000,000 reads/run 400,000,000 reads/run

8 Target gene panels using the MiSeq Suspected inherited SCD n=142 Structural n=20 SUD n=61 SUDI n=9 Unspecific n=52

9 Target gene panels using the MiSeq SUD n=61 SUD n=15 34 genes (387kb) associated with non-structural heart diseases NimbleGen SeqCap EZ Library (custom design) MiSeq Reporter data analysis Alamut Software Suite variant interpretation SUD Likely functional effect n=3 (20%) n=12 Variants associated with heart diseases HGMD phenotype Bioinformatic predictions

10 Target gene panels using the MiSeq Suspected inherited SCD n=142 Structural n=20 SUD n=61 SUDI n=9 Unspecific n= genes (788 kb) associated with non-structural and structural heart diseases Agilent HaloPlex Target Enrichment system (custom design) Agilent SureCall data analysis Alamut Software Suite variant interpretation ~One third had variants with likely functional effects

11 Target gene panels using the MiSeq Suspected inherited SCD n=142 Structural n=20 SUD n=61 SUDI n=9 Unspecific n= genes (788 kb) associated with non-structural and structural heart diseases Agilent HaloPlex Target Enrichment system (custom design) Agilent SureCall data analysis Alamut Software Suite variant interpretation ~One third had variants with likely functional effects

12 Target gene panels using the MiSeq Suspected inherited SCD n=142 Structural n=20 SUD n=61 SUDI n=9 Unspecific n= genes (788 kb) associated with non-structural and structural heart diseases Agilent HaloPlex Target Enrichment system (custom design) Agilent SureCall data analysis Alamut Software Suite variant interpretation ~One third had variants with likely functional effects

13 19/11/ How it is done in Copenhagen today Suspected inherited SCD Structural ABNORMAL heart Structural NORMAL heart Screening of candidate gene Targeted gene panels associated with specific diseases at Copenhagen University Hospital Clinical evaluation of relatives Family history, ECG, physical examination Screening of candidate genes in relatives

14 Molecular autopsy using WES and WGS Suspected inherited SCD n=142 MiSeq/FGx NextSeq 25,000,000 reads/run 400,000,000 reads/run

15 Molecular autopsy using WES and WGS Suspected inherited SCD n=142 Structural n=20 SUD n=61 SUDI n=9 Unspecific n=52

16 Molecular autopsy using WES and WGS Suspected inherited SCD n=142 Structural n=20 SUD n=61 SUDI n=9 Unspecific n=52 Cases with no variant identified using targeted panels SUD/SUDI n=14 WES, WGS, and WTS of FFPE heart tissue 400,000,000 reads/run

17 Molecular autopsy using WES and WGS SUD/SUDI n=14 WES n=14 WGS n=13 WTS n=12 Severely degraded Low DNA/RNA quality n=2

18 Molecular autopsy using WES Agilent SureSelect Clinical Research Exome V2 Capture Capture with probes result in unbalanced coverage and variants may be missed In house analysis pipeline (transparent analysis settings) American College of Medical Genetics and Genomics (ACMG) standards 1 Case ID Sex Age (yrs) Gene Transcript Nucleotide variant Amino acid change Coding effect ACMG classification 2 M 44 BRCA2 NM_ c.7069_7070del p.leu2357valfs*2 Frameshift Likely deletion pathogenic 8 F 0 REN NM_ c.145c>t p.arg49* Stop gain Likely substitution pathogenic Associated phenotype Breast-, ovarian-, and colon cancer Renal tubular dysgenesis in infants and embryos 12 M 0 GJA5 NM_ c.286g>t p.ala96ser Missense substitution Let s dig a little deeper! Likely pathogenic Atrial fibrillation 1 Richards, S., et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, (5): p

19 Molecular autopsy using WGS Illumina TruSeq DNA PCR-Free Library Prep Even coverage, much data and many novel variants per sample, easy laboratory work, expensive Investigate promoter and intronic regions of known heart genes In house analysis pipeline (transparent analysis settings) American College of Medical Genetics and Genomics (ACMG) standards 1 Richards, S., et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, (5): p

20 Molecular autopsy using WGS Identified 1-4 variants (all novel) in regulatory regions of well-known heart genes in 10 out of 12 victims Collect our own evidence to support the importance of the variants (gene expression WTS) Case ID Sex Age (yrs) Associated gene Transcript Nucleotide variant Regulatory region 5 F 47 NEXN NM_ c.-194a>g Promoter SNTA1 NM_ c g>a Enhancer

21 Molecular autopsy using WES and WGS SUD/SUDI n=14 WES found one likely variant (7%) WGS found one likely variant (7%) DNA/RNA quality was low for some samples Improve performance of library kits for WES and WGS Novel variants are challenging (WGS) More studies using WGS will increase knowledge and lead to fewer variants of unknown significance (VUS)

22 Considerations with MPS and molecular autopsy No dedicated analysis softwares and analysis criteria Custom analysis pipeline American College of Medical Genetics and Genomics (ACMG) standards Consider the IT-infrastructure NextSeq runs are ~20x MiSeq/FGx runs Ethical considerations Costs?

23 Acknowledgement The heart group Stine B. Jacobsen (Research ass.) Linea Trudsø (Research ass.) Sofie Lindgren Christiansen (Post.doc) Marie-Louise Kampmann (MPS specialist) Niels Morling (Professor in Forensic Genetics) Jytte Banner (Professor in Forensic Medicine) Christin Hertz