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Whole genome and transcriptome sequencing of post-mortem cardiac tissues from sudden cardiac death victims identifies a gene regulatory variant in NEXN

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Abstract

Background

Sudden cardiac death (SCD) is a major public health problem and constitutes a diagnostic and preventive challenge in forensic pathology, especially for cases with structural normal hearts at autopsy, so-called sudden arrhythmic death syndrome (SADS). The identification of new genetic risk factors that predispose to SADS is important, because they may contribute to establish the diagnosis and increase the understanding of disease pathways underlying SADS. Pathogenic mutations in the protein coding regions of cardiac genes were found in relation to SADS. However, much remains unknown about variants in non-coding regions of the genome.

Methods and results

In this study, we explored the potential of whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) to find DNA variants in SCD victims with structural normal hearts.

With focus on the non-coding regulatory regions, we re-examined a cohort of 13 SADS and sudden unexplained death in infancy (SUDI) victims without disease causing DNA variants in recognized cardiac genes. The genetic re-examination of DNA was carried out using frozen tissue samples and WTS was carried out using five distinct formalin fixed and paraffin embedded (FFPE) cardiac tissue samples from each individual, including anterior and posterior walls of the left ventricle, ventricular papillary muscle, septum, and the right ventricle. We identified 23 candidate variants in regulatory sequences of cardiac genes, including a variant in the promotor region of NEXN, c.-194A>G, that was found to be statistically significantly (p < 0.05) associated with decreased expression of NEXN and cardiac hypertrophy.

Conclusion

With the use of post-mortem FFPE tissues, we highlight the potential of using WTS investigations and compare gene expression levels with DNA variation in regulatory non-coding regions of the genome for a better understanding of the genetics of cardiac diseases leading to SCD.

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Abbreviations

ACMG:

American College of Medical Genetics and Genomics

AWLV:

Anterior walls of the left ventricle

BrS:

Brugada syndrome

CPVT:

Catecholaminergic polymorphic ventricular tachycardia

ENCODE:

Encyclopedia of DNA Elements

DCM:

Dilated cardiomyopathy

FFPE:

Formalin fixed and paraffin embedded

GTEx:

The Genotype-Tissue Expression Project

GTF:

Gene transfer format

HCM:

Hypertrophic cardiomyopathy

LQTS:

Long QT syndrome

Mt.:

Mitochondrial

NUMT:

Nuclear mitochondrial DNA segment

PCA:

Principal component analysis

PM:

Papillary muscle

PMI:

Post-mortem interval

PWLV:

Posterior walls of the left ventricle

SADS:

Sudden arrhythmic death syndrome

SCD:

Sudden cardiac death

SD:

Sudden death

SEP:

Septum

SIDS:

Sudden infant death syndrome

SUDEP:

Sudden unexplained death in epilepsy

SUDI:

Sudden unexplained death in infancy

TFBS:

Transcription factor binding sites

TPM:

Transcripts per kilo base million

VUS:

Variants of unknown significance

WGS:

Whole genome sequencing

WTS:

Whole transcriptome sequencing

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Acknowledgements

We thank Carina Grøntved Jønck for bioinformatics support, Anja Ladegaard Jørgensen for technical assistance in the laboratory, Mikkel M. Andersen for statistical support, and Steffan N. Christiansen and Sofie L. Christiansen for fruitful discussions. This work was supported by Ellen and Aage Andersen’s Foundation.

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Authors and Affiliations

  1. Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Jeppe D. Andersen, Stine B. Jacobsen, Linea C. Trudsø, Marie-Louise Kampmann & Niels Morling

  2. Section of Forensic Pathology, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Jytte Banner

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  1. Jeppe D. Andersen
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Correspondence to Jeppe D. Andersen.

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All procedures performed in the study were in accordance with the ethical standards of the Committees on Health Research Ethics in the Capital Region of Denmark (H-2-2012-017) and the Danish Data Protection Agency (2011-54-1262).

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Supplementary Figure 1

Boxplot of the percentage of mitochondrial (mt)-RNA in cardiac tissue from four sudden unexplained death in infancy (SUDI) and six sudden arrhythmic death syndrome (SADS) victims. mt-RNA was identified using the gene transfer format file of the hg19 reference genome from the GENCODE consortium release 27. The whiskers extends 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). (JPEG 40 kb)

Supplementary Figure 2

Principle component analysis of all protein coding transcripts in four sudden unexplained death in infancy (SUDI) and six sudden arrhythmic death syndrome (SADS) victims. Principle components represents variation between gene expression levels of SUDI and SADS victims. (JPEG 116 kb)

Supplementary Figure 3

Boxplot of the percentage of mitochondrial (mt)-RNA in five mildly decomposed sudden cardiac death victims and five sudden cardiac death victims characterized as not decomposed. Decomposition status was determined by a forensic pathologist at autopsy. mt-RNA was identified using the gene transfer format file of the hg19 reference genome from the GENCODE consortium release 27. The whiskers extends 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). (JPEG 48 kb)

Supplementary Table 1

Detailed description of cases in the study population. (XLSX 18 kb)

Supplementary Table 2

Nuclear mitochondrial DNA segment (NUMT) genes excluded from analysis. Positions are from the human genome assembly GRCh37 (hg19). (XLSX 68 kb)

Supplementary Table 3

Whole transcriptome sequencing and alignment statistics of the anterior and posterior wall of the left ventricle, right ventricle, septum, and ventricular papillary muscle of 10 sudden cardiac death victims. Alignment statistics were obtained from STAR (Spliced Transcripts Alignment to a Reference), and RNA types were extracted from the gene transfer format file of the hg19 reference genome from the GENCODE consortium release 27. Abbreviations: Mb = Megabase, PF = Passing Filter, RIN = RNA Integrity Number. (XLSX 51 kb)

Supplementary Table 4

(DOCX 30 kb)

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Andersen, J.D., Jacobsen, S.B., Trudsø, L.C. et al. Whole genome and transcriptome sequencing of post-mortem cardiac tissues from sudden cardiac death victims identifies a gene regulatory variant in NEXN. Int J Legal Med 133, 1699–1709 (2019). https://doi.org/10.1007/s00414-019-02127-9

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