The rapid evolution of genetic disease understanding and expanding genetic databases behooves clinician specialists to regularly review the status of clinical genetic test panels, reevaluating the status of identified variants. With the increasing use of WES and WGS sequencing, these results likely also need regular review. We present a four-generation family affected by sudden cardiac arrests, ventricular tachycardia and (within the last 2 generations) documented left ventricular non-compaction. An affected member underwent a 38-gene pan-cardiomyopathy clinical genetic panel with no identified pathogenic variants. Therefore, affected members from three surviving generations (*) agreed to undergo genetic testing by whole genome sequencing in 2016. Analysis with Golden Helix SVS identified 69 shared, rare (MAF≤0.00005) coding variants in 40 genes, but none were identified to be associated with cardiac phenotypes. At clinical follow-up of the youngest family member in 2019, we elected to review our WGS results again. A recent review of the genetics and genomics of dilated cardiomyopathy now identifies the SRA1 gene as being potentially involved in this phenotype, although no human cases have yet been reported. Upon review of this families previous WGS sequencing using SVS, we identified a SRA1 c.328_329 ins GAC (p.Val110delinsGlyLeu) rare shared variant. This variant is only present in 2 of 142724 alleles in the gnomAD database and inserts an additional amino acid into a partially conserved two amino acid region within an otherwise highly conserved protein region. Friedrichs and colleagues previously identified SRA1 as one of 3 genes within a 600kb haploblock associated with cardiomyopathy in three independent Caucasian populations. In zebrafish, sra1 morphants display severe pericardial edema. The human SRA1 region also encodes a long noncoding RNA, lnc-SRA1-2, that is modulated in heart failure, attenuates hypoxia-induced injury in experimental cardiomyocytes through PPARγ/NF-κB signalling, and promotes the activation of cardiac myofibroblasts. SRA1 knockdown in C2C12 mouse myoblast cells prevented proper muscle gene expression and cell differentiation. Cardiac genetics assessments by whole genome or whole exome sequencing also need regular review, which can be easily performed by Golden Helix SVS software. Links to new and frequently updated gene tracks and other genetic databases facilitate these reviews.

1. Yearly Clinical Review of a
Patient’s WGS Results Leads
to a New Gene Candidate
for a Complex
Cardiomyopathy Phenotype
Robert M. Hamilton and Priyanka Kugamoorthy

2. Name of presentation
Company name

3. • The rapid evolution of genetic disease understanding and expanding genetic
databases behooves clinician specialists to regularly review the status of clinical
genetic test panels, reevaluating the status of identified variants.
• With the increasing use of WES and WGS sequencing, these results likely also
need regular review.
Yearly Clinical Review of a Patient’s WGS Results Leads to a New Gene
Candidate for a Complex Cardiomyopathy Phenotype
3

4. Family with Left Ventricular Non-Compaction (LVNC)
and Sudden Cardiac Death/Arrest Events
• Sudden Cardiac Death
• Sudden Cardiac Arrest
• LV Non-Compactiom
• LV Non-Compaction4

5. Slide master

2012
ExAC

2009
ESP

2016
GnomAD

2015
ACMG

2017
Sherloc,
Whiffin
& Ware

2008
1KG

2013
ACMG
SF 1.0

2017
ACMG
SF 2.0
5

6. 6

7. ACMG 2015: Reanalysis
• For reports containing variants of uncertain significance in genes related to the primary
indication, and in the absence of updates that may be proactively provided by the
laboratory, it is recommended that laboratories suggest periodic inquiry by health-care
providers to determine whether knowledge of any variants of uncertain significance,
including variants reported as likely pathogenic, has changed.
• By contrast, laboratories are encouraged to consider proactive amendment of cases
when a variant reported with a near-definitive classification (pathogenic or benign) must
be reclassified. Regarding physician responsibility, see the ACMG guidelines on the duty
to recontact.
7

8. • 2020 APHRS/HRS Expert Consensus Statement on the Investigation of
Decedents with Sudden Unexplained Death and Patients with Sudden Cardiac
Arrest, and of Their Families
• For Public Comment Purposes Only. Not for Distribution,Citation,or Dissemination.
• Investigation of Sudden Death: Genetic Evaluation Where the Phenotype Is
Known
• IB NR
• In families affected by sudden cardiac death who have undergone genetic testing, periodic
re-evaluation of the genetic test results is recommended
8

9. Golden Helix Analysis
9

10. 10

11. 11

12. • At clinical follow-up of the youngest family member in 2019, we elected to review our
WGS results again.
• A recent review of the genetics and genomics of dilated cardiomyopathy now identifies
the SRA1 gene as being potentially involved in this phenotype, although no human cases
have yet been reported.
• Upon review of this families previous WGS sequencing using SVS, we identified a SRA1
c.328_329 ins GAC (p.Val110delinsGlyLeu) rare shared variant. This variant is only
present in 2 of 142724 alleles in the gnomAD database and inserts an additional amino
acid into a partially conserved two amino acid region within an otherwise highly
conserved protein region.
12

13. Steroid Receptor RNA Activator 1
(SRA1) Gene
• Both long non-coding RNA (lncRNA) and steroid receptor RNA activator protein (SRAP)
are generated from this gene based on alternative transcription start site usage and
alternatively spliced transcript variants
• Originallydefined as an lncRNA
– Coactivator for many nuclear receptors (NRs)
• Currently found to be involved in dictating many NR and non-NR activities
– Metabolism
– Adipogenesis
– Chromatin organization
• lncRNA transcripts communicate with many different kinds of protein, including the
protein encoded by this gene
Genetics Home Reference
13

14. Chromosomal location
Genetics Home Reference
14

15. Heart Disease. Heart Failure (HF)
AHA
15

16. • HF progressively deteriorates Left Ventricular Function (LVF)
• Cardiovascular morbidity & mortality involve major health and
economic burdens upon patients
• More studiesisolating key underlying mechanismsfor HF are
imperative to improve clinical outcome!
16

17. • Friedrichs and colleagues previously identified SRA1 as one of 3 genes within a 600kb
haploblock associated with cardiomyopathy in three independent Caucasian
populations.
• In zebrafish, sra1 morphants display severe pericardial edema. The human SRA1
region also encodes a long noncoding RNA, lnc-SRA1-2,that is modulated in heart
failure, attenuates hypoxia-induced injury in experimental cardiomyocytes through
PPARγ/NF-κBsignalling, and promotes the activation of cardiac myofibroblasts.
• SRA1 knockdown in C2C12 mouse myoblast cells prevented proper muscle gene
expression and cell differentiation.
17

18. lncRNASRA1 attenuates Hypoxia-Induced Injury in
Experimental Cardiomyocytes through PPARγ/ NF-κB
Signaling Promoting Activation of Cardiac Myofibroblasts
Zhang et. al, 2018
18

19. • C. Zhang and colleagues formed a study aimed to investigate effects &
mechanisms of lncRNA SRA1 on roles in preventing hypoxia-induced
myocardial injury to gain a different perspective in HF prevention induced by
hypoxia
• Cultured hypoxic conditions (3% O2) in H9c2 cardiomyocytes to see whether
hypoxia induced injury could be observed by detecting cell viability, apoptosis,
and autophagy
• Governing relationship between SRA1 and PPARγ evaluated along with the
association between SRA1 and NF-κB
Zhang et. al, 2018
19

20. Results
of Study
• Hypoxia inflicted
injury to H9c2
cardiomyocytes
Zhang et. al, 2018
20

21. Zhang et. al, 2018
21

22. Results
• Hypoxia produced
decreased expression of
SRA1 in H9c2
cardiomyocytes
• Overexpression of SRA1
relieved hypoxia-induced
injury to H9c2
Zhang et. al, 2018
22

23. • Overall evaluation of the effects & mechanisms of SRA1 on
regulating hypoxia-induced injury in H9c2 cardiomyocytes
was observed:
• Results showed that Hypoxia did induce injury to H9c2
cardiomyocytes
– Cell viability inhibition
– Promoted cell apoptosis & autophagy
– Decreased expression of SRA1 in H9c2 cardiomyocytes
– Overexpression of SRA1 alleviated hypoxia-induced injury
– Suppression of SRA1 indicated contrary results
Zhang et. al, 2018
23

24. Peroxisome Proliferator-Activated
Receptor γ (PPARγ)
• PPARγ: ligand-activated transcription factor
– Influences glucose & mitochondrial fattyacid metabolism within tissues & organs
• Study results detected combined effects of suppression of SRA1 and overexpression of
PPARγ on viability, apoptosis, and autophagy of H9c2 cardiomyocytes under hypoxic
conditions
• SRA1 positively regulated PPARγ
– Overexpression of SRA1 alleviated hypoxia injury by activating PPARy
– Suppression of SRA1 activated inhibited its expression
• Overall data indicated SRA1 regulated hypoxia-induced injury to H9c2 cardiomyocytes by
activating PPARγ
Zhang et. al, 2018
24

25. Prior Studies Surrounding PPARγ
• Previous studieshave revealed that lncRNA SRA1 can bind to and
coactivate PPARγ in mice
• PPARγ agonist, pioglitazone, found to have ability to relieve severe
pulmonary HTN & vascular remodeling and prevent Right Heart
Failure in rats
Zhang et. al, 2018
25

26. Prior Studies Surrounding PPARγ
• Other studies have also show nonischemic LVF and deletion of PPARγ cofactor
and coactivator 1 resulted in:
– Decreased mitochondrial fatty acid oxidation alongside lipid homeostasis which
exacerbated overall cardiac function
• Hansmann et al.
– Indicated possible, favourable roles of PPARγ agonists in pressure-overload HF
• Yixinshu (traditional Chinese herbal medication)
– Protects against HF and enhanced hippocampal abnormality induced by HF by
modulating PPARγ pathway
26

27. Nuclear Factor Kappa-light-chain-enhancer of
activated B cells (NF-κB) Signaling Pathway:
• Preservative effects of SRA1 on hypoxia-induced injury in H9c2
cardiomyocytes transpired by regulating the NF-κB signaling
pathway
• NF-κB signaling pathway is widely involved in development of HF
• PPARγ shown to have the ability of inhibiting NF-κB pathway
Zhang et. al, 2018
27

28. • Can SRA1 regulate hypoxia-induced injury in H9c2 cells
by modulating NF-κB signaling?
– Suppression could activate NF-κB signaling in hypoxia-treated H9c2
cardiomyocytes
Nuclear Factor Kappa-light-chain-enhancer of
activated B cells (NF-κB) Signaling Pathway:
Zhang et. al, 2018
28

29. NF-κB Signaling Pathway: Important regulator
of inflammation
• Involved in many biological processes
• Activation is implicated in:
– Ischemia/reperfusion damage
– Myocarditis
– Myocardial Infarction
– Heart Failure
Zhang et. al, 2018
29

30. NF-κB Signaling Pathway
• Volz et al.
– Revealed extracellular heterodimeric protein S100A8/A9 could exasperate
post-ischemic HF by activating the signaling pathway
• Xing et al.
– YiQiFuMai (YQFM) injection (traditional Chinese medicine): could enhance
chronic HF through regulating NF-κB inactivation
30

31. NF-κB Signaling Pathway
• All findings propose a potential strategy for the treatment of HF
could be to single out NF-κB inactivation
• Results show that suppression of SRA1 activated the pathway in
cardiomyocytes
• Can safely assume that suppression of SRA1 might contribute to
progression of HF caused by activation of the NF-κB pathway
Zhang et. al, 2018
31

32. • SRA1 may serve as a promising perspective for HF therapy!
• Findings indicated suppression of SRA1 may aggravate hypoxia-induced injury
to H9c2 cardiomyocytes by positive regulation of PPARγ and activation of NF-
kB pathway
• Requires further analysis in SRA1’s relationship between:
– PPARγ
– NF-κB pathway
Where do we go from here?
32

33. Human Dilated Cardiomyopathy (DCM)
• Most common type that usually
occurs in adults 20-60
• Myocardial disease with dilatation
and impaired systolic function of
ventricles
• One of the prominent causes of
Sudden Cardiac Death, HF and
cardiac transplantation
• Causes considerable morbidity and
mortality
AHA
33

34. SRA1: Cosegregating Genes as Determinants of
Cardiomyopathy
• Genetic factors play a key role in etiology and pathogenesis of DCM
• Friedrich and colleagues’ study shows 600-kb region of linkage disequilibrium (LD) on
5q31.2-3
– Harbouring in 16 genes
– Associated cardiomyopathy in 3 independent Caucasian populations
• Assessment in zebrafish indicate at least 3 genes orthologous to loci in this LD block result
independently in a phenotype of myocardial contractile dysfunction
– Occurred when their expression was reduced with morpholino antisense reagents (gene expression
modifier)
Friedrich et. al, 2009
34

35. SRA1: Cosegregating Genes as Determinants of
Cardiomyopathy
• HF-associated LD block emerged through series of genomic rearrangements
across Amphibian, Aviana and Mammalian genomes
• In humans there is strong evidence for genomic clustering of genes through a set of genomic
arrangements under intense selective pressure
• Observations challenge the idea that disease phenotypes can be traced to altered function of
a single locus within a haplotype
– Indicating further analysis!!
• With the availability of large catalog single nucleotide polymorphisms (SNP) and high level
genotyping technology, LD mapping techniques are now commonly used to dissect the
genetic basis of a complex disease
Friedrich et. al, 2009
35

36. DCM
• Heterogeneous
– Monogenic and multifactorial factors
• Mutations in over 20 genes have been found to cause DCM (Franz et al. 2001)
– About half of these mutations are located in genes that encode sarcomere proteins
• Sarcomeric filament proteins have been reported to harbor missense mutations & restricted
to few families
– Actin (ACTC)
– Myosin (MYH7)
– Titin (TTN)
• Simply not enough studies that evaluate the genetic basis of DCM in a case-control setting
Friedrich et. al, 2009
36

37. SRA1: Cosegregating Genes as Determinants of
Cardiomyopathy
• Study evaluated a genomic region that houses risk alleles for DCM
– Alongside association studies and candidate genetic approach detailed in
proinflammatory mediators
• Provided evidence that multiple loci within the identified region could play a
role in disease & overall cardiac function
• Identified 600-kb LD block on chromosome 5q31.2-3
Friedrich et. al, 2009
37

38. Zebrafish Association Study
• Cardiac phenotypes are readily assessed through direct monitoring
of the heart in living animals
• Gene knockdown experiments identified3 genes orthologous to loci
within this human region
– Caused a distinct cardiac phenotype in an association study with a zebrafish
model
Friedrich et. al, 2009
38

39. • Cardiomyopathy study sample: DCM patients, Ischemic CM patients, healthy controls
– To identify common variants in myocardial dysfunction
• Genotyped 109 additional SNPs
– To fine map underlying genomic region
– Delineate underlying haplotypes
– Define most informative haplotype tagging SNPs for downstream analyses
Friedrich et. al, 2009
39

40. • SNPs were selected from HapMap project to attain genetic variation within and
beyond the main LD block, known coding, and promoter SNPs from the gene
region
• CM cluster: comprises 20 individual haplotypes with moderate to low
frequencies but no common haplotype
• Haplotypes were further analyzed according to evolutionary relationship
• Thus, related haplotype groups may harbour protective variants or replicates
the noted overrepresentation of DCM susceptibility haplotypes
Friedrich et. al, 2009
40

41. CM-cluster SNPs on Cardiac Function
Phenotypes
• Subphenotypes which consider intraventricular conduction delay &
frequently increased in patients with DCM
– LVEF
– LVEDD
– LVESD
– Duration of QRS complex
Friedrich et. al, 2009
41

42. SRA1 - HF in Zebrafish
• To evaluate function activities of cardiomyopathy cluster genes
– Assess function of orthologs in zebrafish
• Identify orthologous loci & conduct functional knockdown studies in zebrafish for 8 genes in
region
• MO antisense experiments performed for Zebrafish for 8 genes
• Of 3, one was SRA1
– All 3 independently resulted in impaired cardiac function phenotypes
– Impaired contractility in the ventricular heart chambers at 72 hours post fertilization (hpf)
• Other Zebrafish HF mutants
– MO-SRA1and 2 other injected embryos also showed pericardial edema at 72hpf
Friedrich et. al, 2009
42

43. Friedrich et. al, 2009
43

44. Cardiomyopathy Cluster Genes - Vertebrate
Evolution
• Findingsindicate at least 3 genes, including SRA1, in this disease
associated LD block play extremely important roles in cardiac
function & participate in disease risk
• Questions the hypothesis that changes in phenotype or disease risk
can be traced to a single functional variant within an allele
Friedrich et. al, 2009
44

45. Cardiomyopathy Cluster Genes - Vertebrate
Evolution
• So evolutionary analysis was conducted to communicate gene
susceptibility collected in one cosegregating genomic segment
– Trace emergence of cardiomyopathy cluster across multiple vertebrae
genomes
– 8 orthologs in zebrafish genome scattered across chromosomes 7, 14, 20, 21
Friedrich et. al, 2009
45

46. Friedrich et. al, 2009
46

47. Further Evaluation!
47

48. Plans to further investigate
• Evaluate BAM files via Golden Helix SVS Software to affirm SRA1
variant is called correctly
• Sangar Sequencing
– Segregate variant vs wild-type across affected and unaffected members of
family well enough
• Hamilton Lab has already entered the gene into “online
genematcher” to see if any other groups are gene-implicatedin
cardiac or noncardiac phenotype
48

49. Plans to further investigate
• Submit for “Rare Diseases Models and Mechanisms” funding
– Generate model in zebrafish by suppressing to see if cardiac phenotype is
expressed and observe abnormalities
– Work alongside zebrafish colleagues at St. Michael’s Hospital
• Expand Family
– Work alongside Toronto General Hospital’s Cardiology division to expand
pedigree in family to see who has SRA1 mutation
– Characterize by regular visits
49

50. The emerging roles of long noncoding RNAs in common
cardiovascular diseases
Jiang & Nang, 201550

51. • Cardiac genetics assessmentsby whole genome or whole
exome sequencing also need regular review, which can be
easily performed by Golden Helix SVS software.
• Links to new and frequently updated gene tracks and other
genetic databases facilitate these reviews.
51

52. Acknowledgements
• The Hospital for Sick Children and Research Institute
• Hamilton Lab
• Golden Helix
52

53. QUESTIONS?