1. Olga Jarinova, PhD, FCCMG
Next Generation Sequencing for Inherited Cardiomyopathies: Implications for Clinical Practices
Olga Jarinova, PhD, FCCMG
Associate Head, Regional Genetics Diagnostic Laboratory
Children’s Hospital of Eastern Ontario

2. Regional Genetics Diagnostic Laboratory (GDL) at the Children’s Hospital of Eastern Ontario
8,000 clinical specimens processed annually
>20 diseases tested, including
Inherited Cardiomyopathies

3. Expanded (45) vs Original (9) CM Panels

4. Expanded Cardiomyopathy Panels
Pan Cardiomyopathy (PanCM) – 45 genes
Dilated Cardiomyopathy (DCM) – 25 genes
Hypertrophic Cardiomyopathy (HCM) – 19 genes
Arrhythmogenic Right Ventricular Dysplasia (ARVC) – 9 genes
Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) – 2 genes

5. Test Development Cohorts
Retrospective Cohort
Previously tested by original panel
14 HCM cases; 10 ARVC cases; 12 presumed healthy individuals
Prospective Cohort
Tested by original and expanded panel
27 HCM cases; 9 ARVC cases

6. Test Development: Quality Metrics
Average read depth ~500x
99.78% of the targeted regions covered at > 20X
254 known variants detected in correct zygosity
100% analytical sensitivity
% false positive rate

7. Richards et al ACMG

9. Test Optimization and System Impact
Optimization cohort: 993 prospective cases with a diagnosis or suspicion of CM
HCM (5 /19 genes)
DCM (25 genes)
PanCM (45 genes)
Diagnostic yield
14.7% / 16.6%
- / 22.7%
- / 16.7%
VUS yield
14.5% / 27.4%
-/ 60.6%
- / 68.1%
This striking increase in rate of VUSs is associated with additional cost, increased need for genetic counselling resources and it is expected to have a significant psychosocial impact on individuals and their families.

10. Strategies for Reducing Proportion of VUSs
Gene panel content
Population frequency cut offs

11. Gene Panel Content
No pathogenic or likely pathogenic variants were identified in 24 out of 45 genes in our optimization cohort
HCM gene selection completed by ClinGen
DCM and ARVC gene selection will be completed in collaboration with ClinGen

12. 24 out 45 in our cohort
ClinGen Expert Panel 2017

13. Population Frequency Cut Offs
Bamshad et al., 2011; ClinGen Expert Panel, 2017; Whiffin et al. 2017

14. Frequency Cut Offs: New Workflow
NGS Data Filtering : frequency from > 1% to > 0.1%
Variant Interpretation Filtering : frequency from > 0.3% to > 0.03%

15. Validation of Reduced Frequency Cut Offs

16. Conclusions
CM panel expansion from 9 to 45 genes achieved more than 45% reduction in cost and led to an increase in diagnostic yield
Rate of inconclusive results increased significantly
New NGS data frequency cut offs reduced proportion of uncertain findings by 11% and had no impact on diagnostic yield
Gene panel redesign and new interpretation frequency cut offs are pending and expected to further improve test performance and system outcomes
Introducing Sanger waving policies achieved additional savings

17. Considerations for Introducing NGS-based Tests in Health-Care Setting
Analytical metrics and test volumes are NOT reliable estimates of overall test performance
Diagnostic yield and rate of uncertain findings are more reliable indicators of system impact
Ongoing monitoring of test performance is essential to achieving maximum diagnostic utility
Technology is advancing faster than understanding of disease mechanism of genes and individual variants

18. Acknowledgements Hussein Daoud, PhD GDL Laboratory Staff:
Mahdi Ghani, MD, PhD
Nasim Vasli, PhD
Caitlin Chisholm, MS, CGC
Amanda Smith, PhD, FCCMG
Ryan Potter, MLT
Shelley Ordorica, MLT
GDL Laboratory Staff:
Molecular Geneticists
Genomic Specialists
Molecular Technologists
Technicians
Jean McGowan Jordan, PhD, FCCMG
Robert Roberts MD, FRCPC
Liz Sinclair-Bourque, DipRCPath
Gabrielle Mettler, MS, GCG