Exome sequencing for disease gene identification and patient diagnostics

1. Exome sequencing for diagnostics
Christian Gilissen
Radboud University Nijmegen Medical Center
Department of Human Genetics
c.gilissen@gen.umcn.nl

2. Published exome sequencing studies
Gilissen et al. Genome Biology, 2011

3. Strategies to prioritize variants from exome studies
SETBP1 – Schinzel Giedion syndrome
Hoischen et al. Nat gen. 2010
WDR35 – Sensenbrenner syndrome
Gilissen et al. AJHG 2010
DYNC1H1 etc. – Intellectual disability
Vissers et al. Nat gen. 2010
SERPIN1F – Osteogenisis Imperfecta
Becker et al. AJHG 2011
ASXL1 – Bohring-Opitz syndrome
Hoischen et al. Nat gen. 2011
IMPAD1 - Chondrodysplasia
Vissers et al. AJHG 2011
ACTB/ACTG – Baraitser-Winter syn.
Riviere et al Nat gen. 2012
ABCC9 - Cantu syndrome
Van Bon et al. AJHG 2012
Gilissen et al. EJHG, 2012

4. Diagnostics
Can we use sequencing technologies for the
diagnosis of genetically heterogeneous
diseases were current tests have a low
diagnostic yield?

5. Targeted versus whole exome
• Targeted:
• Low chance of incidental findings
• Easy analysis
• Cheap (?)
• Exome:
• Unbiased, not dependent on a gene set
• Uniform approach for all diseases
• Synergy

6. Approaches
Gene package approach
• Exome sequence a single individual with a specific
indication
•In vitro enrichment for known disease genes
•Prioritization for obvious candidates outside of known
genes
De novo approach
• Exome sequence patient-parent trio
•In vitro enrichment for known disease genes
•De novo candidates
•X-linked and recessive candidates
•Prioritization for obvious candidates outside of known
genes

7. How to implement Exome sequencing for diagnostics
1.Increase diagnostic yield
3.Prevent incidental findings
5.Quality control of sample
7.Easy and standardized interpretation

8. Results
• Proof of Concept: 50 samples for 5 genetically
heterogeneous disorders

9. Analysis
1.Quality control
3.Coverage of gene packages
5.Interpretation
7.Visualization

10. 1. Quality Control
• QC statistics
• Gender match
• 12 SNP pyrosequencing test
• Trio concordance

11. 2. Coverage of disease gene package

12. 2. Disease gene coverage

13. Disease gene coverage - Agilent SureSelect version 4

14. % reads showing Known
variant SNP
HGMD
Kegg
GO Exonic /
Splice site
OMIM
Protein
Mouse
domains
phenotypes
Other exomes
Grantham
Conservation

15. 3. Interpretation
• Automated annotation per indication
• Database of known disease genes
• Automated prioritization
• Graphical user interface
• Stand-alone web-application
• Flexible with respect to input files
• Prevent unrelated findings
• Flexible with respect prioritization
• Allow for quality control

16. 4. Visualization

17. Results – variants requiring attention

18. Results – diagnostic yield?
• Exact diagnostic yield is still being calculated
• Preliminary results indicate that:
• Yield varies considerably per gene package
• Considerably higher than with current diagnostic tests
• E.g. blindness package: > 40% positive reports

19. Conclusion
• We designed and implemented an analysis for exome
sequencing based genetic diagnosis of genetically
heterogeneous disorders.
• Minimizes risk of incidental findings
• Allows for strict quality control
• Standardized interpretation

20. Acknowledgments
• Rick de Reuver
• Marisol del Rosario
• Nienke Wieskamp
• Yannick Smits
• Joris Veltman
• Marcel Nelen
• Hans Scheffer
• Genomic disorders group
• DNA Diagnostics Nijmegen

22. Prioritization of variants causing genetic disease
• Number of coding variants:
~ 12,000
• Private* non-synonymous
variants:
~ 150-200
*Not detected in dbSNP or other control
data.
How to identify the 1 causative variant?