Van test naar diagnose naar

Transcription

1 Van test naar diagnose naar V therapie op maat Marjolein Kriek, LUMC Joris Veltman, RUNMC

2 Exome diagnostics in genetically heterogeneous disease Joris Veltman, PhD Department of Human Genetics Radboud University Nijmegen Medical Centre

3 Genetic diagnostics of heterogeneous disease Examples: Blindness, deafness, movement disorders, mitochondrial disorders, intellectual disability, heriditary cancer 100s of genes can cause disease when mutated Many of the disease-causing genes unknown Different types of mutations can cause disease Diagnostic yield low with most current methods No information on cause, prognosis, recurrence risk, treatment

4 DNA sequencing becomes cheaper fast! Moore s law From: Stein Genome Biology :207 doi: /gb

5 The ideal scenario to start disease gene identification DNA from blood/saliva Genome sequence with all variation Important: - Accuracy - Speed - Price

6 Whole genome sequencing examples

7 Whole genome sequencing; Ready to be applied? In genome sequencing centers: Yes! Throughput in academic labs too small Price: 2,000-5,000 consumables higher coverage = higher quality = higher price Data-storage and analysis challenging Result: 3-4 million variants per genome How to interpret this variation, especially outside of coding regions? Alternatives?

8 Exome sequencing Exome (all exons of a genome) ~1% of the human genome All coding sequences of a human genome (>180,000 exons) are specifically selected for sequencing and analysis in one experiment

9 Exome sequencing exome-wide mutation detection In liquid exome enrichment: Agilent s SureSelect Next generation sequencing: Life technologies 5500XL exomes/system/week Nijmegen 2012: ~1000 exomes

10 Mapping and annotation of exome sequencing reads seq reads targets genes

11 Previous experience Explorative targeted next gen sequencing studies Assay development can be optimized per disease Regular updating required Exome sequencing in Mendelian disease research Highly reproducible method Coverage not perfect Interpretation can be focused & straightforward Exome sequencing a generic diagnostic test? Hoischen et al. Nat Genet 2010; van Bon et al. AJHG 2012; Hoischen et al. Nat Genet 2011; Rivière et al. Nat Genet 2012 Hoischen et al. Human Mutation 2010 Neveling et al. Human Mutation 2012

12 Exome sequencing practicalities: Aims in diagnostics High diagnostic yield Practical diagnostic workflow Minimize incidental findings All patients entered after pre-test counseling by clinical geneticist

13 Diagnostic workflow Blindness, deafness, movement disorders, Mitochondrial disorders, colorectal cancer Intellectual disability Novel gene Candidate ID gene Known ID gene

14 Informed consent All individuals must agree with the entire procedure All individuals must understand the possibility of unsought for findings and agree to be informed. Unsought for findings will be assessed by independent expert committee

15 Standardized exome sequencing workflow Step 1: Generic exome sequencing Agilent V4 sureselect, SOLiD 5500XL sequencing

16 Quality criteria established for diagnostics Exome sequencing implemented in diagnostic laboratory and accredited by Dutch Accreditation Council in 2011.

17 Next step: Disease-specific interpretation Gene packages developed and updated regularly by team of Clinical Molecular Geneticist, Clinical Geneticist, Research Specialist & Bioinformatician

18 User Interface Important: Use friendly software for interpretation Selected variants with annotation Easy data filtering QC information

19 Filtering variants in known disease genes Median absolute deviation between brackets Follow-up: - Mutational impact by majority vote of SIFT, Poly-phen-2 & Align GVGD - Phylo-P > 2 - Validate variant by Sanger sequencing - Study inheritance & phenotype of patient with literature

20 Efficient analysis and interpretation of germline mutations requires study of patient-parent trios DNA from blood/saliva Genome sequence with all variation Vissers et al. Nature Genetics 2010 Veltman & Brunner. Nat Rev Genet 2012

21 Diagnostic exome sequencing in 100 ID trios

22 # Patients Summary of clinical and genetic work-up of 100 ID patients Total dedicated gene tests: 233 Number of different genes: 56 Range per patient: 0-13 genes 20 Other tests performed: 100 SNP microarrays 79 metabolic screens 34 brain MRIs 4 cerebral CT scans Number of dedicted gene tests

23 Diagnostic yield in 100 ID patients Positive diagnosis All mutations 16 De novo mutations 13 Autosomal dominant 10 X-linked 2 Autosomal recessive 1 Inherited mutations 3 X-linked 3 Autosomal recessive 0 Candidates 19 No. of patients International collaboration & data sharing essential to improve interpretation of mutations in novel genes! de Ligt et al. New Engl J Med. 2012

24 Impact of genetic diagnosis in severe ID End to long search (on average > 2 years) Information about cause Information about prognosis Information for future reproductive choices Prevention of further complications: - ketogenic diet for patients with PDHA1 mutation - antiepileptic treatment, avoiding sodium-channel blockers for patient with SCN2A mutation

25 Is personal genomics already useful? January 2010

26 From association study to personal genomics What do companies claim to offer: - Insight in disease risk - Carrier status for rare recessive disorders - Pharmacogenetics information - Genetics of personal triats - Ancestry information - Paternity testing

27 How do I respond to drugs?

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30 Conclusions Exome sequencing can be used as generic diagnostic test for genetically heterogeneous diseases Analysis can be targeted towards patient s disease, reducing amount of work & incidental findings Improvements in sequencing technology & data interpretation are important for widespread implementation

31 Acknowledgements Clinical genetics Ilse Feenstra Marjolein Willemsen Bregje van Bon Tjitske Kleefstra Sascha Vermeer David Koolen Anneke Vulto-van Silfthout Carlo Marcelis Marlies Kempers Bert de Vries Wendy van Zelst Han Brunner Genome Diagnostics Kornelia Neveling Helger Yntema Marcel Nelen Lies Hoefsloot Eric-Jan Kamsteeg Arjan Mensenkamp Richard Rodenburg Marjolijn Ligtenberg Rolph Pfundt Hans Scheffer Genome Research Christian Gilissen Lisenka Vissers Joep de Ligt Alex Hoischen Jayne Hehir-Kwa Frans Cremers Hannie Kremer Marisol del Rosario Nienke Wieskamp