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The first whole genomes James Watson Craig Venter
Nature 2008
Nature 2008
PloS Biology 2007
Nature 2008
Marjolein Kriek
Ozzy Osbourne genome sequencing project
Jim Lupski
TEDMED 2010
Ozzy Osbourne
Nijmegen Clinical Exome Sequencing 2011- 2015
Clinical Exome Requests 2013-2014
All individuals must agree with the entire procedure
All individuals must understand the possibility of incidental findings
and agree to be informed.
Data will be stored and may be shared with other researchers
Informed consent
Full exome or targeted analysis?
Most families with severe or progressive disease choose full exome e.g. severe ID in child Some patients with milder or stable conditions choose targeted analysis e.g. deafness in adult
Next generation sequencing is a tool for diagnosis and differential diagnosis Accross clinical specialities
- Reanalysis of data 2012-2014 Better mapping algorithms Candidate genes confirmed Better variant interpretation * 27% Diagnosis by 2014 Genome sequencing Gilissen et al. Nature 2014 * 40% Diagnosis
Things get better over time
De Ligt et al NEJM 2012 - Trio exome sequencing in 100 patients with unexplained ID * 16% Diagnosis
Majority of new candidate ID genes already confirmed
YY1
DYNC1H1
DEAF1
CIC
ZMYM6
GRIA1
PHIP
WAC
MIB1
PPP2R5D
KIF5C
COL4A3BP
EEF1A2
MYTL1
CAMIIKG
ASH1L
PSMA7 RAPGEF1
PROX2
LRP1
TANC2
TNPO2
PHACTR
MTF1
GATAD2B
CTNNB1
Additional mutations No additional mutations
DDX3X
Gilissen et al. Nature 2014 De Ligt et al. New England Journal of Medicine 2012
De Vries et al. American Journal Human Genetics 2006
New GeneticTechnologies elucidate Intellectual Disability
More coding de novo mutations with Whole genome Sequencing
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Average number of
coding de novo
mutations per individual
8
125
167 40 79 67
294 305 167 103
23 38
329 242
85 84
Patients Controls Number of identified coding de novo mutations #
De novo structural variants, example 1
•A patient with the clinical suspicion of Rett syndrome
•MECP2 gene tested by Sanger and MLPA but no mutations identified
FW1
RV1
FW2
RV2 MLPA probe
600 bp
MECP2, exon 4 (~1000 bp)
t g a a a c aa t g t ct t t g c g c t c t c c c t c c c c t c g g t g t t t c g c t t c c t g c c g g ggc g t t t g a t c a
Example 2: A de novo duplication on chromosome 4
patient
father
mother
Chromosome 4
Log2
ratio
• ~60 kb duplication on chromosome 4 • Affecting the last 6 exons of the TENM3 gene • TENM3 is associated with coloboma, and microphthalmia
250k SNP microarray
Duplication from chr4 to chrX
chr4
chrX
Array + Exome + Whole Genome ~ 60% diagnosis in ID
Gilissen et al. Nature 2014
The hit rate of diagnostic exome sequencing (including CNVs) Clearly Exceeds The current diagnostic standard of clinician driven investigation
An inconvenient truth
4 years old
- Severe intellectual disability no speech at 4 years
- Normal length, weight, OFC
- MRI: mild cerebral atrophy
- Hypotonia
Negative for: -MECP2 -SNP array -UBE3A
DD: Angelman/Rett like
- Stereotypic movements,
De novo TCF4 mutation
Pitt Hopkins syndrome
Severe intellectual disability no speech at 4 years
- Normal length, weight, OFC
- MRI: mild cerebral atrophy
Hypotonia
- Stereotypic movements,
Severe intellectual disability, autism
Lessons from doing exomes
-Midface hypoplasia -Broad nasal bridge One abnormal ear
Unilateral deafness Unilateral facial palsy
CHD7 mutation > CHARGE syndrome!
Severe intellectual disability, autism -Midface hypoplasia -Broad nasal bridge One abnormal ear
Unilateral deafness Unilateral facial palsy
Targeted analysis across 5 diagnostic groups
Compare with clinical diagnostic practice 2011
Neveling et al. Human Mutation 2013
Exome sequencing 20.000 genes of Ender and his parents Shows that Ender has just 1 de novo mutation. This mutation is in the PACS1 gene
Exome sequencing 20.000 genes of Siebe and his parents Shows that Siebe has just 2 de novo mutations. One mutation is in the PACS1 gene
New genes
Exome sequencing 20.000 genes of Ender and his parents Shows that Ender has just 1 de novo mutation. This mutation is in the PACS1 gene
Exome sequencing 20.000 genes of Siebe and his parents Shows that Siebe has just 2 de novo mutations. One mutation is in the PACS1 gene
And they have the same PACS1 mutation
Schuurs-Hoeijmakers et al. Am J Hum Genet 2012
Now 18 patients with PACS1 Arg203Trp mutation
Some through Matchmaking DDD – Decipher / clinicians Most matches through mother’s Facebook page !
Is exome sequencing costly? ~ $ 2000
Number of patients with dedicated gene tests: 78 of 100 Total dedicated gene tests: 233 Number of different genes: 56 Range per patient: 0-13 genes Also: 100 SNP microarrays 79 metabolic screens 34 brain MRIs 4 cerebral CT scans
0
100
200
300
400
500
600
700
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4
2013 2014 2015
# of
test
s Sanger vs Exomes
Sanger
Exomes
Three reasons why Diagnostic Next Generation Sequencing of all genes (exome) is becoming part of mainstream medicine: • 1500 Euros is not prohibitive, and we are replacing Sanger-based tests • We are not as good at clinical diagnosis as we would like to think • Process not more difficult to handle than current diagnostic strategies Misdiagnosis Missed diagnosis Confusion Harm
The candidate gene problem is rapidly going away NGS diagnostic services should ideally update their diagnoses Seeing all the VUSs teaches us diagnostic humility Matchmaking between experts is highly effective The next step is to involve the patients more
Observations
Acknowledgements Willy Nillesen Dorien Lugtenberg Helger Ijntema Rolph Pfundt Marjolein Willemsen Bregje van Bon Tjitske Kleefstra Bert de Vries Lisenka Vissers Charlotte Ockeloen Tjitske Kleefstra Anneke Vulto – van Silfhout
Human Genetics Christian Gilissen Jayne Hehir-Kwa Joep de Ligt Alex Hoischen Djie Tjwan Thung Maartje van de Vorst Petra de Vries Michael Kwint Irene Janssen Marloes Steehouwer Tan Bo Joris Veltman
Complete Genomics Rick Tearle Robert Klein Richard Leach
Washington University, Seattle Heather Mefford, Gemma Carvill Evan Eichler, Bradley Coe
Many clinical collaborators
