Clinical Application of Whole Exome and Genome

Clinical Application of
Whole Exome and Genome
Sequencing
Elizabeth Chao, MD, FACMG
Department of Pediatrics, Division
of Genetics & Metabolism
UC Irvine
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Disclosures
A. “I have the following financial relationships
with the manufactures(s) of any commercial
product(s) and/or provider(s) of commercial
services discussed in this CME activity:”
• Consultant for: Ambry Genetics
B. I do not intend to discuss an
unapproved/investigative use of commercial
product/device in my presentation.
Diagnostic Exome Sequencing Flow
1
Family’s
samples arrive
2
Prepare exome
libraries and
sequence
4
5
Medical
review
6
10
Medical review:
Secondary findings
3
Informatics
filtering
Cosegregation
analysis
Primer
design
9
7
Secondary findings
report
8
Medical
review
Reporting
Why Order Whole Exome/Genome
Sequencing?
Indications
1. Diagnosis
Rare and heritable disease
2. Treatment
Cancer
Somatic Alteration
3. Prevention
Exome Sequencing To Identify
Mutations in Rare, Heritable Disease
• A genetic disorder is suspected based on
clinical findings, but limited and/or no
comprehensive panels are available.
• The patient's clinical presentation is
unclear/atypical disease and there
are multiple genetic conditions in the
differential diagnosis.
• A novel gene is suspected, but has yet to be
discovered.
14-year old female
• Hypotonia
• Developmental Delay
– Single word vocalization
• Seizures
• Cognitive Impairment
• Severe Ataxia
– Unable to ambulate
• Similarly affected 5yr old
sister
• Years of inconclusive
biochemical, molecular,
cytogenetics, invasive
testing
Exome Result
Likely Molecular Diagnosis: Mitochondrial Complex I Deficiency
• 103 patients
• Isolated complex I
deficiency by
biochemical analysis
• NGS of 103 candidate
genes (complex I
components) plus
2x7kb of mtDNA
Calvo et al Nature Genetics, 2010
NUBPL variants
• Homozygous c.166G>A
(p.G56R)
• Heterozygous intronic
variant c.815-27T>C
Padgett et sl.(Sep 2005) Splice Sites. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net
• Well conserved
• Have we identified the
causative mutations?
– Homozygous missense
– Compound Heterozygous
Calvo et al., Nat Genetics 2010
Interpret with care: Parental Testing
Apparently
^
• Homozygous c.166G>A (p.G56R)
• Inherited from father
• Needs deletion/duplication analysis
– Affymetrix cytoarray
Copy number analysis
• Heterozygous c.166G>A
(p.G56R)
• Array-based copy-number
Padgett et sl.(Sep 2005) Splice Sites. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net
Calvo et al., Nat Genetics 2010
p.G56R has no functional defect
• In-vitro assay import assay
of 35S-labeled protein
• Efficient import of WT and
mutant protein
• No difference in size
suggesting intact protein
processing
• Transduction of lentiviral
vector with mutant and WT
NUBPL into patient
fibroblasts
– Both expressed and stable
– Restored complex I deficiency
to WT levels
Calvo et al., Nat Genetics 2010
Tucker et al. Hum Mut 2012
c.815-27T>C causes aberrant splicing
• Abnormal splicing
– 2 abnormal transcripts
• Heterozygous intronic
variant c.815-27T>C
• Exon 10 skipping
• Partial intron 9 inclusion
Padgett et sl.(Sep 2005) Splice Sites. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net
• Branch Site (-18 to 40bp)
Padgett et al Trends in Genetics 2012
Calvo et al., Nat Genetics 2010
Tucker et al. Hum Mut 2012
Summary of Results
• Mutation 1 (Maternal):
Rearrangement
(Ex1_4del; Ex7dup)
• Mutation 2 (Paternal):
c.815-27T>C
– Branch site mutation
– Exon 10 skipping
– Missense variant in cis
and likely benign
Calvo et al., Nat Genetics 2010
Three cases of NUBPL-linked complex I
deficiency
Presenting
Calvo et al. 2010
Gender
Genetic
Current Age Age of onset Symptom
Alterations
Ataxia
Male
p.G56R
Ins/Del
Developmental
8
yrs
2
yrs
Delay
Yes
c.815-27T>C Rearrangement
Tenisch et al. 2012 p.G56R
Male
c.815-27T>C
23 yrs 2-3 yrsc.205_205delGT Yes
Current Patient
c.815-27T>C
14 yrs 6 mo p.L104P
Delay
Speech Ocular Sx Seizures
Yes
CNS Lactate Muscle Bx
Leukodystrophy
Slurred Nystagmus
T2 hyperinstensity of
cerebellum and
brainstem
Elevated
Impaired Nystagmus Yes
Cerebellar and pons
atrophy
Normal
Elevated
Mutations
Variant, Likely Pathogenic
Variant, Likely Benign
Plasma
Lactate
Nystagmus staring
Impaired Squint
spells
Developmental
Female
MRI
Normal
Elevated
~20% Ragged
Red Fibers
Complex I
Deficiency
Skeletal
Muscle and
Fibroblasts
Skeletal
Muscle
Pending
Pending
Impact on Family
• Molecular Diagnosis
• AR Inheritance pattern
– Known recurrence risk
• Previously reported
patients
– Stable, not progressive
• Awaiting confirmatory
enzyme testing
• Treatment Options
– Mitochondrial Cocktail
– Novel therapies
• EPI-743 (Leigh Syndrome)
Time to Diagnostic Exome Sequencing= 13 years
Time to Molecular Diagnosis= 6 weeks
Rare Gene Findings
Collaborative Effort
Connecting Clinicans in the Genomic Age
http://www.ambrygen.com/cde-genes
• Not every exome result is published today
• Whole exome sequencing is done at many labs
– Both research and clinical
• Are there any other patients out there like me?
• Are there any other patients out there like mine?
Rare Gene Findings
Collaborative Effort
http://www.ambrygen.com/cde-genes
Negative Exome Testing
• Review the differential
diagnosis
• Review the candidate
genes with the testing
laboratory
– What was the coverage?
– Was the gene/locus
considered?
• Disease may still be
heritable
• Consider other options
for clinical diagnosis
– Biochemical testing
– Biopsy, tissue, diagnosis,
etc.
• Additional Molecular
Testing options?
Human Genome: 6 billion basepairs (diploid)
What about Whole
Sequenced Exome: ~95%
Genome?
?
?
Helps but does not Cure
More Targeted
coverage Exome: ~97%
More cost
? More
Exome: ~20K genes
variants
Still limitations
Low Complexity Regions
Triplet repeats
My gene/mutation of interest
- Was it targeted?
- Was it covered?
Why Order Whole Exome/Genome
Sequencing?
Indications
1. Diagnosis
Rare and heritable disease
2. Treatment
Cancer
Somatic Alteration
Cancer Progression and Treatment
“….molecular alterations in
several cancers supported
targeted therapeutic
intervention on clinical trials
with known inhibitors,
particularly for alterations in
the RAS/RAF/MEK/ERK and
PI3K/AKT/mTor pathways.”
9 of 14 triple negative
breast tumors
Recurrent Pediatric Cancer
Hyundai Cancer Institute- CHOC
• Pilot Program
– Enrolling recurrent
refractory pediatric cancers
– Trio Analysis
• Primary, recurrent and
germline
• Whole Genome
Sequencing
• RNA-seq transcriptome
profiling
– Molecular Tumor Board
• Therapeutic Intervention
• Challenges
– Rarity of pediatric tumors
• 14 cases enrolled to date
• N=1 based analysis
– Availability of primary
tissue
• New protocols to bank
primary tissue in case of
recurrence
– Turn- around time
• Molecular analysis
– Therapeutic Availibility
Why Order Whole Exome/Genome
Sequencing?
Indications
1. Diagnosis
Rare and heritable disease
2. Treatment
Cancer
Somatic Alteration
3. Prevention
Healthy Exomes: Are we there yet?
Prevention
Healthy Exomes: Are we there yet?
Newborn Screening
Incidental Exome Findings
Do we have time to care?
Reportable incidental findings mutations include only:
– HGMD or OMIM-defined
– Well-established disease causing genes
– No variants of uncertain significance
– Predefined gene lists
•
•
•
•
ACMG minimum
Cancer predisposition (~90 genes)
Carrier Status (~150 genes)
Adult and Childhood onset disease (~400genes)
Time to Analysis:
–
–
–
–
Average of 30 variants reviewed per patient
Average of 1 disease-causing mutation identified
Carrier Status Range: 0-5
HFE, UROD, SCNN1G, CDH1, DGUOK, TTN, MYH7, etc.
Yes, we do and we should
Thank you
UC Irvine/CHOC
Ambry Genetics
Virginia Kimonis
Moyra Smith
Wenqi Zeng
Sha Tang
Jennifer Wei
Julie Neidich
Aaron Elliott
Sharon Mexal
Shela Lee
Layla Shahmirizadi
Melissa Parra
Kelly Gonzalez
Chad Garner
Lennie Sender
Pierre Baldi
Dan Mercola
Michael McClelland
RARE Global Genes Project
Nicole Boice
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