Exome

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Clinical Exome Sequencing

Pinar Bayrak-Toydemir, M.D., Ph.D.


Rong Mao, M.D.
Outline

1- Exome Sequencing Methodology

2- Guidelines / Recommendations

3- Real Life Experience


“Every dollar we spent to map the human genome has
returned $140 to our economy -- $1 of investment, $140 in
return.”

--President Obama April 2, 2013


Remarks by the President on the BRAIN Initiative and American Innovation,
History

1953 Discovery of DNA structure

1977 Discovery of Sanger sequencing

1985 Development of PCR

1999 First human chromosome sequenced- ch 22

2004 Development of next generation sequencing (NGS)

2008 First individual genome sequenced using NGS


Exome sequencing

A powerful tool for gene discovery

Over 200 genes have been discovered in a couple of years

Now a powerful diagnostic tool !


Next Generation Sequencing Cost Dropping

Cost per base is in free-fall !

http://www.genome.gov/sequencingcosts/
Cost driven test ordering change

$ ≈ $
FBN1 Aortapathy (Marfan and
Sanger sequencing Marfan like syndromes)
10 gene NGS panel
Total Number of Cases in ARUP
Next Generation Sequencing Lab
60

50

40

30
Total number of cases

20

10

0
Jan-Mar 2012 Apr-Jun 2012 Jul-Sep 2012 Oct-Dec 2012 Jan-Mar 2013
What is Exome sequencing ?
The sequence of all exons of the genome

What is missing?
Some protein coding genes
Some exons of some genes
Non-genic control elements
Copy number changes
Structural changes
mtDNA
Some microRNA genes
Why Exome Sequencing?
Focuses on the part of the genome we understand best,
the exons of the genes

Exons comprise 1% of the genome

~85% of all known disease causing mutations are located


on exons

Exome sequencing costs 1/6 of the cost of whole genome


sequencing
Diagnostic Yield

Based on the NIH Undiagnosed Diseases Program


clinical sensitivity of exome sequencing is around 20%

Possibly selection of “best” cases

Gahl et al., Vol14 (1) Jan 2012 | Genetics in medicine


Diagnostic Odyssey

Multiple congenital abnormalities

Intellectual disability

Unexplained developmental delay or declining


Preanalytic Considerations

Patient specific:
- well defined findings
- good evidence for a genetic basis

Family specific:
- affected family members
- inheritance pattern
Analytic Considerations

Limitations of exome testing


- capturing efficiency

Bioinformatic aspects
- variant calling
- filtering
- analyzing genes only in Human Genome
Mutation Database or OMIM
- analyzing genes on mandatory reporting
Postanalytic Considerations

Reporting
- negative, positive, uncertain for primary patient finding

Ethical and counseling issues

Patient consent

Education of consumers (patients, clinicians, payers)


Clinical Exome Sequencing
• Agilent and Nimblegen liquid capturing
• Indexing of samples (barcoding)
• Illumina HiSeq 2000
• Alignment / Variant calling / Phenotype scoring
• Candidate mutation list
• Interpretation
CLINICAL EXOME SEQUENCING
Work flow : Time Frame:
DNA (Sheared DNA)

Library prep 2 days


with automation

Enrichment (RNA or DNA beads in solution)

Barcoding
Cluster generation 1 day

14 days for paired-end


Sequencing

Data Analysis 5-10 days


CLINICAL EXOME SEQUENCING
Work flow :
DNA (Sheared DNA)

Library prep

Enrichment

Barcoding
Cluster generation

Sequencing

Data Analysis
CLINICAL EXOME SEQUENCING
Work flow :
DNA (Sheared DNA) DNA fragments

Library prep
Repair and prepare ends
Ligate adapters
Enrichment

Barcoding
Cluster generation

Adapters attach flow cells for


Sequencing cluster formation

Data Analysis
CLINICAL EXOME SEQUENCING
Work flow :
DNA (Sheared DNA) λmax
157 bp

After Sonication
Library prep 150-200 bp desired

Enrichment

Barcoding 244 bp
Cluster generation After adapter
binding

Sequencing

Data Analysis
Peak shift indicates successful library generation
CLINICAL EXOME SEQUENCING
Work flow :
DNA (Sheared DNA)

Library prep Gene : Ex 1 Ex 2

Coverage
Enrichment

Barcoding
Cluster generation

Sequencing

Data Analysis
Biotinylated RNA library baits covers all exons annotated in the
consensus CDS database as well as flanking sequence for each
targeted region and small non-coding RNAs
Work flow : CLINICAL EXOME SEQUENCING
DNA (Sheared DNA)

Library prep

Enrichment
A flow cell attached Hybridization of enriched Bridging
Barcoding with adaptors DNA to flow cell
Cluster generation

Sequencing

Data Analysis After amplification


x n cycles of amplification clustered fragments
4 reversible incorporate one capture cleave dye
Work flow : dye terminators nt at a time image terminator
DNA (Sheared DNA)
100 cycles

Library prep

Enrichment
100bp
Barcoding

Cluster generation

Sequencing

Data Analysis
T A C G - - -
Work flow :
DNA (Sheared DNA)

Library prep

Enrichment

Barcoding

Cluster generation

T G C A
Sequencing

Image of clusters during sequencing.


Data Analysis
Work flow : Paired-End Reading (2X100 bp)
DNA (Sheared DNA)
Seq primer Seq primer
Reads 100 bp Reads 100 bp
Library prep
Flip

Enrichment

Barcoding
Reference sequence
Cluster generation

Paired end reads


Sequencing

• Increase read coverage per cluster


Data Analysis • More accurate reading and alignment
• Detect small and large insertions, deletions,
inversions, and other rearrangements
Work flow : Sequencing Data, Exon Coverage of a Gene
DNA (Sheared DNA)

Library prep

Enrichment

Barcoding

Cluster generation

Sequencing

Data Analysis
Work flow :
DNA (Sheared DNA)

Library prep

Enrichment

Barcoding

Cluster generation

Sequencing

Data Analysis
GUIDELINES/REGULATIONS
CLIA/CAP/ACMG
Guide validation of samples, analysis and reporting

Genetics in Medicine, 2013


Direct laboratories to return with each genomic
sequencing order results from 57 genes in which
mutations greatly increase risk of 24 serious, but
treatable diseases, even if clinicians do not
suspect patients have them.
What are incidental findings?

Variants found by exome/genome sequencing , which are


unrelated to the disease of interest

- majority of them are benign


- a small number of them (between 1-5) might be well-described,
disease-associated mutations
Incidental Findings

The ACMG Working Group recommended that the laboratory


actively search for the specified types of mutations in the
specified genes listed in these recommendations.

Mandatory reporting known mutations for the disorders:


- Hereditary cancers,
- Marfan syndrome,
- Long QT syndrome,
- Brugada syndrome,
- Certain cardiomypathies
Patient Autonomy?

the ACMG Working Group did not favor offering the


patient a preference as to whether or not to receive the
minimum list of incidental findings described in these
recommendations.

This may be seen to violate existing ethical norms


regarding the patient’s autonomy and “right not to know”
genetic risk information.
Returning incidental findings in children
Recommendations for seeking and reporting incidental findings
not be limited by the age of the person being sequenced.

The ethical concerns about providing children with genetic risk


information about adult-onset diseases were outweighed by the
potential benefit to the future health of the child and the child’s
parent of discovering an incidental finding where intervention
might be possible.
Ambry ARUP Baylor Emory GeneDx UCLA
Name of test Clinical Diagnostic Exome Sequencing Whole Exome EmExome: Clinical XomeDx Clinical Exome
ExomeTM With Symptom- Sequencing Whole Exome Sequencing
Guided Analysis Sequencing
Began offering 09/2011 04/2012 10/2011 06/2012 01/2012 01/2012

Turn around time 8–16 12–16 15 15 12–16 11–12


(weeks)

Method (exome Agilent SureSelect Agilent SureSelect, NimbleGen NimbleGen SeqCap Agilent SureSelect Agilent SureSelect
capture) NimbleGen SeqCap (custom
designed) VCRome
2.1

Coverage: (mean 90–100X >100X >100X 100X 100-120X >100X


depth of coverage)

Coverage (% target 90% 95% >95% 96% 90–95% 95%


bases covered at
10)

Variant + Only primary + Only primary + Primary, some + Primary, all + Only primary + Only primary
confirmation secondary results secondary results

Jamal SM, Yu J-H, Chong JX, Dent KM, Conta JH, Tabor HK, Bamshad MJ. 2013. Practices and policies of clinical exome sequencing providers: Analysis and implications. Am J Med Genet Part A 9999:1–16
Exome Interp Algorithm: weekly meeting
Variants (SNV)s in 20-25,000 genes, ~ 20K-30K

SNVs ~2,000
Bioinformaticist

Inherited De Novo HGMD/OMIM


~ 40-60 Pathogenic ~200-400
~40-60
Symptom guided analysis

Variants interpretation: dbSNP, disease


database, SIFT, Polyphen 2, ARUP frequency, Medical Director/
publication, OMIM and HGMD Genetic Counselor

Sanger Confirm/Report
Bioinformatics Pipeline: NGS
Variant Viewer
Brendan O’Fallon: Bioinformaticist at ARUP
Pop. frequency:
e.g. Exclude all var with pop
frequency greater than 0.01
Exon effect: e.g. Exclude
var intergenic, intragenic,
UTR
Quality & Depth
Deleterious Score: SIFT,
PolyPhen, Mutation Taster
Genes & Regions

HGMD & OMIM

Courtesy of Brendan O’Fallon


Pedigree analysis: IGV viewer Incidental
Including affected findings
fam mem and 57 genes
parents

Courtesy of Brendan O’Fallon


ARUP frequency:

42 total, 2: autovalidation, 7:
noonan, 2: marfan, 31: HHT

Courtesy of Brendan O’Fallon


Case 1: trio
9 mos. boy
Postnatal growth failure, global DD, hypotonia
Mildly distinctive craniofacial features, dysphagia,
sleep disturbance
No fam Hx
Physical exam: global DD
Neuro/muscular
EEG, Echo
Brain MRI, Upper GI Normal
Microarray
Metabolic evaluation
CF Panel
Case 1: bioinformatics
Bioinformatics Data Analysis:
Overall: 44,760
Initial filtering criteria:
Remove var homo in parents: 19,688
Remove var freq >5%: 4,755
Remove synonymous and deep intronic var

Initial filtering yielded


781 missense mutations
105 exonic insertions / deletions
30 potential splice site variants
18 stop gains / losses
Total: 934
Case 1: variants review
Bioinformatics Data Analysis:
Inheritance: autosomal recessive, X-linked,
de novo
Clinical Information: patient symptoms
included hypotonia and fail to thrive
HGMD genes, Variant Ranking (Brendan
O’Fallon)
Yield: 381

Two medical directors review the variants


Case 1: candidate gene/mutation
MOGS gene: two missense variants V62M and
V567I/Sanger confirmed, parents: Het

c. 1699G>A, p.V567I

c. 184G>A, p.V62M

Courtesy to Brendan O’Fallon


Case 1: MOGS
 Autosomal Recessive
Mutation causing Congenital glycosylation disorder type
IIb
 phenotype: affect neonatal, severe hypotonia,
dysmorphic features
Biochemical assay: elevated oligosaccharides (urine)

Consultation with Dr. Longo : not likely


 Typical features of CDG: FTT (Has), strabismus (not listed),
abnormal cerebellum (not listed), inverted nipples (not
listed), abnormal fat pads in the buttocks (not listed) but
abnormal in the fingers.
Case 1: MOGS
Variant c.184G>A (p.V62M): in dbSNP 2.9% freq
Emory University Genetics Mutation database: benign
http://genetics.emory.edu/egl/emvclass/emvclass.php
 classification: Benign
Variant c.1699G>A (p.V567I): 0.9% freq, not in
literature
SIFT: deleterious, Polyphen2: damage
Classification: Variant of unknown significant (VUS)
Case 1: MLL2
De novo variant: c. 6664C>T, p. Q2222X ( 22X coverage)
 Kabuki syndrome
 distinctive facial features “peculiar face”
Skeletal anomalies: brachydactyly,
spinal deformity
Mild-moderate mental retardation
Postnatal growth deficiency

 Sanger sequencing: failed confirmation


Case 1: report
 No pathogenic mutation detected by symptom-
guided analysis
One VUS found in MOGS , V567I

Follow up: Oligosaccharides and transferrin normal

Lesson learned: clinical phenotype plays important


role for data analysis
Case 2: Proband only
Clinical Information:
3 year-old female with intractable epilepsy,
hypotonia, and developmental regression.

aCGH performed at the University of Florida


detected UPD9. One mutation detected in Tpp1
gene. Physician interested in evaluating “neuro”
genes on Chromosomes 9 and X.

Parental samples were not submitted.


Case 2: Bioinformatics
Bioinformatics Data Analysis:
Initial filtering yielded
1093 missense mutations
93 exonic Insertions / deletions
916 potential splice site variants
26 stop gains / losses
Total: 2128

No relatives available for filtering


Clinical Information: Patient symptoms included
epilepsy, intractable seizures, hypotonia and
developmental delay
HGMD genes, Variant Ranking (Brendan O’Fallon):
Yield: 291
Case2: Alexander Disease?
Varian in GFAP:
Gene Variant Inheritance Phenotype

GFAP c. 469G>A, p. Autosomal Alexander Disease


Asp157Asn (D157N), Dominant
Missense

 Alexander disease: AD, early onset seizures,


psychomotor impairment, developmental delay,
macrocephaly Dx: Brain MRI
 Contact MD, normal MRI
 Variant also in 0.5% population
Case2: Report

 2 mutations and 1 VUS detected


Gene Var. Zygosity Inheritance Var. Phenotype
Category

GRIA3 c.381_382insG Homo X-linked Pathogenic X-linked


p.GLy127 fs developmental
delay
TPP1 c.196C>T Hetero Recessive Pathogenic Neuronal ceroid
p.Q66X lipofuscinosis type
2
GABRG2 c.1204G>A Hetero Dominant Variant of Neuortransmitter
p.A402T unknown Generalized
significant epilepsy and febrile
seizures
Case2: Follow up
 Parental specimens received:
 GRIA3c.381_382insG (p.GLy127 fs): Homo-
mother and hemi-father : not causative for
patient pheno
 TPP1 c.196C>T (p.Q66X): Het-father
 GABR2 c.1204G>A (p. A402T): Het-father, AD
not causative

Lesson learned: proband only will be difficult


for data analysis/interpretation and lower the
positive yield
Case 3: trio
 11 yrs. male
 Globe DD, short stature, feeding problem require G-tube,
hypotonia, hypoplastic genitalia, pectus carinatum,
behavioral problems, broad deviated thumbs and great
toes, dysmorphic facial features including a flat face,
posteriorly rotated ears
 Fam history, NO
CMA SNP
FISH for DiGeorge, Prader-Willi, subtelomeric
rearrangements, 16p for Rubinstein-Taybi
Normal
Metabolic screening wit UOA/AA, urine MPSs
Karyotype , 46, XY
EEG and Brain MRI
Case 3: Bioinformatics/Variant review
Bioinformatics Data Analysis:
 Same initial filtering criteria used

Inheritance: autosomal recessive, X-linked, de novo


Clinical Information: patient symptoms included
Globe DD, short stature, feeding problem,
hypotonia, hypoplastic genitalia, behavioral
problems, broad deviated thumbs and great toes,
flat face, posteriorly rotated ears

HGMD genes, Variant Ranking (Brendan O’Fallon)


Case 3: Candidate Gene/mutation
ARID1B gene: de novo variant, c.4204G>T, p.E1402X

c.4204G>T, p.E1402X
Case 3: ARID1B
ARID1B: At-rich interaction domain-containing
protein 1B
Santen et al, 2012, Nature
Genetics:
“de novo truncated mutations
in ARID1B gene in three
individuals with Coffin-Siris
syndrome”
Case 3: Coffin-Siris
Globe developmental delay
Short stature
Feeding difficulties
Hypotonia
Moderate to severe learning difficulties
Broad thumbs and toes
Posterior rotated ears

Mostly AR, can also be sporadic or AD


Case 3: Report
 One pathogenic mutation that is predicted
to be causative to the patient's symptoms
was detected

Gene Var. Zygosity Inheritance Var. Phenotype


Category

ARID1B c.4204G>T Hetero De novo Pathogenic Coffin Siris


p.E1402X syndrome
Conclusion
 Clinical exome sequencing has a great potential for
diagnosing diseases of unknown etiology; possible
leading to improve treatment and patient care.
Quality control measures, data analysis and reporting
of incidental findings will continue to evolve and
improve.
Exome interpretation is optimally performed by
including bioinformaticians, geneticists and clinicians
Acknowledgement
ARUP R&D ARUP Genomics Lab
 Whitney Donahue •Ana Hooker
 Shale Dames •Jennifer Stocks
 Brendan O’Fallon •Tyler Wayman
•Marc Singleton
•Lisa Robles
University of Utah •Josh Raney
•Nicola Longo
•Alan Rope ARUP Genetics Counselors
•Chris Miller
•Patti Krautscheid
ARUP Institute for Clinical & ARUP Laboratories
Experimental Pathology

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