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Clinical next-gen sequencing at CUMC
Laboratory of Personalized Genomic Medicine
Peter L. Nagy M.D., Ph.D. Assistant Professor
Associate Director, Laboratory of Personalized Genomic Medicine
Department of Pathology and Cell Biology
Columbia University
Prevention of unnecessary treatment:
2 month old with multiple congenital anomalies
RSV+ pneumonia, respiratory failure
Splenomegaly, fever, cytopenias, hemophagocytosis
Clinical diagnosis of hemophagocytic lymphohistiocytosis
Early onset likely hereditary HLH
Hereditary HLH
Bone marrow transplant
Secondary HLH
Chemo (x1)
Targeted test (Cinci)$4,980
Based on slide
by Andrew Kung
WES (PGM)
30% of hereditary HLH
not accounted for by this panel
$6,000
MLL2: c.11640delG frame shift
Kabuki Syndrome diagnosis
WES is not sensitive to incomplete differential diagnosis
Avoidance of ineffective bone marrow transplant
Young girl with AML, prolonged thrombocytopenia with therapy
Unable to tolerate conventional therapy, referred for BMT
Sister found to be perfect HLA match, however, PLT 160k…
Based on slide
by Andrew Kung
RUNX1 Splice Mutation [IVS6-2 (808-2 A>G)] 30% Risk
of AML, BOTH PROBAND and SISTER!
Bone Marrow Aspirate
of sister: histology normal
Other targeted tests all
normal
1. SBDS (Shwachman–Bodian–
Diamond syndrome)
2. DEB test
Fanconi anémia
3.PNH (Paroxysmal nocturnal
hemoglobinuria)
4.FISH
WES
WES allows for evaluation of up to 3 relevant samples
Relapsed AML: Finding an actionable mutation
cKIT N655K (tumor on left, normal on right)
Based on slide
by Andrew Kung
White Blood Cell Count (x 109/L)
70
60
50
40
30
20
10
0
0
5
10
15
20
Days on Imatinib
25
30
0
5
10
15
20
Days on Imatinib
25
30
90
80
Blast Cell Count (%)
70
60
50
40
30
20
10
0
Do not limit testing based on anatomic location
Precise diagnosis –optimal treatment
2 year-old boy with congenital left upper extremity
hemimelia who developed an expansile soft tissue
mass at 15 months of age.
Biopsy of the mass revealed a malignant spindle
cell neoplasm with histologic features consistent
with a diagnosis of infantile fibrosarcoma.
However, the tumor was negative for the
characteristic ETV6-NTRK3 fusion [t(12;15)
(p13;q25)].
Transcriptome:
Chr1 Position1 Chr2 Position2 KnownGene1 KnownGene2
2
42472827
15
88576276
EML4
EML4
NTRK3
Based on slide
by Andrew Kung
FusionJunctionSequence
FusionGene
EML4ACAGCCACGGGACctttacttgagac >NTRK3
NTRK3
The fusion that is present is not always the most common one
Cancer Results Summary (Jan-June 2014)
Diagnosis
Samples
Tests
Hepatosplenic T-cell
Lymphoma
Mediastinal Germ Cell
AML/maffuccis/olliers
AML t(6;11)
Alveolar Soft Part Sarcoma
AML
Spleen/ buccal
Tumor/normal
Tumor/normal
BM/buccal
Tumor/ blood
Sorted BM/buccal
WES/ Transcriptome
WES/ Transcriptome
WES/ Transcriptome
Only WES
WES/ Transcriptome
WES
Two post-therapy
tumors/ buccal
Tumor/ blood
Tumor
Tumor/ blood
Tumor/ blood
Tumor/ blood
WES/
Transcriptome
WES/ Transcriptome
Transcriptome
WES/Transcriptome
WES/ Transcriptome
WES
13
14
15
ALL to AML (MLL
associated)
Metastatic Ewings
“Infantile fibrosarcoma"
Metastatic Wilms
Immature Teratoma Gr3
Plexiform Schwannoma
Inflammatory
Myofibroblastic Tumor
AML w/ thrombocytopenia
r/o familial HLH
Tumor/ blood
Blood
Blood
WES/ Transcriptome
WES
WES
16
Neuroblastoma
Tumor/blood
WES/ Transcriptome
17
18
ALCL (ALK+)
Nested stromal tumor
Tumor/ blood
Tumor/ blood
WES
WES
1
2
3
4
5
6
7
8
9
10
11
12
Green: diagnosis & staging
Red: traditional or novel “actionable” target
Yellow:: decisions NOT to act
Blue: stratifies for specific treatment
Highlight of next-Gen findings
STAT5B, JAK1, KRASV14I in recurrent lesion
AURKA (VUS)
IDH1R132C (somatic); NRAS, WT1 mutations
NRAS, WT1
ASPCR translocation; AXIN1 mutation
KIT (N655K) DDX3X mutations; PR to Imatinib
Mutations in multiple pathways: NRAS, TP53
(R248Q, G245S), NOTCH2, TET1, DNMT1 , JAK3,
APC, MLH1…
EWSR1/FLI1 translocation; copy number changes
EML4-NTRK3 fusion; PDX trial of Crizotinib
CREBBP, NF1 and MED12 mutations
TP53 Y163H mutation with LOH
STAG2 A956D; predicted “disease causing”
No ALK Translocation
VCAN-IL23R fusion; therapeutic trial Ruxolitinib
Constitutional RUNX1 mutation
Constitutional MLL2 mutation (Kabuki Syndrome)
NRAS; Loss of 1p; loss of distal 1q; 2p gain w/
amplicon (NMYC); 6qdel; 11qdel; 17q gain;
“breakpoint” distal to ERBB2
No perforin mutation; No sig tumor specific variant
No sig tumor specific variant. Trisomy 5, 12 and 20
Clinical impact goes far beyond
traditional “actionable”
mutations
Based on slide
by Andrew Kung
Hardware: Illumina sequencers
MiSeq x 2
up to 300 bp reads
15 Gb per run
HiSeq 2500 V4 x 2
up to 250 bp reads
10 genomes in 6 days or
one genome in 27 hours
>30x coverage
Constitutional genetics
Well characterized/defined conditions associated with many
different genes
1. Mitochondrial Genome Sequencing (Long range PCR)
2. Columbia Combined Genetic Panel (CCGP) ~ 1300 genes
(Custom Agilent Sureselect)
Conditions with uncertain diagnosis
3. Constitutional Whole Exome Sequencing (WES)
Agilent Sureselect v5 + UTR
4. Constitutional Whole Genome Sequencing (WGS)
Columbia cancer evaluation
Well defined cancers with mutations known to affect therapy
Illumina Truseq cancer panel ~ 40 genes
Well defined cancers to be categorized for clinical studies
based on mutations
Columbia Combined Cancer Panel (CCCP)~ 500 genes
1. Agilent Sureselect capture
>500-fold average coverage using Illumina 2500;
>100ng starting material
Characterization of unique/rare cancer cases
Cancer Whole Exome Sequencing (CWES) has 3 components
Agilent Sureselect version 5+ UTR; >150 fold coverage
1.Predisposing Germline Mutations (WES trio) ;
2.Somatic mutations (Normal and Cancer WES comparison) CNV detection by
EXCAVATOR: Alberto Magi et al. Genome Biology 2013
3.Cancer transcriptome sequencing; Greater than 50 million uniquely mappable
reads
Confirmation of somatic mutations
Translocation detection (FusionMap) Huanyin Ge et. al. Bioinformatics 2011
Detection of overexpression of oncogenes and silencing of tumor suppressors ;
Rankit – PGM developed
Ethical considerations, patient consent
• Testing for heritable conditions requires consent
• Consent requires patient education
• Essential role for geneticists and genetic counselors
– Points to review
• What does a genetic diagnosis mean
• Secondary findings; right to know, right not to know
– ACMG recommendations
– Carrier status
•
•
•
•
•
Recording of results in patient’s electronic records
Storage of genetic information
Reinterpretation of genetic information
Access to raw data
Storage of DNA
Quality metrics for WES
C1-mother
P-proband
C2-father
Individual dataset processing
NextGENe
Softgenetics
FASTQ
Mapping
BAM
Mutation
calling
VCF
Comparative analysis: “SNP-catcher”database
– Windows sql server
– Integrates gene description, allele frequencies and functional
predictions from the internet (GeneCards, CLINVAR, OMIM, MSV3D)
– Integrates frequency calculations from 1000 genome, EVS and internal
database
– Prioritizes mutations based on phenotype and pattern of inheritance
– Search function based on phenotype, model system phenotype,
molecular system associations
SNP catcher output
All mutations in protein coding regions (+/- 5bp) listed in vcf file
(coverage >10 fold; allele frequency >10%)
Reference range filter
CATEGORY 1
Known pathogenic
mutations
(Clinvar/HGMD)
Reportable range filter
CATEGORY 2
Known pathogenic
mutations not covered
(Clinvar/HGMD)
CATEGORY 3
Mutations in known
Disease associated genes
(Clinvar/HGMD)
CATEGORY 4
Mutations in
non-disease
associated genes
ACMG secondary findings
Frequency filter
(<1% allele frequency in 1000 genome project and EVS and internal database)
CATEGORY 5
Rare known
pathogenic
mutations
(Clinvar/HGMD)
CATEGORY 6
Rare known
pathogenic
mutations not covered
(Clinvar/HGMD)
Missense Compound het
CATEGORY 7
Rare mutations in known
disease associated genes
De novo
SS, FS, SC
CATEGORY 8
Rare mutations in
non-disease
associated genes
Homozygous
Standards and guidelines for the interpretation of sequence
variants: a joint consensus recommendation of the American
College of Medical Genetics and Genomics and the Association for
Molecular Pathology.
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm
HL; ACMG Laboratory Quality Assurance Committee.
Genet Med. 2015 May;17(5):405-24. doi: 10.1038/gim.2015.30. Epub 2015 Mar 5.
PMID: 25741868
Discovery of new
disease genes
Patient phenotype
information
OMIM terms
Laboratory value
associated
phenotypes
information
OMIM terms
15 core phenotype categories
Gene/system
associated
phenotype
information
OMIM terms
Mouse phenotype
information
International Mouse
Phenotyping
Consortium
terms
Clinical presentation with fulminant hepatic
failure
• Few months old boy
• Severe reduction of mtDNA copy number in blood, liver,
and muscle (75%, 85%, 80% respectively)
• No mutation found in POLG1
Mitochondrial
disease,
Progressive
external
ophthalmopleg
ia,
Developmental
delay,
Lateonset
ptosis
C
624
myopathy;OMI D
925 N
POLG2 M604983P
S 17 43NA A
ho
mo
zyg
20
ous 202 2 27.2G A
Provean:
60
Deleterious;
49
SIFT:
R> 83:
Damaging W P
1
Functional follow-up with
Bill Copeland’s group
When model organisms are the key osteogenesis imperfecta
•
•
•
•
•
Prenatal intrauterine fracture of femur
Osteopenia
Wormian bones
Blue sclera
OI panel negative: COl1a1, COL1a2, CRTAP, LEPRE1,
PPIB,FKBP10, SERPINF1, PLOD2, SERPINH1, SP7, BMP1, WNT1,
TMEM38B, ALPL negative
Mother
ANK
S1B NA CDS
Father
ANK
S1B NA CDS
Patient
ANK
S1B NA CDS
Het
991
eroz
293
ygo
AGTG AGT
12 62NA NA us 136 59NA TGT GT .
Het
991
eroz
293
ygo
AGTG AGT
12 62NA NA us 154 75NA TGT GT .
991
hom
293
ozyg
AGTG AGT
12 62NA NA ous 286 267NA TGT GT .
607
NA>
815:
NA NA P
607
NA>
815:
NA NA P
607
NA>
815:
NA NA P
0.43
0.49
0.93
Mouse phenotype associated with
ANKS1B deletion
Should we be aware of our Achilles’ heel(s)?
Case in point
• 5 y.o male with T-ALL and sibling who passed away
from medulloblastoma.
• Patient is from Saudi Arabia.
• No information on consanguinity.
PMS2 (p.S459*, c.1376C>G)
Constitutional mismatch repair deficiency (CMMR-D)
Vasen HFA, Ghorbanoghli Z, Bourdeaut F, et al. J Med Genet
2014;51:283–293.
Opportunities for collaborative innovation
• Doctors dealing with patients with genetic disease and cancer and data
science/medical informatics experts
– translational studies supported by comprehensive variant and clinical
databases
• Scientists studying specific processes, genes, pathways
– Adopt a gene – adopt a pathway – act as consultant for interpretation
• Structural biologist
– Map variants identified in clinical samples onto protein and RNA structures
to define functional domains and protein-protein and protein-nucleic
acid and protein-lipid interaction surfaces
• Analytical biochemists
– Comparing contrasting peptide signatures and metabolite levels with
genome and transcriptome data
• Computer scientist interested in data display and visualization
– Google Earth –Google Cell ; Facebook -Genebook
• Business majors
– Working out models for making these diagnostics tools accessible to all
Long term goal: synthesis and cures
Metabolomics/regulatory networks
Transcript sequencing
to define regulatory
consequences of
genetic diversity
Proteomics/
Ribonucleoproteomics;
Structural
consequences
of genetic diversity
Genomic sequencing
to map genetic and epigenetic diversity
Summary
Genome level diagnosis of human conditions is a
transforming event in history of medicine and humanity
The greatest decade of medicine is upon us
Next generation sequencing
based diagnosis of hearing loss
Peter L. Nagy MD, PhD
Director, Laboratory of Personalized
Genomic Medicine
Columbia University
Background
• 1 in 500 newborns ; 360 million people worldwide
• Greater than 80 genes with more than 1000 reported
deafness-causing mutations
• Importance of testing:
– Rare actionable mutations
– Prognostication
– Heritability information to patients
– Exclusion of syndromic causes
– Prevention of unnecessary and costly testing
Causes of prelingual hearing loss in
children; over 400 loci identified
Autosomal Dominant Hearing Loss
Syndromes
• Waardenburg syndrome
– Hearing loss plus pigmentary abnormalities
– PAX3, MITF, EDNRB, EDN3,SOX10
• Branchiootorenal syndrome
– Developmental abnormality of branchial pouches
– EYA1, SIX1, SIX5
• Stickler syndrome
– Skeletal and eye abnormalities
– COL11A1, COL11A2, Col1A1
• Neurofibromatosis 2
– Various malignancies- e.g. acoustic Swannomas
– NF2
Autosomal Recessive Hearing Loss
Syndromes
• Usher syndrome; 50% of deaf-blind
– Vestibular problems and retinitis pigmentosa
• Pendred syndrome
– Thyroid abnormalities and enlarged vestibular aquaduct
– SLC26A4
• Jervell and Lange –Nielsen sy.
– Elongated QT interval
• Biotinidase deficiency
– Complex metabolic problems; seizures, developmental
delays, ataxia
• Refsum disease
– Retinitis pigmentosa and phytanic acid abnormalities
X-linked deafness syndromes
• Alport syndrome
– Renal problems
• Mohr-Tranebjaerg syndrome
– Deafness-dystonia-optic atrophy
– TIMM8A
• Mitochondrial deafness syndrome
– Association with diabetes
– MTTL1 – Japanese patients
– Same mutation as MELAS
Genes associated with nonsyndromic
hearing loss
• Autosomal dominant; > 27 genes – none
predominant
• Autosomal recessive; >35 genes –GJB2
responsible for 50%
• X-linked; 3 genes
• Mitochondrial; 3 mutations
• 20 studies included in the review analysis
• Total of 426 control samples and 603 patients with
unknown causes of hearing loss
• Sensitivity and specificity 99%
• Variation in genes tested
– Deafness genes are still being discovered
– Some authors combine syndromic and nonsyndromic
testing
– Inclusion of candidate genes
Diagnostic rate
• 41% (range,10%-83%)
– Varies with
• mode of inheritance; autosomal dominant inheritance
is higher (60%) than autosomal recessive (40%)
• prescreening prior to comprehensive testing (GJB2)
• the number and type of genes included
• whether copy number variations were examined
– Lowest yield
• in adults - potential environmental causes
• sporadic cases with no family history
• Copy number changes might be responsible for over 10 % of
hearing loss (STRC region) – few labs test for it (30%)
Traditional and Nextgen Sequencing
Comparison
• Sanger
– all exons of a single gene may be sequenced with this method at
a cost in the clinical laboratory ranging from $1000 to $3000 per
gene
– turnaround time of about 3 months per gene
• Next generation sequencing
– Columbia combined genetic panel (CCGP); Proband only
• Up to 20 genes
• Up to 40 genes
• Greater than 40 genes
• parents are tested for free if testing required to establish
pathogenecity
– Exome – trio tested
Laboratories performing testing in US
Columbia PGM Website:
http://pathology.columbia.edu/diagnostic/PGM
Clinical dilemmas
• Which panel to use?
– Number of genes tested vary from 20 to 139
• Should exome sequencing be used as a first
line
• How to deal with incidental findings?
• Who will provide genetic counseling to the
patient and the patient’s family?
Conclusion
• Comprehensive testing provides a better
overall diagnostic rate on varying ethnicities
than single gene testing
• Is not significantly more expensive than single
gene testing
• It is now considered the standard of care for
genetic diagnosis of sensori-neural hearing
loss.