1. 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

2. 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
Secondary HLH
Bone marrow transplant
Chemo (x1)
Targeted test (Cinci) $4,980
Based on slide
by Andrew Kung
WES (PGM) $6,000
MLL2: c.11640delG frame shift
Kabuki Syndrome diagnosis
30% of hereditary HLH
not accounted for by this panel
WES is not sensitive to incomplete differential diagnosis

3. 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

4. White Blood Cell Count (x 109/L)
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)
Days on Imatinib
Blast Cell Count (%)
Days on Imatinib
Do not limit testing based on anatomic location

5. FusionJunctionSequence
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)].
Based on slide
by Andrew Kung
Transcriptome:
Chr1
Position1
Chr2
Position2
KnownGene1
KnownGene2
FusionJunctionSequence
FusionGene 2 15
EML4
NTRK3
ACAGCCACGGGACctttacttgagac
EML4->NTRK3
EML4
NTRK3
The fusion that is present is not always the most common one

6. Highlight of next-Gen findings
Cancer Results Summary (Jan-June 2014)
Diagnosis
Samples
Tests
Highlight of next-Gen findings 1
Hepatosplenic T-cell Lymphoma
Spleen/ buccal
WES/ Transcriptome
STAT5B, JAK1, KRASV14I in recurrent lesion 2
Mediastinal Germ Cell
Tumor/normal
AURKA (VUS) 3
AML/maffuccis/olliers
IDH1R132C (somatic); NRAS, WT1 mutations 4
AML t(6;11)
BM/buccal
Only WES
NRAS, WT1 5
Alveolar Soft Part Sarcoma
Tumor/ blood
ASPCR translocation; AXIN1 mutation 6
AML
Sorted BM/buccal
WES
KIT (N655K) DDX3X mutations; PR to Imatinib 7
ALL to AML (MLL associated)
Two post-therapy tumors/ buccal
WES/
Transcriptome
Mutations in multiple pathways: NRAS, TP53 (R248Q, G245S), NOTCH2, TET1, DNMT1 , JAK3, APC, MLH1… 8
Metastatic Ewings
EWSR1/FLI1 translocation; copy number changes 9
“Infantile fibrosarcoma"
Tumor
EML4-NTRK3 fusion; PDX trial of Crizotinib 10
Metastatic Wilms
WES/Transcriptome
CREBBP, NF1 and MED12 mutations 11
Immature Teratoma Gr3
TP53 Y163H mutation with LOH 12
Plexiform Schwannoma
STAG2 A956D; predicted “disease causing” 13
Inflammatory Myofibroblastic Tumor
No ALK Translocation
VCAN-IL23R fusion; therapeutic trial Ruxolitinib 14
AML w/ thrombocytopenia
Blood
Constitutional RUNX1 mutation 15
r/o familial HLH
Constitutional MLL2 mutation (Kabuki Syndrome) 16
Neuroblastoma
Tumor/blood
NRAS; Loss of 1p; loss of distal 1q; 2p gain w/ amplicon (NMYC); 6qdel; 11qdel; 17q gain; “breakpoint” distal to ERBB2 17
ALCL (ALK+)
No perforin mutation; No sig tumor specific variant 18
Nested stromal tumor
No sig tumor specific variant. Trisomy 5, 12 and 20
Green: diagnosis & staging
Red: traditional or novel “actionable” target
Yellow:: decisions NOT to act
Blue: stratifies for specific treatment
Clinical impact goes far beyond traditional “actionable” mutations
Based on slide
by Andrew Kung

7. Hardware: Illumina sequencers
MiSeq x 2 HiSeq 2500 V4 x 2
up to 300 bp reads up to 250 bp reads
Three next-generation sequencing platforms
Roche 454
Pyrosequencing  chemiluminescent detection of pyrophosphate
-longer read lengths
Illumina genome analyzer
Reversible dye terminators
ABI Solid
Sequencing by ligation
10 genomes in 6 days or
one genome in 27 hours
>30x coverage
15 Gb per run 7

10. 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)

11. 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
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
Predisposing Germline Mutations (WES trio) ;
Somatic mutations (Normal and Cancer WES comparison) CNV detection by EXCAVATOR: Alberto Magi et al. Genome Biology 2013
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

12. 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

13. Quality metrics for WES

14. C1-mother
P-proband
C2-father

15. Individual dataset processing
NextGENe
Softgenetics
FASTQ
Mapping
BAM
Mutation
calling
VCF

16. 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

17. SNP catcher output Reference range filter Reportable range filter
All mutations in protein coding regions (+/- 5bp) listed in vcf file
(coverage >10 fold; allele frequency >10%)
Reference range filter
Reportable range filter
CATEGORY 1
Known pathogenic
mutations
(Clinvar/HGMD)
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)
CATEGORY 7
Rare mutations in known
disease associated genes
CATEGORY 8
Rare mutations in
non-disease
associated genes
Missense
Compound het
De novo
SS, FS, SC
Homozygous

18. 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 May;17(5): doi: /gim Epub 2015 Mar 5. PMID:

19. Discovery of new disease genes
Laboratory value
associated
phenotypes
information
OMIM terms
Patient phenotype
information
OMIM terms
15 core phenotype categories
Mouse phenotype
information
International Mouse
Phenotyping
Consortium
terms
Gene/system
associated
phenotype
information
OMIM terms

20. 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
POLG2
Mitochondrial disease, Progressive external ophthalmoplegia, Developmental delay, Lateonset ptosis myopathy;OMIM604983P
CDS 17 NA
homozygous
202
27.2 G A
Provean:
Deleterious;
SIFT: Damaging
R>W
604983:P 1

21. Functional follow-up with
Bill Copeland’s group

22. 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
ANKS1B NA
CDS 12
Heterozygous
136 59
AGTGTGT
AGTGT .
NA>NA
607815:P
0.43
Father
154 75
0.49
Patient
homozygous
286
267
0.93

24. Mouse phenotype associated with ANKS1B deletion

25. Should we be aware of our Achilles’ heel(s)?

26. 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.

28. PMS2 (p.S459*, c.1376C>G)

29. Constitutional mismatch repair deficiency (CMMR-D)
Vasen HFA, Ghorbanoghli Z, Bourdeaut F, et al. J Med Genet 2014;51:283–293.

30. 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

31. Long term goal: synthesis and cures
Metabolomics/regulatory networks
Proteomics/
Ribonucleoproteomics;
Structural
consequences
of genetic diversity
Transcript sequencing
to define regulatory
consequences of
genetic diversity
Genomic sequencing
to map genetic and epigenetic diversity

32. The greatest decade of medicine is upon us
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

33. Next generation sequencing based diagnosis of hearing loss
Peter L. Nagy MD, PhD
Director, Laboratory of Personalized Genomic Medicine
Columbia University

34. 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

35. Causes of prelingual hearing loss in children; over 400 loci identified

36. 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

37. 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

38. 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

39. 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

40. 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

41. 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%)

42. 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

43. Laboratories performing testing in US
Columbia PGM Website:

44. 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?

45. 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.