Clinical next-gen sequencing at CUMCLaboratory of Personalized Genomic Medicine

Peter L. Nagy M.D., Ph.D. Assistant ProfessorAssociate Director, Laboratory of Personalized Genomic Medicine

Department of Pathology and Cell BiologyColumbia University

MLL2: c.11640delG frame shift Kabuki Syndrome diagnosis

Prevention of unnecessary treatment: 2 month old with multiple congenital anomaliesRSV+ pneumonia, respiratory failureSplenomegaly, fever, cytopenias, hemophagocytosisClinical diagnosis of hemophagocytic lymphohistiocytosisEarly onset likely hereditary HLH

Targeted test (Cinci)$4,980

30% of hereditary HLHnot accounted for by this panel

Secondary HLHHereditary HLH

Bone marrow transplant Chemo (x1)

WES (PGM) $6,000

WES is not sensitive to incomplete differential diagnosis

Based on slide by Andrew Kung

RUNX1 Splice Mutation [IVS6-2 (808-2 A>G)] 30% Risk of AML, BOTH PROBAND and SISTER!

Avoidance of ineffective bone marrow transplantYoung girl with AML, prolonged thrombocytopenia with therapyUnable to tolerate conventional therapy, referred for BMTSister found to be perfect HLA match, however, PLT 160k…

Bone Marrow Aspirateof sister: histology normal

Other targeted tests all normal1. SBDS (Shwachman–Bodian–Diamond syndrome) 2. DEB test Fanconi anémia3.PNH (Paroxysmal nocturnal hemoglobinuria)4.FISH

WES

WES allows for evaluation of up to 3 relevant samples

Based on slide by Andrew Kung

cKIT N655K (tumor on left, normal on right)Relapsed AML: Finding an actionable mutation

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Based on slide by Andrew Kung

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)].

Precise diagnosis –optimal treatment

Chr1 Position1 Chr2 Position2 KnownGene1 KnownGene2 FusionJunctionSequence FusionGene

2 42472827 15 88576276 EML4 NTRK3 ACAGCCACGGGACctttacttgagacEML4-

>NTRK3

Transcriptome:

EML4 NTRK3

The fusion that is present is not always the most common one

Based on slide by Andrew Kung

Diagnosis Samples Tests Highlight of next-Gen findings

1Hepatosplenic T-cell Lymphoma Spleen/ buccal WES/ Transcriptome STAT5B, JAK1, KRASV14I in recurrent lesion

2 Mediastinal Germ Cell Tumor/normal WES/ Transcriptome AURKA (VUS)3 AML/maffuccis/olliers Tumor/normal WES/ Transcriptome IDH1R132C (somatic); NRAS, WT1 mutations4 AML t(6;11) BM/buccal Only WES NRAS, WT15 Alveolar Soft Part Sarcoma Tumor/ blood WES/ Transcriptome ASPCR translocation; AXIN1 mutation6 AML Sorted BM/buccal WES KIT (N655K) DDX3X mutations; PR to Imatinib

7ALL 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 Tumor/ blood WES/ Transcriptome EWSR1/FLI1 translocation; copy number changes9 “Infantile fibrosarcoma" Tumor Transcriptome EML4-NTRK3 fusion; PDX trial of Crizotinib

10 Metastatic Wilms Tumor/ blood WES/Transcriptome CREBBP, NF1 and MED12 mutations11 Immature Teratoma Gr3 Tumor/ blood WES/ Transcriptome TP53 Y163H mutation with LOH12 Plexiform Schwannoma Tumor/ blood WES STAG2 A956D; predicted “disease causing”

13Inflammatory Myofibroblastic Tumor Tumor/ blood WES/ Transcriptome

No ALK TranslocationVCAN-IL23R fusion; therapeutic trial Ruxolitinib

14 AML w/ thrombocytopenia Blood WES Constitutional RUNX1 mutation15 r/o familial HLH Blood WES Constitutional MLL2 mutation (Kabuki Syndrome)

16 Neuroblastoma Tumor/blood WES/ TranscriptomeNRAS; Loss of 1p; loss of distal 1q; 2p gain w/ amplicon (NMYC); 6qdel; 11qdel; 17q gain; “breakpoint” distal to ERBB2

17 ALCL (ALK+) Tumor/ blood WES No perforin mutation; No sig tumor specific variant 18 Nested stromal tumor Tumor/ blood WES No sig tumor specific variant. Trisomy 5, 12 and 20Green: diagnosis & stagingRed: traditional or novel “actionable” targetYellow:: decisions NOT to actBlue: stratifies for specific treatment

Cancer Results Summary (Jan-June 2014)

Clinical impact goes far beyond traditional “actionable” mutations

Based on slide by Andrew Kung

Hardware: Illumina sequencers

MiSeq x 2 HiSeq 2500 V4 x 2

up to 300 bp reads up to 250 bp reads

10 genomes in 6 days or one genome in 27 hours

>30x coverage15 Gb per run

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 diagnosis3. Constitutional Whole Exome Sequencing (WES)

Agilent Sureselect v5 + UTR4. Constitutional Whole Genome Sequencing (WGS)

Columbia cancer evaluationWell defined cancers with mutations known to affect therapy

Illumina Truseq cancer panel ~ 40 genesWell defined cancers to be categorized for clinical studies based on mutations

Columbia Combined Cancer Panel (CCCP)~ 500 genes1. Agilent Sureselect capture

>500-fold average coverage using Illumina 2500;>100ng starting material

Characterization of unique/rare cancer casesCancer 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 mutationsTranslocation 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

C2-fatherP-proband

Individual dataset processing

FASTQ

VCF

BAM

Mapping

Mutation calling

NextGENeSoftgenetics

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 outputAll mutations in protein coding regions (+/- 5bp) listed in vcf file

(coverage >10 fold; allele frequency >10%)

Reference range filter Reportable range filter

CATEGORY 1Known pathogenicmutations (Clinvar/HGMD)

CATEGORY 3Mutations in known Disease associated genes (Clinvar/HGMD)

ACMG secondary findings

CATEGORY 4Mutations in non-disease associated genes

Frequency filter (<1% allele frequency in 1000 genome project and EVS and internal database)

CATEGORY 7Rare mutations in known disease associated genes

CATEGORY 8Rare mutations in non-disease associated genes

SS, FS, SCDe novo HomozygousCompound hetMissense

CATEGORY 2Known pathogenicmutations not covered(Clinvar/HGMD)

CATEGORY 5Rare known pathogenicmutations (Clinvar/HGMD)

CATEGORY 6Rare known pathogenicmutations not covered(Clinvar/HGMD)

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

Patient phenotype informationOMIM terms

Mouse phenotype information

International Mouse Phenotyping Consortium

terms

Gene/system associated phenotype informationOMIM terms

Laboratory value associated phenotypesinformation

OMIM terms

15 core phenotype categories

Discovery of new disease genes

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

624925

43NANA

homozygous 202

202 27.2G A

Provean:Deleterious;SIFT: Damaging

R>W 60

4983:P

1

Functional follow-up withBill 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

MotherANKS1B NA CDS 12

991293

62NA NA

Heterozygous 136 59NA

AGTGTGT

AGTGT .

NA>NA NA

607815:P 0.43

FatherANKS1B NA CDS 12

991293

62NA NA

Heterozygous 154 75NA

AGTGTGT

AGTGT .

NA>NA NA

607815:P 0.49

Patient ANKS1B

NA CDS 12

991293

62NA NA

homozygous 286 267NA

AGTGTGT

AGTGT .

NA>NA NA 607

815:P

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)

Vasen HFA, Ghorbanoghli Z, Bourdeaut F, et al. J Med Genet 2014;51:283–293.

Constitutional mismatch repair deficiency (CMMR-D)

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

Genomic sequencingto map genetic and epigenetic diversity

Transcript sequencingto define regulatory

consequences of genetic diversity

Metabolomics/regulatory networks

Proteomics/Ribonucleoproteomics;

Structural consequences

of genetic diversity

SummaryGenome 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, PhDDirector, Laboratory of Personalized Genomic

MedicineColumbia 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.