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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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Quality metrics for WES
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C1-mother P-proband C2-father
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Individual dataset processing
NextGENe Softgenetics FASTQ Mapping BAM Mutation calling VCF
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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
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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
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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:
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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
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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
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Functional follow-up with
Bill Copeland’s group
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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
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Mouse phenotype associated with ANKS1B deletion
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Should we be aware of our Achilles’ heel(s)?
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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.
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PMS2 (p.S459*, c.1376C>G)
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Constitutional mismatch repair deficiency (CMMR-D)
Vasen HFA, Ghorbanoghli Z, Bourdeaut F, et al. J Med Genet 2014;51:283–293.
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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
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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
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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
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Next generation sequencing based diagnosis of hearing loss
Peter L. Nagy MD, PhD Director, Laboratory of Personalized Genomic Medicine Columbia University
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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
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Causes of prelingual hearing loss in children; over 400 loci identified
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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
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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
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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
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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
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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
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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%)
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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
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Laboratories performing testing in US
Columbia PGM Website:
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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?
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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.
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