This Lecture On “Clinical Use Of Whole Genome And Whole .
Welcome to the PHC Webinar Series on“Hot Topics in Pathology”This lecture on “Clinical Use of Whole Genome andWhole Exome Sequencing Today”presented by David Bick, MD andPaula E. North, MD, PhD FCAP.Your host is Jill Kaufman, PhD.For comments about this webinaror suggestions for upcomingwebinars, please contactJill Kaufman at jkaufma@cap.orgTHE WEBINAR WILL BEGIN MOMENTARILY. ENJOY! 2011 College of American Pathologists. All rights reserved.1
David Bick, MD Professor of Pediatrics andObstetrics & Gynecology atMedical College of Wisconsin Section Chief of the Division ofGenetics in the Department ofPediatrics at Medical College ofWisconsin Medical Director, Genetics atChildren's Hospital of Wisconsin Director of the AdvancedGenomics Laboratory in theDepartment of Pediatrics at theMedical College of Wisconsin 2010 College of American Pathologists. All rights reserved.2
Paula E. North, MD, PhD FCAP 2010 College of American Pathologists. All rights reserved. Professor of Pathology, TraditionalPathway, Medical College ofWisconsin (MCW) Chief of Pediatric Pathology,Department of Pathology, MCW Medical Director of Pathology andLaboratory Medicine, Children‟sHospital of Wisconsin Associate Director of the Children‟sResearch Institute (CRI) Director of three researchsupportive CRI Core facilities(Histology, Imaging, and PediatricBioBank/Tissue Analytical Core)3
DisclaimerThe College does not permit reproduction of any substantial portion of thematerial in this Webinar without its written authorization. The College herebyauthorizes attendees of the CAP Webinar to use the pdf presentation solely foreducational purposes within their own institutions. The College prohibits use ofthe material in the Webinar – and any unauthorized use of the College’s nameor logo – in connection with promotional efforts by marketers of laboratoryequipment, reagents, materials, or services.Opinions expressed by the speaker are the speaker’s own and do notnecessarily reflect an endorsement by CAP of any organizations, equipment,reagents, materials or services used by participating laboratories. 2010 College of American Pathologists. All rights reserved.4
Clinical Use of Whole Genome and Whole ExomeSequencing TodayDavid Bick, MD and Paula North, MD, PhDDecember 14, 2011www.cap.orgv. #
Topics David Bicko First clinical case at Children‟s Hospital of Wisconsin/Medical College ofWisconsin (CHW/MCW)o WGS program initiated in 2010 at CHW/MCWo Key counseling issues Paula Northo WGS vs WESo Quality management strategieso Technical challenges in a CAP/CLIA environment Instrumentation Software Reporting 2010 College of American Pathologists. All rights reserved.6
DisclosuresThe following relationship(s) exist related to this presentation:o Children‟s Hospital of Wisconsin (CHW) and Medical College of Wisconsin(MCW) provide whole genome sequencing (WGS) for clinical use, andthe patient is billed for these technical and professional diagnosticservices.o WGS is not an FDA approved test; the FDA has determined that suchapproval is not necessary.o Testing (sequencing and analysis) is performed in a CLIA/CAP approvedlaboratory. Audience participation:o Information collected today by polling the audience may be used beused in future presentations & publications.o All information collected is anonymous.o You are not required to participate in the polling process.– polling process.1Footnote:Century Gothic, 9 pt, sentence case 2010 College of American Pathologists. All rights reserved.7
Whole Genome Sequencing (WGS) & Whole Exome Sequencing(WES) is in clinical practiceBaylor College ofMedicine offersWhole exomesequencing:
WGS/WES isnow inclinicalpracticePartners HealthCareSystem, which is affiliatedwith Harvard MedicalSchool and includesMassachusetts GeneralHospital and Brigham andWomen's Hospital, hasenrolled its first family inthe sequencing programand plans to follow thepilot effort by introducingthe technology to itshospitals early in 2012.
Polling Question #1Would you want to have your genome sequenced? yes no 2010 College of American Pathologists. All rights reserved.10
Polling Question #2Education?1.Pathologist2.Non-pathologist attending MD3.Medical student, resident or fellow4.Ph.D.5.Laboratory staff 2010 College of American Pathologists. All rights reserved.11
Nic‟s story Presented at 15 months: poorweight gain and a perianalabscess Progressed: Inflamation entirecolon & developed fistulae to theskin Severe Crohn‟s Bowel rest, immunosuppression andother rx - failed In 3 yr: 142 anesthesia for varioussurgeries and treatments 2010 College of American Pathologists. All rights reserved.12
Difficult to treat a condition if you do not know the cause The cause of Crohn's disease isunknowno chronic inflammatory reaction ofthe intestinal mucosa directedagainst microbiota of the gut ingenetically susceptible individualso Identified over 50 susceptibilitygeneso Immune system „over-reacts‟ to gutflorao Medications suppress the immunesystem Nic‟s severity required a differentapproacho Sequence the genomeo Hope to find a treatable geneticdisorder 2010 College of American Pathologists. All rights reserved.13
Next – generation (NexGen) sequencing also called massivelyparallel sequencingRoche – 454sequencerIllumina – Hi-SeqLife Technology –Ion TorrentPacificBioSciences - RS500 Mb for 15K9000 Mb for 15K19 Mb for 9932 Mb for 502nd generation2nd generation3rd generation3rd generation 2010 College of American Pathologists. All rights reserved.14
Analyzed Nic‟s Exome 16,124 variants (SNP, small dup, small del)o Map to reference genome 7,157 non-synonymous (changed an amino acid)o Filter thru variant database – dbSNP 878 novel variants (not in dbSNP)o Unaffected parents – filter for recessive & X-linkedo Filter thru programs that predict whether change damages proteinfunction 136 geneso Filter AA change based on evolutionary conservation program 2010 College of American Pathologists. All rights reserved.15
More analysis 35 geneso Filter for genes that are not frequently inactive in the general population 5 geneso Filter for genes know to cause disease that are biologically relevance topatient 1 gene: XIAP - an X-linked diseaseo Developed an informatics package to do this – analysis took months! 2010 College of American Pathologists. All rights reserved.16
Phylogenetically conserved AA in XIAP – Nic had a tyr instead ofa cys 2010 College of American Pathologists. All rights reserved.17
XIAP (inhibitor of apoptosis protein 3) mutations cause X-linkedlymphoproliferative (XLP) syndrome Fatal or near-fatal EBV infection - lymphadenopathy, hepatosplenomegaly,fulminant hepatitis, hepatic necrosis, and profound bone marrow failure Hypogammaglobulinemia Lymphomas (cancer of lymphocyte) or other lymphoproliferative disease 70% of individuals with XLP die by the age of 10 years Only possible cure is a bone marrow transplant Nic’s symptoms did not match so XLP was not considered!! 2010 College of American Pathologists. All rights reserved.18
Nic had a bone marrow transplant – all of his findings resolved 2010 College of American Pathologists. All rights reserved.19
MCW/CHW Whole Genome Sequencing program startedsummer 2010 Goal: Clinical utilization of WGS for diagnostic purposes in a pediatricpopulation Purpose: Define molecular etiology of complex, rare, likely monogenicdiseases for medical decision-makingo Employ whole genome sequencing Key to success: Senior leadership of CHW & MCW involved at the beginning 2010 College of American Pathologists. All rights reserved.20
MCW/CHW WGS Program Case Nomination Review Committee – MDs, ethicists, scientistso Assure that all reasonable testing already done, testing will advanceclinical care and is medically necessaryo 41 reviewed, 14 in process Genetic counseling 2010 College of American Pathologists. All rights reserved.21
Counseling regarding results Primary Result (1 )o Likely pathogenic change(s) felt to be responsible for the patient‟sphenotype Secondary Result (2 ) or “incidental finding”o Result likely unrelated to the patient‟s phenotypeo BUT felt to cause a different disease/greatly increase risk for a differentdisease 2010 College of American Pathologists. All rights reserved.22
Incidental findings:Sequencing finds a genetic condition that was unexpected „Medically actionable‟:o refers to a variant in a genewhere knowledge of theparticular variant will affectmedical decision making suchas initiation of a treatment 2010 College of American Pathologists. All rights reserved. „Not medically actionable‟:o refers to variants that increasethe individual‟s risk for adisease where no treatment isproven to significantly changemedical decision making.23
Examples – childhood onsetMedically actionable Biotinidase deficiencyo unable to recycle the vitaminbiotino Seizure, hypotonia, ataxia,developmental delayo Biotin rx prevents all problemso Childhood onset – treatable 2010 College of American Pathologists. All rights reserved.Medically not actionable Tay-Sachs diseaseo Hexosaminidase A deficiencyo Unable to degradeglycosphingolipid GM2ganglioside in the braino progressive neurodegenerationo starting at 6 mo of ageo Death before age four yearso Childhood onset – nottreatable24
Examples – adult onsetMedically actionable BRCA1 – autosomal dominantbreast and ovarian cancero Common before 50 yoo 57% breast by 70 yoo 40% ovarian by 70 yoo Can has mastectomy &oophorectomy- Reduces risk 90%o Adult onset – treatableMedically not actionable Familial Alzheimer – autosomaldominanto PSEN1, PSEN2, APPo Onset in 40‟s & 50‟so Severe memory failureeventually incapacitatingo Confusion, poor judgment,language disturbance,agitation, withdrawal,hallucinationso Adult onset – not treatableSequencing a child can giveinformation about the parents ! 2010 College of American Pathologists. All rights reserved.25
Categorical Model of Choicemandatorydisclosuresoptionaldisclosures2 1 treatablechildhooddiagnostic2 none 2010 College of American Pathologists. All rights reserved.2 notactionablechildhood2 actionableadulthood2 notactionableadulthood26
Counseling TimeClinical geneticsevaluation9%Formal g19% 2010 College of American Pathologists. All rights reserved.Exploration ofexpectations16%Inheritance14%6-10 hours totalTestMethodology9%CategoricalModel of Choice18%27
Polling Question #3Your child is having their genome sequenced. Would you want to be told ofan incidental finding that is childhood onset & treatable (e.g., Biotinidasedef.)?1.yes2.no 2010 College of American Pathologists. All rights reserved.28
Polling Question #4Your child is having their genome sequenced. Would you want to be told ofan incidental finding that is childhood onset & not treatable (e.g., Tay-Sachsdisease)?1.yes2.no 2010 College of American Pathologists. All rights reserved.29
Polling Question #5Your child is having their genome sequenced. Would you want to be told ofan incidental finding that is adult onset & treatable (e.g., BRCA1 – early onsetbreast cancer)?1.yes2.no 2010 College of American Pathologists. All rights reserved.30
Polling Question #6Your child is having their genome sequenced. Would you want to be told ofan incidental finding that is adult onset & not treatable (e.g., FamilialAlzheimer – autosomal dominant )?1.yes2.no 2010 College of American Pathologists. All rights reserved.31
Who should decide? A parent decides to find out about adult onset diseases in their child Now the child has lost the right to decide NOT to knowo Perhaps a parent will not leave an inheritance to the child who willdevelop Alzheimer diseaseo Perhaps the child would not want to know that they will get Alzheimerdisease 2010 College of American Pathologists. All rights reserved.32
Polling Question #7Your child is having their genome sequenced. Should you be excluded fromknowing an incidental finding that is adult onset & not treatable (e.g., FamilialAlzheimer – autosomal dominant)?1.yes2.no 2010 College of American Pathologists. All rights reserved.33
Decision-MakingParental DecisionsPrimaryChild-No RxAdult-ActionAdult-No ActionYES – 9YES – 9YES - 9YES - 7NO – 2NO – 0NO – 0NO – 2Families have follow up each year because theirdecision may change and variant may changecategories 2010 College of American Pathologists. All rights reserved.34
Laboratory Considerations in WGS/WESo WES vs WGSo Considerations in a CAP/CLIA environment Instrumentation Software Reporting Quality Management 2010 College of American Pathologists. All rights reserved.35
Exome sequencing – sequence of the coding region Human Genome: 20K to 25Kgenes Geneso Genes composed of exonsthat code for AA of aproteino Introns are spacer regionsthat are spliced outo Can interpret a change inAA sequence such as anArg to a stop codon 2010 College of American Pathologists. All rights reserved.36
Exons are shown as colored part ofgene Capture array has complementarysequence of each exon bound tosolid support Single strand DNA of exonshybridize Selected DNA sequenced 1% of the genome 2010 College of American Pathologists. All rights reserved.37
Whole genome sequencing (WGS) Human genome 3.1 billion basepairs WGS – determining the sequenceof an individuals genomeo Includes sequence of thegenes – exons & intronso Includes sequence of regionsbetween genes 2010 College of American Pathologists. All rights reserved.38
WES vs WGSAdvantages of WES Exome is 1% of genomeo WES costs much less than WGS Exome includes only the codingregiono Tools to interpret changes bestdeveloped for exons 2010 College of American Pathologists. All rights reserved.Advantages of WGS WGS has better coverageo Exome capture array does notcapture all exons Certain parts of introns and regionsbetween genes can be used tomake a diagnosis of disease39
How does it work?Next Generation (NexGen) technologies produce millions of shortlengths of DNA sequenceRoche – 454sequencerIllumina – Hi-SeqLife Technology –Ion TorrentPacificBioSciences - RS500 Mb for 15K9000 Mb for 15K19 Mb for 9932 Mb for 502nd generation2nd generation3rd generation3rd generation 2010 College of American Pathologists. All rights reserved.40
A short length of sequence is called a read Example: Illumina Hi- Seq Each read is 100 bp 160M of these reads! Jigsaw Puzzle .o Need to connect sequences toeach other 2010 College of American Pathologists. All rights reserved.41
Aligning each „read‟ against sequence of reference humangenome17 reads include thisnucleotide therefore17X coverage 2010 College of American Pathologists. All rights reserved.42
Major challenges to the use of genomic data There is no reference/normal humangenomeo More than 2 million SNPs aredifferent between any twounrelated individualso Software cannot map all reads NCBI (National Center forBiotechnology Information)maintains:o Reference assembly of thehuman genome that is derivedfrom many individualso Databases of variants 2010 College of American Pathologists. All rights reserved.43
Other challenges The current reference humangenome (called Hg19 or Build 37)has 250 gaps Much variation not in Hg19o WGS can end up withunmapped readso Read with an insertion ordeletion may not map to refgenome Ref genome & database ofvariants improvingCurrent NexGen devices works wellfor single bp substitutionsOthers variation problematic 2010 College of American Pathologists. All rights reserved. Partial list of genomic variants andstructureso Single base-pair substitutionsoInsertionsoDeletionsoAdjacent insertion and deletionoGC rich regionsoTrinucleotide repeatsoCopy number variantsoHomopolymer tractsoTranslocationsoInversionsoShort tandem repeatsoPseudogenesoHighly polymorphic regions (e.g. HLAlocus)44
Instrumentation considerations Roche 454, Life Technologies SOLiD, and Illumina most frequently usedo Each with pros/conso Machines generate reads & quality score for each nucleotide of eachreado Reads mapped to human genomeo Differences between the reference human genome and the patientrecorded in a file: variant file Clinical laboratory: validationo Must validate the test for each type of variant that the system is designedto detecto Include DNA isolation, library preparation, sequencing run and dataanalysiso Validate using samples with known mutations Clinical laboratory: controls in each run and proficiency testing 2010 College of American Pathologists. All rights reserved.45
Unresolved instrument issues Manufacturers frequently improve reagents and steps in the processo Changes require re-validation before clinical use.o This re-validation is expensive.o As an option, a lab can send the sample to a CAP/CLIA lab forsequencing then analyze the resulting variant file in-house. Currently there are no reference materials for validation, proficiency testing orquality control for each runo Labs currently choose samples from their own institution or Coriell Institute Clinical Laboratory: establishing precisiono Repeatability - testing sample multiple timeso Reproducibility – testing by multiple operators during different runso The optimal number of runs and samples is not established. 2010 College of American Pathologists. All rights reserved.46
Software considerations Each manufacturer uses different program foro Nucleotide quality scoreo Mapping reads to human genomeo These are frequently updated & require revalidation Depth of coverageo An error can occur in a nucleotide in a reado Must have a number of reads with same result at a nucleotideo Entire genome not covered evenly by readso Test accuracy depends on depth of coverage Sensitivity & Specificityo Need to assess variants across entire genome in each runo Labs currently compare SNP array data from patient with NexGEN resultsfrom patient 2010 College of American Pathologists. All rights reserved.47
Unresolved software considerations Adequate depth of coverage for a given nucleotide has not beenestablishedo Recent study suggested 95% of genome is callable at 40X averagecoverageReference materials composed of read files can be used for softwarevalidation, quality control and proficiency testingo Currently labs used variant tables from other laboratories 2010 College of American Pathologists. All rights reserved.48
Evaluation of variants generated by instrument/software Each variant evaluated info in a variety of databases & prediction software:o Known mutations Human Gene Mutation Database Online Mendelian Inheritance in Mano Stop, readthough, missense NCBI , Ensembl SIFT, Polyphen2o Splice-site GeneSplicero Evolutionary conservation PhastConso Novel /rare variants dbSNP, Exome Variant Server 2010 College of American Pathologists. All rights reserved.49
Tools combined in Carpe-Novo interfaceInsert gene listSet filtersResult – click to get details 2010 College of American Pathologists. All rights reserved.50
Reporting results Recommend confirmation with another method (Sanger sequencing, qpcr ) Patients/Families provide input to lab regarding reporting „incidental findings‟ Start with a list of genes connected to patient‟s phenotypeo Expand to entire genome if this failso This can limit the „incidental findings‟ Report genes that have insufficient coverageo 7X coverage of a nucleotide has 0.99 theoretical power to detect aheterozygous allele in a di-allelic systemo This process can generate a „reportable range‟ for a patient‟s genomeo Allows physician to know what genes were not coveredReference range types of variation NexGen finds o At present: only single nucleotide substitutionsErrors in research literature for disease causing variants 2010 College of American Pathologists. All rights reserved.51
Polling Question #7Would you want to have your genome sequence
Baylor College of Medicine offers Whole exome sequencing: Whole Genome Sequencing (WGS) & Whole Exome Sequencing (WES) is in clinical practice . WGS/WES is now in clinical practice Partners HealthCare System, which is affiliated with Harvard Medical School and includes Massachusetts General
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