Clinical Exome Sequencing At Baylor Whole Genome .

Clinical Exome Sequencing at Baylor WholeGenome Laboratory: Molecular Diagnosisand Disease Gene DiscoveriesYaping Yang, Ph.D.Associate Professor, Department of Molecularand Human GeneticsLaboratory Director, Whole Genome Laboratory

Dept of Molecularand Human GeneticsBaylor College ofMedicine

BCM WGL Launches ClinicalExome Sequencing Oct 2011 5200 samplesreceived, 4200cases finalized 85% peds; 15%adult Mostly neurologic In addition: skeletaldisorders, pulmonaryartery hypertension,cardiovascular dz Variety of referralsources – academicmedical centers,private hospitalsN 4200 25% molecular Dx fits with clinical picture - Pts now givenoption to consent to research analysis for 75% no Mol. Dx. rendered

Clinical Exome Sequencing at WGL Discoveries made– Diagnostic rates– Rare genetic events identified– New disease genes Lessons learned– Key elements of clinical exome

WES-Workflow for Proband ExomeFatherMotherExtraction of Patient DNAExome sequencingPatientData filtering and annotationVariant interpretationSanger sequencingReportingTrio exome sequencing is also available from our laboratory now

Molecular Diagnosis Rate: Overalland by Phenotypic GroupsOverall (n 2000)Non-neurologic (n 244)Neurologic plus (n 1147)Neurologic only (n 526)Specific Neurologic (n 83)0%10%20%30%40%50%Diagnostic rate ( /- 95% CI)504/2000 25%25% Diagnostic Rate Maintained for 3384 patientsYang, et al., JAMA, 2014

Inheritance Manners among the 504 Positivesin WES 2000X-LINKED, 13% Mito, 0.2%de novo,74%AD, 53%AR, 36%inherited, 11%unknown, 14%de Novo(AD: 74%; XL: 62%)

Findings against Textbook Expectations:Cases with Two Molecular Diagnoses (23/504) 5%

Findings against Textbook Expectations:Uniparental Disomy Detected in 5/504Positive CasesCase123Age/Sex1.1/MMatUPD 29.6/MPatUPD 220/F44/M5UPDPatUPD 9MatUPD22IsodisomyTypeCausal Genes/diseaseMat age/Pat agePartialSCN9A(epilepsy, insen. Pain)36/41CompleteCHRNG(pterygium, lethal)19/18CompleteSIGMAR1(ALS 16, juvenile)32/28CompleteUPD 3a15/FCompletea Parental samples not availablePLA2G6(neuraxonal dystrophy)SLC25A38(anemia, sideroblastic)27/33n.a./n.a. a

Medically actionable incidental findings(95/2000: 5%) Unrelated to the phenotype but with immediateimplications ACMG recommended genes (56): Cancerpredisposition, Cardiomyopathy, Long QT Non-ACMG: G-6-PD, Fabry disease, mt mutationconferring risk for hearing loss33%ACMG Genes66%Non ACMGGenes

Examples of New Gene DiscoveriesLeading to Updated ReportingCaseDateOriginalReportDate-DiseaseGene DiscoveryDateUpdatedReportGeneDisease1-3Dec 2012Sep 2013Oct 2013MAGEL2Prader-Willi-like, intellectualdisability, autism4Feb 2013Sep 2013Sep 2013FBXL4Mitochondrial Encephalopathy5-6Oct 2012Dec 2012Jul 2013WDR45Neurodegeneration with brainiron accumulation 57Mar 2013May 2013Jul 2013DEPDC5Familial focal epilepsy withvariable foci8April 2012Jun 2012Jul 2013SERAC13-methylglutaconic aciduria withdeafness, encephalopathy, andLeigh-like syndrome9Dec 2013May 2014May 2014ADHC1Xia-Gibbs syndrome10Jan 2013Oct 2014Oct 2014PURANeonatal hypotonia, seizures andencephalopathy (5q31.3microdeletion syndrome)

Clinical Exome Sequencing on ProbandOct 2011-Jun 2012Jun 2012-Nov 2013CurrentlyFirst 250 SamplesAdditional 2000 SamplesN Engl J Med.JAMA 2014 200samples/monthOct. 2013Oct. 2014WES Version 3

Clinical Exome Sequencing at WGL Discoveries made– Diagnostic rates– Rare genetic events identified– New disease genes Lessons learned– Key elements of clinical exome

Key Elements of Clinical Exome Optimization wet lab assays– Improve exome coverage and turn-aroundtime (TAT) Variant interpretations and classifications– SNVs, CNVs and AOH analyses– Don’t stop at one diagnosis, the patient could haveblended phenotypes resulting from two single genedefects– Incorporating clinical expertise in exome reporting Building and sharing knowledge database New disease gene discoveries

Wes Version 1: ‘WGL’ – VCRome2.1 is ‘just right’ Coding Exons from: Vega, CCDS, RefSeq, Predicted coding exons from: Contrast and GenScan. 197K targets, 42Mb genomic region; NimbleGen Rebalanced x2% 20X Coverage100%95%90%Whole Exome Sequencing85%Total samples: 5100; Avg: 96.6% at 20X Coverage80%050010001500Observation:Some regions not covered .still need ‘polishing’!What about comparison with clinical panels?What about ‘Medical Exome’2000

Exome “Spike-in” design contentSpike-in PKv1 (WES Version 2)1977 Genes (0.220 Mbp)GeneTests21 Clinical PanelsPKV1PKV2VCRome 2.1Exome42 MbpSpike-in PKv2 (WES Verson 3)3643 Genes (2.5 Mbp)PKv1 designOMIMSelected Cancer GenesSolved Clinical CasesBy Donna Muzny et al.

Evaluation of 100 PositiveSamples Tested by WES Version 2 Would the molecular diagnoses for the 100 cases havebeen made definitively if the samples had been testedby WES Version 1?SDHAF1DOK7①Start with causal variantsin the 100 cases testedby WES V2②Identify genomiccoordinates for thecausal variants③Plot sequence coverageacross target regions ofWES V1④Flag regions wherecoverage 20X

WES Version 1 Would Have Missed MolecularDiagnoses for Three Cases 1.7 yr old male SDHAF1 Mitochondrial complex II deficiency [MIM:252011] Homozygous pathogenic variant: c.156C A (p.Y52X), 1/1:50:1:51 19.3yr old male DOK7 Familial limb-girdle myasthenia (LGM) [MIM: 254300]Fetal akinesia deformation sequence [MIM:208150] Compound heterozygous c.1138dup (p.A380fs), 1/0:45:40:85, andc.1476 1485dup (p.G496fs), 13 year old female ADCY5, Dyskinesia, familial, with facial myokymia [MIM 606703] c.1253G A (p.418Q), 0/1:8:18:26, de novo

WGL Whole Exome SequencingHistorical SummaryWhole Exome SequencingTotal samples: 5,100; Avg: 96.6% at 20X Coverage100%98%% 20X Coverage96%94%92%90%88%HiSeq2000from GAII:86%84%MitochondrialGenome Exome:Oct 2012HiSeq2500Rapid Runs:July 2013LibraryAutomation:Aug 2013WES KapaLibraries:Feb 2014WES version 2Apr 2014WES version 3:Sep 201482%80%05001000150020002500WES Samples3000350040004500

PKv2 (WES Version 3) Design PerformanceIncludes GeneTests and OMIM (n 3643)11Gbp, VCRome 2.1 exome PKv2 Spike-in Design3 500Number of Genes3 0002 5002 000Standard Exome ControlExome 2Exome 3 3200 Genes at 100%Polished 700-800 Genes to 100% 1000 ClinVar sites recovered 2000 HGMD sites recovered1 5001 0005000 90%90-97%97-100%100%Percent of gene covered 20XBy Donna Muzny et al.

Lightning Capture:Reduced turnaround time in the wet labStandard Exome: 20 daysLibraryCaptureSequencingMercuryAnalysis3 days4 days12 days24 hrsAnnotation, filtering,prioritization15 min.Mapping thruVariant callsOptimized workflowKapa enzymeOptimizedHybridizationsHiSeq 25004.5 hrs8 hrs27 hrs24 hrsDemonstrated using three sample dataset, 11Gbp 98% target bases at 20x; 94% target bases at 40x;15 min.Lightning Exome: 64 hrs ( 3 days)By Donna Muzny et al.

Key Elements of Clinical Exome Optimization wet lab assays– Improve exome coverage and turn-around time (TAT) Variant interpretations and classifications– Analyses of single nucleotide variants (SNVs),as well as copy number variants (CNVs) andabsence of heterozygosity (AOH) regions– Thorough data analyses Explore all possible inheritance manners Don’t stop at one diagnosis, the patient could haveblended phenotypes resulting from two single gene defects– Incorporating clinical expertise in exome reporting Building and sharing knowledge database New disease gene discoveries

Rare Genetic Events: SCID due to compoundheterozygous IL7R Mutations (SNV CNV) Detectedby WES and CMAc.361dupA/ wtEx3 Deletion/ wtc.361dupA / Ex3 deletionSporadic caseof SCID:CNV SNV Aut Recess SCIDThe case is solved combininggenomic analysis by BOTH WES and CMA

Case example 5 year old male Father is 39 yrs old, mother is 27 yrs old Clinical Presentation: Global developmental delay (a history of hypotonia, rolled at12 mo, sat at 12 mo, walked at 2 yr, first words at 2.5 yr, stillreceives ST) Overweight (at 4y 8 mo, weight 90-95th ile) Mild joint laxity Genital anomalies Mild facial dysmorphisms Behavioral problems (aggression) Tested negative for Prader-Willi Syndrome (PWS)

Absence of Heterozygosity (AOH)on chromosome 14Concurrent Illumina HumanExome-12v1 (cSNP) array analysisrevealed contiguous regions of copy neutral Absence ofHeterozygosity (AOH) on chromosome 14 (approximate 39 Mb,14q11.2-14q22)

Case 5

18478039UPD(14)mat Resembles PWSThisPatientUPD(14)mat(n 36)IUGR-12/13Low birth weight20ile18/21Short statue25-50ile20/24Obesity90-95ile10/15Hypotonia 18/21Feeding difficulties 9/25Joint laxity 7/11Facial dysmorphisms 16/20Motor delay 17/25Mental delayLanguagedelay8/24Premature pubertyToo young11/12Mitter et al, Am JMed Genet A, 2006

Key Elements of Clinical Exome Optimization wet lab assays– Improve exome coverage and turn-around time (TAT) Variant interpretations and classifications– SNVs, CNVs and AOH analyses– Thorough data analyses Explore all possible inheritance manners Don’t stop at one diagnosis, the patient could haveblended phenotypes resulting from two single genedefects– Incorporating clinical expertise in exome reporting Building and sharing knowledge database New disease gene discoveries

Mosaicism in a parent causingrecurrent AD condition in the family Clinical Presentation: Twin brother delayed speech, developmental regression, autism/autisticspectrum, intellectual disability, seizure disorder, short stature,microcephaly, dysmorphic features, and congenital heart disease. WES was requested on the proband only Samples from twin brother, unaffected sister and parents were available for Sanger studies46 y.o.51 y.o.?15 y.o. 12.1 y.o. 12.1 y.o.SHANK3 SH3 and multiple ankyrin repeatdomains 3, c.3329 3332del (p.I1110fs), chr.22q13.33.Both parents are negative, the twin brother isalso heterozygous, the unaffected sister isnegative,Associated disease: Phelan-McDermidsyndrome [MIM:606232], AD

Blended Phenotypes with Two DiagnosesCase Disease IDisease II1Ataxia-telangiectasiaSpastic paraplegia 502Carpenter SyndromeNeurofibromatosis, type 13Nicolaides-Baraitser syndromeDravet syndrome4Contractural arachnodactyly,congenitalRenpenning syndrome5Epilepsy, progressive myoclonic 5Rubinstein-Taybi syndrome6Leigh syndrome, X-linkedBardet-Biedl syndrome 107Mental retardation, autosomaldominant 12Mental retardation, X-linked 948CardiomyopathyDuchenne muscular dystrophy9Malformations of corticaldevelopment and microcephalyPitt-Hopkins-like syndrome 210Rothmund-Thomson syndromeXeroderma pigmentosum,group C11Epilepsy, juvenile absence,susceptibility to, 1Cornelia de Lange syndrome 2

Key Elements of Clinical Exome Optimization wet lab assays– Improve exome coverage and turn-around time (TAT) Variant interpretations and classifications– SNVs, CNVs and AOH analyses– Explore all possible inheritance manners– Don’t stop at one diagnosis, the patient could haveblended phenotypes resulting from two single genedefects– Incorporating clinical expertise in exomereporting Weekly WGL meeting Monthly WES sign-out meeting: accessibleworldwide from the internet Building and sharing knowledge database New disease gene discoveries

Key Elements of Clinical Exome Optimization wet lab assays– Improve exome coverage and turn-around time (TAT) Variant interpretations and classifications– SNVs, CNVs and AOH analyses– Explore all possible inheritance manners– Don’t stop at one diagnosis, the patient could haveblended phenotypes resulting from two single genedefects– Incorporating clinical expertise in exome reporting Building and sharing knowledge database– Data submission from WGL to ClinVar, etc. New disease gene discoveries

New Gene Discoveries

New Disease Gene Discoveries Opportunity for unsolved exome negativecases to join research studies

Global Collaborations are Essential

Clinical Whole Exome Sequencing(WES) Sign-Out ?PMID 21319Tel: 1-800-411-GENE / Fax: 713-798-2787e-mail: genetictest@bcm.edu

Clinical Exome Sequencing at Baylor Whole Genome Laboratory: Molecular Diagnosis and Disease Gene Discoveries Yaping Yang, Ph.D. Associate Professor, Department of Molecular and Human Genetics Laboratory Director, Whole Genome Laboratory