Outline. Next-Generation Sequencing. The Application of Genomic Medicine in Clinical and Laboratory Practice

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1 Outline The Application of Genomic Medicine in Clinical and Laboratory Practice Jerry Feldman, MD, PhD, FACMG Medical Director, Division of Laboratory Genetics and Molecular Pathology, Detroit Medical Center University Laboratories Director, Clinical Genetics Services and Professor Molecular Medicine and Genetics, Pediatrics and Pathology Wayne State University School of Medicine 23 rd Annual Symposium on Molecular Pathology, William Beaumont Hospital September 17, 2014 Next Generation Sequencing (NGS) - Workflow/chemistry - NGS panels vs. Whole Exome Sequencing (WES) - Rationale for use clinically» Case examples - Overall process (laboratory and clinical) - Test ordering procedures and a reality check Future of NGS Identification of Genetic Abnormalities Paradigm Shift in Test Utilization Is there anything different or unique about NGS? Clinicians (mostly geneticists - Single Gene Test - Test based on suspected clinical diagnosis Oncologists/Pathologists - Single Gene Test - Test based on tumor markers or type of tumor Small (one gene) data set (genotype) Rare disease testing performed in only a few labs; large commercial or academic laboratories with larger test menus Potentially All Clinicians - Gene Panels - Whole exome sequencing - Unknown diagnosis Large and complex data (genomic) Additional expertise, manpower and technology..$$$$ Next-Generation Sequencing Next-generation sequencing (NGS) platforms perform massively parallel sequencing, during which millions of DNA fragments are sequenced in unison Whole Exome Sequencing - Only need to sequence 1% of genome - 180,000 exons - 30Mb of sequence Identifies base substitutions and deletions which likely make up ~85% of pathogenic variants NATURE VOL MARCH

2 Next Generation Sequencing NGS Workflow Genomic DNA sample is fragmented Binding/ligation of each fragment Capture of ligated segments In-vitro shotgun library created; - Library enriched for sequences corresponding to exons Fragments hybridized and captured Processing of millions of pairs of sequence reads simultaneously ( massively parallel sequencing ) sample ready for high-throughput sequencing, using a variety of methods, each with advantages and disadvantages Data Analysis.. Sample Preparation (1-3 days) Genomic DNA fragmentation Library preparation and target enrichment A.PCR amplification of targets followed by adapter ligation OR B. Adaptor ligation followed by target capture by probe hybridization Sequencing (1-6 days) Hybridization to flow cell Cluster generation Sequencing by synthesis Data Analysis Read alignment Quality assessment Variant calling Variant Annotation Sanger confirmation Data Analysis (Identification of Variants = Bioinformatics) Sequence alignment and comparison Alignment of raw sequence to reference sequence Comparison to identify variants» Identification of clinically important variants (filtering)» What about new variants in known genes or variants in genes without known function? Fig. 3 An example of whole exome sequencing data showing the number of variants from the reference sequence identified and how many remain at each filtering step. Missense mutations result in a change in the amino acid sequence. Nonsense mutations are base changes that result in a premature stop codon or nonsense codon, causing the protein product to be truncated and incomplete Br Med Bull (2011) 99 (1): Clinical Applications of NGS Is it an affordable clinical tool? - End of genetic odyssey for patient and family? - More value for the health care dollar? - Diagnosis rate ~25% Whole-exome sequencing - Used in gene discovery research - Facilitates the discovery of disease-causing mutations Targeted sequencing (gene panels) - Targets regions of genome of interest - Target disease-causing mutation hotspots WGS/WES: Indications for Diagnostic Testing - ACMG Policy Statement, May 2012 High suspicion of unknown genetic disorder Phenotype has high genetic heterogeneity such that WES/WGS more practical approach (e.g., severe intellectual disability) Phenotype likely genetic but current testing has not found underlying etiology Fetus with likely genetic disorder in which specific testing has failed to provide diagnosis 2

3 NGS: Panel vs. Exome Panel Exome - Hypothesis driven, less genes are interrogated - Sanger sequencing to fill in missing/poorly covered exons - 100% of genes covered at 20X - Del/dup by exon array may be included - No secondary findings - Less expensive/faster TAT - Custom panel or exome slice? - Hypothesis free, covers most exons or exons and UTRs (platform dependent) - Some exons missed or insufficiently covered % of genes covered at 10-20X - Secondary findings - Variants and genes of unknown significance Secondary (Incidental) Findings Primary Finding pathogenic alterations in gene(s) relevant to diagnostic indication sequencing was ordered Secondary Finding results of a deliberate search for pathogenic or likely pathogenic alterations in genes that are apparently unrelated to diagnostic indication - Patient referred for chest X-ray to R/O pneumonia» Radiologist identifies no pneumonia but notes a suspicious lung mass and enlarged heart. Should finding be reported? - Patient referred to dermatologist for rash/eczema» Dermatologist identifies lesion on skin suspicious for melanoma (cancer). Should finding be reported? Secondary Findings Case Study 1a Recommendations of American College of Medical Genetics and Genomics and the President s Commission on Bioethics Minimum list of 57 actionable genes and specific mutations - Hereditary cancer genes, Marfan and related syndromes, inherited cardiomyopathies & arrhythmias, familial hypercholesterolemia, malignant hyperthermia and others Pathogenic variants in this gene list should be reported regardless of indication for exome sequencing - Additional genes may be analyzed for incidental variants - Minimal list should be reported regardless of patient age - Patients/parents may opt out at time of consent Two year old male with developmental delay Maternal uncle is reported to be slow Normal Fragile X and microarray results Is NGS the next test to order? If so. What NGS test to order? - Panel, but which one? - Panel and then if negative reflex to WES? - Directly to WES? Case Study 1b A 25 year old recently married woman comes in with her husband, mother, and 29 year old brother The brother has severe mental retardation (intellectual disability) The couple wants whatever testing can be done to ensure that they won t have a similarly affected child Case Study 1b Family history reveals one case of mental retardation in the patient s maternal great uncle. History and physical exam in brother does not elicit a unifying diagnosis: non-syndromic MR Fragile X, microarray, and baseline metabolic screening in brother is unremarkable Further work-up? 3

4 Case Study 1a and 1b Option 1: X-linked mental retardation panel Factors to consider - Do all XLMR panels test for the same genes? - Are the costs the same? - Is the depth of coverage the same? - What is the turnaround time? - Which lab should you use? X-Linked Intellectual Disability Panels Emory U Chicago Ambry GeneDx Exome Slice # of genes Clinician determined TAT weeks 8-10 weeks weeks weeks List price $3200 $4500 $4170 $3000 Del/Dup included No No No Customizable? Limit to 30 or 60 genes No No Coverage of XLID Genes by WES Baylor WES Ambry WES (% of CDS with >20x (% of CDS with >10x coverage) Gene coverage) ABCD ARX ATP6AP CDK HPRT IDS MID NAA NHS NXF SOX SYN SYP UBE2A Emory panel only U Chicago panel only Ambry panel only Emory and U Chicago Emory and Ambry U Chicago and Ambry XLID Panel Gene Content PANEL SPECIFIC GENES CDK16, CNKSR2, DMD, NAA10, RBM10 EIF2S3, SLC6A8 AGTR2, NXF5, ZNF674 AFF2, CCDC22, FMRPD4, HCFC1, IGBP1, IQSEC2, KLF8, NSDHL, UPF3B, ZDHHC15 ABCD1, ATP7A, DKC1, FANCB, FGD1, GK, GPD3, HCCS, IDS, LAMP2, MTM1, NDP, NDUFA1, OTC, PGK1, PORCN, SOX3, TIMM8A ARHGEF9, SRPX2, ZNF81 Case Study 2 Answer: Sequence the Exons! What to do with that impossible to diagnose patient.significant cognitive impairment, seizures, birth defects. Every reasonable and some not-so-reasonable genetic tests you send are normal Parents want to have more children but are scared to without the ability to perform prenatal diagnosis for this severe condition Genotype = Phenotype 4

5 What are the Steps in the Process? Who should order test? Primary care physician v. specialist (geneticist, neurologist?) - Qualified to discuss options? interpret results? - Genetic counseling essential Review insurance coverage; obtain preauthorization Obtain informed consent (usually laboratory has consent form which describes testing options) Test performed, results returned Interpret and discuss results with patient/guardian - Develop plan based on results Total time = 3-6 months Baylor- WES List Price $7,000 TAT mtdna included? Platform Coverage Exome of parents Sanger Confirmation 15 weeks Agilent SureSelect All Exon v4 95% of exome at 20X WES Laboratories Ambry- ExomeNext $5,800 or $15, or 2-5 weeks Nimblegen, Agilent or Illumina 90-95% of exome at 20X GeneDx- XomeDx $5000 or$9000 (for trios) Medical College of WI $4,875 or $7,500 (for trios) Emory- Medical EmExome* $6,000 or $6,700 (for trio) weeks 12 weeks 16 weeks Only with XomeDx Plus Agilent SureSelect All Exon v % of exome at 10X? 97% of exome at 10X Agilent SureSelect v5 Plus 97% of exome at 20X No If ordered If ordered If ordered Clinical Experience One Lab (Baylor) >1200 cases - 85% pediatric patients, mostly for intellectual disability and autism spectrum disorder ~200,000 variants/wes - filter to ~ to evaluate by clinical team ~25% positive pathogenic variant related to disease phenotype - 52% de novo dominant variant - Broadening of phenotypic spectrum for some classic syndromes - Requires team to interpret results, including clinicians Limitations of NGS Variants of Uncertain Significance (VUS) Secondary findings Will not detect - Pathogenic variants in non-coding areas - Pathogenic variants in exons (~5%) not covered by WES - Triplet repeats - Some insertions/deletions/duplications - Abnormal methylation NGS - Clinical Challenges Which variants should be reported? - Health risks? - Reproductive risks? What about secondary findings? - Health risks? - Reproductive risks? Data storage/electronic medical record Turnaround time 3-6 months Billing Options Insurance bill - Outside lab will directly bill the patient s insurance - Patient s insurance info must be provided to the outside lab Institutional bill - Outside lab will bill your institution, then your institution will bill the patient/patient s insurance - Some institutions mark-up the pricing of the bill to cover shipping/processing/non-reimbursed testing cost (illegal in Michigan) Self-pay - Outside lab will directly bill the patient (not insurance) - Most labs require checks/credit card # - Some labs offer price discount for self-pay patients who pay upfront - Patient may not have insurance, or choose not to use insurance 5

6 Case Report Born at full term after uncomplicated pregnancy Feeding difficulty and irritability in infancy Developmental concerns at 3 months began therapies 7 months old FTT and generalized stiffness - EEG, brain MRI normal - Dx of Hyperkplexia (Stiff Baby Syndrome) considered» 1 VUS identified Lab and medical workup non-diagnostic, including clinical microarray Management symptomatic only 20 months old not sitting up, poor head control, no words (only sounds) Whole exome sequencing.. What Payers Want to Know: Did Management Change? Pathogenic variant identified fit phenotype - Went back to prove this - Uric Acid levels (high) Management - Referral to Nephrology - Allopurinol treatment begun - Placed on low purine diet - Monitor for self-injurious behaviors - Counseling cause, recurrence risk 6

7 Next Generation Sequencing and Cancer? Hereditary tumor panels Somatic tumor panel testing - Treatment options - Tumor classification Non-invasive method of diagnosis? Is the $1000 genome possible? - Technically, yes - Bioinformatics, not yet 3 rd Generation Sequencing - single molecule sequencing with longer read lengths in shorter run times at a lower cost Future of NGS Summary and Conclusions Next generation sequencing is here to stay Methodologies will continue to improve Costs will continue to decrease Better understanding of variants will develop as more data becomes available and publically shared Patients will benefit from all of the above. 7