Advances in Genomic Testing Wendy Chung, MD PhD Director of Clinical Genetics Columbia University Disclosure Statement of Financial Interest I, (Wendy Chung, MD PhD) DO NOT have a financial interest/arrangement or affiliation with one or more organizations that could be perceived as a real or apparent conflict of interest in the context of the subject of this presentation. Learning Objectives • Review advances in non-invasive prenatal testing • Review chromosome microarrays methods and when to order the test • Review advances in carrier screening • Understand the new approach to evaluating multiple genes simultaneously, even all genes • Understand how to read a genetic test report Why a Diagnosis Matters • Prognosis, ability to tailor health maintenance • Identifies treatment options • Risk of recurrence, ability to prevent having other affected children if desired • Closure about how this happened • Ends the diagnostic odyssey which may involve invasive/expensive tests Cell free fetal DNA in the Maternal Circulation Relative Chromosome Dosage (RCD) Ratio Chromosome 21:1 Euploid Trisomy 21 In Placenta: Euploid Trisomy 21 V VV VV 1:1 XX X X 3:2 In Maternal Circulation 10% Fetal DNA/90%MaternalDNA Chromosome 21 Chromosome 1 Euploid 1:1 Trisomy 21 1.05:1 Mass Parallel (Shotgun) Sequencing Analysis of Fetal DNA Zhong, X, Holzgreve, W, Glob. libr. women's med 2009 Down syndrome screening threshold Fan et al, 2008 Cell free fetal DNA improves the screening for Down syndrome Previous Technology: Karyotype & FISH • Karyotype – A method of GLOBALLY visualizing chromosomes – Allows visualization of the STRUCTURE of the chromosomes – Resolution is >5 mega bases (MB) • FISH – A method of visualizing smaller, but targeted deletions – Hypothesis driven – Better at visualizing deletions than duplications Microdeletion Syndromes Associated with CHD • DiGeorge syndrome (1/4000) – Velocardiofacial syndrome – Interrupted aortic arch type B, truncus arteriosus, tetralogy of Fallot, VSD, right aortic arch, aberrant right subclavian artery, aortopulmonary window • William syndrome – Developmental delay, social, hypercalcemia Chromosome Microarray: Mega FISH • A method of detecting genetic deletions or duplications • Resolution can be as high as 2 KB • Does not detect balanced translocations and inversions Typical Patterns for Constitutional Deletion/Duplication Genetic Test Results • Positive • Negative • Variant of uncertain clinical significance AJHG;86(5):749-64, 2010. Available evidence strongly supports the use of chromosome microarray in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with developmental delay/intellectual disability, autism spectrum disorder, or multiple congenital anomalies. AJHG;86(5):749-64, 2010. Indications for a Post Natal Chromosome Microarray • • • • • • Developmental Delay Autism spectrum disorder Seizures Major birth defect (s) Dysmorphic features Failure to thrive • 5-15% yield depending on the presentation • Should be the first line test before a karyotype Access to Testing • Cost of testing is increasingly covered by health insurance, including medicaid, for common tests • Many genetic testing laboratories will assist with insurance pre-authorization (testing for common indications: seizures, mitochondrial disorders, cardiomyopathies, intellectual disabilities, whole exome sequencing) Recommendations Prenatal Chromosome Microarray • All fetuses with a structural anomaly should have CMA as the primary cytogentic/genomic test – 6% incremental detection • All patients wishing to undergo invasive testing for standard indications (AMA, positive screen, choice) be offered microarray – 1-1.5% incremental detection • Pre Test Counseling is mandatory if CMA offered – CMA can reveal results of uncertain significance (as can karyotype) – CMA does NOT test for single gene disorders, such as CF, SMA and Fragile X – CMA cannot detect balanced rearrangements Preimplantation Genetic Diagnosis Seizures • 9 year old female • Seizures and dyskinesia at birth, microcephaly, intellectual disability • No known family history of similar symptoms Epilepsy Testing • Comprehensive Epilepsy Panel (53 Genes) – Infantile Epilepsy Panel (38 Genes) – Childhood Epilepsy Panel (40 Genes) – Adolescent Epilepsy Panel (21 Genes) – Progressive Myoclonic Epilepsy Panel (12 Genes) • Chromosome microarray Positive for a mutation in SLC2A1 Causing GLUT1 deficiency syndrome • GLUT1 deficiency syndrome is due to the inability to transport glucose to the brain • Diagnostic Implications: – In individuals with SLC2A1 mutations, a ketogenic diet often improves seizure control and reduces paroxysmal events, although cognitive impairment persists – Mutation-specific testing for the SLC2A1 mutation showed this was a de novo mutation Gene Components & Structure Exons Start Transcription Promoter 5’ 1 DNA 3 2 4 Transcribed Enhancer 3’ Silencer AATAAA polyadenylation signal Locus control region Introns Stop Poly A tail CAP Site mRNA G 1 2 3 AUG: Start Translation 4 AAAAAAAA UAG: Stop Translation Translated microRNAs Exome sequencing • Sequencing of the exome (all coding exons of all genes) – ~1.5% of the genome (30Mb) – ~20,500 genes • Capture of the exons • Sequence using NextGen technology • Generates a massive amount of data which needs to be filtered Indications for Clinical Exome Sequencing • Patients who have undergone an extensive diagnostic odyssey, with no molecular basis identified – Individual gene tests negative – Targeted panels negative • Patients with a clinical phenotype that could be explained by one of many, many genes (ID/cognitive disability/developmental delay) where sequencing each individual gene is prohibitive • Higher yield if familial condition and/or consanguinity • Severe disorder in a child with no known family history Characteristics of Columbia SeriesIndication for Referral • • • • • • 62 neurological 8 cardiac 8 birth defects 6 syndrome 6 deceased family members 15 other – Cancer, recurrent fetal malformation, undiagnosed disorder, rare disorder, lipodystrophy, EB, RP, hearing loss Results in our Series • 35 definitive positive – Role of de novo mutations • 25 possible answer DNA Banking • • • • DNA sample stored for future use Can be saved for many years Alternative for uninformative families Consider when testing is currently unavailable - especially for patient at risk for premature death “Incidental” Results • Variants that are not related to the patient’s indication for testing are called “incidental findings” or “secondary findings” ACMG Guidelines for CLINICAL exome sequencing • 56 genes – Cancer: BRCA1/2, Lynch syndrome, Li-Fraumeni syndrome, FAP, VHL, MEN, PTEN, RP, TSC, NF2 – Cardiac: LQT, CPVT, Cardiomyopathy, ARVC, Marfan/aortic dissection, familial hypercholesterolemia – Malignant hyperthermia • Regardless of age of patient, including children JAMA. 2013;310(4):367-368. doi:10.1001/jama.2013.41700 JAMA. 2013;310(4):369-370. doi:10.1001/jama.2013.41709 JAMA. 2013;310(4):365-366. doi:10.1001/jama.2013.41703 Limitations of WES • Many variants, even in causative genes, are novel and the association with the disease is unknown or unclear. N of 1 • Some variants may be in genes whose function is unknown • Functional follow-up is required to determine if the variant affects the protein and is causative • ~50% of families who should have identifiable mutations do not Resources to assist with Genetic Testing • GeneTests/GeneReviews • Genetic Test Registry • • • • • • • • • • • • • • • • • • • • • • • Acknowledgements Julia Wynn Ashley Wilson Donna Russo Lan Yu Lijiang Ma Patrick Cheung Patricia Lanzano Liyong Deng Jiancheng Guo Charles LeDuc Jimmy Duoung Yan Zhang Josue Martinez Paul Appelbaum Robert Klitzman Roslyn Yee Dorothy Warburton Emma Marquez Katrina Celis Ismee Williams Jennie Kline Teresa Lee Yufeng Shen •Arianne Perez-Garcia •Michael Hadler • Isaura Rigo •Kara Kelly •Chaim Jalas • Elisabeth Paietta •Janis Racevskis •Jacob M. Rowe •Martin S. Tallman • Maddalena Paganin •Giuseppe Basso Wei Tong •Adolfo A. Ferrando •Elizabeth E. Crouch •Jay Lefkowitch •Mirjam M.C. Wamelink •Cornelis Jakobs •Gajja S Salomons •Xiaoyun Sun •Rocky Kass •Danilo Roman-Campos •Mélanie Eyries •Kevin Sampson •Florent Soubrier •Marine Germain •David-Alexandre Trégouët •Alain Borczuk •Erika Berman Rosenzweig •Barbara Girerd •David Montani •Marc Humbert •James E. Loyd • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Gudrun Aspelund Mark Arkovitz Ken Azarow Brian Bucher Dai Chung Tim Crombleholme Foong Yen Lim George Mychaliska Doug Patoka John Pietsch Bard Warner Charles Stolar Usha Krishnan Eric Austin Jeff Delaney Scott Fletcher Rob Gajarski Mark Grady Eunice Hahn Shelby Kutty Eric Michelfelder Donald Moore Erika Rosenzweig Christiana Farkouh Annette Zygmunt Jennifer Butcher Kate Brennan Mary Michaeleen Cradock Bob Drongowski Teresa Gratton Barbra Jackson Howard Needleman Questions? Wendy Chung, MD PhD 212-305-6731 [email protected]