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Genetic Testing for Hearing Impairment: Current State and Future Directions

Genetic Testing for Hearing Impairment: Current State and Future Directions. Tracy Stockley, PhD, FCCMG Associate Director, Molecular Genetics Laboratory The Hospital for Sick Children Assistant Professor Dept Laboratory Medicine & Pathobiology University of Toronto. Molecular Diagnostics.

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Genetic Testing for Hearing Impairment: Current State and Future Directions

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  1. Genetic Testing for Hearing Impairment: Current State and Future Directions Tracy Stockley, PhD, FCCMG Associate Director, Molecular Genetics Laboratory The Hospital for Sick Children Assistant Professor Dept Laboratory Medicine & Pathobiology University of Toronto

  2. Molecular Diagnostics Research (Cochlear Implant Program) Future Directions

  3. Causes of Congenital Deafness • Prenatal infection • (toxoplasmosis, rubella, • CMV, herpes, syphillis) Non-Genetic (~35-45%) • Meningitis • Low birth weight • Prematurity Genetic (~55-65%) • Hyperbilirubinemia • Ototoxic Drugs • Traumas/ • Exposures

  4. Causes of Congenital Deafness Non-Syndromic: 60 identified genes, >100 loci Most significant: GJB2 (Cx26) GJB6 (Cx30) SLC26A4 (Pendrin) Syndromic: More than 400 syndromes Individually rare, but collectively common Non-Genetic (~35-45%) Genetic (~55-65%) Autosomal recessive Autosomal dominant X linked Mitochondrial

  5. How can we translate this into patient care? What are the next steps?

  6. Molecular Diagnostics • Clinical lab testing for genetic changes • Report to health care providers • Quality control, accreditation: • College of American Pathologists/CLIA • Ontario Laboratory Accreditation • Ontario Ministry of Health License • Standardized protocols, tracking of samples and reagents, certified staff • Volume 20,000 samples/yr - automation www.sickkids.ca/molecular

  7. Non-Syndromic Deafness an extremely heterogeneous condition Loci/year

  8. DFNB Non-Genetic (~35-45%) DFNA DFN Genetic (~55-65%) (DFNM)

  9. Molecular Diagnostics and Deafness How should the genetic testing be performed? What genes? What methods? What additional tests? (e.g. CMV virus) Who should be offered genetic testing? How can genetic test results help us better manage patients with hearing loss?

  10. What deafness genes do we test? • Genes and mutation types must be well described • Mutations must have a relatively high frequency in deaf population • Must be able to design a reliable, effective test

  11. Genetic Test Methods • Genetic testing identifies mutations in known genes • Provides: • Diagnosis • Carrier status • Risk assessment • Various methods • Different costs • Different sensitivities

  12. THE CAP IS RED THE CAT IS RED

  13. GJB2 Mutation: 35delG Normal Sequence 35delG Mutation DNA sequencing

  14. From DOE

  15. Gene Mutations and Deafness Gene/protein function is absent Gene/protein are 50% of normal function Gene/protein function are nearly normal GJB2 mutations: 35delG/35delG 35delG/M34T M34T/M34T

  16. Genetics vs. Environment Cause is mostly due to gene mutations Cause is both gene mutations and environment Cause is mostly environment Down Syndrome Cystic Fibrosis Chicken pox Lung cancer Diabetes mellitus Heart disease Deafness GJB2 mutations Deafness Mitochondria A1555G Deafness CMV infection

  17. Non-syndromic and Syndromic Deafness • Non-syndromic Deafness: • no other associated features (not a syndrome) • Example: GJB2 mutations • Syndromic Deafness: • Deafness AND other features = Syndrome • Gene/protein important in many cell types so different organs/tissues affected by mutation • Example: Branchial-oto-renal (BOR) syndrome, Pendred syndrome

  18. Non-syndromic Genes (recessive) • > 65 genes, 110 possible genes mapped • Hereditary Hearing Loss homepage:webh01.ua.ac.be/hhh/

  19. Syndromic Deafness • Clinical geneticist assessment • Genetic testing of relevant genes • Red flags of syndromes: • More than one organ or system involved? • Deafness + heart, kidney, eye, thyroid problems • Similar features in other family members? • May be different severity in family members • Similar unusual facial features or characteristic in family members? • White forelock, ear shape, ear/neck pits

  20. Deafness Syndromes Severe mutation or Syndrome Fully Expressed Mild mutation or Syndrome Variable Mild features, Family members affected to different degrees All or many features of the syndrome

  21. Why have a genetic test? • A definite cause • Family members can find out if they are carriers, and know risk factors for future children • May help to find appropriate treatment/ management (syndromic), or avoid unnecessary management (non-syndromic) • Future: therapy based on mutation type

  22. Why decide not to have a genetic test? • May not necessarily find the answer • Severity, course may not be predicted • Interpretation of certain mutations may not be clear

  23. Molecular Diagnostics and Deafness How should the genetic testing be performed? What genes? What methods? What additional tests? (e.g. CMV virus) Who should be offered genetic testing? How can genetic test results help us better manage patients with hearing loss?

  24. Research Questions • How does the molecular test information inform medical management and interventions? • - Cochlear implants? • - Management of syndromic patients? • What are the best strategies for gene testing? • - Test strategies and methods? • - point mutations vs. deletions • - Which genes in which patients? • - audiological features • - clinical features • - ethnic background • What are we missing by looking only at DNA? • - Analysis of message RNA

  25. 2002-2008 GJB2 • Cochlear Implant Program, Sick Kids • (B. Papsin, K. Gordon) • 970 probands tested for GJB2 (Cx26) • (cochlear implant candidates, non-syndromic, severe to profound) 58% 30% 12%

  26. Evaluation of neural activity in GJB2-related hearing loss in children with cochlear implantsEvan Propst, Karen Gordon, Blake Papsin, Cochlear Implant Program; Sandi Sidhu, Genetic CounsellorQuestion: Is audiotory nerve spiral ganaglion survival more consistent along the length of the cochlea in GJB2-deafness?

  27. Evoked Compound Action Potential (ECAP) Study Group:39 GJB2-HI, 58 C

  28. Visual threshold Slope of amplitude growth tNRT P2 N1 Evoked Compound Action Potential Analysis Cascade Pane Analysis Pane Graph Pane

  29. Evoked Compound Action Potential (ECAP) Propst et al.2006

  30. Implications for cochlear implants: • MAPping dynamic range of loudness limits (C- to T- level) • - Test fewer electrodes and assume equal for rest • More equal distribution of frequencies along electrode array in GJB2-deafness than non-GJB2 Implications For Implantation Maximum Comfortable Loudness Level (C-level) For diagnostics: GJB2 heterozygotes? Threshold of Hearing Level (T-level)

  31. Patients with two GJB2 mutations can also have specific inner ear abnormalities • Semicircular canal dehiscence • Large vestibular aqueduct • Small bony island Implications for CI

  32. Research Questions • How does the molecular test information inform medical management and interventions? • - Cochlear implants ? • - Management of syndromic patients? • What are we missing by looking only at DNA? • - Analysis of message RNA • What are the best strategies for gene testing? • - Test strategies and methods? • - point mutations vs. deletions • - Which genes in which patients? • - audiological features • - clinical features • - ethnic background

  33. Syndromic Diagnostic Challenges • Not genetic heterogenetity • Syndromes can be distinguished as unique • May have several genes of large size, few patients for testing • Eg. Usher syndrome: 6 genes, up to 69 exons • Entire gene needs to be tested • Multiple types of mutations • Point mutations • Large deletions of parts of gene • Inheritance patterns always not clear • All family members not affected to same degree

  34. Can mutations in syndromic deafness be related to the clinical phenotype?EYA1 mutations in Branchio-oto-renal syndromeDivision of Clinical Genetics: Roberto Mendonza-Londono, Lucie Dupuis, Sandi Sidhu, Cochlear Implant Program: Evan Propst, Blake Papsin

  35. Branchio-oto-renal syndrome (BOR) • Sensorineural, conductive or mixed hearing loss • Branchial pits, cysts and/or fistulae • Renal dysplasia or aplasia • Malformed pinnae • Ear pits and/or tags Photograph of a malformed pinna fromRichard JH Smith, MD.

  36. Branchio-oto-renal syndrome (BOR) • Incidence: ~1 in 40,000 • Autosomal dominant • Genes: • EYA1 (8q13.3) • SIX1 • SIX5 Photograph of a branchial fistula provided byRichard JH Smith, MD.

  37. EYA1 protein is a transcription factor • Interacts with genomic DNA, other proteins to activate other genes • Interacts with Six and Dach-type proteins, in developmental cascade EYA protein EYA protein regulates other genes and proteins

  38. Mutations in EYA1 and Molecular Testing Two-stage molecular testing: • Direct sequencing of all exons and boundaries • 2) Dosage analysis for large deletions (MLPA method) From Pendred/BOR Homepage, MORL

  39. BOR Syndrome and EYA1 mutations • How many patients referred for BOR syndrome testing have EYA1 mutations? • Used as a rule-out test? • Are the EYA1 mutations inherited or new (de novo) in the families? • Many patients did not have a family history • Is there correlation of mutations with certain clinical features?

  40. 14/17 probands had EYA1 gene mutations identified (82%) • Previously unreported (novel) mutations in bold

  41. Phenotype correlations?

  42. BOR Syndrome • Some EYA1 mutations may reoccur • Testing implications • Indicate important region of the gene/protein • Clinical features of BOR? • May allow prediction of clinical severity or organ involvement by better comprehension of mutation effects

  43. Research Questions • How does the molecular test information inform medical management and interventions? • - Cochlear implants ? • - Management of syndromic patients? • What are we missing by looking only at DNA? • - Analysis of message RNA • What are the best strategies for gene testing? • - Test strategies and methods? • - point mutations vs. deletions • - Which genes in which patients? • - audiological features • - clinical features • - ethnic background

  44. Why do so many deaf patients have only one GJB2 mutation?Karen Gordon, Blake Papsin, Cochlear Implant Program

  45. 2002-2008 GJB2 • Cochlear Implant Program, Sick Kids • (B. Papsin, K. Gordon) • 970 probands tested for GJB2 (Cx26) • (cochlear implant candidates, non-syndromic, severe to profound) 58% 30% 12%

  46. Is the second mutation not in DNA?DNA→ RNA→ Protein Replication Transcription Translation

  47. 2002-2008 GJB2 • Current study: • - Testing RNA in deaf GJB2 heterozygotes 58% 30% 12%

  48. Research Questions • How does the molecular test information inform medical management and interventions? • - Cochlear implants ? • - Management of syndromic patients? • What are we missing by looking only at DNA? • - Analysis of message RNA • What are the best strategies for gene testing? • - Test strategies and methods? • - point mutations vs. deletions • - Which genes in which patients? • - audiological features • - clinical features • - ethnic background

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