1. Genetic examination for hearing loss and its clinical applications 8 th International Symposium of Rare Diseases 2014/9/19 Chen-Chi Wu, MD, PhD. Department of Otolaryngology, National Taiwan University Hospital

2. Contents Genetic examination Clinical applications Newborn screenings Future perspectives

3. Structure of the Ear ConductiveSensorineural Hearing impairment in humans Permanent hearing loss in children

4. Etiologies of deafness in children (Dahl et al., 2013)

5. Genetics of hereditary hearing impairment SNHI in children ~ 3/1000: > mod. SNHI ~ 1/100: mild SNHI > 50% : hereditary > 70% Non-syndromic ~ 50 genes related to non-syndromic HHI

6. Genetic screening of deafness in Taiwan % 14.4% 3.2% SLC26A4GJB2m. 12S rRNA 12.1% c.919-2A>G 4.5% c.235delC 6.3% 15.4% p.V37I 2.9% m.1555A>G p.H723R NTUH-1 NTUH-2 H H H H H NTUH: NTU hospital : other hospitals R R R: rehabilitation facility 10 100 200 H H > 2000 families > 4000 patients (Audio Neurotol, 2008)

7. OTOF mutations in auditory neuropathy Epidemiology of AN 2000~2009 13/850 (1.5%) SNHI children Genetic study 22 AN families 5/22: p.E1700Q (c.5098G>C) OTOF & otoferlin 2p23.1, 49 exons 1977 a.a. Synapse of IHCs Auditory neuropathy (Audio Neurotol, 2010)

8. stopSSQ c.466C>T (p.Q156X) c.1735delG (p.D579fs) EYA1 mutations in branchio-oto-renal syndrome Screened EYA1, SIX1 and SIX5 Novel EYA1 mutations in 2/12 BOR families NSHS (Laryngoscope, 2012)

9. Simplex families: 1/3 Multiplex families: 1/2 Screening for common mutations Genetic diagnosis Mutations in uncommon deafness genes? Massively parallel sequencing (MPS) or Next-generation sequencing (NGS)

10. Direct sequencing DNA PCR amplification of specific exons Sanger sequencing > 50 non-synd. HHI genes ! > 100 synd. HHI genes ! Massively parallel sequencing (MPS) DNA Target capture: a large amount of DNA fragments DNA sequencing Sequence analysis

11. >2000 families with idiopathic SNHI Data analysis Screening of 3 common deafness genes MPS ~130 deaf genes + whole mt. genome + ~1500 miRNA coverage: ~ 200 X ~100 multiplex families w/o common mutations Select 25 multiplex families: 15 AD, 7 AR & 3 XR The MPS panel for deafness Select 39 simplex families w/o common mutations

12. DE2721: MYH9 p.E1256K (PLoS One, 2013) Detection of rare mutations in non-syndromic HHI

13. 64 SNHI families w/o common mutations 7 undiag.8 diag. GJB2 GJB3 MYO7A GATA3 KCNQ4 MYH9 POU4F3 WFS1 6 undiag.1 diag.1 undiag. 2 diag. 7 AR families MPS panel 3 XR families 15 AD families LRTOMT MYO3A ESRRB PCDH15 DFNB59 POU3F4 22/64 (34%) diagnosed TMC1 39 simplex families 11 diag. 28 undiag. Exome sequencing to identify novel deafness genes MPS panel

14. (Choi BY et al, 2013)

15. 5 y/o boy Brought to your clinic for hearing loss 0 10 20 30 40 50 60 70 80 90 100 110 120 1252505001K1K2K2K4K4K 8K8K Frequency (Hz) Hearing level (dBHL) R: 72dBHL L: 85dBHL Why performing genetic test? The parents often ask: Will his hearing get worse? Will our next kid also have hearing problems?

16. Basic research Clinical medicine 1.Genetic diagnosis and prognosis prediction 2.Prenatal diagnosis - amniocentesis or chorionic membrane biopsy - pre-implantation genetic diagnosis (PGD) 3. Prevention: avoid oto-toxic medication 4. Predict the outcome of therapy (eg. CI) Clinical applications

17. Genetic counseling & prognosis prediction Children with SNHI Genetic mutations GJB2 mutations SLC26A4 mutations m.1555A>G mutation Other mutations Stable or slowly progressive Fluctuating Progressive, ototoxicity Specific phenotypes Simplex family: 1/3 (MPS: 1/2) Multiplex family: 1/2 (MPS: 2/3)

18. c.919-2A>G/wt c.919-2A>G/ c.919-2A>G SLC26A4 c.919-2A>G mutation c.919-2A>G/wt c.919-2A>G/ c.919-2A>G wt/wt Prenatal genetic diagnosis (PND) - Amniocentesis or chorionic villus biopsy

19. Pre-implantation genetic diagnosis (PGD) (Nature Education, 2008)

20. Figure 3 (Audio Neurotol, 2010) Normal hearing after birth EVA Potentially goiter Fluctuating SNHI SLC26A4 mutations

21. Prevention: avoid oto-toxic medications Mr./Ms. is a patient with mitochondrial 12S rRNA m.1555A>G mutation. Please do not give aminoglycoside to him/her.

22. Cochlear implant (CI) - Still expensive - Invasive - Long-term (>2 y) (re)habilitation  Variable performance !

23. Long-term CI outcome vs. genotypes (Laryngoscope, 2011) Multicenter study 743 unrelated children; 180 with CI 110 CI children: > 3 years F/U 20.6 % CI children: definite genetic diagnosis Better CAP scores in children with mutations

24. Cochlear implant (CI)

25. Children with cochlear implants 12 cases (poor CI outcome) 30 matched controls (good CI outcome) CAP<5 & SIR<3CAP=7 & SIR=5 Inclusion criteria: CIs in use for > 3 years No mutations in common deafness genes Exclusion criteria: Syndromic hearing loss Acquired hearing loss Cochlear nerve deficiency in the implanted ears Additional cognitive or psychological defects Comprehensive genetic analyses with MPS Comparison of genotypes Phenotype characterization of audiological features, imaging findings and CI performances MPS in CI children

26. DFNB59 and PCDH15 mutations  poor CI outcome (Paper under review) PCDH15DFNB59 (Hereditary Hearing Loss Homepage)

27. Screening for deafness in newborns Newborn hearing screening Newborn genetic screening Newborn CMV screening

28. Early identification of hearing loss in children 1. Hearing loss occurs more frequently than any other birth disorders. 2. Undetected hearing loss has serious negative consequences. 3. There are dramatic benefits associated with early identification of hearing loss.

29. Incidence per 10,000 of congenital disorders/diseases 1. Hearing loss occurs more frequently than other birth disorders

30. Reading comprehension scores of hearing and deaf students Age in Years (Schildroth & Karchmer, 1986) Grade Equivalents 2. Undetected hearing loss has serious negative consequences

31. Language of early- and later-identified children with HL (Yoshinaga-Itano, 1998, Pediatrics) 3. Dramatic benefits associated with early identification

32. Earlier Hearing Detection and Intervention (EHDI) (NEJM, 2006) Hearing screening before 1 m Audiological evaluation before 3 m Early intervention before 6 m

33. Universal newborn hearing screening (UNHS) In all newborns Screening tools: - OAEs (Oto-acoustic emissions) - AABR (automated auditory brainstem response) (Int J Pediatr Otorhinolaryngol. 2014) In 2013: >95% in Taiwan

34. Newborn hearing screening at D1 (OAEs or AABR) Reassurance & education PassRefer OAEs or AABR at D3 Reassurance & education PassRefer OAEs or AABR + tympanogram at 1m Reassurance & education PassRefer Diagnostic tests at 3m ABR (tone bursts) ASSR or BOA The algorithm for UNHS in Taiwan

35. (Arch Otolaryngol Head Neck Surg, 2011) Retrospective review of 417 pediatric implant recipients born in Illinois ~30%: passed UNHS because of progressive or late- onset SNHI These children may not benefit from improved outcomes conferred by early identification and intervention

36. (Int J Pediatr Otorhinolaryngol. 2013)

37. 80 NTUH 1.54 Severe SNHI years Mod. SNHI Newborn hearing screening Newborn genetic screening Median age of genetic diagnosis Mild SNHI ? Late-onset SNHI ? Babies without access to NHS ? Inherent limitations of NHS

38. 1017 Newborns were subjected to NHS & NGS 38 (3.7%) Failed NHS979 (96.3%) Passed NHS 9 With potential hearing deficits 8 Carried 2 GJB2 mutations 3 Were p.V37I/p.V37I 5 Were p.V37I/c.235delC 1 Carried the m.1555A>G mutation NGS results 9 Carried 2 GJB2 mutations 9 Carried 1 GJB2 mutation 20 No mutation detected 970 With only 1 recessive mutation or with no mutation detected 172 With 1 GJB2 or SLC26A4 mutation 166 Carried 1 GJB2 mutation 6 Carried 1 SLC26A4 mutation 798 With no mutation detected 8 With hearing loss 7 With bilateral hearing loss 1 With unilateral hearing loss Diagnostic ABR at 3m 3 With slight/mild hearing loss (PLoS One, 2011)

39. Bloodspot-based genetic testing for common GJB2 alleles should be considered as second tier testing for bedside newborn hearing screening. (Genet Med, 2011)

40. Pediatric SNHI Newborn hearing screening False positive Newborn genetic screening Slight/mild SNHI Late-onset or progressive SNHI No access to UNHS Earlier diagnosis Earlier intervention with hearing aids Prevent recurrence Avoid ototoxic medication Avoid head trauma Newborn genetic screening for deafness

41. Etiologies of deafness in children (Dahl et al., 2013)

42. Congenital CMV infection (0.2-2.3% of live births) Symptomatic neonates Asymptomatic neonates ~1/8~7/8 IUGR Microcephaly Petechiae Hepatosplenomegaly Encephalitis

43. Symptomatic neonates Asymptomatic neonates ~1/8~7/8 Sequelae 40-50% Sequelae 13.5% Hearing loss: most common Congenital or progressive Bilateral or unilateral Treatable w/ antiviral mx Congenital CMV infection (0.2-2.3% of live births)

44. Screening of newborns for CMV infection will permit early identification of at-risk congenitally infected infants for purposes of targeted monitoring and intervention during critical stages of speech and language development. (Boppana et al, NEJM, 2011)

45. Newborn hearing screening at D1 (OAEs or AABR) Reassurance & education PassRefer OAEs or AABR at D3 Reassurance & education PassRefer OAEs or AABR + tympanogram at 1m Reassurance & education PassRefer Diagnostic tests at 3m ABR (tone bursts) ASSR or BOA Newborn CMV screening CMV screening

46. Newborn hearing screening at D1 (OAEs or AABR) Reassurance & education PassRefer OAEs or AABR at D3 Reassurance & education PassRefer OAEs or AABR + tympanogram at 1m Reassurance & education PassRefer Diagnostic tests at 3m ABR (tone bursts) ASSR or BOA Newborn CMV screening CMV screening

47. Screening for deafness in newborns Newborn hearing screening Newborn genetic screening Newborn CMV screening Long-term cost-effectiveness: e.g. parental anxiety, ethical issues, etc.

48. Genetic diagnosis What next? Treatment? Genetic examination Residual hearing Time Intervention? Natural course of SLC26A4 mutations Transgenic mice Slc26a4 c.919-2A>G Slc26a4 p.H723R Gjb2 p.V37I

49. Mice with Slc26a4 c.919-2A>G Autosomal recessive inheritance Profound hearing loss 46%: head-tilting/circling Enlarged vestibular aqueduct Endolymphatic hydrops Stria vascularis atrophy Giant otoconia (PLoS One, 2011)

50. Mice with Gjb2 p.V37I Autosomal recessive inheritance Progressive, mild/mod. hearing loss Vestibular function: normal Inner ear morphology: normal Cx26 expression: normal Dye transfer: compromised Noise exposure  delayed recovery (Paper submitted)

51. Knock-in mice Slc26a4 c.919-2A>G Slc26a4 p.H723R Gjb2 p.V37I Other models Characterization of audiovestibular features, inner ear morphology & electrophysiology Novel therapeutic strategies Pharmacogenetic or gene therapy Pathophysiology of HHI Future perspectives Genome editing TALEN CRISPR

52. Acknowledgement Grants: - National Health Research Institute - National Science Council - National Taiwan University - National Taiwan University Hospital Transgenic Mouse Models Core (TMMC), National Taiwan University Department of Medical Genetics, National Taiwan University Lab of Molecular Genetics in Otology, Department of Otolaryngology, NTUH.

53. (Sun-Moon Lake, Nantou)(The Grand Hotel, Taipei) Welcome to Taiwan