Xuezhong Liu, M.D., PhD., F.A.C.S.

July 11, 2019

Global Impact and Screening Strategy for Deafness Genes in Individuals with CI

Disclosures

No conflicts of interest to disclose in relation to this presentation.

Grant support: NIH/NIDCD

Second largest medical Center in USA

Jackson Memorial

UMHC/Sylvester Cancer Cent

Veterans AdministrationAnne Bates Leach HospitalUniversity of Miami Hospital

Serving a diverse patient population from the South Eastern United States to Latin America and the Caribbean

University of Miami Ear Institute

Center for Hereditary Deafness

Cochlear Implant Program

Skull Base Treatment Center

Clinical Audiology

Hearing Aid Center

The Center for Advanced Treatment of Meniere's Disease and Balance Disorders

Facial Nerve Disorders

Microsurgery Training Center (RMSB)

Barton G CI Family Resource Center

Thomas Balkany, MD Simon Angeli, MD

Christine Dinh, MD Adrien Eshraghi, MD

Fred Telischi, MD Xue Z. Liu, MD, PhD

University of Miami Ear Institute

Hillary Snapp, AuD, PhD Michael Hoffer, MD

• Extremely heterogeneous with at leas >800 genes involved

• Only >100)Identified genes with diverse function

• 2-3 genes accounting for major component of human deafness

• 20-60% patients identified to have mutations in known genes

Gene Identification

Genetic mapping

Candidate genesPositional/functional candidate genes

Gene

Mouse models

Sequential screening

Gene

Inbred/large families

Cochlear cDNA libraries

Small families

NGS - Exome Sequencing

The discovery that genes at only 2-3 loci account for a major component of human deafness suggested to us that the sequential screening of DNA samples from probands in multiplex sibships for mutations at selected candidate loci would be an effective strategy for identifying new genes for hereditary deafness. And mutations

Nance WE, Liu XZ, Pandya A, Lancet 2000

Sequential Screening Strategy

SequentialScreening ofNon syndromic

DeterminePhenotypicFrequencies &Distribution ofMutantAlleles.

Screening forMYO7A

Screen forCx26 &MitochondrialMutations

Screen forotherconnexinmutations

Cx26Het

Mit-Cx26- Cx26+ Mit+ Mit+ Cx26+ Mit-

Cx26- Cx26 het

Non-syndromic

Screen for otherHuman/mousecloned genes

_

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_

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Reserve for future genomic tech available

Screening forUSH2A

Screen forotherconnexinmutations

Estimate %genetic deafness

Multiplex Simplex

Identify newDeafness genes

Liu et al, 2003

Full sequence analysis by direct sequencing of individual genes -Dideoxy or Sanger chain termination method

Multigene SNP mutation chips-selected number of mutations in a small number of genes

Next Generation Sequencing (NGS)+Targeted Sequence Capture (TSC)-fully sequence all known deafness genes

NGS + exome sequencing -fully sequqence all coding regions of genome

Third generation Sequencing introduced in a research setting - used with TSC; exome or whole genome sequencing approaches

NGS in widespread use across research setting

whole genome sequencing by third generation sequencing technology may be available in clinical setting

60-70 known genes associated with hearing loss Developments in the speed/cost of

sequencing enable knowledge in the field of genetic hearing loss to proceed at a rapid pace

Potentially all 500 genes involved with hearing loss identified

Sequencing of all 20,000-25,000 genes in the genome may be possible a reasonable cost. Identify all conditions with a genetic basis - issue for all services within the NHS

2010/2011 2013

2015-2016 2020

2012

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0 yrs

5yrs

3yrs

10yrs

Sequencing technology

Knowledge of genetic hearing loss

Timeline of developments in genetic screening for hearing loss

Screening methods available for genes causing hearing loss in USA

- Sanger sequencing; DNA Array; NGS

Treatment of Hearing Loss

• Ultimate goal is to customize HL treatment based on the particular mutation

• Prevention and early treatment of HL– Prior to giving known ototoxic drugs, screen for pre-disposing

mutations (e.g. m.A1555G)– Early rehab w/ HA or CI and speech therapy– More predictable response to CI or ABI

• Gene therapy• Stem cells• Preimplantation genetic diagnosis

DNA sample collection, population-based cohort data, gene mapping, &

whole exome sequencing

In vitro and in vivo therapeutic Gene/cell/drug-based studies

Clinical Management & Diagnosis

diagnosis, counseling, personalized sequence profile

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MiamiOtoGenomic Diagnostic and Therapeutic Platform

Multidisciplinary collaboration effort

Center for Hereditary Deafness (CHD)

Department of Otolaryngology

Department of Human

Genetics

Department of Biology

HIHG-iPSc Miami OtogeneticCenter

Div of Audiology ECHO

Ear InstituteHearing Loss Clinics (Clinical Services, clinical research)

HIHG

Cochlear implant research Labs

CI Program

Multidisciplinary collaborations & integrate biomedical research and medicine

The Miami Hearing Loss Clinic

The Miami Hereditary Hearing Loss Clinic

Molecular Genetic Lab

The Miami Hearing Loss Clinic

CHD

Miami Molecular Otogenetic Program Miami Clinic Otogenetic Program

Gene Function Lab

HIHG

Case Presentation

• 10 y/o boy with congenital profound HL• CI when 3 y/o, but outcome was poor• USH1B with MYO7A mutations 2010• Autism 2010• Using implant more for environment contact

and preparing worsening eye-sight deteriorates

For 7 Cutler Bay Siblings Born Deaf from genetic etiology, Sweet Sounds

Most families would take the buzz of four kids chirping away over homework for granted, but for the Guillou children, sound was never heard until recently. NBC 6's Keith Jones reports. (Published Monday, Nov 25, 2013)

GJB2/GJB6

Blood SampleTumor tissue Skin Biopsy

Genetic Testing Establish Cell Lines

Therapy

Evaluate Mutations

Test Efficacy of Gene and Drug TherapiesCreate Transplantable Cells

Therapeutic Strategies for genetic deafness

iPSCVirus based therapies

Genome based based therapies

Stem cell based

iPSC

Etiology of Hearing LossIncreasing Importance in Cochlear Implantation

• There are differences in auditory performance that are not attributable to age at implantation or auditory training

• In DFNB1, cochlear nerve and spiral ganglion cells are preserved and this suggests the potential for excellent performance

• Most common identified etiology of congenital deafness in children receiving CI (18-40%)

• Positive GJB2 screening results establish an etiologic diagnosis and provide prognostic, genetic, and therapeutic information

• Better speech performance/preservation of central cognition functionfrom different countries

Vivero et al, Int J of Ped Otol 2010

Vivero… Liu, Int J of Ped Otol 2010

CI in Cx26 Related Deafnessn=31 of 44 Pediatric CI recipients (10/98 – 4/05)

• Mean duration of follow-up: 32 months (12-102)• 1 Med-el, 2 Clarion, 28 Nucleus-24• Verbal comprehension score:

DFNB1: 80%Non-DFNB1: 64% (p<0.001)

• Expressive language scoreDFNB1 75%Non-DFNB1 65% (p=0.04)

• DFNB1 patients show higher scores in Language Development tests, andfaster and more uniform gains on speech perception tests than non-DFNB1patients

Connell…Liu et al Otolaryngol Head Neck Surg 137:596-602 (2007)

Language Skills

• DFNB1 children who use cochlear implants show greater gains in expressive language than non-DFNB1 children, independent of age at implantation and duration of implant use

CI in Usher Syndrome

• Most common deaf-blind disorder

• 7% in congenital deaf patients with CI

• Virtually all members of the deaf community view

the visual impairment as a devastating handicap

• ERG for early diagnosis, genetic tests available now

• Studies showing the best perceptive results found in

children implanted before 9 y/o *Liu XZ, Angeli SI, et al. Cochlear implantation in individuals with usher type 1 syndrome. Int J PediatrOtorhinolaryngol. 2008;72(6):841-847.

Genetic variants in the peripheral auditory system significantly affect cochlear implant performance

Shearer, 2017Etiologic diagnosis INCLUDING neural vs sensory genetic defect is the biggest predictor of post-implant performance (18%). Further work: would different CI programming/rehab work better for neural defects?

Nine Common Deafness Mutations Screening – CapitalBioMiamiArray for Caucasian Populations (Yan et al, PLOS ONE, 2017)

Gene Mutation(s) Frequency in patients (%)

Occurrence in the gene (%)

GJB2 35delC 25.4 Up to 88W44C 0.38 0-3.6L90P 0.29 0-1.47

SLC26A4 L236P 16T416P 15

MT-RNR1 1555A>G 0-3.6 37.6-100

7444G>A 1.6-1.84GJB6 309 kb deletion

Monogenic0.5

309 kb deletion Digenic with GJB2

16-21

A custom capture panel of 180 known deafness geneswith a target size of approximately 1MB (Agilent SureSelect DNA Design). These results prove the accuracy andreliability of the custom capture experiment.

27%

52%

7% 6% 8%

DominantRecessiveBothX-LinkedResearch Purpose

39%44%

7%

2%

8%

Syndromic

Non-syndromicBoth

miRNA

Identifying Causative Gene VARIANTS for Hearing Loss Using a Target Enrichment/Next Generation Strategy – MiamiOtoGenes (MOG)

Tekin et al, 2015; Yan et al, 2016

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USA IRAN INDIA TURKEY TUNISIA NIGERIA SOUTH AFRICA

Ethnic-Based Sequential Screening Strategy to diagnose genetic deafness and to identifying new genes.

YES

NO

Probands/Pedigrees

Screen GJB2, SLC2CA4, MTRN1

Homozygous or Compound Heterozygous

Pathogenic Variants

YES

Stop

Targeted Sequence All Deafness Genes(MiamiOtoGenes Panel)

NO

Gene Identified

Whole Exome Sequencing

Novel new genes

Screen Repository

In vitro and In vivo Studies

Molecular Epidemiology

YES

Confirm pathogenic Variants CLIA Lab

Miami Clinical Otogenic Program

Sanger SequencingPopulation/Ethnic based GenechipsCapitalBioMiamiArray

MiamiOtoGenes (MOG) used for identification of the remaining cases.

Who should be offered genetic screening in CI patients?

1) Expert opinion by the members of the International Pediatric Otolaryngology Group (IPOG), 2016. After audio, comprehensive genetic testing has highest diagnostic yield of any test for SNHL (62% agree)

2) Non-syndromic children with unilateral HL should NOT be offered genetic testing with initial workup (62% agree)

3) Identification of HL caused from the peripheral auditory system

• CI team will need to become increasingly familiar with available tests and their interpretation in order to use them effectively and counsel patients regarding prognosis and treatment

Conclusions

• Genetic deafness affects 50% of all individuals with hearing loss• > 70% genetic deafness is nonsyndromic• Connexin 26 is the most common etiology• Usually CI given good to excellent outcomes are favorable An appropriate

management algorithm facilitates effective patient careLearn to recognize syndromes and their special considerations

• CI team will need to become increasingly familiar with available tests and their interpretation in order to use them effectively and counsel patients regarding prognosis and treatment

Thank You