CMV and Connexin 26 Frontiers of Research and Therapy for

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CMV and Connexin 26 Frontiers of Research and Therapy for Congenital Hearing Loss

Dylan K. Chan, MD, PhD, FAAPAssistant ProfessorPediatric Otolaryngology-Head and Neck SurgeryDirector, Children’s Communication CenterUniversity of California, San [email protected]

UCSF Audiology Update XIINovember 4, 2016

CMV and Cx26 Hearing Loss

Background – Congenital hearing lossPrevalence and impactDiagnostic testing

CMV-associated SNHLOverviewDiagnostic testingTreatmentCurrent management

Cx26-associated SNHLClinical overviewResearch





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Childhood hearing loss Definition

Permanent Childhood Hearing Impairment (PCHI)

Childhood hearing loss Definition

Permanent Childhood Hearing Impairment (PCHI)

Mild to profoundBilateral or unilateral

All frequencies or some frequenciesStable or progressive

Sensorineural or conductive

All are eligible by law for services to prevent developmental delay

Childhood hearing loss Prevalence

How common is PCHI?

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Childhood hearing loss Prevalence

Prevalence depends on your exact definition:

From newborn/school/pediatrician screening

1:500 newborns (> 40 dB HL)1:300 by age 9 (> 40 dB HL)

From NHANES survey

1:100 by age 19 (> 25 dB HL, bilateral)1:5 by age 19 (> 15 dB HL, any kind, laterality, or frequency)

Shargorodsky, 2010

Mild/unilateral hearing loss Impact

Children with mild or unilateral hearing loss have impaired school performance, behavior, and psychosocial well-being

Mild/moderate hearing lossBess 1998

1200 students, 5.5% with mild SNHLWorse on general education, educational risk, behavior, physical/emotional/social function

Unilateral hearing lossLieu 2010/2012

74 children, case-control studyChildren with UHL had significantly worse speech and language with persistent delays in academic and behavioral performance after 6 years

Childhood hearing loss Economic impact

Economic cost to the public for an untreated child with hearing loss:

• $652 million: total cost in the US for education for DHH children ($11,006/ child)• $115,600: lifetime direct educational costs for 1 DHH child• $420,000: additional direct educational costs (if untreated)• $1,000,000: total lifetime (educational costs + lost productivity)• $2.1 billion: economic cost to the public for a one-year cohort of DHH children• $10 billion: cost if untreated

• Estimated lifetime costs for all 380,000 children born in one year with asthma• $7 billion

Johnson, 1993Corso, 2009http://www.cdc.gov/ncbddd/hearingloss/data.html

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Early Intervention Timing matters

Yoshinaga-Itano, 1998

Newborn Hearing Screening 1-3-6

Birth AABR or DPOAE- based screen

By 1 month Outpatient screen/rescreen

By 3 monthsDiagnostic audiology evaluation (ABR)

By 6 monthsEarly intervention services initiatedOtolaryngology evaluationOngoing audiology management

REFER

REFER

Confirmed hearing

loss

Goal Diagnosis, medical treatment, and early intervention initiated by 6 months





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All children with congenital SNHL

50% acquired 50% genetic

67% non-syndromic33% syndromic

50% GJB2

50%Non-GJB2

Congenital SNHL Diagnostic workup

Pendred’sUsher’s

CMV

TORCH infections

Congenital SNHL Why test?

Knowledge Why can’t my child hear?

Prognosis Is the hearing going to get worse?

Exclusion of other causes What else is wrong with my child?

Family counseling What does this mean for my other kids?

Intervention What can we do about it?

hearing.siemens.com

Congenital SNHL Diagnostic workup

HistoryPhysical ExamDiagnostic Tests

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History Syndromic hearing loss

Associations with syndromic SNHL apparent from personal or family history

Visual impairment Usher’sBranchial cleft anomalies Branchio-oto-renalThyroid dysfunction/goiter Pendred’sSudden death/arrhythmia Jervell Lange-NielsenRenal/urinary complaints Alport’s, branchio-oto-renalPigmentation disorders Waardenburg

Unsure?? Refer to genetics

Physical Exam Syndromic hearing loss

Associations with syndromic SNHL apparent from physical exam

GoiterPendred’s

Craniofacial abnormalitiesMultiple

Branchial cleft anomalies/preauricular pitsBranchio-oto-renal

Pigmentation abnormalitiesWaardenburg’s

adhb.govt.nz

www.motownsports.comwww.chargesyndrome.org

www.thyroidmanager.org

History Non-syndromic hearing loss

Causes of non-syndromic SNHL apparent from history

Infectious Toxoplasmosis, syphilis, rubella, CMV, HSV, meningitis

Ototoxic drugs Gentamicin, cisplatinGenetic Family history, consanguinityHyperbilirubinemia JaundiceTrauma EVA-related sudden hearing loss

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Idiopathic Non-syndromic SNHL Core diagnostic tests

1) Imaging1) CT2) MRI

2) Genetic testing1) Connexin 26/302) Complete hearing loss gene array testing

3) CMV testing1) Urine PCR (active infection)2) Newborn blood spot PCR (congenital infection)





Imaging Impact

How does information gleaned from imaging affect patient management?

Cochlear nerve hypoplasia cochlear implant candidacy

Temporal-bone abnormalities surgical planning

EVA recommendations regarding head traumaYan et al., 2013

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EVA Head trauma

2015 Meta-analysis of EVA, head trauma, and progressive hearing loss

- EVA is associated with 40% risk of long-term progressive SNHL- There is no association between head trauma and risk for long-term

progression of hearing loss in people with EVA- I do NOT recommend special precautions for head trauma relating to EVA

Alemi, 2015

Imaging Recommendations

1) Cochlear implant candidates MRI with or without CT

2) All others MRI or CT after discussion with parents(age, cost, anxiety)

3) No special precautions for head trauma relating to EVA





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Genetic Testing Impact

Any cause identified genetic counseling/family planning

Syndromic association identified careful screening for associated phenotype

Specific mutations increased risk of progressive hearing loss, ototoxicity

Future therapeutic options gene therapy

Genetic testing Available tests

Test Location #genes tested

Turnaround time

Cost/insurance Indication

GJB2/GJB6 (Connexin26/30)

Multiple (Quest)

2 1-3 wks $300 / yes Non-syndromicSNHL

Pendred’s/ SLC26A4

Stanford, Cincinnati, Boston

1-3 4 wks $1100 / yes SNHL + EVA or thyroiddysfunction

Usher’s panel Cincinnati,Boston

9 8-12 wks $2500 / yes SNHL + visual dysfunction

OtoScope Iowa 133 3 months $1500 / no Any congenital hearing loss

Exome sequencing

Referral to genetics

All Varies Varies / no Any congenital hearing loss



Turnaround time



Multiple (Quest)








Exome sequencing



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Turnaround time



Multiple (Quest)








Exome sequencing





Turnaround time



Multiple (Quest)








Exome sequencing





Turnaround time



Multiple (Quest)








Exome sequencing



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CMV Congenital Hearing Loss

15-20% of all congenital hearing loss

More common in lower socioeconomic strata

Often progressive

May be syndromic

Can be treated

Core diagnostic tests How to choose?

Neonates < 2 weeks old CMV testing ASAP

Cochlear implant candidate MRI -> Cx26/CMV*

Unilateral CMV* -> imaging at appropriate age -> Cx26

All others Cx26/CMV* -> imaging at appropriate age

* CMV testing only by dried blood spot if > 2 weeks. If unable, skip** If no findings from core testing, consider full genetic array testing

Order and type of testing depends on many patient factors, systems consideration, and family preference

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CMV-associated SNHL noted to progress and/or newly develop up to teenage years

20-30% of CMV(+) will have SNHL0.2-0.6/1000 live births = 1000 new cases/year in the US

May be vastly underestimated

Take home point 1 – CMV-associated hearing loss

• Highly variable

• Frequently progressive

• Onset/progression throughout childhood

• Occurs with or without other symptoms

• ONLY occurs with prenatal transmission

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Prenatal screening healthy pregnant women - not routinely done

Newborn screening saliva PCR assays - not routinely done

Neonatal (up to 2-3 weeks) culture or PCR from urine or blood- gold standard

Postnatal PCR assay from dried newborn blood spots- 99% specificity- 30% sensitivity

CMV Testing

SNHL CMV DBS testing

Boppana et al., JAMA 2010

DBS CMV PCR (vs saliva culture)

34.4% sensitivity99.9% specificity

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Take home point 2 – CMV testing

• CMV testing (CMV PCR/culture)

• is sensitive and specific before 3 weeks• Is sensitive but NOT specific after 3 weeks

• CMV DBS testing

• Is specific but NOT sensitive• Is the only specific test after 3 weeks

Question:

What is the best testing/screening paradigm to efficiently and accurately identify congenital CMV-associated hearing loss?

1) Universal neonatal CMV screening2) Hearing-targeted CMV screening

NHS and CMV screening CHIMES data

• Ross (UAB) – AAP NCE 2014

• CMV and newborn hearing screening (NHS) in 100,000 newborns

CMV - CMV +

NHS PASS

97,571 429

NHS REFER

1967 33 21 SNHL12 No SNHL

Hearing unknown

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NHS and CMV screening CHIMES data +

63% SNHL21

All asymptomatic neonates100,000

2% refer NHS2000

1.6% + CMV33

98% pass NHS98,000

0.4% + CMV429

98.4% ‐ CMV1967

99.6% ‐ CMV97,571

15% SNHL(295)

7‐15% SNHL(30‐60)

0.1‐0.3% SNHL(100‐300)

NHS and CMV screening CHIMES data +

63% SNHL21


2% refer NHS2000

1.6% + CMV33

98% pass NHS98,000

0.4% + CMV429

98.4% ‐ CMV1967

99.6% ‐ CMV97,571

15% SNHL(295)

7‐15% SNHL(30‐60)

0.1‐0.3% SNHL(100‐300)

NNT = 100 to identify one case of congenital CMV‐associated SNHL for treatment

NHS and CMV screening Criticism

63% SNHL21


2% refer NHS2000

1.6% + CMV33

98% pass NHS98,000

0.4% + CMV429

98.4% ‐ CMV1967

99.6% ‐ CMV97,571

15% SNHL(295)

7‐15% SNHL(30‐60)

0.1‐0.3% SNHL(100‐300)

This method will miss CMV +children who pass NHS and later develop progressive SNHL

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NHS and CMV screening Criticism

63% SNHL21


2% refer NHS2000

1.6% + CMV33

98% pass NHS98,000

0.4% + CMV429

98.4% ‐ CMV1967

99.6% ‐ CMV97,571

15% SNHL(295)

7‐15% SNHL(30‐60)

0.1‐0.3% SNHL(100‐300)

Is this method an effective means for identifying babies at risk for congenital CMV?Yield of CMV + is 1.6% vs 0.4%. But still misses 93% of CMV + babies.

Take home point 3 – identification of CMV-associated hearing loss

• Universal CMV screening would be ideal

• Hearing-targeted CMV screening misses many potential cases, but is the best current option





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Treatment Valganciclovir for CMV hearing loss

NIAID Collaborative Antiviral Study GroupKimberlin et al., NEJM 372(10):933-43

Multinational 31-institution Phase III randomized, controlled clinical trial

109 infants < 30 days old Symptomatic congenital CMV43% with baseline hearing loss

6 wks vs. 6 mos PO valganciclovir24-month follow up

Significantly increased odds of hearing improvement or stabilization of normal hearing with 6-month course (OR (1.02-6.91) at 24 months)

V-GCV for congenital CMV Utah study

HT-CMV screening-directed treatment for congenital CMV-associated hearing loss

Study sites25 sites; coordinator Albert Park (OHNS, University of Utah);

InclusionReferred NHS and subsequent CMV + by urine/saliva PCR or culture

GoalDetermine hearing, speech, language, developmental outcomes of infants with referred NHS entered into a pathway of early CMV screening and subsequent treatment (placebo vs. valganciclovir)

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NCT01649869 (Valgan Toddler Study)

Multi-institution Phase II randomized, controlled clinical trial

6 wks PO valganciclovir vs. placeboAge 1 month – 4 years with sensorineural hearing loss

Congenital CMV by neonatal urine CMV or dried blood spot CMV

CMV-associated SNHL New Kimberlin study

Take home point 4

• CMV treatment (6 months valganciclovir)

• Can prevent progression of hearing loss

• Is of unknown efficacy in kids with isolated CMV-associated hearing loss AND in older kids





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Babies under 3 weeks of age with referred NHS- CMV testing (urine/saliva PCR or culture)- Diagnostic audiologic testing

Babies over 3 weeks of age with referred NHS- Diagnostic audiologic testing

Babies and children over 3 weeks of age up to 4 years of age with confirmed SNHL- CMV DBS testing (as part of diagnostic workup for SNHL)- Consider V-GCV if positive

Children over 4 years of age with confirmed SNHL- CMV DBS testing (purely for diagnostic workup for SNHL)

CMV-associated SNHL Practice guidelines

1) Babies under 3 weeks of age with referred NHS- CMV testing (urine/saliva PCR or culture)- Diagnostic audiologic testing

2) Babies over 3 weeks of age with referred NHS- Diagnostic audiologic testing

3) Babies and children over 3 weeks of age with confirmed SNHL- CMV DBS testing (as part of diagnostic workup for SNHL)- Consider V-GCV if positive

Hearing-targeted CMV screening

State law in UT, CT, MN, IL

CMV-associated hearing loss Take-home

If you see a baby < 3 weeks old for a referred newborn hearing screen, recommend discussion with pediatrician/OHNS about CMV testing

If you are involved with your hospital’s NHS program, consider hearing-targeted CMV testing

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Cx26 hearing loss Prevalence among all hearing impaired

54 countries141 studies23093 hearing-loss probands4064 GJB2 biallelic probandsFrequency = 18%

0-10%

>40%

10-20%20-30%30-40%

Chan and Chang, 2015

0%

50%

100%

T/T NT/T NT/NT

profound

severe

moderate

mild

1531 probandsSnoeckx et al., 2006

Per

cent

of i

ndiv

idua

ls

Genotype

Cx26 hearing loss Severity

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Initial audiogram

Final audiogram

0

20

40

Pure‐tone threshold (dB)

60

0.5 1 2 4 8

Frequency (kHz)

Thresholds averaged from 7 V37I/V37I individuals with progressive hearing loss

Cx26 hearing loss Progression and V37I





gene GJB2 (gap junction protein, beta 2)Chromosome location13q11-q12

protein Connexin 26Expressed in cochlea and epidermisForms heteromeric/heterotypic gap-junction channels

and apical hemichannels with Connexin 30

Ions, small molecules, electrical currents, cell adhesion

Cx26 GJB2

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Connexin function K+ recycling

K+

K+ K+

K+

Connexin independentConnexin dependent

Noise

Connexin function Noise detection

ATP

Ca2+ waves MAPKConnexin independentConnexin dependent

Cx26 dysfunction Animal models

Mouse (genetic model) Gerbil (physiologic model)

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CreER loxP loxP

Cx26

TMX

Mouse Inducible Cx26 KO

ER-Cre/Cx26-lox

TMX-inducible Cx26 cKO Progressive hearing loss

Zhu, 2015

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TMX-inducible Cx26 cKO Noise-induced hearing loss

Baseline Days Permanent shift

Clic

k A

BR

thre

shol

d (d

B S

PL)

103 dB, 2 hr octave-band noise

20

30

40

50

60

70

80

0 2 4 6 8 10 12 14 16

WT

Cx26

Wild-typeConnexin-26 knockout


Connexin 26‐deficient mice have increased susceptibility to age‐ and noise‐induced

hearing loss



hearing loss

Connexin 26 may protect against cochlear trauma

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hearing loss

Connexin 26 may protect against cochlear trauma

How?



Acoustic stimulus

Endolymph

Perilymph

Endocochlearpotential

Microphonicpotential

Calcium imagingCochlear stroboscopy

Adult gerbil cochlear physiology in vitro

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DorsalVentral

Lateral

Medial

50 µm

In vitro Cochlear explant

Chan and Hudspeth, 2005

10 µm

Lateral

Medial

In vitro Cochlear explant



Noise-exposure Cochlear stroboscopy

1000 Hz, 100 dB SPL

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Noise exposure Intercellular Ca2+ signalling

AAA

0.4

0.5

0.6

0.7

0.8

0.9

Rat

io (

340

/387

)

slope = 6.2 m/s

0

10

20

30

40

50

60

70

80

90

0 5 10 15Time (s)

Dis

tanc

e (m

m)

10 s

OHCs

50 M

A B DC

E F

Adult gerbil cochlear explant


Connexin 26 protects against cochlear

trauma

Calcium signalling in cochlear supporting cells is involved

Cx26 dysfunction From rodents to humans

Are people with Connexin 26 mutations more susceptible to

NIHL?

Could calcium regulation in the cochlea be a potential drug

target?

What about gene therapy?

Connexin 26 protects against cochlear

trauma

Calcium signalling in cochlear supporting cells is involved

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Connexin 26 Gene therapy

What about gene therapy?

Normal cochlea Long-deafened cochlea


The risks and benefits of intervention now are unclear with respect to future therapies

We need to support your child’s hearing and communication development now


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Connexin 26 is the most common gene affected in congenital SNHL and has highly variable presentation

Connexin 26-associated hearing loss may be due to changes in calcium regulation in the cochlea that make it break down more easily

This may lead to new drug targets or gene therapy in the future

Let’s take care of our kids now

Cx26 hearing loss Summary

Thanks

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Cochlear implantation Cost effectiveness

Unilateral Cochlear Implant

Estimated cost per QALY: $9,000

Once accounting for indirect costs (including reduced educational expenses): Savings of $53,000 per child

Cheng, 2000Lammers, 2011

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Early Intervention Timing matters

Cochlear implantation at< 18 months18-36 months>36 months

Niparko, JAMA 2010

Receptive language Expressive language

How was hearing loss detected?

Unresponsive to sound

Speech/language

Education/behavior

Newborn Infant Preschooler Schoolchild

Profound Moderate Mild/unilateral

Age

Degree

Sign

How was hearing loss detected?

Unresponsive to sound

Speech/language

Education/behavior

Newborn Infant Preschooler Schoolchild

Profound Moderate Mild/unilateral

Age

Degree

Sign

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Diagnostic tests Congenital hearing loss

What next?

1) Work up potential causes revealed in H&P• Visual loss -> ophthalmology consult• Urinary problems -> renal US• Branchial cleft anomalies -> renal US• Goiter or thyroid problems -> TFTs, genetic testing (SLC26A4), imaging

(EVA)• History of syncope or arrhythmia -> EKG• ANY syndromic association -> genetics consult

Unsure?? Refer to genetics

Diagnostic tests Congenital hearing loss

What next?

1) Work up potential causes revealed in H&P• Visual loss -> ophthalmology consult• Urinary problems -> renal US• Branchial cleft anomalies -> renal US• Goiter or thyroid problems -> TFTs, genetic testing (SLC26A4), imaging

(EVA)• History of syncope or arrhythmia -> EKG• ANY syndromic association -> genetics consult

2) If H&P/genetics evaluation unrevealing:• do NOT empirically pursue these tests for specific syndromic causes.• DO consider one of three core diagnostic tests

Imaging CT vs MRI

Right: http://www.nidcd.nih.gov/health/hearing/pages/eva.aspx

CT

Better bony anatomy+ radiationLess sedationLess expensiveLess incidental findings

MRI

Better neural anatomyNo radiationMore sedationMore expensiveMore incidental findings

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Genetic testing Recommendations

1) GJB2/Connexin 26 testing

2) Refer to genetics for consideration of other testing

Documents

CMV and Connexin 26 Frontiers of Research and Therapy for