Purpose of the proposed investigation and statement of scientific value

1. To characterise the hearing status of the ALSPAC cohort at age 7, 9 and 11 years

2. To investigate the impact of the 35delG mutation on audiological measures at age 7, 9 and 11 years

3. To investigate the impact of the M34T mutation on audiological measures at age 7, 9 and 11 years

Background

The human gap junction b-2 gene (GJB2) that encodes the protein connexin-26 was the first autosomal gene for non-syndromic deafness to be identified(Kelsell et al, 1997). Connexin-26 is involved in recycling of potassium ions from the endolymph of the cochlea(Kikuchi et al, 2000) and mutations in this gene are by far the commonest cause of autosomal recessive non-syndromic sensorineural hearing loss (NSSNHL) (~50% in Europe). In addition mutations also contribute to both autosomal dominant NSSNHL and syndromic forms of hearing loss as well (Denoyelle et al, 1998; Gasparini et al, 2000, see references on http://davinci.crg.es/deafness/index.php?seccion=mut_db&db=synd&synd=cx26synd

). The mutation, 35delG, accounts for the majority of the mutations in the gene in Caucasian populations with an estimated carrier frequency of 1 in 80 in northern and central Europe (Gasparini et al, 2000).

The high frequency of the 35delG and the small size of the GJB2 gene (~ 5.5kb, 2 exons only 1 of which is coding) has facilitated the development of clinical mutation screening, which in turn has led to a fuller characterisation of the common variants in GJB2 than in other NSSNHL genes. A case in point is the GJB2 sequence variant 101 T(registered trademark)C (M34T), which has an UK heterozygote frequency of about 2% and changes a hydrophobic methionine residue in the first transmembrane region of the protein to a polar threonine. Functional data generated using the Xenopus laevis paired oocyte assay showed that M34TCx26 protein is a dominant disrupter of normal channel formation (White et al, 1998). Human genetic studies have shown that it is not a dominant mutation, but the role of M34T as a contributing recessive allele in NSSNHL is controversial and remains unclear despite both functional as well as clinical studies.

There have been few attempts to make carefully controlled audiological assessment of obligate heterozygotes from families with autosomal NSSNHL. Cohen and colleagues (Cohen, 1999; Cohen et al, 1997) found an increase in abnormalities in pure tone audiometry and otoacoustic emissions (OAE), but these did not correlate with the presence of the 35delG mutation. A small family study of Ashkenazi Jews (Morell et al, 1998) suggested that heterozygous carriers of GJB2 mutations (principally 167delT) with apparently normal hearing had significantly lower amplitudes of OAE than controls. A subsequent study of the same population showed significantly reduced suppression of OAE than control subjects, although no significant difference in OAE amplitude (Hood et al, 2000).

Within the ALSPAC cohort, Bitner-Glindzicz and colleagues have detected the 35delG and the M34T mutations of the GJB2 gene (unpublished). 1% (125 out of 9348) were heterozygote for the 35delG mutation and 2% (255 out of 9324) were heterozygote for the M34T mutation. Pilot analysis showed that the hearing status of the heterozygotes at age 7 and 9 were no different to the normal population. However the 35delG heterozygotes (but not M34T) had significantly reduced TEOAE amplitude indicating reduced cochlear function, at age 9 compared to the normal population.

The significance of the findings from the pilot study needs further investigation. We aim to evaluate whether being a carrier for 35delG and M34T has any functional significance on hearing.

Research questions:

1. Does being a carrier increase your risk of developing hearing loss?

a. Do carriers show significant changes in OAE between age 9 and 11 years?

b. Do carriers show significant changes in hearing (conventional and extra-high frequency thresholds) between 7 and 11 years?

2. Does being a carrier convey advantages in hearing function?

a. Are carriers more sensitive to sound (hyperacusis)?

b. Do carriers have lower acoustic reflex thresholds?

c. Are carriers less likely to report tinnitus?

ALSPAC measures required

1. Hearing thresholds at age 7, 9 and 11 years (child measure; focus clinic)

2. Otoacoustic emissions at age 9 and 11 years (child measure; focus clinic)

3. Acoustic reflex thresholds (ipsilateral) at age 7, 9 and 11 years (child measure; focus clinic)

4. Child report of tinnitus and hyperacusis at age 11 years (child measure; focus clinic)

5. Children in Focus hearing and tympanometry measures (child measure; focus clinic)

6. Tympanometry at age 7, 9 and 11 years (child measure; focus clinic)

7. Noise measures at age 11 years (child measure; focus clinic)

8. Connexin 26 genotypes

9. Measures of socio-economic status (maternal education status and others to be determined with statistician)

Research plan

1. To put the genetic results in context, we intend to categorise the normal hearing sensitivity of the ALSPAC cohort at age 7, 9 and 11 years. Most of this work was completed by Amanda Hall during her employment at ALSPAC, but requires additional funding to complete.

2. We intend to repeat the connexin 26 analysis using the age 11 year hearing and otoacoustic emission results. This will allow the repeatablity of the 9 year results to be checked.

3. We intend to investigate changes in the hearing and OAE for the carriers/non-carriers between age 7, 9 and 11 years.

4. We intend to compare measures of hearing function (tinnitus and hyperacusis) for the carriers/non-carriers.

Resources

* We require funding to process and prepare the 11 year otoacoustic emission and hearing data

* We require statistical support for the analysis

References

Cohen M, Francis M, Coffey R, Pembrey ME, Luxon LM. Abnormal audiograms and elevated acoustic reflex thresholds in obligate carriers of autosomal recessive non-syndromic hearing loss. Acta Otolaryngol 1997; 117: 337-42.

Cohen M. Auditory and vestibular studies in families with autosomal recessive non-syndromic hearing loss. PhD University of London 1999

Denoyelle F, Lina-Granade G, Plauchu H, Bruzzone R, Chaib H, Levi-Acobas F, Weil D, Petit C. Connexin 26 gene linked to a dominant deafness. Nature 1998; 393(6683): 319-320

Gasparini P, Rabionet R, Barbujani G, Melchionda S, Petersen M, Brondum-Nielsen K, Metspalu A, Oitmaa E, Pisano M, Fortina P, Zelante L, Estivill X. High carrier frequency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG. Eur J Hum Genet 2000; 8:19-23

Hood L, Berlin C, Morlet T, Goforth-Barter L, Tedesco S, Bordelon J, Picton T, Keats B, Friedman T, Morell R, Griffith A. Otoacoustic emissions in obligate carriers of genes for recessive hereditary hearing loss. Abstracts Association Research Otolaryngology 2000

Houseman MJ, Ellis LA, Pagnamenta A, Di WL, Rickard S, Osborn AH, Dahl HH, Taylor GR, Bitner-Glindzicz M, Reardon W, Mueller RF, Kelsell DP. Genetic analysis of the connexin-26 M34T variant: identification of genotype M34T/M34T segregating with mild-moderate non-syndromic sensorineural hearing loss. J Med Genet 2001; 38: 20-25

Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 1997; 387 (6628): 80-83

Kikuchi T, Kimura RS, Paul DL, Takasaka T, Adams JC. Gap junction systems in the mammalian cochlea. Brain Res Brain Res Rev 2000; 32: 163-166

Morell RJ, Kim HJ, Hood LJ, Goforth L, Friderici K, Fisher R, Van Camp G, Berlin CI, Oddoux C, Ostrer H, Keats B, Friedman TB. Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness. New England Journal of Medicine 1998; 339: 1500-1505

White TW, Deans MR, Kelsell DP, Paul DL. Connexin mutations in deafness. Nature. 1998 Aug 13;394(6694):630-1.