Aim.

To investigate whether genetic polymorphisms/mutations in the FSCN2 gene, or in genes encoding proteins known to be present in hair cell stereocilia and associated physically or functionally with fascin-2 protein, are correlated with early-onset hearing loss within the ALSPAC study population.

Hypotheses

1. The human population includes mutations/polymorphisms in FSCN2 that are associated with early-onset hearing loss.

2. The human population includes mutations/polymorphisms in genes encoding proteins that are linked physically or functionally with FSCN2 for assembly and mechanotransduction by stereocilia of hair cells, that are associated with early onset hearing loss.

Rationale

The hair cells of the inner ear have essential roles in mechanotransduction of sound waves into intracellular signals that result in nerve cell impulses. Crucial to mechanotransduction are the hair bundles on the apical surfaces of hair cells. These are made up from many actin-rich stereocilia, fine projections of the cell surface that are made rigid by a central bundle of cross-linked actin filaments. It has recently become appreciated that major components of a protein complex (tip complex) located at the tip of stereocilia are important for gating ion channels in response to mechanical movements of the stereocilia, and that function-perturbing mutants in multiple members of the the tip complex lead to deaf/blindness syndromes such as Usher syndrome (Bonnet and El-Amraoui, 2012). An example of one of these proteins is cadherin-23. Mis-sense mutations in cadherin-23 are also associated with non-syndromic deafness (Schultz et al., 2011). The range of phenotypes associated with cadherin 23 mutations suggest that additional interactions between components of the stereocilia and potential genetic associations with early-onset hearing loss remain to be discovered.

Fascin-2 is an actin-crosslinking protein that until recently was considered to be located specifically in photoreceptor cells of the retina (Lin-Jones and Burnside, 2007; Hashimoto et al., 2011). New data demonstrate that fascin-2 is a component of inner and cochlear hair cell stereocilia in humans, mice and fish (Shin et al., 2010, Chou et al., 2011). Furthermore, studies of very early onset hearing loss (phenotype: increased 16kHz hearing threshold by 5 weeks of age) in the inbred DBA/2J strain of mice have identified a casual role for a mutant variant of fascin-2, fascin-2R109H, in synergy with a mutation in cadherin-23 (Johnson et al., 2008, Shin et al., 2010). This phenotype of these mice can be rescued by transgenesis of wild-type fascin-2 (Shin et al., 2010).

Many SNPs, including multiple non-synonymous coding single nucleotide polymorphisms (SNPs) have been identified in human FSCN2 (NCBI SNP database). Thus a major goal of this pilot study is to identify if any polymorphism in human FSCN2 is associated with early onset hearing loss. We will also examine possible contributions of fascin-2 associated proteins. The large ALSPAC cohort of children who have had hearing measurements taken, including the very sensitive measurements of 16kHz audiometry and otoacoustic emissions and tympanometry variables for middle ear function, can enable us to address this question.

Design

1. The first hypothesis we will test is that SNPs in human FSCN2 and CAD23 are associated with early onset hearing loss in humans, with emphasis on results of extra-high frequency audiometry and otoacoustic emissions, but also including tympanometry variables for middle ear functions.

2. Secondly, we will examine for possible associations of SNPs in genes that encode other proteins of the stereocilia that associate physically or functionally with fascin-2 or cadherin-23. These will include:

-protocadherin-15, that acts with cadherin-23 in the tip links of the tip complex

-other actin crosslinking proteins that are present within stereocilia, as determined by proteomic analyses of isolated stereocilia (plastin-1 as the second most abundant cross-linker, also espin, plastin-2, plastin-3, fascin-1, espin-like protein and xin-related protein 2) (Shin et al., 2010)

-the ion channel HCN2, reported in a biochemical study to associate physically with fascin-2 in cochlear hair cells (Ramakrishnan et al., 2012)

-the human orthologues of six gene products from a candidate interval on mouse chromosome 18 that appears to account for broader spectrum, low frequency hearing losses that occur in the DBA/2J mouse strain by 2-3 months of age (Nagtegaal et al., 2012). The syntenic region of the human genome is 18q21.1 and the six genes are all conserved in this region (NCBI Genes database); MYO5B, ACAA2, c18orf32, DYM, SMAD7 and CTIF

Associations will be examined for each individual gene, and we will also examine if any coding or other SNPs identified occur in the same individual as any SNPs identified in fascin-2 or cadherin-23.

3. To make these analyses, we request access to all SNPs examined within the children in the ALSPAC hearing data studies for the following genes:

Priority 1:

FSCN2

CDH23

HCN2

PCDH15

PLS1

Priority 2:

ESPNL

FSCN1

PLS2

PLS3

XIRP2

MYO5B

ACAA2

c18orf32

DYM

SMAD7

CTIF

References

Bonnet C, El-Amraoui A. (2012) Curr Opin Neurol. 25:42-9.

Schultz, JM et al. (2011) J Med Genet. 48:767-75

Lin-Jones J, Burnside B. (2007) Invest Ophthalmol Vis Sci. 48:1380-8.

Chou SW et al. (2011) PLoS One. 2011;6(4):e14807

Johnson KR et al. (2008) Genomics 92: 291-225.

Shin JB et al., (2010) J. Neurosci 30: 9683-94.

Hashimoto, Y, Kim DJ and Adams, JC (2011). J. Pathology 224:281-300.

Ramakrishnan NA et al (2012) J Biol Chem. 287:37628-46.

Nagtegaal AP et al. (2012) Genes Brain Behav. 2012 Sep 18. doi: 10.1111/j.1601-183X.2012.00845.x.