WO2014190422A1 - Methods and compositions for detecting progressive hearing loss - Google Patents

Abstract

Methods and products for identifying a human subject as having or having an increased likelihood to develop otosclerosis hearing loss are provided. The method comprises (a) obtaining a suitable sample from the subject; (b) assaying the sample for the presence or absence of a mutation in: i) a FOXL1 polynucleotide encoding an amino acid corresponding to position 326 to position 330 of SEQ ID NO:2 or ii) a FOXL1 polypeptide encoded by said polynucleotide; and (c) identifying the subject as having or having an increased likelihood to develop otosclerosis hearing loss if said mutation is detected.

Description

Title: Methods and Compositions for Detecting Progressive Hearing Loss

Related Applications

[0001] This is a Patent Cooperation Treaty Application which claims the benefit of 35

U.S.C. § 1 19 based on the priority of U.S. Provisional Patent Application No. 61/829,050, filed May 30, 2013 which is incorporated herein by reference in its entirety.

Field of the Disclosure

[0002] The disclosure relates to methods and products for detecting susceptibility of a subject of to having and/or developing hearing loss and specifically to methods of detecting a mutation in FOXL1 associated with otosclerosis.

Introduction

[0003] Hearing loss due to otosclerosis is relatively common and is estimated to contribute to 0.3-1 % of cases of late onset hearing loss ([1 , 2]). Both sporadic and hereditary cases are currently recognized, and at least eight known loci for otosclerosis inherited as a dominant trait (OTSC1, OTSC2, OTSC3, OTSC4, OTSC5, OTSC7, OTSC8 and OTSC10) and have been mapped [3-10].

[0004] Otosclerosis of the temporal bone is characterized by abnormal bone metabolism, with focal resorption and formation of abnormal bone (greater density, cellularity and vascularity and eventually thick sclerotic bone) in the otic capsule. Otosclerotic foci occurring anterior to the oval window can cause stapes fixation and conductive hearing loss, and more rarely sensorineural hearing loss caused by otosclerotic foci within the cochlea. Certain diagnosis of otosclerosis requires surgical confirmation and is based on the finding of otosclerotic foci on the oval window with stapedial fixation during surgery.

[0005] The decision to undergo surgical exploration of the middle ear for suspected otosclerosis is based on the clinical findings of conductive hearing loss with a normal tympanic membrane and absent/ abnormal stapedial reflexes. Although high resolution CT scan is proving to be useful for assisting with presurgical diagnosis, it is not widely available clinically, and the results appear to be false negatives in a significant proportion of cases. Most importantly, relying on currently available clinical tests (high resolution CT, audiological testing) means that the disease has progressed significantly before surgical treatment is considered and a diagnosis of otosclerosis can be confirmed.

[0006] Early detection of the disease through genetic testing, prior to the development of advanced clinical symptoms, will facilitate earlier treatment, with the potential for improved surgical outcomes and the reduction of surgical complications. Furthermore, insights provided by identifying a genetic etiology may elucidate the underlying pathophysiological mechanisms associated with this metabolic bone disorder and improve understanding of the onset and natural progression of otosclerotic disease processes, thus facilitating identification of ways for the prevention of this disorder through targeted treatments.

Summary of the Disclosure

[0007] An aspect includes a method for identifying a human subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss, the method comprising (a) obtaining a suitable sample from the subject; (b) assaying the sample for the presence or absence of a mutation in. i) a FOXL 1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues in FOXL1 polypeptide C-terminus; ii) a FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; iii) a FOXL1 polypeptide at one or more amino acid residues in FOXL1 polypeptide C-terminus; or iv) a FOXL1 polypeptide at one or more amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; and (c) identifying the subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss if said mutation is detected.

[0008] Another aspect includes a method of determining if a human subject is a carrier of a mutation that increases the likelihood of developing otosclerosis and/or hearing loss, the method comprising (a) obtaining a suitable sample from the subject; (b) assaying the sample for the presence or absence of a mutation in: i) a FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues in FOXL1 polypeptide C- terminus; ii) a FOXL 1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; iii) a FOXL1 polypeptide at one or more amino acid residues in FOXL1 polypeptide C-terminus; or iv) a FOXL1 polypeptide at one or more amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; and (c) identifying the subject as a carrier of a mutation that increased the likelihood of developing otosclerosis and/or hearing loss if said mutation is detected.

[0009] Another aspect includes a method for identifying a human subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss, the method comprising (a) obtaining a suitable sample from the subject; and (b) assaying the sample for the presence or absence of a mutation in: i) a FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues in FOXL1 polypeptide C-terminus; ii) a FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; iii) a FOXL1 polypeptide at one or more amino acid residues in FOXL1 polypeptide C-terminus; or iv) a FOXL1 polypeptide at one or more amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; ; wherein detection of the presence of said mutation indicates that the subject has, or has an increased likelihood, to develop otosclerosis and/or hearing loss.

[0010] Yet another aspect includes an isolated polynucleotide comprising the sequence of SEQ ID NO: 2 or 5.

[0011] Another aspect includes an isolated polypeptide comprising the sequence of

SEQ ID NO: 4 or 6.

[0012] Another aspect includes an isolated antibody or binding fragment that binds

SEQ ID NO: 6 or that binds an epitope comprising amino acid residues 325 and 331 of SEQ ID NO: 4 that does not bind an epitope comprising amino acids 326 to 330 of SEQ ID NO: 3.

[0013] In an embodiment, the isolated polynucleotide, isolated polypeptide or isolated antibody or binding fragment thereof is conjugated to and/or comprises a heterologous moiety, optionally a detectable label.

[0014] A further aspect includes a composition comprising: i) a polynucleotide comprising the sequence of SEQ ID NO. 2 or 5; ii) a polypeptide comprising the sequence of SEQ IN NO: 4 or 6; or iii) an antibody that binds SEQ ID NO: 6 or that binds an epitope comprising amino acids 325 and 331 of SEQ ID NO: 3 that does not bind an epitope comprising amino acids 326 to 330 of SEQ ID NO: 3 and a diluent.

[0015] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

[0016] An embodiment of the present disclosure will now be described in relation to the drawings in which:

[0017] Figure 1 : Partial pedigree of family 2081 segregating a form of autosomal dominant otosclerosis.

[0018] Figure 2: Audiological series of the family 2081 proband.

[0019] Figure 3: Partial pedigree (right hand side) of NL family 2081 co-segregating a 9Mb green haplotype that located 2Mb downstream of OTSC4. [0020] Figure 4: Electropherogram showing 15 base pair deletion causing in frame deletion.

[0021] Figure 5: Multiple alignment of FOXL1 showing the deletion of 5 amino acids when compared across Eukaryotic and Prokaryotic species: XP 001231599.2 (G.gallus), NP 032050.2 (M.musculus), XP 002694802.1 (B.taurus), XP 851625.1 (C.lupus), NP 005241.1 (Homo species) and XP 51 1 154.2 (p.troglodytes).

[0022] Figure 6: Gene structure of FOXL1.

[0023] Figure 7: Expression of FOXL1 gene from lymphoblastoid cell line in subjects with otosclerosis.

[0024] Figure 8: Effect of 15bp deletion on FOXL1 expression.

[0025] Figure 9: Expression of FOXL1 -mutant protein in nuclear extract from HEK

293A cell line transfected FOXL1 (WT and Mutant).

[0026] Figure 10: Transient transfection of FOXL1 (WT and Mutant) in osteoblast cell line and measuring FOXL1 by real time PCR.

[0027] Figure 11 : Nuclear localization of FOXL1 (WT and Mutant) in osteoblast cell line.

[0028] Figure 12: Transient transfection of FOXL1 (WT and Mutant) in osteoblast cell line and measuring protein expression.

[0029] Figure 13: Expression of predicted genes involved in otosclerosis.

[0030] Table 1 : Sequenced functional candidate genes at extended OTSC4 region.

[0031] Table 2: Variants detected from sequencing of 13 genes in extended OTSC4.

[0032] Table 3: Sequences

Detailed description of the Disclosure

I. Definitions

[0033] The term "FOXL1 " as used herein means forkhead box protein L1 and optionally refers to the gene, and/or its gene products including nucleic acid transcripts and translated polypeptide as determinable from its context usage and includes all naturally occurring variants thereof. FOXL1 is an 345-amino acid protein with one isoform (NM_005250) and one known functional domain (Fork head domain). The polynucleotide sequence of wildtype human FOXL1 is shown in SEQ ID NO: 1 and the amino-acid sequence of wildtype human FOXL1 is shown in SEQ ID NO: 3. The polynucleotide sequence of mutant human FOXL1 is shown in SEQ ID NO:2 and the amino acid sequence of mutant human FOXL1 is shown in SEQ ID NO: 4. The FOXL1 gene is found at positions 866121 15 86615304 of chromosome 16 (Accession number NM_005250, Genomic BUILD . hg19).

[0034] The term "c.976_990het_delGGGATCCCCTTCCTC" or

"del976_990GGGATCCCCTTCCTC" as used herein means a nucleotide mutation in a FOXL1 polynucleotide, the mutation being a deletion of the GGGATCCCCTTCCTC (SEQ ID NO: 5) nucleotides corresponding to position 976 to position 990. SEQ ID NO:2 is a nucleic acid sequence that shows the polynucleotide sequence of FOXL1 deleted for this sequence.

[0035] The term "p.Gly326_Leu330del" as used herein means an amino acid mutation in a FOXL1 polypeptide, the mutation comprising mutation of the amino acids corresponding to position 326 to amino acid at position 330 from Glycine (Gly) to Leucine (Leu) (the deleted amino acid sequence is shown in SEQ ID NO:6). SEQ ID NO:4 is a polypeptide sequence that shows the polypeptide sequence of FOXL1 deleted for this sequence.

[0036] The term "c.976_990het_delGGGATCCCCTTCCTC: p.Gly326_Leu330del" as used herein means both or either the c.976_990het_delGGGATCCCCTTCCTC FOXL1 polynucleotide mutation or the p.Gly326_Leu330del FOXL1 polypeptide mutation. Deletion of the GGGATCCCCTTCCTC from position 976_to position 990 of SEQ ID NO: 1 results in a mutated FOXL1 polypeptide comprising p.Gly326_Leu330del.

[0037] The term "mutated FOXL1 " as used herein refers to a FOXL1 molecule, including a FOXL1 polynucleotide encoding a polypeptide deleted for one or more or all amino acids corresponding to position 326 to position 330 of SEQ ID NO:3 and/or a FOXL1 polypeptide encoded by such a polynucleotide.

[0038] The term "corresponding to" as used herein includes a residue situated in a different sequence position but having sequence characteristics in common, including identical, or substantially identical, nucleotide sequence flanking the mutation (e.g. substantial identity is optionally at least 100% identity over four or more contiguous nucleotides). For example the deleted nucleotide positions are given relative to SEQ ID NO: 1 , which is the cDNA sequence of wildtype human FOXL1 and the deleted amino acid positions are given relative to SEQ ID NO: 3. A person skilled in the art would readily be able to determine the amino acids corresponding to position 326-330 of SEQ ID NO:3 in a different FOXL1 polypeptide e.g. different species, or naturally occurring variant . Similarly in methods involving genomic DNA, a person skilled in the art would readily be able to determine the genomic residue corresponding to position 976-990 of SEQ ID NO: 1. [0039] The term "heterologous moiety" as used herein means a molecule, optionally a nucleotide or amino acid sequence that is not a FOXL1 sequence, such as a targeting moiety that targets the sequence when expressed in a cell to a particular cell compartment and/or a signal sequence that directs cellular localization of a fused polypeptide. The heterologous moiety can also be a detectable label that is conjugated to an isolated polypeptide or isolated polynucleotide such as a fluorescent polypeptide or polynucleotide encoding a fluorescent polypeptide or an enzyme etc. The detectable label can also be comprised in the polynucleotide or polypeptide, for example when the detectable label is a radioactive nucleotide or amino acid.

[0040] The term "nucleic acid" and/or "oligonucleotide" as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, and is intended to include DNA and RNA which can be either double stranded or single stranded, represent the sense or antisense strand as well as cDNA. The term also includes modified or substituted oligomers comprising non- naturally occurring monomers or portions thereof, which function similarly, which are referred to herein as "chemical analogues" and/or "oligonucleotide analogues" such as "peptide nucleic acids". Such modified or substituted nucleic acids may be preferred over naturally occurring forms because of properties such increased stability in the presence of nucleases.

[0041] The term "isolated nucleic acid sequence", and/or "isolated polynucleotide" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.

[0042] The term "isolated DNA" as used herein means genomic or cDNA that is purified sufficiently for a method of detecting a mutation, for example sufficiently purified for PCR or other method described herein.

[0043] The term "probe" as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to a mutated FOXL1 polynucleotide such as a RNA or DNA FOXL1 nucleic acid deleted or substituted for GGGATCCCCTTCCTC (SEQ ID NO. 5) corresponding to position 976 to position 990 of SEQ ID NO: 1 or a nucleic acid sequence complementary to said RNA or DNA. For example, the probe can comprise residues complementary to 5' and 3' sequence flanking the deletion sequence, for example comprising at least 8 contiguous nucleotides of SEQ ID NO: 2 , the at least 8 contiguous nucleotides including residues corresponding to position 975 and 976. The length of probe depends on the hybridize conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length. In an another embodiment, the probe nucleotide length is any whole number between 8 and 500 nucleotides.

[0044] The term "primer" as used herein refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less. In an embodiment, the primer is about 12 nucleotides. The primer can be from about 15 to about 30 nucleotides in length or any whole number in between 12 and 30 nucleotides. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.

[0045] As used herein "all or part of of a probe or primer refers to the portion sufficient for in the case a probe, sufficient to specifically hybridize to the intended target and in the case of a primer, sufficient to prime amplification of the intended template.

[0046] The term "hybridize" refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. One aspect of the application provides an isolated nucleotide sequence, which hybridizes to a RNA product of FOXL1 or a nucleic acid sequence which is complementary to an RNA product of a gene of FOXL1 . In one embodiment the hybridization is conducted under at least moderately stringent conditions. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C for 15 minutes, followed by a wash of 2.0 x SSC at 50°C for 15 minutes may be employed.

[0047] The stringency may be selected based on the conditions used in the wash step. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C for 15 minutes. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C for 15 minutes. [0048] By "at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5°C - 16.6 (Log10 [Na+]) + 0.41 (%(G+C) - 600/I), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1 % mismatch may be assumed to result in about a 1 °C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% sequence identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's solution/1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC/0.1 % SDS at 60°C for 15 minutes. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C for 15 minutes. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. , 1989, 6.3.1-6.3.6 and in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2000, Third Edition.

[0049] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , % identity=number of identical overlapping positions/total number of positions.times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. , 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. , for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. In an embodiment, the isolated nucleic acids are useful as primers.

[0050] As used herein "NASBA" refers to a sensitive isothermal transcription-based amplification method used for example for RNA research. NASBA technology is optionally applied to single nucleotide polymorphism (SNP) analysis using human genomic DNA as a template. For example combination of DNA NASBA with multiplex hybridization of specific molecular beacons makes it possible to discriminate the presence of mutations of interest (Berard, C, Cazalis MA, Leissner P, Mougin B., DNA nucleic acid sequence-based amplification-based genotyping for polymorphism analysis. Biotechniques. 2004, 37:680-2, 684, 686).

[0051] The term "isolated polypeptide" as used herein refers to a proteinaceous agent, such as a peptide, polypeptide or protein, which is substantially free of cellular material or culture medium when produced recombinantly, or chemical precursors, or other chemicals, when chemically synthesized. [0052] The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include Fab, Fab', F(ab')₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab')₂ fragments can be generated by treating the antibody with pepsin. The resulting F(ab')₂ fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0053] To produce human monoclonal antibodies, antibody producing cells

(lymphocytes) can be harvested from a human having cancer and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g. the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al. , Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Methods Enzymol, 121 : 140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246: 1275 (1989)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with mutated FOXL1 polypeptide and the monoclonal antibodies can be isolated.

[0054] Specific antibodies, or antibody fragments, reactive against mutated

FOXL1 antigen, may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with cell surface components. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341 :544-546 (1989); Huse et al., Science 246: 1275-1281 (1989); and McCafferty et al., Nature 348:552-554 (1990)).

[0055] The term "amplifying a portion of a FOXL1 coding sequence, the portion comprising minimally FOXL1 residues corresponding to positions 976 to position_990 of SEQ ID NO: 1 " as used herein means amplifying a portion of exon 1 of FOXL1 in genomic DNA or in FOXL1 cDNA, the portion including minimally residues corresponding to positions 976 to 990 of SEQ ID NO: 1 , optionally all of SEQ ID NO: 1. In a subject carrying the mutation, amplifying minimally positions 976 to 990 produces no amplified product. The amplified FOXL1 polynucleotide if larger for example comprising an amplified product corresponding to positions 900 to 999, produces an amplified product of 100 nucleotides in a wild type carrier and 85 nucleotides in a FOXL1 c.976_990het_delGGGATCCCCTTCCTC mutant carrier.

[0056] A "microarray" as used herein refers to an ordered set of probes fixed to a solid surface that permits analysis such as gene analysis of a plurality of genes. A DNA microarray refers to an ordered set of DNA fragments fixed to the solid surface. For example, in one embodiment the microarray is a gene chip. A tissue microarray refers to an ordered set of tissue specimens fixed to a solid surface. For example, in one embodiment the tissue microarray comprises a slide comprising an array of arrayed tumor biopsy samples in paraffin. Tissue microarray technology optionally allows multiple specimens, such as biopsy samples, to be analyzed in a single analysis at the DNA, RNA or protein level. Tissue microarrays are analyzed by a number of techniques including immunohistochemistry, in situ hybridization, in situ PCR, RNA or DNA expression analysis and and/or morphological and clinical characterization or a combination of techniques. The specimens are optionally from the same subject or from a plurality of subjects. Methods of detecting gene expression using arrays are well known in the art. Such methods are optionally automated.

[0057] The term "subject" as used herein includes all members of the animal kingdom including multicellular organisms, including mammals, and preferably means humans.

[0058] The term "suitable sample" as used herein means a sample that can be assayed for FOXL1 protein and/or polynucleotide sequence. Suitable samples for detecting the FOXLI deletion are blood, saliva, throat swab and any DNA accessible tissue.

[0059] As used herein, "a relative" or "blood relation" is a relative genetically related, or related by birth, and includes without limitation 1 st, 2nd, 3rd , 4th, 5th, 6th , 7th, 8th, 9th and 10th degree relations, for example but not limited to parents, children, grandchildren, grandparents, cousins and/or 2nd cousins related by blood.

[0060] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies

[0061] In understanding the scope of the present disclosure, the term "consisting" and its derivatives, as used herein, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.

[0062] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0063] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

II. Methods

[0064] A novel 15bp deletion, c.976_990het_delGGGATCCCCTTCCTC, in FOXL1 deleted in subjects with otosclerosis and/or hearing loss is disclosed herein. The deletion causes an inframe deletion of 5 amino acids (p.Gly326_Leu330).

[0065] Accordingly, an aspect includes a method for identifying a human subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss, the method comprising (a) obtaining a suitable sample from the subject; (b) assaying the sample for the presence or absence of a mutation in: i) a FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues in FOXL1 polypeptide C-terminus; ii) a FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; iii) a FOXL1 polypeptide at one or more amino acid residues in FOXL1 polypeptide C-terminus; or iv) a FOXL1 polypeptide at one or more amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; and (c) identifying the subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss if said mutation is detected.

[0066] Another aspect includes a method for identifying a human subject as having or having an increased likelihood to develop a otosclerosis and/or hearing loss, the method comprising (a) obtaining a suitable sample from the subject; (b) assaying the sample for the presence or absence of a mutation in: i) a FOXL1 polynucleotide encoding an amino acid corresponding to position 326 to position 330 of SEQ ID NO:3 or ii) a FOXL1 polypeptide encoded by said polynucleotide; and (c) identifying the subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss if said mutation is detected.

[0067] A further aspect includes a method of determining if a human subject is a carrier of a mutation that increases the likelihood of developing otosclerosis and/or hearing loss, the method comprising (a) obtaining a suitable sample from the subject; (b) assaying the sample for the presence or absence of a mutation in: i) a FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues in FOXL1 polypeptide C- terminus; ii) a FOXL 1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; iii) a FOXL1 polypeptide at one or more amino acid residues in FOXL1 polypeptide C-terminus; or iv) a FOXL 1 polypeptide at one or more amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3; and (c) identifying the subject as a carrier of a mutation that increased the likelihood of developing otosclerosis and/or hearing loss if said mutation is detected.

[0068] In another aspect the method comprises: (a) obtaining a suitable sample from the subject; (b) assaying the sample for the level of a FOXL1 polypeptide wherein an increased level of is indicative the subject is a carrier of a mutation and/or has or has an increased likelihood of developing otosclerosis and/or hearing loss.

[0069] In an embodiment, the presence or absence of a mutation in a FOXL1 polynucleotide is assessed by detecting deletion of the sequence in SEQ ID NO: 5. For example SEQ ID NO: 1 comprises the 15 nucleotide sequence in SEQ ID NO: 5 whereas SEQ ID NO:2 is deleted for this sequence. Detecting a FOXL1 polynucleotide sequence deleted for SEQ ID NO: 5 is indicative that the subject has or has an increased likelihood to develop otosclerosis and/or hearing loss.

[0070] Deafness research has demonstrated that there are many examples where a single deafness gene can cause several, distinct forms of hearing loss. For example, a particular gene mutation can cause both syndromic and non-syndromic forms of hearing loss; both dominant and recessive patterns of inheritance in families; mild and severe forms; early and late onset; various patterns as detected by audiogram. The detected deletion mutation occurs in a highly conserved region of the gene and at the c-terminus of the FOXL1 transcription factor. The c-terminus of transcription factors is typically involved in binding to genomic targets (other genes) in order to regulate downstream effects.

[0071] The suitable sample can be any sample comprising DNA containing cells, such as a saliva sample, blood sample or DNA accessible tissue sample. In an embodiment, said sample is a blood sample or fraction thereof. For example the blood can be any fraction such as a plasma fraction or a serum fraction. In an embodiment, the sample comprises leukocytes. In an embodiment, the sample comprises DNA and/or is a DNA sample. For protein and RNA transcript related applications, the sample comprises and/or is derived from cells that express FOXL1 , for example bone cells and/or gastric tissue cells. In yet another embodiment, the sample comprises protein or is a protein fraction.

[0072] An aspect includes a method for detecting the presence of a mutation identified as c.976_990het_delGGGATCCCCTTCCTC in FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues corresponding to positions 326 to 330 of SEQ ID NO:3, in a suitable sample, the method comprising: a) contacting the sample with a pair of primers, wherein the oligonucleotide primers hybridize under stringent conditions with a region flanking the mutation, under conditions permitting hybridization of the primers with the DNA contained in the sample; b) amplifying the region of interest of the FOXL1 gene or transcript; c) detecting the mutation in the amplification products.

[0073] In another aspect, the method comprises a method for detecting the presence or absence of a mutation identified as c.976_990het_delGGGATCCCCTTCCTC in FOXL1 polynucleotide at a nucleotide sequence encoding one or more of amino acid residues corresponding to positions 326 to 330 of SEQ ID NO: 3, in a suitable sample, the method comprising: a) contacting the sample with a probe, wherein the probe hybridizes with a FOXL1 gene or transcript comprising a c.976_990het_delGGGATCCCCTTCCTC deletion mutation, and does not hybridize with a wild-type FOXL1 probe, under conditions permitting hybridization of the probe with the DNA contained in the sample; and b) detecting the presence or absence of hybrids formed between the probe and the DNA contained in the sample, thereby indicating the presence or absence, respectively, of the mutation. [0074] Either a FOXL1 polynucleotide or FOXL1 polypeptide can be assayed for said mutation. Methods are known for obtaining a sample from a subject that can be assayed for a mutated FOXL1 polynucleotide (e.g. DNA or RNA) or a mutated FOXL1 polypeptide (e.g. protein fraction). In an embodiment the subject is pre-symptomatic. In another embodiment, the subject exhibits or is suspected of having hearing loss. In another embodiment, the subject has one or more blood relative carriers of the c.976_990het_delGGGATCCCCTTCCTC: p.Gly326_Leu330del mutation.

[0075] In an embodiment, said sample or DNA or RNA isolated therefrom or DNA amplified therefrom, is assayed for the presence or absence of said mutation in said FOXL1 polynucleotide.

[0076] In an embodiment, the presence or absence of said mutation can be determined by assessing in the FOXL1 polynucleotide comprises a mutation in one or more of the codon that encodes glycine 326 to the codon that encodes leucine 330 of SEQ ID NO: 3. The nucleotides that encode G326, I327, P328, F329 and L330 are nucleotides 976- 990of SEQ ID NO: 1 (e.g. GGGATCCCCTTCCTC SEQ ID NO:5).

[0077] Said mutation in said polynucleotide can correspond to deletion of one or more nucleotides that encodes glycine, isoleucine, proline, phenylalanine and/or leucine at position 326,327,328,329 and/or 330 of SEQ ID NO:3. For example, as shown here, the mutation can be deletion of the nucleotides corresponding to positions 976 to position 990 of SEQ ID NO: 1.

[0078] In an embodiment, said mutation in said FOXL1 polynucleotide comprises mutation of the nucleotides corresponding to position 976 to position 990 of SEQ ID NO: 1 (e.g. mutation of nucleotides in SEQ ID NO:5).

[0079] In an embodiment, said mutation in said FOXI1 polynucleotide is

976_990het_delGGGATCCCCTTCCTC. The wildtype sequence of FOXL1 transcript is provided is SEQ ID NO: 1 and comprises three guanine (G) at position 976, 977, and 978, adenine at position 979, thymine (T) at position 980, four cytosine ( C) at position 981 - 984, two thymine (T) at position (985-986), two cytosine ( C) at position (987-988), thymine (T) at position 989 and thymine (T) at position 990. The corresponding polypeptide sequence is provided in SEQ ID NO: 3 and comprises glycine, isoleucine, proline, phenylalanine and leucine at position 326,327,328,329 and 330. The mutated polynucleotide sequence FOXL1 which is deleted for GGGATCCCCTTCCTC (SEQ ID NO:5) corresponding to position 976 to position 990 as found in SEQ ID NO: 1 , is provided in SEQ ID NO:2. Deletion of the GGGATCCCCTTCCTC (SEQ ID NO:5) at position 976 to position 990 causes an in frame deletion which is reflected in the mutant polypeptide sequence provided in SEQ ID NO:4. SEQ ID NO: 3 comprises a glycine, isoleucine, proline, phenylalanine and leucine at position 326,327,328,329 and 330 consecutively and is deleted in SEQ ID NO: 4 in comparison to the wildtype sequence in SEQ ID NO:3.

[0080] Methods for detecting a mutation in a polynucleotide or nucleic acid alteration within a sample, include genotyping, microarrays, Restriction Fragment Length Polymorphism, Southern Blots, single-strand conformation polymorphism (SSCP), dHPLC, single nucleotide primer extension, allele-specific hybridization, allele-specific primer extension, oligonucleotide ligation assay, and invasive signal amplification, Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and Fluorescence polarization (FP). Such methods optionally employ the isolated nucleic acid primers, probes compounds and compositions of the disclosure described herein.

[0081] Some methods employ polymerase chain reaction (PCR), real time PCR, multiplex ligation dependent probe amplification (MLPA), nucleic acid sequence based amplification (NASBA) and/or real time NASBA.

[0082] As an example, one assay strategy comprises gene amplification analysis such as polymerase chain reaction (PCR) analysis, optionally followed by sequencing, and comparing the amplification profile or identified sequence to a wild-type amplification profile or wild-type FOXL1 sequence as found for example in SEQ ID NO: 1 . Methods of sequencing are well known in the art. In an embodiment, one or more FOXI1 exons are amplified by PCR, and analyzed for gene mutations, for example by SSCP.

[0083] In an embodiment, DNA or RNA is isolated from and/or DNA is amplified said sample and the DNA or RNA is assayed for the presence or absence of said mutation.

[0084] In an embodiment, the mutation in FOXL1 polynucleotide is detected by a PCR based method and/or a hybridization based method.

[0085] In an embodiment, said DNA is genomic DNA.

[0086] In another embodiment, assaying said sample for the presence or absence of the mutation in FOXL1 corresponding to positions 976-990 comprises: a) amplifying a portion of a FOXL1 coding sequence, the portion spanning minimally FOXL1 residues corresponding to position 976 to position 990 of SEQ ID NO: 1 , to produce amplified FOXL1 polynucleotide, b) sequencing the amplified FOXL1 polynucleotide and determining if said mutation is present. [0087] In an embodiment, the portion comprises at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 1 10 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 1800 nucleotides, at least 200 nucleotides, at least 220 nucleotides, at least 240 nucleotides, at least 260 nucleotides, at least 280 nucleotides, at least 300 nucleotides, or at least 320 nucleotides.

[0088] In another embodiment, mRNA is isolated from said sample, for example a bone cell sample or gastric cell sample, and the mRNA is assayed directly for the presence or absence of said mutation and/or cDNA is prepared using said mRNA and the cDNA is assayed for the presence or absence of said mutation.

[0089] In an embodiment, the assay comprises: a) isolating mRNA from said sample; b) deriving corresponding DNA (cDNA) from said isolated mRNA; c) amplifying all or a portion of FOXL1 coding sequence, the portion spanning minimally FOXL1 nucleotide residues corresponding to position 976 to position 990 of SEQ ID NO: 1 to produce amplified FOXL1 polynucleotide, d) sequencing the amplified FOXL1 polynucleotide; e) determining if said mutation is present; and f) identifying the subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss if said mutation is detected.

[0090] The amplification step can comprise use of one or more primers described herein. For example, FOXL1 specific primers flanking said FOXL1 mutation are selected, for example primers which amplify all or part of FOXL1 coding sequence either in genomic sequence and/or corresponding transcripts, are used to amplify the gene region comprising said FOXL1 mutation.

[0091] The amplified portion can comprise for example a 490 bp fragment as described below. The amplified portion can be about or at least 50 nucleotides, about or at least 100 nucleotides, about or at least 150 nucleotides, about or at least 200 nucleotides, about or at least 250 nucleotides, about or at least 300 nucleotides, about or at least 350 nucleotides, about or at least 400 nucleotides, or about or at least 450 nucleotides or any whole number between for example 15 nucleotides and the full length sequence.

[0092] Instead of sequencing the amplified FOXL1 polynucleotide, in certain embodiments, the mutation is detected using a probe that hybridizes to the mutated FOXL1 polynucleotide. In an embodiment, the probe comprises a probe described herein. For example, a probe specific for the mutant FOXL1 polynucleotide can comprise for example residues complementary to 5' and 3' sequence flanking the deletion sequence, for example comprising at least 8 contiguous nucleotides of SEQ ID NO: 2 , the at least 8 contiguous nucleotides including residues corresponding to positions 975 and 976. For example the probe specific for detecting mutant FOXL1 can comprise residues 972, 973, 974, 975, 976, 977, 978 and 979 of SEQ ID NO:2.

[0093] The probe can also be specific for the wildtype FOXL1 sequence. A probe specific for the wildtype FOXL1 comprises for example all or part of the sequence of SEQ ID NO: 5. In an embodiment, detecting mutatnt FOXL1 comprises using a wildtype FOXL1 probe, wherein hybridization identifies the subject as not having or not having an increased likelihood of developing otosclerosis and/or hearing loss and lack of hybridization identifies the subject as having or having an increased likelihood of developing otosclerosis.

[0094] In an embodiment, the method comprises assaying for the presence or absence of mutant FOXL1 and assaying for the presence or absence of wildtype FOXL1.

[0095] In an embodiment, assaying the sample for the presence or absence of a mutation in a FOXL1 polynucleotide encoding an amino acid in FOXL1 polypeptide C- terminus comprises:

1. a) contacting the sample with a pair of primers, wherein the oligonucleotide primers hybridize under stringent conditions with a region flanking the C-terminus, optionally wherein an upstream primer binds upstream of the DNA binding forkhead domain and a downstream primer binds in the 3' UTR, under conditions permitting hybridization of the primers with the DNA contained in the sample; b) amplifying the region of interest of the FOXL1 gene or transcript; c) detecting the mutation in the amplification products; or

2. a) contacting the sample with a probe, wherein the probe hybridizes with a FOXL1 gene or transcript comprising a C-terminus deletion mutation, and does not hybridize with a wild-type FOXL1 probe, under conditions permitting hybridization of the probe with the DNA or RNA contained in the sample; and b) detecting the presence or absence of hybrids formed between the probe and the DNA or RNA contained in the sample, thereby indicating the presence or absence, respectively, of the mutation.

[0096] In an embodiment, the C-terminus end is the 70, 60, 50, 40 or 30 C-terminal coding amino acids and/or the corresponding nucleotides encoding said coding region.

[0097] FOXL1 polypeptide can also be assayed for said mutation. A sample can also be assayed for the level of FOXL1 polypeptide. As demonstrated herein, the level of FOXL1 protein is increased. In an embodiment, the mRNA level is not increased. [0098] Accordingly in an embodiment, a sample from the subject, for example a bone sample, and/or a protein fraction derived therefrom, is assayed for the presence or absence of said mutation in said FOXL1 polypeptide and/or the level of FOXL1 polypeptide.

[0099] In an embodiment, assaying the sample for the presence or absence of a mutation in a FOXL1 polynucleotide encoding an amino acid corresponding to position 326 to position 330 of SEQ ID NO:3 comprises:

1. a) contacting the sample with a pair of primers, wherein the oligonucleotide primers hybridize under stringent conditions with a region flanking the mutation, under conditions permitting hybridization of the primers with the DNA contained in the sample; b) amplifying the region of interest of the FOXL1 gene or transcript; c) detecting the mutation in the amplification products; or

2. a) contacting the sample with a probe, wherein the probe hybridizes with a FOXL1 gene or transcript comprising a c.976_990het_delGGGATCCCCTTCCTC deletion mutation, and does not hybridize with a wild-type FOXL1 probe, under conditions permitting hybridization of the probe with the DNA or RNA contained in the sample; and b) detecting the presence or absence of hybrids formed between the probe and the DNA or RNA contained in the sample, thereby indicating the presence or absence, respectively, of the mutation.

[00100] In an embodiment, the method comprises assaying for a level of FOXL1 expression, optionally mRNA expression or protein expression, wherein an increased level of FOXL1 expression compared to a control is indicative that the subject is a carrier of a mutation and/or has or has an increased likelihood of developing otosclerosis and/or hearing loss.

[00101] In another embodiment said mutation in said FOXL1 polypeptide is p.Gly326_Leu330del.

[00102] Methods that can be used to assess if mutated FOXL1 is in the sample. As GGGATCCCCTTCCTC corresponding to position 976 to position 990 in SEQ ID NO: 1 results in an encoded polypeptide that is missing five amino acids, analysis of FOXL1 polypeptide size can indicate whether the subject has a mutated FOXL1 polypeptide. For example, an antibody that binds specifically to the sequence in SEQ ID NO: 6 detects wild type FOXL1 , and an antibody that is specific for an epitope comprising contiguous amino acids 325-327 of SEQ ID NO: 4 is specific for the mutant FOXL1. [00103] In an embodiment, assaying the sample for the presence or absence of mutated FOXL1 , e.g., assaying the sample for the presence or absence a mutation in a FOXL1 polypeptide at position 326 to position 330 of SEQ ID NO:3 comprises contacting the sample obtained from the subject with an antibody having specific binding affinity for FOXL1 deleted for amino acids 326, 327, 328, 329 and/or 330, thereby forming a complex of the antibody and FOXL1 deleted for amino acids 326, 327, 328, 329 and/or 330.

[00104] In an embodiment, the method comprises assaying the sample for the level of FOXL1 compared to a control, wherein an increased protein level compared to a control is indicative the subject is a carrier of a mutation and/or has or has an increased likelihood of developing otosclerosis and/or hearing loss.

[00105] In an embodiment, the antibody is conjugated to and/or comprises a heterologous moiety. In an embodiment, the antibody is conjugated to and/or comprises a detectable label. In another embodiment, the detectable signal is detectable indirectly, for example, using a secondary antibody. In an embodiment, the secondary antibody is conjugated to and/or comprises a detectable label.

[00106] The detectable label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³l, ¹²⁵l, ¹³¹1; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzymatic label, such as alkaline phosphatase, beta-galactosidase or peroxidase such as horseradish peroxidase; an imaging agent; or a metal ion. In an embodiment, the detectable label is a hapten label such as dintrophenol, biotin or digoxigenin.

[00107] In an embodiment, the antibody contacted with the sample can be complexed to a solid support. In an embodiment the antibody FOXL1 complex is coupled to a solid support prior to separating.

[00108] In an embodiment, the method comprises separating the complex formed from antibody not complexed of the mutated FOXL1 ; detecting or quantifying a signal from the detectable label of the antibody, optionally the signal being proportional to an amount of mutated FOXL1.

[00109] Detecting a signal proportional to the amount of mutated FOXL1 identifies the subject as having or having an increased likelihood to develop otosclerosis and/or hearing loss. [00110] In an embodiment, the method comprises detecting wildtype FOXL1 polypeptide, wherein the detection of wildtype polypeptide identifies the subject has not having an increased likelihood to develop otosclerosis and/or hearing loss (e.g. mutated FOXL1 related otosclerosis and/or hearing loss.

[00111] Detection of mutant FOXL1 is achieved in antibody based binding methods and/or hybridization based methods when for example the signal measured is at least 2X, at least 3X at least 4X assay background levels (e.g. in an assay control where binding is not expected). A positive control (e.g. assay test known to comprise mutant FOXL1 polypeptide, wildtype FOXL1 polypeptide, mutant FOXL1 polynucleotide or wildtype FOXL1 polynucleotide, assayed with a mutant detecting probe or antibody (e.g. for mutant molecules) or assayed with a wildtype detecting probe or antibody (e.g. for wildtype molecules).

[00112] In an embodiment, the detectable label comprises a peroxidase conjugate or digoxigenin.

[00113] In an embodiment, the antibody is coupled to a solid support and/or conjugated to a component configured for coupling to a solid support.

[00114] In an embodiment, the assaying comprises contacting the sample obtained from the subject with a capture antibody having specific binding affinity for mutated FOXL1 , thereby forming a complex of the capture antibody and the mutated FOXL1 , the capture antibody being coupled to a solid support or comprising a component configured for coupling the capture antibody to a solid support; contacting the sample with a detection antibody having specific binding affinity for mutated FOXL1 , thereby forming a complex of the capture antibody, mutated FOXL1 , and the detection antibody, the detection antibody having a detectable label; contacting the complex of the capture antibody, mutated FOXLI and the detection antibody with a solid support, whereby the complex of the capture antibody, mutated and the detection antibody couples to the solid support; separating the complex of the capture antibody, mutated FOXL1 and the detection antibody coupled to the solid support from detection antibody, capture antibody and mutated FOXL1 not coupled to the solid support; exposing the complex of the capture antibody, mutated FOXL1 and the detection antibody to a substrate, thereby producing a signal from the detectable label of the detection antibody comprising the complex of the capture antibody, FOXL1 and the detection antibody which couples to the solid support in said step of contacting; detecting or quantifying the signal produced in said step of exposing the complex of the capture antibody, FOXL1 and the detection antibody, the signal being proportional to an amount of FOXL1 in the sample from the subject.

[00115] In an embodiment, the second epitope is different than the first epitope. In an embodiment, the step of contacting the sample with the capture antibody and the step of contacting the sample with the detection antibody occur at substantially the same time.

[00116] In an embodiment, the solid support is a mircrowell.

[00117] Further, in some embodiments, one or more mutations in FOXL1 in addition to a mutation in p.Gly326_Leu330 (and/or the corresponding GGGATCCCCTTCCTC deletion) are assessed.

[00118] The control is for example a suitable sample from a subject without otosclerosis and/or hearing loss.

[00119] In an embodiment, the assay is conducted as part of a multiplex assay. In an embodiment, the method further comprises audiological testing, optionally measuring conductive and/or sensorineural hearing, and/or surgical diagnosis. In an embodiment, a subject identified with a FOXL1 mutation and decreased conductive and/or sensorineural hearing is identified as having otosclerosis.

[00120] Surgery and hearing aids are the typical treatments for otosclerosis. Otosclerosis is heterogeneous and can involve both conductive and sensorineural components. As hearing loss progresses, and particularly with significant sensorineural components, hearing aids may be less effective and in these cases cochlear implantation requiring surgery may be necessary.

[00121] A further aspect includes a method of treating a subject, the method comprising determining if the subject is a carrier of c.976_990het_delGGGATCCCCTTCCTC: p.Gly326_Leu330del for example in FOXL1 polynucleotide and/or polypeptide, for example a mutation corresponding to positions 326 to position 330 of SEQ ID NO: 3 optionally p.Gly326_Leu330 del FOXL1 as described herein; and providing subjects determined to be carriers of the mutation, optionally p.Gly326_Leu330 del FOXL1 , with a hearing loss therapy.

[00122] In an embodiment, the hearing loss therapy comprises providing the subject with a suitable hearing aid, FM hearing system or cochlear implant.

III. Products and Kits [00123] Also provided isolated nucleic acids, isolated polypeptides, nucleic acid primers and probes, antibodies, compounds, compositions as well as kits comprising said products which can for example be used in a methods described herein.

[00124] Accordingly in another aspect is provided an isolated polynucleotide encoding at least 5 and a maximum 50, 60, 70 or 80 contiguous amino acids of SEQ ID NO:3, 4 or 6, the at least 5 contiguous amino acids comprising the amino acid residues of SEQ ID NO:6 and/or excluding the amino acid residues in SEQ ID NO: 6. In an embodiment, the polynucleotide comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides of SEQ ID NO: 5, 1 or 2. In an embodiment, the polynucleotide comprises the nucleotides of SEQ ID NO: 5, 1 or 2.

[00125] A further aspect includes an isolated polypeptide comprising at least 5 and a maximum 50, 60, 70 or 80 contiguous amino acids of SEQ ID NO:3, 4 or 6, the at least 5 contiguous amino acids comprising the amino acid corresponding to position 326-330 of SEQ ID NO:3 or comprising the amino acids of SEQ ID NO:6. and/or excluding the amino acid residues in SEQ ID NO: 6. In an embodiment, the polypeptide comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at lest 50 amino acids of SEQ ID NO: 3, 4 or 6.

[00126] In an embodiment, the isolated polynucleotide encodes and/or the isolated polypeptide comprises at least 5, at least 10, at least 12, at least 15 or at least 20 amino acids.

[00127] A further aspect includes a compound comprising a polynucleotide encoding at least 5 and a maximum 50, 60, 70 or 80 contiguous amino acids of SEQ ID NO:3, 4 or 5, the at least 5 contiguous amino acids comprising the amino acid corresponding to residue 326-330 of SEQ ID NO: 3and/or excluding the nucleotides encoding the amino acids in SEQ ID NO: 6. In an embodiment, the compound comprises a polynucleotide comprising at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides of SEQ ID NO: 5, 1 or 2. In an embodiment, the polynucleotide comprises the nucleotides of SEQ ID NO: 5, 1 or 2.

[00128] In an embodiment, the FOXL1 polynucleotide encoding the at least 5 and a maximum of 50, 60 , 70 or 80 contiguous amino acids comprises least 15 nucleotides of SEQ ID NO: 1 , 2 or 5, optionally said at least 15 nucleotides comprising the nucleotide corresponding to position 976 to 990 of SEQ ID NO:1. GGGATCCCCTTCCTC. [00129] Yet another aspect includes a compound comprising a polypeptide comprising at least 5 and a maximum 50, 60, 70 or 80 contiguous amino acids of SEQ ID NO: 3, 4 or 6, optionally the at least 5 contiguous amino acids comprising the amino acid corresponding to position 326 to position 330 of SEQ ID NO:3 .

[00130] In an embodiment, the polynucleotide encodes and/or the polypeptide comprises at least 5, at least 10 at least 12, at least 15 or at least 20 amino acids.

[00131] In a further embodiment, the isolated polynucleotide or the isolated polypeptide is conjugated to and/or the compound comprises a heterologous moiety. In an embodiment, the isolated polynucleotide or the isolated polypeptide is conjugated and/or the compound comprises a detectable label.

[00132] In an embodiment, the compound comprises a detectable label. For example, the label can be conjugated to a polynucleotide, primer, probe, polypeptide, antibody, or compound described herein.

[00133] Also provided in another aspect is a nucleic acid primer for diagnosing otosclerosis and/or hearing loss which hybridizes under stringent conditions to a portion of the nucleic acid sequence of SEQ ID NO: 1 or complement thereof, wherein the primer amplifies all or a portion of the coding sequence of FOXL1 such that one or more nucleotides encoding an amino acid corresponding to position 326 to position 330 in SEQ ID NO: 3 is amplified.

[00134] The amplified portion can comprise for example a 490 bp fragment as described below. The amplified portion can be about or at least 50 nucleotides, about or at least 100 nucleotides, about or at least 150 nucleotides, about or at least 200 nucleotides, about or at least 250 nucleotides, about or at least 300 nucleotides, about or at least 350 nucleotides, about or at least 400 nucleotides, or about or at least 450 nucleotides or any whole number between for example 15 nucleotides and the full length sequence.

[00135] In an embodiment, the primer is at least 12 nucleotides in length, for example about 15 to 30 nucleotides, or any whole number in between.

[00136] In an embodiment, the primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 7 or 8. In another embodiment, the primer consists of at least 12 consecutive nucleotides of SEQ ID NO: 7 or 8.

[00137] In an embodiment, the primer is conjugated to and/or comprises a heterologous moiety, optionally a detectable label. [00138] A further aspect includes a nucleic acid probe for diagnosing otosclerosis hearing disorder which hybridizes under stringent conditions to a portion of the complement of SEQ ID NO: 1 or 2 comprising residues encoding amino acid corresponding to position 326 to position 330 of SEQ ID NO: 3 or a nucleic acid sequence that differs from the SEQ ID NO: 1 at one or more nucleotides encoding arginine at residue 326 to position 330 of SEQ ID NO:3, and which detects the presence or absence of a substitution or lesion at one or more nucleotides that encode the Glycine, Isoleucine, Proline, phenylalanine and Leucine at position 326,327,328,329 and 330 consequence of SEQ ID NO:3.

[00139] In an embodiment, the probe detects a substitution or lesion at a nucleotide corresponding to position 976 to position 990 of SEQ ID NO: 1.

[00140] In a further embodiment, the probe is at least 12 nucleotides in length. In an embodiment, the probe is a maximum of 50, 100 or 150 nucleotides in length. A probe for detecting the mutant FOXL1 polynucleotide can comprise none of the deleted sequence. A wildtype probe for example comprising at least 12 nucleotides of SEQ ID NO: 5, can be used to detect the wildtype sequence. Accordingly another aspect includes a method using both a probe specific for the deletion mutant and a probe specific for the wild type allele.

[00141] In yet a further embodiment, the probe comprises at least 12 consecutive nucleotides of SEQ ID NO:1 or 2.

[00142] In another embodiment, the probe hybridizes to or hybrids to the complement of at least 50, at least 100 or at least 150 consecutive nucleotides of SEQ ID NO: 1 or 2.

[00143] In yet another embodiment, the probe comprises and/or is conjugated to a detectable label.

[00144] In an embodiment, the isolated polynucleotide, compound, nucleic acid primer, nucleic acid probe, is comprised in a composition with a suitable diluent or carrier.

[00145] In an embodiment the diluent is a saline such as PBS, water, such as sterile water, or hybridization solution.

[00146] A further aspect is an immunogen comprising SEQ ID NO: 6. A further aspect is an immunogen comprising contiguous amino acid residues 326-327 or 325-328 of SEQ ID NO: 4. The immunogen can comprise a tag such as KLH to improve immunogenicity, can be administered for example with an adjuvant. An antibody specific for SEQ ID NO: 6 detect wild-type FOXL1. An antibody specific for amino acid residues 326-327 or 325-328 of SEQ ID NO: 4 would be predicted to recognize mutant FOXL1. [00147] Another aspect is an isolated antibody specific for SEQ ID NO:6 or specific for an epitope comprising amino acid residues 326-327 or 325-328 of SEQ ID NO: 4. The antibody specific for said epitope comprising amino acid residues 326-327 or 325-328 binds said epitope with at least 2 fold, 3 fold or 4 fold specificity over SEQ ID NO:6 and/or 3.

[00148] A further aspect includes a kit for diagnosing a subject at risk for a otosclerosis hearing loss, comprising: at least two nucleic acid primers which hybridize under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1 or complement thereof, wherein the primers amplify all or a portion of SEQ ID NO: 1 or 2, such that minimally one or more nucleotides encoding an amino acid residue corresponding to position 326 to position 330 of SEQ ID NO:3 is amplified; and instructions for diagnosing a otosclerosis hearing loss by detecting a substitution or deletion of one or more nucleotides encoding amino acid residues at 326 to position 330.

[00149] In an embodiment, the kit comprises a primer that is described herein.

[001 SO] In an embodiment, the kit further comprises a nucleic acid probe which hybridizes under stringent conditions to the complement of SEQ ID NO: 2 at one or more nucleotides encoding Glycine, Isoleucine,, Proline, phenylalanine and Leucine at residue 326 to position 330 of SEQ ID NO:3 and which detects the presence or absence of a substitution or lesion at one or more nucleotides that encode the Glycine, Isoleucine,, Proline, phenylalanine and Leucine at residues 326 to position 330 of SEQ ID NO:3.

[00151] In an embodiment, the kit comprises a probe that is described herein.

[00152] In an embodiment, the kit comprises more than one probe.

[00153] In a further embodiment, one or more of the probes is conjugated to and/or comprises a heterologous moiety optionally a detectable label. In an embodiment, one or more of the probes is a labeled probe.

[00154] The kit can also comprise in an embodiment, one or more primers or probes for detecting additional mutations in FOXL1 ,

[00155] In an embodiment, the kit comprises a probe that comprises at least 12 nucleotides in length.

[00156] In yet another embodiment, the kit comprises a probe that comprises at least 12 consecutive nucleotides of SEQ ID NO:1 or 2.

[00157] In a further embodiment, the kit comprises a probe that comprises at least 50 consecutive nucleotides of SEQ ID NO: 1 or 2. [00158] In an embodiment, the kit comprises a primer that is at least 12 nucleotides in length.

[00159] In yet another embodiment, the kit comprises a primer that comprises at least 12 consecutive nucleotides of SEQ ID NO: 7 or 8. In an embodiment, the kit comprises a primer wherein the primer is labeled.

[00160] In an embodiment, the kit comprises one or more of 10X Buffer, dNTPs, MgCI₂ and a DNA polymerase such as Taq polymerase.

[00161] In certain embodiments, the kit is a diagnostic kit for medical use. In other embodiments, the kit is a diagnostic kit for laboratory use.

[00162] In another aspect the disclosure provides a commercial package comprising an isolated nucleic acid or composition described herein and instructions for use.

[00163] In an embodiment, the kit comprises a microarray, for example a DNA microarray such as a gene chip, or a tissue array.

[00164] For example, in an embodiment the tissue microarray comprises a slide comprising an array of arrayed biopsy samples in paraffin. Tissue microarray technology optionally allows multiple specimens, such as biopsy samples, to be analyzed in a single analysis at the DNA, RNA or protein level. Tissue microarrays are analyzed by a number of techniques including immunohistochemistry, in situ hybridization, in situ PCR, RNA or DNA expression analysis and and/or morphological and clinical characterization or a combination of techniques. The specimens are optionally from the same subject or from a plurality of subjects. Methods of detecting gene expression using arrays are well known in the art. Such methods are optionally automated. In an embodiment, the sample from the subject is analyzed using a tissue microarray.

[00165] In an embodiment the polypeptide, optionally the isolated polypeptide, polynucleotide optionally isolated polynucleotide, primer, probe, antibody or antibody fragment is conjugated to and/or comprises a detectable label.

[00166] The detectable label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³l, ¹²⁵l, ¹³¹1; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. [00167] In another embodiment, the detectable signal is detectable indirectly, for example, using a secondary antibody.

[00168] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[00169] The following non-limiting examples are illustrative of the present disclosure:

Examples

Example 1

[00170] Otosclerosis hearing loss is a common form of hearing loss contributing to 0.3-1 % of all cases worldwide. We identified a multiplex family from Newfoundland (Family 2081 ) segregating otosclerosis inherited as an autosomal dominant trait. Haplotyping of the 9Mb region at chr16q23.1-q24.2 and sequencing positional candidate genes revealed a 15 bp deletion (c.976_990het_delGGGATCCCCTTCCTC) in the FOXL1 gene which encodes a Forkhead protein. This novel deletion causes an in frame deletion of five amino acids: glycine, isoleucine, proline, phenylalanine and leucine (p.Gly326_Leu330). To investigate the role of FOXL1 in other patients, FOXL1 was sequenced in 17 probands from NL with otosclerosis but did not identify a mutation in FOXL1. Further screening of otosclerosis families for this mutation will help in identifying other families with the mutation.

Example 2

[00171] A multiplex, extended pedigree (Family 2081 ) segregating non-syndromic otosclerosis hearing loss from the founder population of Newfoundland, Canada (Figure 1 ) was identified. Family 2081 is a multiplex family spanning 4 generations and includes 36 available family members. Otosclerosis in this family appears to segregate in an autosomal dominant pattern, both parents of the proband have a medical history of hearing loss (subject I-3 and I-4 ) (Figure 1 ). The diagnosis of otosclerosis in this family is based on; the surgical report of otosclerosis in seven affected member (subjects II-4, II-5, II-8, 11- 1 , 11-13, 111-1 ) and the audiological profile of conductive hearing loss with absence of stapedial reflex and low compliance of the tympanic membrane in subject III-5. The hearing loss in the all affected family members starts around the age of 12 years as conductive hearing loss in six out of seven affected (subjects II— 4, II-8, 11- 1 , 11-13, 111-1 , III-5) and as a pure SNHL in the seventh member (11-5) (Figure 2). Genomic DNA was isolated from peripheral leukocytes of 7 affecteds (11-4, 11-5, 11-8, 11-12, 11-13, 111-1 , III-5) and also from four relatives with no history of hearing loss (II-6, ll-7. lll-2,lll-6, 111-10 and 111-12) and three relatives with hearing loss uncategorized (11-9, 11-14, 11-15) as previously described (Miller, et al. , 1988). Ethics approval was obtained (Human Investigation Committee #01.186). Genotyping the 9Mb region at chr16q23.1 -q24.2 was performed on all available DNA samples spanning two generations. Six microsatellite markers were amplified and run on an ABI 3130x1 or 3730 DNA Analyzer and analyzed using GeneMapper v4.0 (Applied Biosystems). Haplotype analysis revealed that all affected members (11-4, 11-5, 11-8, 11-12, 11-13, 111-1 , III-5) inherited the green (presumably disease) haplotype (Figure 3). This 9Mb contains 79 positional candidate genes. Thirteen positional candidate genes (Table 1 ) were sequenced based on a biological role in bone remodeling. All sequencing variants were tested for co-segregation with the otosclerosis phenotype and a 15 bp deletion in FOXL1 (c.976_990het_delGGGATCCCCTTCCTC) (SEQ ID NO: 5) was shown to segregate with the disease haplotype. This in frame deletion in the coding region of FOXL1 is absent in 1 16 chromosomes tested from NL population controls.

[00172] In order to evaluate the effect of FOXL1 deletion on the mRNA expression, RNA was extracted from lymphoblastoid cell line of 4 deletion carriers (II-4, II-5, II-8 and II- 12) and compared to controls (11-1 and TA09). No amplification from the lymphoblastoid cell line RNA extract using RT-PCR analysis was detected, which was further confirmed by real time PCR. These data suggested that FOXL1 is normally not expressed in lymphocyte (Figure 7).

[00173] To determine the effect of the FOXL1 deletion on the mRNA level, HEK293A cells were transiently transfected with FOXL1 (both wild and-mutant forms). RNAs were isolated and the level of FOXL1 expression compared between the transfectants by RT- PCR. No significant difference in the level of FOXL1 expression between HEK-FOXL1 wild type and HEK-FOXL1-mutant was detected (Figure 8).

[00174] The localization and expression of FOXL1 at the protein level were next examined in HEK293-FOXL1-wildtype and HEK293 FOXL1 -mutant cells to explore the possibility of protein degradation as a result of post-transcription or post-translational modification. Nuclear and cytoplasmic extracts were prepared from HEK-23-FOXL1 wild- type cells and HEK-293-FOXL1 -mutantcells. FOXL1 expression was determined using Anti- FOXL1 rabbit polyclonal IgG and Anti-GFP mouse IgG for the FOXL1 -GFP tagged transcripts. No significant difference was detected in the FOXL1 protein level between the mutant FOXL1 and wild type in the cytoplasm. However, the mutant FOXL1 did show an up regulation in the nuclear extract (Figure 9).

[00175] Functional studies showed that the FOXL1 deletion up regulated the level of the FOXL1 protein. To confirm the result from HEK293A and exclude any other confounding factor that related to type of cell line (hFOB 1.19), Human fetal osteoblast femur HEK293A cells were transiently transfected with FOXL1 wild and-mutant forms. No significant difference in the level of FOXL1 expression between hFOB-FOXL1 wild type and hFOB- FOXL1 -mutant as detected by RT-PCR (Figure 10). Up regulation of the FOXL1 mutant protein was observed using the nuclear extract of FOXL protein (Figure12). The nuclear localization of FOXL1 was visualized using living cells that were transfected with mock, wild, or mutant types FOXL1 -GFP expression plasmids. Living cells were examined under the fluorescence microscope and showed that nuclear localization of FOXL1 was not altered by the mutation (Figure 1 ). As FOXL1 is a known transcription factor, it was hypothesized that FOXL1 deletion could affect the FOXL1 protein structure by interfering with signaling of downstream gene targets. Expression of 13 genes that have been predicted to be involved in bone formation (Table 4) was tested by RT-PCR, using RNA from HEK293A transfected with wild FOXL1 and HEK293A transected by the mutant FOXL1. No significant change in the expression of these genes between the wild-type and mutant FOXL1 transfected cell line was observed (Figure 13).

[00176] A positional mapping approach and a large extended family from NL were used to identify a 15bp deletion in FOXL1 gene that segregates with the otosclerosis in the study family. Family 2081 is a large multiplex five-generation family. Linkage analysis excluded linkage of family 2081 to any previously mapped loci and suggest linkage of the family to 9Mb at chr16q23.1-q24.2. This new locus is located 2Mb telomeric to the published OTSC4 locus. The 9Mb region contains 79 annotated genes. Bidirectional Sanger sequencing of the coding region and intron/exon boundaries of 13 functional candidates within the region identified a 15 bp deletion in FOXL1

(c.976_990het_delGGGATCCCCTTCCTC) that segregates with the disease haplotype and is absent from dbSNP and not found in 16 NL control chromosomes. The deletion mutation occurs in a highly conserved region of the gene and at the c-terminus of the FOXL1 transcription factor.

[00177] A potential NL founder effect for the 15bp deletion in FOXL1 was explored by screening 17 NL otosclerosis families for the 15 bp deletion but none was found. As well, the full gene was sequenced in these other otosclerosis families and no variants identified. The prevalence of otosclerosis in NL is unknown. [00178] Human Forkhead (FKH)-box (FOX) gene family consist of at least 43 members. This gene family was named after the discovery of Drosophila FKH gene in 1989. FOX proteins act as a transcription factor and are characterized by sharing a common element of 1 10 amino acid DNA binding site, the fork head domain (FHD). FOX genes are important during embryogenesis and cell differentiation [1 1 , 12] and play an important role in the regulation of other genes in different species from yeast to human and have been involved in different biological process including tumorigenesis and speech acquisition in human [13]. Mutations have been identified indifferent FOX genes, for example, mutations in FOXC1 cause a range of developmental defects associated with glaucoma, as in Axenfeld anamoly, Rieger anomly, iris hyperplasia and Rieger syndrome[14] [15]

[00179] FOXL1 gene (Accession number; NM_005250) consists of one coding exon, a small 3UTR and a huge 5UTR. FOXL1 protein as other members of FOX family has the conserved sequence domain FHD which is the DNA binding site. FOXL1 is highly expressed in otic vesicles in vertebrates and is important in their development by acting as a negative regulator to the SHH signaling [16] and has a role in embryonic transcriptional regulator. Previous in vivo studies has shown that Foxfl and Foxl1 are target genes of HHL pathway in developing stomach and their expression is controlled by Gli2 and Gli3 transcription factors that harbour a binding site on the 5UTR of FOXI1 gene and this finding shows that FOXL1 genes involve in HHL pathway [17] [18]. HHL signaling pathway is important in regulating many cell processes including cell proliferation, differentiation, angiogenesis, cellular matrix remodeling and stem cell homeostasis and disregulation of this pathway lead to development a different forms of tumors[19]. FOXL1 has no identified role in bone regulation. The FOXL1 protein has been identified as one of a number of proteins associated with the BMR2 (bone morphogenic receptor2) receptor as seen with two-dimensional gel electrophoresis and mass spectrometry [20]. FOXL1 has also been implicated in the expression of BMP4 as has shown in the study by Madison etal [18] FOXL1 and FOXF2 mutants show reduced expression of BMP4 in the gut. [18]. The mechanism under development of otosclerosis as a result of a mutation in FOXL1 is not known yet. The deletion mutation didn't cause any change in the RNA level but did cause up regulation of the FOXL1 mutant protein. The identified mutation is located at the c-terminus end of the FOXL1 protein and the up regulation of the FOXL1 mutant protein could be as result of FOXL1 protein misfolding. The effect of FOXL1 mutant transcription factor on the some genes that known to be involved in bone metabolism was tested. The expression of ten genes (SCD1 , BMP2, BMP4, BMP7, CDKN1 A, LTB, ATP1 B3, TGB1 , TNFSF1 1 and BMPR2) were chosen for their expression in four cell lines (hFOB-empty, hFOB-non- transfected, hFOB-FOXL1 mutant and hF0B-F0XL1wild) using RT-PCR. No difference in expression between the hFOB- FOXL1 mutant and hFOB- FOXL1 wild type was found.

Conclusion

[00180] This study has identified a novel 15 bp deletion in FOXL1 gene that co segregates in a Newfoundland family with otosclerosis and support association of FOXL1 to the newly identified locus (extended OTSC4). The level of FOXL1 carrying the c.976_990het_delGGGATCCCCTTCCTC mutation results in up regulated FOXL1 protein levels. Further screening of patient with otosclerosis will help in identifying families with this deletion.

[00181] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00182] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and/or biomarker sequences ( e.g. protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

Table 1 : sequenced functional candidate genes at extended OTSC4 region

The homology between ZNF469 and collagen genes predicts that ZN469 can function s regulatory factor for synthesis and organization o collagen fibers.

5

10

Table 2: variants detected from sequencing of 13 genes in extended OTSC4

OSGIN C.-950T 5'UTR Not predicted rs4782864 No

C.170T Coding (EX1) p.P6L Not listed No c.101A>C Coding (EX2) P.N34T rs28555129 No c.169A>C Coding (EX2) p.M57L rs2244899 No

C.1750G Coding (EX2) p.L59V rs2244898 No c.218-104C>T Intron 2 not predicted no No

C.316+115T>C Intron 3 not predicted rs824396 No c.316+116T>G Intron 3 not predicted rs862200 No c.316+153 316+156 Intron 3 not predicted Not listed No het delATTT

c.317-80G>A Intron 3 not predicted rs2665296 No

C.454-480A lntron4 not predicted rs2665294 No

C.454-210T Intron 4 not predicted Not listed No c.646-17G>T Intron 5 not predicted rs2136662 No c.646-38G>A Intron 5 not predicted rs2741876 No c.1332C>A Coding (EX7) P.L444LL rs35132222 No

C.^*205T>C 3'UTR not predicted rs4782574 No c.^*41A>G 3'UTR not predicted rs77204347 No

SLC7A5 c.-21G>T 5'UTR Not predicted Not listed No c.345C>A Coding (EX1) P.G115GG rs17853938 No c.770+114T>A Intron 3 not predicted Not listed No c.770+124T>A Intron 3 not predicted Not listed No c.770+125G>A Intron 3 not predicted Not listed No c.770+127C>A Intron 3 not predicted Not listed No c.770+135A>G Intron 3 not predicted Not listed No c.770+145T>A Intron 3 not predicted Not listed No c.770+146G>T Intron 3 not predicted Not listed No

C.[939+61 T>G Intron 6 not predicted rs750950 No c.[1044-35C>T Intron 6 not predicted rs2287120 No c.[1140+85C>G Intron 7 not predicted rs3815559 No

SLC38A8 c.[189+11G>T Inton 1 not predicted rs1105355 No c.[189+151A>T Intronl not predicted rs13339651 No c.[190-9T>G Intron 1 not predicted rs1876960 No c.[190-5C>T Intron 1 not predicted rs1876962 No c.[195G>C Coding (Ex2) P.S65SS rs1317524 No c.[273G>A Coding (Ex2) P.V91VV Not listed No c.[388+69G>T Intron 2 not predicted rs8057543 No c.[388+128A>T Intron 2 not predicted rs34702590 No c.[531-147C>T Intron 3 not predicted rs907044 No c.[531-137T>C Intron 3 not predicted rs907043 No c.[690+65C>T Intron 5 not predicted Not listed No c.[690+127G>C Intron 5 not predicted Not listed No c.805+53 805+54het Intron 6 not predicted Not listed No delAA

c.[1162+87C>G Intron 8 not predicted rs 12929392 No c.[1214+116C>T lntron8 not predicted rs 1864162 No c.[1214+174T>C lntron8 not predicted rs1 864037 No c.[1214+194A>G lntron8 not predicted rs11149615 No c.[1214+204C>G lntron8 not predicted rs11864124 No PLCG2 c.565-22delG Intron 6 not predicted Not listed No c.[648+134T>C Intron 7 not predicted rs7192509 No c.[648+171T>A Intron 7 not predicted rs7192535 No

C.[648+237A>T Intron 7 not predicted rs7187837 No c.[648+2420T Intron 7 not predicted rs7187163 No

C.[648+319T>C Intron 7 not predicted Not listed No c.[692+25C>T Intron 8 not predicted rs11865395 No c.[692+61A>C Intron 8 not predicted rs41305761 No c.[765+137T>A Intron 9 not predicted Not listed No c.[770A>T Coding EX10 His257Leu rs45443101 No c.[802C>T Coding EX10 p.H257H rs17537869 No c.[987-97G>C Intron 11 P.R268RW rs4889430 No c.[1073-33G>A Intron 12 not predicted rs13333348 No c.[1149C>T Coding EX12 not predicted rs1143688 No c.[1188C>G Coding EX12 p.D383D rs13333716 No c.[1467+38G>C Intron 15 p.T396TT rs4435248 No

C.[1467+45G>T Intron 15 not predicted rs11859176 No c.[1497C>T Coding EX16 P.A499A rs1143689 No c.[1557+65C>T Intron 16 not predicted rs71400183 No c.[1557+87T>A Intron 16 not predicted Not listed No

C.[2054+7G>A Intron 19 not predicted Not listed No c.[2054+47A>C Intron 19 not predicted rs41305765 No c.[2055-8T>C Intron 19 not predicted rs12448130 No c.[2236-14C>G Intron 20 not predicted rs12446127 No

C.[3093T>C Coding EX28 p.N1031 NN rs1071644 No c.[3198+74A>G Intron 28 not predicted rs1071644 No

C.[3314-55T>C Intron 29 not predicted rs11150425 No c.[3314-23C>A Intron 29 not predicted rs4405546 No c.[3798+35T>C 3'UTR not predicted rs45554137 No

ST3GAL2 c.*25G>A] 3'UTR not predicted Not listed No

ZDHHC7 c.[315+55G>A Intron 3 not predicted rs931713 No c.316-366het delT Intron 4 not predicted Not listed No c.316-57het deIC Intron 4 not predicted Not listed No c.316-50het delT Intron 4 not predicted Not listed No c.375C>T Coding EX5 p.P125PP rs16975086 No c.538-11 C>T Intron 6 not predicted rs8044638 No c.750+77T>C Intron 8 not predicted rs80330099 No c.906T>C Coding EX9 p.G302GG rs7195377 No

ZNF469 c.1069T>C Coding EX1 P.S357P rs11648572 No

C.1098A>C Coding EX1 P.R366S rs11640794 No

C.1529G>C Coding EX1 P.G510A rs7199961 No c.1776A>G Coding EX1 P.P592P rs12927001 No c.2130C>T Coding EX1 p.P710PP rs12918876 No c.2717C>T Coding EX1 P.P906L rs77951481 No

C.3438G>A Coding EX1 p.P1146P rs9938800 No c.3484A>G Coding EX1 p.K116GLU rs7197071 No c.7072G>C Coding EX2 P.G2358R rs12598474 No c.8520C>T Coding EX2 p.R2840RR rs3812953 No

Coding EX2 P.H2848R rs1983014 No c.8543A>G Coding EX2 E3630EQ rs904783 No c.10888G>C Coding EX2 P.T3636A rs904783 No c.10906A>G Coding EX2 P.S3924S rs4782362 No c.11772C>T Coding EX2 P.P3891 P rs4782301 No c.1 1673A>G 5'UTR not predicted rs45504291 No

^*8G>A

Table 3: Sequences

SEQ ID NO: 1 Polynucleotide sequence for wildtype FOXI1

ATGAG TCACCTCTTC GATCCCCGGC TGCCTGCCCT 250

GGCCGCCTCG CCCATGCTGT ATCTGTACGG TCCCGAGAGA CCCGGCCTCC 300

CTCTGGCCTT CGCCCCCGCG GCTGCTCTAG CTGCCTCGGG CCGGGCCGAG 350

ACCCCGCAGA AGCCTCCCTA CAGCTACATC GCGCTCATCG CCATGGCGAT 400

CCAGGACGCG CCCGAGCAGA GGGTCACGCT CAACGGCATC TACCAGTTCA 450

TCATGGACCG CTTCCCCTTC TACCACGACA ACCGGCAGGG CTGGCAGAAC 500

AGCATCCGCC ACAACCTCTC GCTCAACGAC TGCTTCGTCA AGGTGCCCCG 550

CGAGAAAGGG CGGCCGGGCA AGGGCAGCTA CTGGACGCTG GACCCCCGCT 600

GCCTGGACAT GTTTGAGAAC GGCAACTACC GGCGCCGGAA GAGGAAGCCC 650

AAGCCGGGCC CCGGGGCCCC GGAGGCCAAG AGGCCCCGCG CCGAGACGCA 700

CCAGCGCAGC GCGGAGGCGC AGCCGGAGGC GGGGAGCGGG GCAGGGGGCT 750

CGGGCCCCGC AATCTCCCGC CTGCAGGCAG CGCCCGCGGG CCCCTCGCCC 800

CTCCTGGACG GCCCCTCTCC GCCGGCGCCC CTCCACTGGC CGGGGACCGC 850

GTCCCCGAAC GAGGACGCTG GTGACGCTGC CCAGGGCGCA GCGGCCGTGG 900

CGGTCGGCCA GGCAGCGCGC ACAGGGGACG GCCCGGGGTC CCCTCTGCGC 950

CCCGCCTCCC GCAGCTCTCC GAAGAGCTCC GACAAGTCCA AGAGCTTCAG 1000

CATAGACAGC ATCCTGGCGG GAAAGCAGGG CCAGAAGCCG CCTTCAGGGG 1050

ACGAACTCCT AGGGGGTGCC AAGCCTGGGC CCGGCGGCCG TCTGGGTGCC 1100

TCGCTCCTGG CCGCCTCCTC CAGCCTCCGT CCGCCTTTCA ACGCTTCCCT 1150

GATGCTCGAC CCGCATGTCC AGGGCGGCTT TTACCAGCTC GGGATCCCCT 1200

TCCTCTCTTA TTTCCCCCTG CAGGTTCCCG ACACGGTACT CCACTTCCAG 1250

TAA

SEQ ID NO:2 Polynucleotide sequence for mutant F0XL1 comprising c.976_990het_delGGGATCCCCTTCCTC . Note . dashes represent the deleted 15 bp sequence

ATGAG TCACCTCTTC GATCCCCGGC TGCCTGCCCT 250

GGCCGCCTCG CCCATGCTGT ATCTGTACGG TCCCGAGAGA CCCGGCCTCC 300

CTCTGGCCTT CGCCCCCGCG GCTGCTCTAG CTGCCTCGGG CCGGGCCGAG 350

ACCCCGCAGA AGCCTCCCTA CAGCTACATC GCGCTCATCG CCATGGCGAT 400

CCAGGACGCG CCCGAGCAGA GGGTCACGCT CAACGGCATC TACCAGTTCA 450

TCATGGACCG CTTCCCCTTC TACCACGACA ACCGGCAGGG CTGGCAGAAC 500

AGCATCCGCC ACAACCTCTC GCTCAACGAC TGCTTCGTCA AGGTGCCCCG 550

CGAGAAAGGG CGGCCGGGCA AGGGCAGCTA CTGGACGCTG GACCCCCGCT 600

GCCTGGACAT GTTTGAGAAC GGCAACTACC GGCGCCGGAA GAGGAAGCCC 650

AAGCCGGGCC CCGGGGCCCC GGAGGCCAAG AGGCCCCGCG CCGAGACGCA 700

CCAGCGCAGC GCGGAGGCGC AGCCGGAGGC GGGGAGCGGG GCAGGGGGCT 750

CGGGCCCCGC AATCTCCCGC CTGCAGGCAG CGCCCGCGGG CCCCTCGCCC 800

CTCCTGGACG GCCCCTCTCC GCCGGCGCCC CTCCACTGGC CGGGGACCGC 850

GTCCCCGAAC GAGGACGCTG GTGACGCTGC CCAGGGCGCA GCGGCCGTGG 900

CGGTCGGCCA GGCAGCGCGC ACAGGGGACG GCCCGGGGTC CCCTCTGCGC 950

CCCGCCTCCC GCAGCTCTCC GAAGAGCTCC GACAAGTCCA AGAGCTTCAG 1000 CATAGACAGC ATCCTGGCGG GAAAGCAGGG CCAGAAGCCG CCTTCAGGGG 1050

ACGAACTCCT AGGGGGTGCC AAGCCTGGGC CCGGCGGCCG TCTGGGTGCC 1100

TCGCTCCTGG CCGCCTCCTC CAGCCTCCGT CCGCCTTTCA ACGCTTCCCT 1150

GATGCTCGAC CCGCATGTCC AGGGCGGCTT TTACCAGCTC 1200

TCTTA TTTCCCCCTG CAGGTTCCCG ACACGGTACT CCACTTCCAG 1250

TAA

Amino acid sequence Sequences

[00 83] SEQ ID NO:3 Amino acid sequence for wildtype FOXI1

>NP_005241 length=345

MSHLFDPRLPALAASPMLYLYGPERPGLPLAFAPAAALAASGRAETPQKP PYSYIALIAMAIQDAPEQRVTLNGIYQFIMDRFPFYHDNRQGWQNSIRHN LSLNDCFVKVPREKGRPGKGSYWTLDPRCLDMFENGNYRRRKRKPKPGPG APEAKRPRAETHQRSAEAQPEAGSGAGGSGPAISRLQAAPAGPSPLLDGP SPPAPLHWPGTASPNEDAGDAAQGAAAVAVGQAARTGDGPGSPLRPASRS SPKSSDKSKSFSIDSILAGKQGQKPPSGDELLGGAKPGPGGRLGASLLAA SSSLRPPFNASLMLDPHVQGGFYQLGIPFLSYFPLQVPDTVLHFQ

[00184] SEQ ID NO:4 Amino acid sequence for mutant FOXL1 comprising deletion of 5 amino acid p.Gly326_Leu330. Note: dots represent the deleted 5 amino acid sequence

MSHLFD RLPALAASPMLYLYGPERPGLPLAFAPAAALAASGRAETPQKP PYSYIALIAMAIQDAPEQRVTLNGIYQFIMDRFPFYHDNRQGWQNSIRHN LSLNDCFVKVPREKGRPGKGSYWTLDPRCLDMFENGNYRRRKRKPKPGPG APEA RPRAETHQRSAEAQPEAGSGAGGSGPAISRLQAAPAGPSPLLDGP SPPAPLHWPGTASPNEDAGDAAQGAAAVAVGQAARTGDGPGSPLRPASRS SPKSSDKSKSFSIDSILAGKQGQKPPSGDELLGGAKPGPGGRLGASLLAA SSSLRPPFNASLMLDPHVQGGFYQL SYFPLQVPDTVLHFQ Deleted sequences

[00185] SEQ ID NO: 5 GGGATCCCCTTCCTC.

[00186] SEQ ID NO: 6 GIPFL [00187] Primer sequence that detect the FOXL1 deletion. FOXL1_F:AACGAGGACGCTGGTGAC SEQ ID NO: 7

FOXL1 R:CCCAGGCAAAGATCATTTTA SEQ ID NO: 8

[00188] The amplified product is about 490bp.

XP 001231599.2 275 QVHGRLYHIGIPFLSCFPFHFSE-AVFNFQ 303 SEQ ID NO: 9

NP 032050.2 307 HVQGGFSQLGIPFLSYFPLQVPEATVLRFH 336 SEQ ID

NO: 10

XP 002694802.1 318 HVQGGFYQLGIPFLSYFPLQLPE-AVLHFQ 346 SEQ ID

NO: 1 1

XP 851625.1 328 HVQGGFYQLGIPFLSYFPLQLPD-TVLHFQ 356 SEQ ID NO:

12

NP 005241.1 317 HVQGGFYQLGIPFLSYFPLQVPD-TVLHFQ 345 SEQ ID NO:

13

XP 51 1 154.2 317 HVQGGFYQLGIPFLSYFPLQLPD-TVLHFQ 345 SEQ ID NO: 14

References:

1 . de Souza C, G.M., Otosclerosis and stapedectomy. Thieme, Stuttgart, 2004.

2. Somers, T.D., F. Kuhweide, R. andRobillard, Th. , Otosclerosis. B-ENT.3, Suppl. 6, 3- 10, 2007.

3. Bel Hadj Ali, I., et al. , A new locus for otosclerosis, OTSC8, maps to the pericentromeric region of chromosome 9. Hum Genet, 2008. 123(3): p. 267-72.

4. Ealy, M. and R.J. Smith, The Genetics of otosclerosis. Hear Res, 2010. 266(1 -2): p.

70-4.

5. Chen, W., et al., Linkage of otosclerosis to a third locus (OTSC3) on human chromosome 6p21.3-22.3. J Med Genet, 2002. 39(7): p. 473-7.

6. Thys, M. , et al., A seventh locus for otosclerosis, OTSC7, maps to chromosome 6q13-16. 1. Eur J Hum Genet, 2007. 15(3): p. 362-8.

7. Van Den Bogaert, K., et al. , A second gene for otosclerosis, OTSC2, maps to chromosome 7q34-36. Am J Hum Genet, 2001. 68(2): p. 495-500.

8. Tomek, M.S. , et al., Localization of a gene for otosclerosis to chromosome 15q25- q26. Hum Mol Genet, 1998. 7(2): p. 285-90.

9. Van Den Bogaert, K., et al., A fifth locus for otosclerosis, OTSC5, maps to chromosome 3q22-24. J Med Genet, 2004. 41 (6): p. 450-3.

10. Brownstein, Z. , et al., Chromosomal mapping and phenotypic characterization of hereditary otosclerosis linked to the OTSC4 locus. Arch Otolaryngol Head Neck Surg, 2006. 132(4): p. 416-24.

11. Carlsson, P. and M. Mahlapuu, Forkhead transcription factors: key players in development and metabolism. Dev Biol, 2002. 250(1 ): p. 1-23.

12. Kaufmann, E. and W. Knochel, Five years on the wings of fork head. Mech Dev, 1996. 57(1 ): p. 3-20.

13. Lai, C.S., et al., A forkhead-domain gene is mutated in a severe speech and language disorder. Nature, 2001. 413(6855): p. 519-23.

14. Nishimura, D.Y. , et al., The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nat Genet, 1998. 19(2): p. 140-7. 5. Mirzayans, F., et al. , Axenfeld-Rieger syndrome resulting from mutation of the FKHL7 gene on chromosome 6p25. Eur J Hum Genet, 2000. 8(1 ): p. 71 -4.

16. Nakada, C, et al., Transcriptional repressor foxl1 regulates central nervous system development by suppressing shh expression in zebra fish. Mol Cell Biol, 2006. 26(19): p. 7246-57.

17. Hallikas, O., et al., Genome-wide prediction of mammalian enhancers based on analysis of transcription-factor binding affinity. Cell, 2006. 124(1 ): p. 47-59.

18. Madison, B.B., et al., FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine. J Biol Chem, 2009. 284(9): p. 5936-44.

9. Bale, A. E., Hedgehog signaling and human disease. Annu Rev Genomics Hum Genet, 2002. 3: p. 47-65.

0. Hassel, S., et al., Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry. Proteomics, 2004. 4(5): p. 1346-58.

1. Schrauwen, I., et al. , Association of bone morphogenetic proteins with otosclerosis. J Bone Miner Res, 2008. 23(4): p. 507-16.

2. Teng, Y.H., R.S. Aquino, and P.W. Park, Molecular functions of syndecan-1 in disease. Matrix Biol, 2012. 31(1 ): p. 3-16.

3. Perreault, N., et al., Foxl1 controls the Wnt/beta-catenin pathway by modulating the expression of proteoglycans in the gut. J Biol Chem, 2001. 276(46): p. 43328-33.

Claims

Claims:

1. A method for identifying a human subject as having, or having an increased likelihood to develop, otosclerosis hearing loss, the method comprising (a) obtaining a sample from the subject; (b) assaying the sample for the presence or absence of a mutation in: i) a FOXL1 polynucleotide encoding one or more of amino acid residues in FOXL1 polypeptide C-terminus and/or encoding an amino acid corresponding to position 326 to position 330 of SEQ ID NO:2, or ii) a FOXL1 polypeptide encoded by said polynucleotide; or iii) a FOXL1 polypeptide level; and (c) detecting the presence or absence of the mutation; wherein detection of the presence of said mutation and/or an increase of FOXL1 polypeptide compared to a control indicates that the subject has, or has an increased likelihood, to develop otosclerosis and/or hearing loss.

2. The method of claim 1 , wherein said sample is blood or a fraction thereof, saliva, throat swab or a DNA accessible tissue or said sample is a bone cell or gastric cell sample.

3. The method of claim 2, wherein said sample is blood or a fraction thereof.

4. The method of any one of claims 1 to 3, wherein the sample is assayed for the presence or absence of said mutation in said FOXL1 polynucleotide.

5. The method of any one of claims 1 to 4, wherein said mutation in said FOXL1 polynucleotide comprises deletion of one or more nucleotides corresponding to 976 to position 990 of SEQ ID NO: 1.

6. The method of any one of claims 1 to 5, wherein said mutation in said FOXL1 polynucleotide is c.976_990het_delGGGATCCCCTTCCTC.

7. The method of any one of claims 1 to 6, wherein the mutation in FOXL1 polynucleotide is detected by a PCR based method and/or a probe based method.

8. The method of any one of claims 1 to 7, wherein DNA is isolated from said sample and the DNA is assayed for the presence or absence of said mutation.

9. The method of claim 9, wherein said DNA is genomic DNA.

10. The method of any one of claims 1 to 9, wherein assaying said sample for the presence or absence of the mutation in FOXL1 corresponding to position 976 to position_990 comprises: a) amplifying all or a portion of FOXL1 coding sequence, the portion comprising minimally FOXL1 residue corresponding to position 976 to position_990 of SEQ ID NO:1 , to produce amplified FOXL1 polynucleotide, b) sequencing the amplified FOXL1 polynucleotide or measuring the length of said amplified FOXL1 polynucleotide to determine if said mutation is present.

11. The method of any one of claims 1 to 7, wherein mRNA is isolated from said sample and the mRNA is assayed for the presence or absence of said mutation.

12. The method of any one of claims 1 to 7 and 11 , wherein the method comprises: a) isolating mRNA from said sample; b) deriving corresponding DNA (cDNA) from said isolated mRNA; c) amplifying all or a portion of FOXL1 coding sequence, the portion comprising minimally FOXL1 residue corresponding to position 976 to position_990 of SEQ ID N0.1 or SEQ ID NO:3, to produce amplified FOXL1 polynucleotide, d) sequencing the amplified FOXL1 polynucleotide; and e) determining if said mutation is present; wherein detection of the presence of said mutation indicates that the subject has, or has an increased likelihood, to develop otosclerosis and/or hearing loss.

13. The method of any one of claims 1 to 3, wherein said sample is assayed for the presence or absence of said mutation in said FOXL1 polypeptide.

14. The method of any one of claims 1 to 3 and 13, wherein said mutation in said FOXL1 polypeptide is p.Gly326_Leu330.

15. The method of any one of claims 1 to 3, 13 and 14, wherein said mutation in said FOXL1 polypeptide is detected with an antibody specific for an epitope comprising residues 325 and 326 of SEQ ID NO: 4.

16. A method of treating a human subject, the method comprising determining if the subject is a carrier of a mutation corresponding to position 976 to position_990 of SEQ ID NO: 2, optionally p.Gly326_Leu330 deletion FOXL1 ; and providing subjects determined to be carriers of the mutation, optionally p.Gly326_Leu330 FOXL1 , with a hearing loss therapy.

17. The method of claim 18, wherein the hearing loss therapy comprises providing the subject with a suitable hearing aid or cochlear implant.

18. An isolated polynucleotide comprising the sequence of SEQ ID NO: 2 or 5.

19. An isolated polypeptide comprising the sequence of SEQ ID NO: 4 or 6.

20. An isolated antibody or binding fragment that binds SEQ ID NO: 6 or that binds an epitope comprising amino acids 325 and 331 of SEQ ID NO: 3 that does not bind an epitope comprising amino acids 326 to 330 of SEQ ID NO: 3, optionally wherein the antibody is labelled.

21. A compound comprising a polynucleotide encoding at least 5 and a maximum

50, 60, 70 or 80 contiguous amino acids of SEQ ID NO:3, 4 or 6, the at least 5 contiguous amino acids optionally comprising the amino acid residues of SEQ ID NO: 6.

22. The compound of claim 21 , wherein the FOXL1 polynucleotide encoding the at least 5 and a maximum of 50, 60 , 70 or 80 contiguous amino acids comprises least 15 nucleotides of SEQ ID NO: 1 , 3 or 5, said at least 15 nucleotides optionally comprising the nucleotide corresponding to SEQ ID NO:5.

23. A compound comprising a polypeptide comprising at least 5 and a maximum 50, 60, 70 or 80 contiguous amino acids of SEQ ID NO:2, 4 or 6, the at least 5 contiguous amino acids optionally comprising the amino acid residues of SEQ ID NO:6.

24. The compound of any one of claims 21 to 23, wherein the compound comprises a detectable label.

25. A nucleic acid primer for diagnosing a otosclerosis hearing loss comprising a polynucleotide which hybridizes under stringent conditions to a portion of the nucleic acid sequence of SEQ ID NO: 1 or complement thereof, the nucleic acid primer binding a portion within nucleotides 1 - 975 or 991-1250 of SEQ ID NO:1 , wherein the primer is for amplifying all or a portion of a FOXL1 coding sequence such that one or more nucleotides encoding an amino acid at residue 976 to position_990 of SEQ ID NO: 1 can be amplified.

26. The nucleic acid primer of claim 25, wherein the primer is at least 12 nucleotides in length.

27. The nucleic acid primer of claim 25 or 26, wherein the primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 7 or 8.

28. The nucleic acid primer of any one of claims 25 to 27, wherein the primer is labelled.

29. A nucleic acid probe for diagnosing otosclerosis hearing loss comprising a polynucleotide which hybridizes under stringent conditions to a portion of SEQ ID NO: 1 or the complement of SEQ ID NO: 1 encoding amino acid corresponding to position 326-330 of SEQ ID NO. 3 or a polynucleotide which hybridizes under stringent conditions to a portion of SEQ ID NO: 2 or the complement of SEQ ID NO: 2 encoding amino acids sequence that differs from the SEQ ID NO: 1 at one or more nucleotides encoding Glycine, Isoleucine,, Proline, phenylalanine and Leucine at residue 326,327,328,329,330 of SEQ ID NO:3, and which detects the presence or absence of a substitution or lesion at one or more nucleotides that encode the Glycine, Isoleucine,, Proline, phenylalanine and Leucine at residue 326,327,328,329,3304 of SEQ ID NO:3.

30. The nucleic acid probe of claim 29, wherein the probe detects a substitution or lesion at a nucleotide corresponding to position 976-990 of SEQ ID NO: 1.

31. The nucleic acid probe of claim 29 and 30, wherein the probe is at least 12 nucleotides in length.

32. The nucleic acid probe of any one of claims 29 to 31 , wherein the probe comprises at least 12 consecutive nucleotides of SEQ ID NO:1 or 2.

33. The nucleic acid probe of any one of claims 29 to 32, wherein the probe comprises at least comprises at least 50 consecutive nucleotides of SEQ ID NO: 1 or 2.

34. The nucleic acid probe of any one of claims 29 to 33, wherein the probe is labeled.

35. A composition comprising the polynucleotide of claim 8, polypeptide of claim 19, antibody of claim 20, compound of any one of claims 21-24, nucleic acid primer of any one of claims 25-28, nucleic acid probe of any one of claims 29- 34; and a diluent.

36. A kit for diagnosing a subject at risk for a otosclerosis hearing loss comprising: i) at least two nucleic acid primers which hybridize under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1 or complement thereof, wherein the primers amplify all or a portion of the coding sequence of SEQ ID NO: 1 or 2, such that minimally one or more nucleotides encoding an amino acid residue corresponding to position 326-330 of SEQ ID NO:3 can be amplified; ii) a nucleic acid probe; which hybridizes under stringent conditions to SEQ ID NO: 1 or2 and/or the complement of SEQ ID NO: 1 or 2 and which detects the presence or absence of a substitution or lesion at one or more nucleotides that encode the Glycine, Isoleucine, Proline, phenylalanine and Leucine at residue 326,327,328,329,330 of SEQ ID NO:3 or iii) an antibody that specifically binds SEQ ID NO: 6 and/or an epitope comprising residues 326 and 327 of SEQ ID NO: 4 and optionally instructions for use, and optionally instructions for diagnosing otosclerosis and/or hearing loss by detecting a substitution or deletion of one or more nucleotides encoding amino acid residue 326-330 of SEQ ID NO: 3, a receptacle for receiving a sample; a standard and/or a tissue solution.

37. The kit of claim 36, comprising a nucleic acid probe which hybridizes under stringent conditions to SEQ ID NO: 1 , 2 or 5 and/or the complement of SEQ ID NO: 1 , 2 or 5 which detects the presence or absence of a substitution or lesion at one or more nucleotides that encode the Glycine, Isoleucine, Proline, phenylalanine and Leucine at residue 326,327,328,329,330 of SEQ ID NO:3.

38. The kit of claim 36 or 37, wherein the kit comprises more than one probe.

39. The kit of any one of claims 36 to 38, wherein one or more of the probes is a labeled probe.

40. The kit of any one of claims 36 to 39, wherein the probe comprises at least 12 nucleotides in length.

41. The kit of any one of claims 36 to 10, wherein the probe comprises at least 12 consecutive nucleotides of SEQ ID NO: 1 , 2 or 5.

42. The kit of any one of claims 35 to 39, wherein the probe comprises at least comprises at least 50 consecutive nucleotides of SEQ ID NO: 1 or 2.

43. The kit of claim 35, wherein the primer is at least 12 nucleotides in length.

44. The kit of claim 35 or 42, wherein the primer comprises at least 12 consecutive nucleotides of SEQ ID NO:7 or 8.

45. The kit of any one of claim 44, wherein the primer is labelled.