1

Introduction

The COCH gene, located on the long arm of chromosome 14 (14q12-q13), is one of the few genes causative of late onset bilateral sensorineural hearing impairment (BLSNHI) when mutated. Mutations in this gene were first reported in 1996 . The protein product of this gene, cochlin, is expressed in the cochlea and the vestibular system of the inner ear. The COCH protein makes up 70% of inner ear proteins . It assists in structural support, sound processing and maintenance of balance. Due to the large contribution COCH makes to inner ear proteins, it follows that mutations in this protein were found to interfere with disulfide bonds and proper protein folding and results in general structural disturbances affecting critical cochlear functions that are essential for hearing . Effects of mutations in the COCH gene have been found to cause non-syndromic autosomal dominant sensorineural deafness type 9 (DFNA9). Mutations were initially identified in exon 4 , resulting in a form of progressive BLSNHI caused by disruption of neural receptors in the inner ear and nerve pathways to the auditory centers of the brain receiving sound information. Mutant cochlin proteins may also lead to abnormal acidophilic deposits in the inner ear that could contribute to DFNA9 BLSNHI by cutting off, or causing degeneration of, dendrite fibers . Individuals with DFNA9 BLSNHI exhibit progressive late onset hearing impairment with onset usually prior to the fourth or fifth decade. Onset typically is in the high frequencies, progressing to include lower frequencies, and generally resulting in severe hearing impairment by the sixth decade of life. COCH mutations also frequently lead to vestibular dysfunction and balance problems.

The COCH gene is routinely screened for mutations in patients with late onset hearing impairment. Clinical mutational analysis for this gene until recently only encompassed exons 4 and 5, both of which encode the Limulus factor C, Coch-5b2, and Lg11 [LCCL] domain, since these were the only exons in which mutations had been found. The first mutation found outside of the LCCL domain was a c.1625 G > T transversion in exon 12 which is in the von Willebrand factor A2 (vWFA2) domain . Recent clinical mutational analysis has extended to include this exon . This protein domain is best known for its involvement in cell adhesion in extracellular matrix proteins, such as collagen . The von Willebrand Factor is itself a protein secreted from endothelial cells that works as a coagulation protein in blood . Cysteines flanking the vWF domain, A1 and A2, form a disulfide bond and mutations in the vWFA2 domain are likely to disrupt this bond, interfering with normal platelet binding activity . Mutations in this domain of the COCH gene would disrupt interactions with collagens and other extracellular matrix proteins and affect structural stability in the inner ear. This is in keeping with the effect of other COCH mutations, which also demonstrate various deleterious effects on inner ear integrity.

To date nine mutations have been identified in the COCH gene, all localized to within exons 4, 5, and 12 . In this study, we report a 3 generation extended family in which a novel mutation was identified in the COCH gene in exon 11, in the vWFA2 protein domain, the same domain also encoded by exon 12. The mutation is a heterozygous 18 base pair deletion, NM_001135058:c.1196_1213del, resulting in an in-frame deletion of 6 amino acids, p.Ile399_Ala404del. This novel mutation has not previously been implicated in DFNA9 related BLSNHI and is not currently screened for by most clinically available tests for COCH mutations. Additionally, all previously reported mutations in the COCH gene have been point mutations, while this mutation is an in frame deletion of 6 amino acids.

Consistent with other mutations in the COCH gene, the hearing impairment present in the family reported here is late onset, first occurring in the third decade, and progressive, initially involving the higher frequencies and progressing to severe encompassing all frequencies over the next several decades. Tinnitus is also a common finding in affected family members. They had no noted balance problems or other common medical problems; all other mutations in this gene have led to some sort of vestibular impairment, making this family an exception.

2

Materials and methods

2.1

Research subjects and controls

All affected individuals from the studied family ( Fig. 1 ) and controls were enrolled in the study under a protocol of informed consent approved by the Institutional Review Board at The Children’s Hospital of Philadelphia. Blood samples were obtained from a total of 17 members of an extended family with hereditary hearing impairment. The DNA was extracted and DNA samples were prepared using the Puregene genomic DNA isolation kit (Qiagen, cat. 158467). Three affected subjects, known to have hearing impairment, are all from one nuclear family (III 6, IV 10, IV 12). One member of the extended family, subject (III 4), had an atypical form of hearing impairment from the rest of the family. Four individuals with available samples are below the expected age of onset of hearing impairment as seen in the affected family members. Samples were also available from nine individuals from the extended family who are above the age of expected onset, but do not exhibit symptoms. Additionally, 49 ethnically matched unrelated controls were sequenced.

Family pedigree. Black squares/circles indicate affected individuals, all of whom presented with the mutation when sequenced. One circle with slashes represents a family member with hearing impairment believed to not be caused by the same mutation. She did not show the mutation when sequenced. * indicates samples available for linkage studies and mutational analysis. Affected status of generation V is unknown due to all members of this generation being below the age of 20 years ( COCH -related hearing loss usually does not manifest below this age), and therefore none were used in the linkage analysis.