2E4/Kaptin (KPTN)--a candidate gene for the hearing loss locus, DFNA4

Abstract

Stereocilia of the inner ear play an integral role in the mechanotransduction of sound. Their structural support is derived from actin filaments and actin-binding proteins. We have identified a novel actin-binding protein, 2E4-kaptin (KPTN), which appears to be involved in this structural network. Using double label immunofluorescence, we now show that KPTN extends beyond the barbed ends of actin filaments at the tips of stereocilia, and using cloned human cDNA, we mapped KPTN to chromosome 19q13.4. A combination of FISH, radiation hybrid mapping and YAC screening localized KPTN between markers D19S412 and NIB1805, making this gene an excellent functional and positional candidate for DFNA4, a form of autosomal dominant non-syndromic hearing loss. We identified a second family with inherited deafness that also maps to the DFNA4 region. To screen KPTN for deafness-causing mutations, we first determined its genomic structure and then completed a mutational analysis by direct sequencing and SSCP in affected family members. Although no deafness-causing mutations were identified in the coding region, KPTN remains an excellent candidate gene for hearing loss; by synteny, its murine orthologue also remains a candidate gene for the Nijmegan waltzer (nv) mouse mutant, which has vestibular defects and a variable sensorineural hearing loss.

Fig. 1

(A) Stereocilia projecting from single hair cells stained with polyclonal anti-KPTN antibody (top panel, red) or monoclonal anti-KPTN, Mab 2E4 (bottom panel, red) and double labeled for actin (both panels, green). Bar = 5 μm. (B) KPTN is encoded on chromosome 19 by fluorescence in situ hybridization. Arrows indicate the duplicated gene on both replicated chromatids of the two chromosome 19s.

Fig. 2

Pedigree of a second family, with sensorineural hearing loss, that has been mapped to an interval which overlaps with the DFNA4 locus. Black and yellow chromosomes are inherited from the affected father, solid blue and white from the unaffected mother, hatched green and hatched blue contributed by unaffected spouse in the second generation. Obligate crossovers in individuals 10 and 11 define the flanking markers. (squares, male; circles, female; black filling indicates affected individual, and black chromosome co-segregates with defect). Numbers beside each chromosome indicate alleles for each marker.

Fig. 3

Region of interest within chromosome 19 showing the common intervals between KPTN and DFNA4 (Family 1070) and between DFNA4 and Family 1030. Drawing is not to scale. Distances are indicated in cRays according to the Whitehead RH map.

Fig. 4

Genomic structure of KPTN. Dark areas of boxes indicate the coding sequence. The dashed area of exon 1 indicates that the 5′ end of the gene was not resolved here, although the full length cDNA including the untranslated 5′ end for KPTN has been obtained (Bearer & Abraham, 1999). The indicated lengths of exons correspond to the coding sequence.