Add mice are deaf and exhibit vestibular dysfunction. All experiments were carried out with mice ≈8 weeks of age. All values are given as mean ± SD. (A) Add mice demonstrated no acoustic startle response to a 120 dB sound burst (n = 10 mice for each genotype). (B) The ABR thresholds to a click stimulus were elevated in add mice (n = 4 for control mice; n = 20 for add). (C) Representative ABR recordings in response to click stimuli. ABR waves I–V are indicated for recordings obtained from a wild-type mouse. (D) Representative DPOAE response spectra for a wild-type and add mouse at 40 dB (top traces) and 70 dB (bottom traces). Note that the 2f1-f2 product (arrow) was absent in recordings from add mice. (E) DPOAE measurements were performed at 16 kHz using stimulus levels between 20 and 70 dB. Add mice were severely affected at all intensity levels (n = 4 wild-type mice; n = 6 add mice). (F) DPOAE thresholds in 8-week-old add mice were elevated at all frequencies analyzed (n = 4 wild-type mice; n = 6 add mice). (G) Add mice show increased small-diameter rotations (n = 14 wild-type mice; n = 5 add mice). (H) Horizontal beam test. None of the add mice tested were able to walk across the horizontal beam to reach the platform on one end (n = 6 mice for each genotype).

Degeneration of the organ of Corti in add mice. (A–D) Semithin sections stained with toluidine blue. The organ of Corti appeared normal in add mice, and IHC and OHC could be detected at P5. By 8 weeks of age, the organ of Corti had degenerated. (E–J) SEM analyis revealed that stereociliar bundles of hair cells in the medial and basal part of the cochlea at P5 showed structural abnormalities (arrows). (Scale bars: A–D, 40 μm; E–J, 4 μm.)

Add mice carry a mutation in COMT2 that affects catecholamine methyltransferase activity in vitro. (A) Coarse mapping of the add locus. The locus was mapped to distal chromosome 7 by 18 meioses with panels of 59 microsatellite markers. (B) The intron/exon structure of Comt2. The coding region is shown in black and the noncoding region in red. Sequence analysis using the SMART program (http://smart.embl-heidelberg.de/) identifies a methyltransferase domain between amino acids 55 and 247. (C) Identification of add mutation. A single nucleotide transversion (G→T) resulted in R48L of the polypeptide chain. (D) In vitro methyltransferase assay with cell lysates prepared from HEK 293 cells transfected with expression constructs for mouse COMT1, COMT2, COMT1^R51L, and COMT2^add (n = 4 separate transfections, mean ± SD). The methyltransferase activity of COMT2^add is significantly diminished from that of COMT2 (P = 0.0346). At the bottom, a Western blot of COMT1, COMT2, COMT1^R51L, and COMT2^add expression in HEK 293 cells is shown.

COMT2 is expressed in hair cells. Sagital section from P4 wild-type mice were analyzed for COMT2 expression by in situ hybridization. (A and B) COMT2 was expressed in the cochlea in IHCs and OHCs. (C) No signal was observed with the sense control probe. (D and E) COMT2 was also expressed in hair cells in the utricle (D) and cristae (E). (F and G) Coronal brain sections at P4. No expression was observed in the brain. Arrows point to ventricles. The anterior commissure (aca) and the accumbens nucleus (acb) are indicated. (Scale bars: A, 30 μm; B–E, 20 μm; F, 250 μm; G, 100 μm.)

Mutations in the human COMT2 gene that cause deafness. (A and C) Pedigree of Iranian families L-1013 and L-714. (Open symbols, unaffected; filled black symbols, affected; double line, consanguineous event; diagonal line, deceased.) (B and D) Mutations in COMT2. (B) The c.213C>G (p.Y71X) stop mutation in homozygous state in affected individual VI:1 (family L-1013) compared with control. (D) The c.47T>C (p.L16P) missense mutation in affected individual IV:2 (family L-714) compared with control. (E) Alignment of amino acid sequences from human COMT1 and mouse COMT2. Identical residues are indicated between the two. Y71, mutated in family L-1013, is conserved in mouse and human COMT2.