Characterization of a spontaneous, recessive, missense mutation arising in the Tecta gene

Abstract

The TECTA gene encodes alpha-tectorin (TECTA), a major noncollagenous component of the tectorial membrane (TM). In humans, mutations in TECTA lead to either dominant (DFNA8/A12) or recessive (DFNB21) forms of nonsyndromic hearing loss. All missense mutations in TECTA that have been reported thus far are associated with the dominant subtype, whereas those leading to recessive deafness are all inactivating mutations. In this paper, we characterize a spontaneous missense mutation (c.1046C > A, p.A349D) arising in the mouse Tecta gene that is, unlike all previously reported missense mutations in TECTA, recessive. The morphological phenotype of the Tecta (A349D/A349D) mouse resembles but is not identical to that previously described for the Tecta(deltaENT)/(deltaENT) mouse. As in the Tecta(deltaENT/(deltaENT) mouse, the TM is completely detached from the surface of the organ of Corti and spiral limbus, lacks a striated-sheet matrix, and is deficient in both beta-tectorin (Tectb) and otogelin. A significant amount of Tecta is, however, detected in the TM of the Tecta (A349D/A349D) mouse, and numerous, electron-dense matrix granules are seen interspersed among the disorganized collagen fibrils. Mutated Tecta (A349D) is therefore incorporated into the TM but presumably unable to interact with either Tectb or otogelin. The Tecta (A349D/A349D) mouse reveals that missense mutations in Tecta can be recessive and lead to TM detachment and suggests that should similar mutations arise in the human population, they would likely cause deafness.

FIG. 1

Lineage diagram illustrating how the Tecta^A349D/A349D mouse was derived. A chimeric male mouse (TG28/1) transmitting the allele through the germ line was mated with two wild-type C57BL6J mice, A and B. Three breeding pairs () set up from the resultant F1 mice produced some F2 mice that had detached tectorial membranes. Inbreeding the Tecta^+/+ F1 mice resulted in some Tecta^+/+ F2 mice with detached tectorial membranes. F2 mice from this cross that were judged by the Preyer reflex to be hearing impaired were set up as breeding pairs, and all resulting F3 progeny were found to have detached tectorial membranes. Preyer reflex tests were not performed on the chimeric male mouse, its two female C57BL6J partners, or the F1 generation, and their inner ears were not retained for histological analysis.

FIG. 2

Phenotype of the Tecta^A349D/A349D mouse. Toluidin blue-stained 1-μm-thick sections (A–C), anti-Tecta- (D–F), anti-Tectb- (G–I), and anti-otogelin (J–L) stained cryosections from A, D, G, J wild-type mice, B, E, H, K mice (litter U), and C, F, I, LTecta^A349D/A349D mice (litter A1). Arrows indicate the tectorial membrane. Arrow in J shows otogelin staining in the sulcal region of the wild-type tectorial membrane. Scale bars = 100 μm.

FIG. 3

Reverse transcriptase PCR analysis and ultrastructure of the tectorial membrane. A Tecta, Tectb, and Otog messenger RNAs (mRNA) are expressed in Tecta^A349D/A349D cochleae. Randomly primed first-strand cDNA was prepared from total cochlear RNA from Tecta^+/+ (WT) and Tecta^A349D/A349D (A349D) mice, and products specific for Tecta, Tectb, and Otog mRNAs were amplified by PCR. C1 Reverse transcriptase reaction control, in which the reverse transcription reaction was performed without reverse transcriptase and then amplified by the PCR, C2 control PCR performed without an aliquot of the reverse transcriptase reaction. B Electron micrographs illustrating the ultrastructural appearance of the tectorial membrane in wild-type (a, b), (c, d), and Tecta^A349D/A349D (e, f) mice at postnatal days P80, P77, and P82, respectively. Collagen fibrils (arrows) are embedded in a striated-sheet matrix (SSM) in the tectorial membrane of wild-type mice (a, b). In Tecta^ΔENT/ΔENT (c, d) and Tecta^A349D/A349D (e, f) mice, the striated-sheet matrix is absent, and large electron dense granules (arrowheads) are visible. These granules are more numerous in the Tecta^A349D/A349D mouse than in the mouse (compare c and e). Scale bars for a, c, e = 1 μm, for b, d, f = 200 nm.

FIG. 4

Phenotype of compound and double heterozygotes. Toluidin blue-stained 1-μm sections (A, D, G, J, and M), anti-Tecta-stained cryosections (B, E, H, K, and N), and anti-Tectb-stained cryosections (C, F, I, L, and O) from the following double heterozygotes Tecta^+/A349D, Tectb^+/− mice (litter D, A–C); , Tectb^+/− mice (litter F, D–F); Tecta^Y1870C/+, Tectb^+/− mice (litter I, G–I); and from the mice (litter J, J–L) and Tecta^Y1870C/A349D mice (litter G, M–O) compound heterozygotes. Arrows indicate the tectorial membrane. Scale bars = 100 μm.

FIG. 5

Identification of the p.A349D mutation in Tecta. A Electropherogram depicting the mouse Tecta exon 6 fragment that contains the homozygous recessive mutation c.1046C > A (p.A349D) responsible for the Tecta^A349D/A349D phenotype. B Schematic representation of the Tecta protein domains. D0–D4 units represent the von Willebrand factor (vWFD) type D repeats (Zonadhesin-like). The asterisk indicates the position of the p.A349D mutation at the vWFD (D1) repeat. The initial and last amino acid (AA) residues for each domain are indicated. C Multiple protein alignment of alpha-tectorin and homologous sequences in the von Willebrand type D1 repeat partial region. The A349 residue, which is mutated in the Tecta^A349D/A349D mouse, is indicated in bold. The alignment comprises the following sequences: Mus musculus (NP_033373), Rattus norvegicus (XP_136124), Homo sapiens (NP_005413), and Gallus gallus (NP_990204).