Integration of human and mouse genetics reveals pendrin function in hearing and deafness

Abstract

Genomic technology has completely changed the way in which we are able to diagnose human genetic mutations. Genomic techniques such as the polymerase chain reaction, linkage analysis, Sanger sequencing, and most recently, massively parallel sequencing, have allowed researchers and clinicians to identify mutations for patients with Pendred syndrome and DFNB4 non-syndromic hearing loss. While thus far most of the mutations have been in the SLC26A4 gene coding for the pendrin protein, other genetic mutations may contribute to these phenotypes as well. Furthermore, mouse models for deafness have been invaluable to help determine the mechanisms for SLC26A4-associated deafness. Further work in these areas of research will help define genotype-phenotype correlations and develop methods for therapy in the future.

Fig. 1

A general anatomical structure of the auditory system is demonstrated by schematic illustration. A: The ear is divided into three distinct anatomical compartments, the outer, the middle and the inner ear. Sound waves captured by the auricle of the external ear passes through the ear canal reaching the tympanic membrane leading to its vibrations. The middle ear conducts the mechanical energy of the sound vibration and transfers it to the inner ear. B: A cross section through the auditory portion of the inner ear (cochlea) reveals its associated fluid field compartments. C: A specialized sensory organ (organ of Corti) resides within the scala media along the coil shape of the cochlea from base to apex. The inner and outer hair cells are constantly elicited by mechanical forces driven by the acoustic stimulus. D: The apical surfaces of the hair cell contain hair bundle structures assembled by actin rich stereocilia with a typical staircase structure. Upon deflection of the hair bundle, mechanoelectrical channels position at the tip of two adjacent stereocilia are open and leads to hair cell depolarization. Complex innervations at the basal pole of the sensory cells propagate the electrical signal through the auditory nerve into the brain. E: A scanning electron micrograph showing hair bundle of an outer hair cell isolated from a newborn mouse. F: A cross section through the organ of Corti shows the arrangement of the inner hair cells with respect to three rows of outer hair cells. Florescent markers were applied for myosin VI (red) and nuclear DAPI staining (blue). Scale bars equal 2 μm in panel E, 4 μm in panel. Adapted from [37] with permission.

Fig. 2

Auditory phenotypic characterization of Slc26a4^loop is common to all known Slc26a4 mouse models. A: A representative image of Slc26a4^loop mice shows the typical unsteady gait of the mutant mice that was determined by a panel of vestibular behavioral tests. B: Paint-filled inner ears of Slc26a4^loop P0 mice show a bulged fluid filled compartment, with a prominent volume increase in the cochlea and the vestibular system. C: Auditory brainstem response (ABR) test on 8-week old mice reveals that Slc26a4^loop mutants are profoundly deaf at three frequencies that were tested, 8Khz, 16Khz and 32Khz. An ABR recording for 8Khz is shown (red). D: Cross sections through the cochlea of Slc26a4^loop mice reveals hydrops of the endolymphatic spaces of the scala media (sm), whereas the perilymphatic spaces of the scala vestibuli (sv) and scala tympani (st) are smaller. E: Histogram graphic representation shows the quantified area of the different cochlear compartments as compared between normal and mutant mice. A prominent enlargement of the endolymph field scala media is apparent in Slc26a4^loop mice, at the expense of the smaller scala vestibuli and scala tympani. Scale bars equal 500 μm in panel B and D. Adapted from [42] with permission.

Fig. 3

The sensory maculae of the vestibular system. Out of the five sensory organs of the vestibular system, the utricle and saccule share similar anatomical and morphological structures. Top: A cross section through the utricular sac illustrates the thick sensory epithelium creating the floor of the maculae. The hair cells (green) surrounded by supporting cells (brown) are connected into nerve fibers (yellow) in their basilar pole. The apical surface of the hair cells is composed of hair bundles of actin rich stereocilia (black) that protrude into dense gelatinous matrix. A large number of calcite crystals, known as otoconia (white), are positioned on top of the epithelium. The inertial mass of these mineralized crystals triggers the hair cells by transferring mechanical energy in response to linear movement of the body. The sac is surrounded by epithelial cells and contains endolymphatic fluids. Bottom: SEM showing the hair cells, dense gelatinous matrix and otoconia. Adapted from [42] with permission.

Fig. 4

Calcium oxalate minerals in the Slc26a4^loop vestibular system is a unique phenotype among Slc26a4 mouse models. Left panel: Schematic illustration of the inner ear summarizing the morphological differences of the inner ear stones at the age of 10 months old. The two sensory maculae of the vestibular system, utricle and saccule are indicated (arrows). A, B: SEM images of the utricle and saccule of a wild-type mouse shows the small (~7 μm) otoconia with typical morphology (inset in A and B). C: The spectrum of wild-type otoconia, showing the typical calcite vibrations at 713, 875 and 1422 cm^-1. D, E: Minerals extracted from the utricle and saccule of Slc26a4^loop mutant mice. The utricle minerals are larger relative to wild-type otoconia. In the saccule, a new type of mineral can be found with crystal morphology typical of Ca-oxalate dihydrate (weddelite) crystals. F: Spectrum of the mineral extracted from the saccule in Slc26a4^loop mutant mice shows vibrations at 1324 and 776 cm^-1 that are typical of weddelite crystals. Scale bars equal 50 μm in panel A applies to B, 100 μm in panel D applies to E. Adapted from [42] with permission.