De novo variants in GREB1L are associated with non-syndromic inner ear malformations and deafness

Abstract

Congenital inner ear malformations affecting both the osseous and membranous labyrinth can have a devastating impact on hearing and language development. With the exception of an enlarged vestibular aqueduct, non-syndromic inner ear malformations are rare, and their underlying molecular biology has thus far remained understudied. To identify molecular factors that might be important in the developing inner ear, we adopted a family-based trio exome sequencing approach in young unrelated subjects with severe inner ear malformations. We identified two previously unreported de novo loss-of-function variants in GREB1L [c.4368G>T;p.(Glu1410fs) and c.982C>T;p.(Arg328*)] in two affected subjects with absent cochleae and eighth cranial nerve malformations. The cochlear aplasia in these affected subjects suggests that a developmental arrest or problem at a very early stage of inner ear development exists, e.g., during the otic pit formation. Craniofacial Greb1l RNA expression peaks in mice during this time frame (E8.5). It also peaks in the developing inner ear during E13-E16, after which it decreases in adulthood. The crucial function of Greb1l in craniofacial development is also evidenced in knockout mice, which develop severe craniofacial abnormalities. In addition, we show that Greb1l^-/- zebrafish exhibit a loss of abnormal sensory epithelia innervation. An important role for Greb1l in sensory epithelia innervation development is supported by the eighth cranial nerve deficiencies seen in both affected subjects. In conclusion, we demonstrate that GREB1L is a key player in early inner ear and eighth cranial nerve development. Abnormalities in cochleovestibular anatomy can provide challenges for cochlear implantation. Combining a molecular diagnosis with imaging techniques might aid the development of individually tailored therapeutic interventions in the future.

Fig. 1. Genetic and imaging data of the families enrolled in this study

A. Pedigree of family 1 with Sanger sequencing traces. B. Pedigree of family 2 with Sanger sequencing traces. Both affected subjects have a de novo loss-of-function mutation in the GREB1L gene. C. Affected subject one’s MRI shows dysplastic SCCs, enlarged vestibules, absent cochlea on the right (yellow arrowhead), and incomplete partition type I (IP-I) on the left (yellow arrow). The diameters of his IACs were too narrow to determine the neural contents on imaging (red arrows). D. Affected subject two’s MRI shows bilateral absent cochleae, enlarged vestibules, and dysplastic SCCs (yellow arrowheads). E. Oblique cross-sectional imaging of the IAC on MRI showed that he has two nerves in the right IAC (shown, orange arrows), and one on the left (not shown), normal = 4.

Fig. 2. Greb1l RNA expression in various craniofacial tissues and the inner ear during the development of the mouse

A/B. RNA expression data from various laser capture microdissected (LCM) tissues during early mouse craniofacial development (Brunskill et al. 2014). A. RNA-Seq profiling shows that Greb1l is expressed with preference during E8.5 in early development. This expression decreases over E9.5 and E10.5. In the above panel, tissue expression is plotted together with Pax2 and Zic2 that show a similar expression profile, both important genes in mouse inner ear development. A higher expression is observed in the craniofacial mesoderm, the caudal brain neural epithelium, and flanking neuroepithelium, including floorplate and more dorsal non-floor plate compared to other craniofacial tissues. The lower panel shows the average Greb1l expression per developmental stage over all tissues in TPM values to illustrate decreased expression over time. Blue = high expression; Green=intermediate expression; Yellow=low expression. B. Micro-array RNA expression analysis of mouse craniofacial tissues during early development demonstrates a widespread and non-compartmentalized expression of Greb1l. This in contrast to Six1, 2 and Sox2, but similar to Fgf3 and Wnt1 (given as an example). The highest expression is also seen during E8.5, similar to the RNA-seq data in A. An intermediate expression in the otic vesicle is seen at E9.5. Blue = high expression; Green=intermediate expression; Yellow=low expression. Greb1l_1 and Greb1l_2 indicate the two different probes used for greb1l on the microarray respectively (10453797, 10453811). For A/B data were downloaded and further analyzed from study GSE55967 (Brunskill et al. 2014) from the Gene Expression Omnibus (GEO) database (National Institutes of Health). C. Micro-array RNA expression data of Greb1l in Spiral and vestibular ganglion neurons collected at six developmental stages (Lu et al. 2011). This shows that Greb1l (probe set 1439341) is expressed in the inner ear ganglia during development and upregulated during E13-E16 after which it slowly decreases in expression. D. RNA-Seq data from cochlea and utricles from mice expressing EGFP under the Pou4f3 promoter (hair cell marker). Hair cells (GFP+) and surrounding cells (GFP-) were separately collected using FACS for RNA extraction (Scheffer et al. 2015). Greb1l is expressed in both the hair cells and surrounding cells of the utricle and cochlea with the highest expression during E16. The Y-axis represents the normalized read counts from the DEseq package. C/D Data were obtained from the Shared Harvard Inner Ear Laboratory Database (SHIELD) database (Lu et al. 2011; Shen et al. 2015; Scheffer et al. 2015), and further analyzed here. Craniofacial tissue codes: B. NE: Neuroepithilium; NC: Neural Crest; PM: Paraxial mesoderm; CNE (E8.5): caudal neural epithelium; FPNE: floor plate neural epithelium; ME: mesenchyme; NFPNE: non-floor plate neural epithelium; CM: Cranial Mesenchyme; EE: epidermal ectoderm; MANA: mandibular arch; MAXA: maxillary arch; OTICV: Otic vesicle; CNM: central neuroepithelium midline; CNFFE: control neuroepithelium not flanking facial emincences; LNE: lateral nasal eminence; MANAE: mandibular arch epidermal ectoderm; MAXAE: maxillary arch epidermal ectoderm; MNP: medial nasal prominence; NLNP: neuroepithelium underlying lateral nasal prominence; NMNP: neuroepithelium underlying medial nasal prominence; OP: olfactory pit; RP: rathke’s pouch; LNP: lateral nasal prominence; CNE (E10.5): central neural epithelium; CDNE: dorsal control neural epithelium; LPNE: lateral prominence neural epithelium.

Fig 3. Greb1l^−/− zebrafish show sensory epithelia innervation abnormalities

A. The stereotyped positions of lateral line neuromasts (red circles), the cristae of the inner ear (purple), and their nerves (green) in 72 hpf zebrafish (Raible and Kruse 2000). B. Loss of the anterior cristae nerve (nAC), and misrouting of the posterior nerve (nPst) of the occipital neuromast (OC) in a portion of greb1l mutant zebrafish. C. Wildtype zebrafish: arrows identifying normal nPst and nAC. D. Mutant greb1l zebrafish representing both abnormalities (absent nAC and abnormal nPst pathway). Blue: Hoechst, Green: Acetylated Tubulin.