ADAMTS1, MPDZ, MVD, and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

Abstract

Hearing impairment (HI) is a common disorder of sensorineural function with a highly heterogeneous genetic background. Although substantial progress has been made in the understanding of the genetic etiology of hereditary HI, many genes implicated in HI remain undiscovered. Via exome and Sanger sequencing of DNA samples obtained from consanguineous Pakistani families that segregate profound prelingual sensorineural HI, we identified rare homozygous missense variants in four genes (ADAMTS1, MPDZ, MVD, and SEZ6) that are likely the underlying cause of HI. Linkage analysis provided statistical evidence that these variants are associated with autosomal recessive nonsyndromic HI. In silico analysis of the mutant proteins encoded by these genes predicted structural, conformational or interaction changes. RNAseq data analysis revealed expression of these genes in the sensory epithelium of the mouse inner ear during embryonic, postnatal, and adult stages. Immunohistochemistry of the mouse cochlear tissue, further confirmed the expression of ADAMTS1, SEZ6, and MPDZ in the neurosensory hair cells of the organ of Corti, while MVD expression was more prominent in the spiral ganglion cells. Overall, supported by in silico mutant protein analysis, animal models, linkage analysis, and spatiotemporal expression profiling in the mouse inner ear, we propose four new candidate genes for HI and expand our understanding of the etiology of HI.

Fig. 1. Pedigrees and audiometry for affected family members.

A Pakistani pedigrees 4457, 4876, 4140, and 4444 displaying genotypes for the candidate genes for each family member that has an available DNA sample. A star indicates the affected family member whose DNA underwent exome sequencing. Females are represented by circles and males by squares, those individuals with solid symbols have NSHI while those with clear symbols are unaffected. B Air conduction thresholds for affected members of the four families. All individuals had profound bilateral NSHI. Circles with smooth connecting lines represent the right ear and crosses with dotted connecting lines, the left ear. The audiogram of each affected individual is represented by a different color.

Fig. 2. Structural modeling of wild type and mutant proteins.

Panel A: Structural model of the wild type ADAMTS1, MPDZ, MVD, and SEZ6 proteins. Panel B: Interacting bond of the wild type proteins for the four candidate genes. Panel C: Mutant type proteins highlighting the change due to the variant. The variants ADAMTS1 [p.(Ser135Ala)] and MPDZ [p.(Pro775Leu)] may result in a difference in interaction pattern with the solvent molecules. As the position of the variant residue is at the loop region, they do not involve a direct interaction, but the native fold of the protein may be affected. MVD [p.Pro379His] mutated protein shows a difference in interacting bond distance in the mutant type from the wild type and may perturb the amino acid side chain. The SEZ6 [p.Val698Ile] mutant protein displayed less interaction distance due to substitution of Val to Ile at position 698, while the SEZ6 wild type protein has a weak H-bond and hydrophobic interaction with Ile635 and Trp609. This variation could possibly change the orientation of the amino acid residue. Structures are displayed as ribbon while residue is represented by stick model. The structure illustrations were created with the PyMOL program. Black dotted lines represent hydrogen bonds.

Fig. 3. RNA expression of the four candidate genes in the mouse inner ear cells at developing and adult stages.

A RNA expression of the four candidate genes in the cochlea and utricle during mouse development. RNA-sequencing data of hair cells (GFP+) and surrounding cells (GFP−) from the cochleae and utricles of mice expressing EGFP under the Pou4f3 promoter. Data were collected at four developmental stages: E16, P0, P4, and P7 and RNA expression is represented as normalized counts. Adamts1, Mpdz, Mvd, and Sez6 were expressed in the hair cells and surrounding cells of the cochlea and utricle during the E16, P0, P4, and P7 stages. B RNA expression of the four candidate genes in the adult inner ear cells. The figure represents expression of the genes of interest in Deiters’ cells (Deiters), Pillar cells (Pillar), Inner Hair Cells (IHC), and Outer Hair Cells (OHC) of adult CBA/J mice. The four candidate genes Adamts1, Mpdz, Mvd, and Sez6 were expressed in the IHC, OHCs, and Deiter’s and Pillar cells. The y-axis represents the gene expression normalized to transcripts per million (TPM). The dataset was obtained from the GEO database (accession number GSE111347).

Fig. 4. Immunostaining of Adamts1, Mpdz, Mvd, and Sez6 in the inner ear of wildtype mice.

A Whole mount immunostaining of Adamts1, Mpdz, and Sez6 in wildtype mice at P12. Immunoreactivity was visualized with a fluorescently labeled secondary antibody (red) and F-actin was stained with phalloidin 488 nm (green). Immunolabeling of Adamts1 and Sez6 is observed in stereocilia as well as cytoplasm of outer hair cells. Mpdz is observed in the cytoplasmic region of hair cells. B Immunostaining of the organ of Corti at the apical, medial and basal turns of the cochlea at P4. C Immunostaining of Mvd in wildtype mice at P1, P4, and P28. Immunoreactivity of Mvd was visualized with a fluorescently labeled secondary antibody (green), F-actin was stained with rhodamine-phalloidin (red) and nuclear bodies were stained with DAPI (blue). The anti-neurofilament (NF-200, purple) was used to mark the neurons. Immunolabeling of Mvd is observed in spiral ganglion cells (SG). HP Habenula perforata, OC Organ of Corti.