Proposed therapy, developed in a Pcdh15-deficient mouse, for progressive loss of vision in human Usher syndrome.

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9-cis retinal improved ERG amplitudes in Pcdh15R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients.

Vestibular phenotype-genotype correlation in a cohort of 90 patients with Usher syndrome.

Usher syndrome has been historically categorized into one of three classical types based on the patient phenotype. However, the vestibular phenotype does not infallibly predict which Usher genes are mutated. Conversely, the Usher syndrome genotype is not sufficient to reliably predict vestibular function. Here we present a characterization of the vestibular phenotype of 90 patients with clinical presentation of Usher syndrome (59 females), aged 10.9 to 75.5 years, with genetic variants in eight Usher syndromic genes and expand the description of atypical Usher syndrome. We identified unexpected horizontal semicircular canal reactivity in response to caloric and rotational stimuli in 12.5% (3 of 24) and 41.7% (10 of 24), respectively, of our USH1 cohort. These findings are not consistent with the classical phenotypic definition of vestibular areflexia in USH1. Similarly, 17% (6 of 35) of our cohort with USH2A mutations had saccular dysfunction as evidenced by absent cervical vestibular evoked myogenic potentials in contradiction to the classical assumption of normal vestibular function. The surprising lack of consistent genotypic to vestibular phenotypic findings as well as no clear vestibular phenotypic patterns among atypical USH cases, indicate that even rigorous vestibular phenotyping data will not reliably differentiate the three USH types.

Noncoding Microdeletion in Mouse Hgf Disrupts Neural Crest Migration into the Stria Vascularis, Reduces the Endocochlear Potential, and Suggests the Neuropathology for Human Nonsyndromic Deafness DFNB39.

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival, and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an overexpression of HGF, causes neurosensory deafness. In humans, noncoding variants in HGF are associated with nonsyndromic deafness DFNB39 However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10 bp deletion (del10) in Hgf Male and female mice homozygous for del10 exhibit moderate-to-profound hearing loss at 4 weeks of age as measured by tone burst auditory brainstem responses. The wild type (WT) 80 mV endocochlear potential was significantly reduced in homozygous del10 mice compared with WT littermates. In normal cochlea, endocochlear potentials are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that, in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in Hgf leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and a reduction in the EP, recapitulating human DFNB39 nonsyndromic deafness.SIGNIFICANCE STATEMENT Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously, we reported that human deafness DFNB39 is associated with noncoding variants in the 3'UTR of a short isoform of HGF encoding hepatocyte growth factor. For normal hearing, HGF levels must be fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a Hgf mutant mouse with a small 10 bp deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

A mutation of MET, encoding hepatocyte growth factor receptor, is associated with human DFNB97 hearing loss.

BACKGROUND: Hearing loss is a heterogeneous neurosensory disorder. Mutations of 56 genes are reported to cause recessively inherited non-syndromic deafness. OBJECTIVE: We sought to identify the genetic lesion causing hearing loss segregating in a large consanguineous Pakistani family. METHODS AND RESULTS: Mutations of GJB2 and all other genes reported to underlie recessive deafness were ruled out as the cause of the phenotype in the affected members of the participating family. Homozygosity mapping with a dense array of one million SNP markers allowed us to map the gene for recessively inherited severe hearing loss to chromosome 7q31.2, defining a new deafness locus designated DFNB97 (maximum logarithm of the odds score of 4.8). Whole-exome sequencing revealed a novel missense mutation c.2521T>G (p.F841V) in MET (mesenchymal epithelial transition factor), which encodes the receptor for hepatocyte growth factor. The mutation cosegregated with the hearing loss phenotype in the family and was absent from 800 chromosomes of ethnically matched control individuals as well as from 136 602 chromosomes in public databases of nucleotide variants. Analyses by multiple prediction programmes indicated that p.F841V likely damages MET function. CONCLUSIONS: We identified a missense mutation of MET, encoding the hepatocyte growth factor receptor, as a likely cause of hearing loss in humans.

Cone responses in Usher syndrome types 1 and 2 by microvolt electroretinography.

PURPOSE: Progressive decline of psychophysical cone-mediated measures has been reported in type 1 (USH1) and type 2 (USH2) Usher syndrome. Conventional cone electroretinogram (ERG) responses in USH demonstrate poor signal-to-noise ratio. We evaluated cone signals in USH1 and USH2 by recording microvolt level cycle-by-cycle (CxC) ERG. METHODS: Responses of molecularly genotyped USH1 (n = 18) and USH2 (n = 24) subjects (age range, 15-69 years) were compared with those of controls (n = 12). A subset of USH1 (n = 9) and USH2 (n = 9) subjects was examined two to four times over 2 to 8 years. Photopic CxC ERG and conventional 30-Hz flicker ERG were recorded on the same visits. RESULTS: Usher syndrome subjects showed considerable cone flicker ERG amplitude losses and timing phase delays (P < 0.01) compared with controls. USH1 and USH2 had similar rates of progressive logarithmic ERG amplitude decline with disease duration (-0.012 log µV/y). Of interest, ERG phase delays did not progress over time. Two USH1C subjects retained normal response timing despite reduced amplitudes. The CxC ERG method provided reliable responses in all subjects, whereas conventional ERG was undetectable in 7 of 42 subjects. CONCLUSIONS: Cycle-by-cycle ERG showed progressive loss of amplitude in both USH1 and USH2 subjects, comparable to that reported with psychophysical measures. Usher subjects showed abnormal ERG response latency, but this changed less than amplitude with time. In USH syndrome, CxC ERG is more sensitive than conventional ERG and warrants consideration as an outcome measure in USH treatment trials.

Genetic analysis through OtoSeq of Pakistani families segregating prelingual hearing loss.

OBJECTIVE: To identify the genetic cause of prelingual sensorineural hearing loss in Pakistani families using a next-generation sequencing (NGS)-based mutation screening test named OtoSeq. STUDY DESIGN: Prospective study. SETTING: Research laboratory. SUBJECTS AND METHODS: We used 3 fluorescently labeled short tandem repeat (STR) markers for each of the known autosomal recessive nonsyndromic (DFNB) and Usher syndrome (USH) locus to perform a linkage analysis of 243 multigenerational Pakistani families segregating prelingual hearing loss. After genotyping, we focused on 34 families with potential linkage to MYO7A, CDH23, and SLC26A4. We screened affected individuals from a subset of these families using the OtoSeq platform to identify underlying genetic variants. Sanger sequencing was performed to confirm and study the segregation of mutations in other family members. For novel mutations, normal hearing individuals from ethnically matched backgrounds were also tested. RESULTS: Hearing loss was found to co-segregate with locus-specific STR markers for MYO7A in 32 families, CDH23 in 1 family, and SLC26A4 in 1 family. Using the OtoSeq platform, a microdroplet PCR-based enrichment followed by NGS, we identified mutations in 28 of the 34 families including 11 novel mutations. Sanger sequencing of these mutations showed 100% concordance with NGS data and co-segregation of the mutant alleles with the hearing loss phenotype in the respective families. CONCLUSION: Using NGS-based platforms like OtoSeq in families segregating hearing loss will contribute to the identification of common and population-specific mutations, early diagnosis, genetic counseling, and molecular epidemiology.

Perrault syndrome is caused by recessive mutations in CLPP, encoding a mitochondrial ATP-dependent chambered protease.

Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.

Allelic hierarchy of CDH23 mutations causing non-syndromic deafness DFNB12 or Usher syndrome USH1D in compound heterozygotes.

BACKGROUND: Recessive mutant alleles of MYO7A, USH1C, CDH23, and PCDH15 cause non-syndromic deafness or type 1 Usher syndrome (USH1) characterised by deafness, vestibular areflexia, and vision loss due to retinitis pigmentosa. For CDH23, encoding cadherin 23, non-syndromic DFNB12 deafness is associated primarily with missense mutations hypothesised to have residual function. In contrast, homozygous nonsense, frame shift, splice site, and some missense mutations of CDH23, all of which are presumably functional null alleles, cause USH1D. The phenotype of a CDH23 compound heterozygote for a DFNB12 allele in trans configuration to an USH1D allele is not known and cannot be predicted from current understanding of cadherin 23 function in the retina and vestibular labyrinth. METHODS AND RESULTS: To address this issue, this study sought CDH23 compound heterozygotes by sequencing this gene in USH1 probands, and families segregating USH1D or DFNB12. Five non-syndromic deaf individuals were identified with normal retinal and vestibular phenotypes that segregate compound heterozygous mutations of CDH23, where one mutation is a known or predicted USH1 allele. CONCLUSIONS: One DFNB12 allele in trans configuration to an USH1D allele of CDH23 preserves vision and balance in deaf individuals, indicating that the DFNB12 allele is phenotypically dominant to an USH1D allele. This finding has implications for genetic counselling and the development of therapies for retinitis pigmentosa in Usher syndrome. ACCESSION NUMBERS: The cDNA and protein Genbank accession numbers for CDH23 and cadherin 23 used in this paper are AY010111.2 and AAG27034.2, respectively.

Usher syndrome: hearing loss with vision loss.

Usher syndrome (USH) is a clinically heterogeneous condition characterized by sensorineural hearing loss, progressive retinal degeneration, and vestibular dysfunction. A minimum test battery is described as well as additional clinical evaluations that would provide comprehensive testing of hearing, vestibular function, and visual function in USH patients. USH is also genetically heterogeneous. At least nine genes have been identified with mutations that can cause USH. The proteins encoded by these genes are thought to interact with one another to form a network in the sensory cells of the inner ear and retina.

Noncoding mutations of HGF are associated with nonsyndromic hearing loss, DFNB39.

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3' untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.

Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development.

Mutant alleles of the gene encoding cadherin 23 are associated with Usher syndrome type 1 (USH1D), isolated deafness (DFNB12) in humans, and deafness and circling behavior in waltzer (v) mice. Stereocilia of waltzer mice are disorganized and the kinocilia misplaced, indicating the importance of cadherin 23 for hair bundle development. Cadherin 23 was localized to developing stereocilia and proposed as a component of the tip link. We show that, during development of the inner ear, cadherin 23 is initially detected in centrosomes at E14.5, then along the length of emerging stereocilia, and later becomes concentrated at and subsequently disappears from the tops of stereocilia. In mature vestibular hair bundles, cadherin 23 is present along the kinocilium and in the region of stereocilia-kinocilium bonds, a pattern conserved in mammals, chicks, and frogs. Cadherin 23 is also present in Reissner's membrane (RM) throughout development. In homozygous v(6J) mice, a reported null allele, cadherin 23 was absent from stereocilia, but present in kinocilia, RM, and centrosomes. We reconciled these results by identifying two novel isoforms of Cdh23 unaffected in sequence and expression by the v(6J) allele. Our results suggest that Cdh23 participation in stereocilia links may be restricted to developing hair bundles.

Modification of human hearing loss by plasma-membrane calcium pump PMCA2.

Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.