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1.  Heritability of Non-Speech Auditory Processing Skills 
Recent insight into the genetic bases for autism spectrum disorder, dyslexia, stuttering and language disorders suggest that neurogenetic approaches may also reveal at least one etiology of auditory processing disorder (APD). A person with an APD typically has difficulty understanding speech in background noise despite having normal pure-tone hearing sensitivity. The estimated prevalence of APD may be as high as 10% in the pediatric population, yet the causes are unknown and have not been explored by molecular or genetic approaches. The aim of our study was to determine the heritability of frequency and temporal resolution for auditory signals and speech recognition in noise in 96 identical or fraternal twin-pairs, aged 6-11 years. Measures of auditory processing of non-speech sounds included backward masking (temporal resolution), notched noise masking (spectral resolution), pure-tone frequency discrimination (temporal fine structure sensitivity), and nonsense syllable recognition in noise. We provide evidence of significant heritability, ranging from 0.32-0.74, for individual measures of these non-speech based auditory processing skills that are crucial for understanding of spoken language. Identification of specific heritable auditory processing traits such as these serve as a basis to pursue the genetic underpinnings of APD by identifying genetic variants associated with common auditory processing disorders in children and adults.
PMCID: PMC4872837  PMID: 26883091
Auditory processing; heritability; twin study; frequency discrimination; backward masking
2.  Challenges and solutions for gene identification in the presence of familial locus heterogeneity 
European Journal of Human Genetics  2014;23(9):1207-1215.
Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3% (95% confidence interval: 11.9%, 19.9%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.
PMCID: PMC4538203  PMID: 25491636
3.  A mutation of MET, encoding hepatocyte growth factor receptor, is associated with human DFNB97 hearing loss 
Journal of medical genetics  2015;52(8):548-552.
Hearing loss is a heterogeneous neurosensory disorder. Mutations of 56 genes are reported to cause recessively inherited nonsyndromic deafness.
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 LOD score of 4.8). Whole-exome sequencing revealed a novel missense mutation c.2521T>G (p.F841V) in MET, which encodes the receptor for hepatocyte growth factor. The mutation co-segregated 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 programs indicated that p.F841V is likely damaging to MET function.
We identified a missense mutation of MET, encoding the hepatocyte growth factor receptor, as a likely cause of hearing loss in humans.
PMCID: PMC4529444  PMID: 25941349
Hearing Loss; Deafness; Hepatocyte Growth Factor Receptor (HGFR); MET; Pakistan
4.  The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing 
eLife  null;4:e08627.
The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.
eLife digest
Sound is detected by the cochlea, a coiled structure encapsulated within the inner ear of humans and other mammals. Inside this organ, intricate arrays of sensory hair cells are stimulated by sound to generate neural signals that are transmitted to the brain. The ‘hairs’ that give hair cells their name are actually structures called stereocilia that act like antennas to detect sound waves. Damage to these delicate mechanical sensors, through genetic mutations or loud noise, are a significant cause of hearing loss in humans.
A protein called myosin 15 is a molecular motor needed for stereocilia to develop and grow to their normal height. Mutations of this protein cause hereditary deafness in humans. Hair cells produce two versions of myosin 15, which are identical except for one version having an extra region called the N-terminal extension.
Using genetic engineering, Fang et al. created mutant mice that only produce the smaller version of myosin 15 that lack the N-terminal extension. These mutant mice helped reveal that when hair cells are young, they mostly produce the smaller version of myosin 15, and this is sufficient for stereocilia to grow normally. Once hair cells mature however, they switch to producing the larger version of myosin 15 that contains the N-terminal extension. In the mutant mice that lacked the larger version of myosin 15, stereocilia ultimately deteriorate, leaving the hair cells unable to detect sound.
Myosin 15 was previously known to help stereocilia grow, but Fang et al. now show that this protein is also required to maintain stereocilia throughout life. The next challenge is to understand how the N-terminal extension enables myosin 15 to preserve the structure of adult stereocilia, and to investigate whether this activity might be stimulated to prevent hearing loss.
PMCID: PMC4592939  PMID: 26302205
deafness; stereocilia; myosin; actin; inner ear; hearing; mouse
5.  Molecular genetics of MARVELD2 and clinical phenotype in Pakistani and Slovak families segregating DFNB49 hearing loss 
Human genetics  2015;134(4):423-437.
Pathogenic mutations of MARVELD2, encoding tricellulin, a tricelluar tight junction protein, cause autosomal recessive non-syndromic hearing loss (DFNB49) in families of Pakistan and Czech Roma origin. In fact, they are a significant cause of prelingual hearing loss in the Czech Roma, second only to GJB2 variants. Previously, we reported that mice homozygous for p.Arg497* variant of Marveld2 had a broad phenotypic spectrum, where defects were observed in the inner ear, heart, mandibular salivary gland, thyroid gland and olfactory epithelium. The current study describes the types and frequencies of MARVELD2 alleles and clinically reexamines members of DFNB49 families. We found that MARVELD2 variants are responsible for about 1.5% (95% CI: 0.8 – 2.6) of non-syndromic hearing loss in our cohort of 800 Pakistani families. The c.1331+2T>C allele is recurrent. In addition, we identified a novel large deletion in a single family, which appears to have resulted from non-allelic homologous recombination between two similar Alu short interspersed elements. Finally, we observed no other clinical manifestations co-segregating with hearing loss in DFNB49 human families, and hypothesize that the additional abnormalities in the Marveld2 mutant mouse indicates a critical non-redundant function for tricellulin in other organ systems.
PMCID: PMC4561550  PMID: 25666562
Deafness; genetic etiology; inner ear; locus heterogeneity; moderate hearing loss; tight junction; MARVELD2; tricellulin
6.  Genetic Analysis through OtoSeq of Pakistani Families Segregating Prelingual Hearing Loss 
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
Research laboratory
Subjects and Methods
We used three 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 multi-generational 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.
Hearing loss was found to co-segregate with locus-specific STR markers for MYO7A in 32 families, CDH23 in one family and SLC26A4 in one 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.
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.
PMCID: PMC4030297  PMID: 23770805
Hearing loss; Usher syndrome; microdroplet PCR; next generation sequencing; clinical diagnosis; genetic etiology; PDS; MYO7A; CDH23
7.  Challenges and solutions for gene identification in the presence of familial locus heterogeneity 
European Journal of Human Genetics  2014;23(9):1207-1215.
Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3% (95% confidence interval: 11.9%, 19.9%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.
PMCID: PMC4538203  PMID: 25491636
8.  Cone Responses in Usher Syndrome Types 1 and 2 by Microvolt Electroretinography 
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.
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.
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.
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.
Cone electroretinography signals in Usher (USH) syndrome demonstrate poor signal-to-noise ratio when measured by standard methodology. The cycle-by-cycle ERG provides for a more reliable measurement with potential as an indicator of disease progression and as an outcome measure in USH treatment trials.
PMCID: PMC4288141  PMID: 25425308
cone function; microvolt electroretinogram; Usher syndrome; Usher genes
9.  Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear 
Nature Communications  2015;6:6873.
The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips.
Precise control of stereocilia length by auditory hair cells is vital for normal hearing. Drummond et al. follow in real-time the incorporation of actin into these structures and show that while the actin core is remarkably stable, and actin polymerization is limited to their distal tips.
PMCID: PMC4411292  PMID: 25898120
10.  Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome 
PLoS Genetics  2015;11(3):e1005097.
Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.
Author Summary
Mitochondrial respiratory chain (MRC) disease represents a large and heterogeneous group of energy deficiency disorders. Here we report three mutations in NARS2, a mitochondrial asparaginyl-tRNA synthetase, associated with non-syndromic hearing loss (NSHL) and Leigh syndrome in two independent families. Located in the predicted catalytic domain of the protein, missense mutation p.(Val213Phe) results in NSHL (DFNB94) while compound heterozygous mutation (p.Tyr323*; p.Asn381Ser) is leading to Leigh syndrome with auditory neuropathy. In vivo analysis deemed p.Tyr323* mutant protein to be unstable. Co-immunoprecipitation assays show that p.Asn381Ser mutant disrupts the dimerization ability of NARS2. Leigh syndrome patient fibroblasts exhibit a decreased steady-state level of mt-tRNAAsn. In addition, in these cells, the mitochondrial respiratory chain is deficient, including significantly decreased oxygen consumption rates and electron transport chain activities. These functions can be partially restored with over-expression of wild-type NARS2 but not with p.Val213Phe mutant protein. Our study provides new insights into the genes that are necessary for the function of brain and inner ear sensory cells in humans.
PMCID: PMC4373692  PMID: 25807530
11.  A null mutation of mouse Kcna10 causes significant vestibular and mild hearing dysfunction 
Hearing research  2013;300:1-9.
KCNA10 is a voltage gated potassium channel that is expressed in the inner ear. The localization and function of KCNA10 was studied in a mutant mouse, B6-Kcna10TM45, in which the single protein coding exon of Kcna10 was replaced with a beta-galactosidase reporter cassette. Under the regulatory control of the endogenous Kcna10 promoter and enhancers, beta-galactosidase was expressed in hair cells of the vestibular organs and the organ of Corti. KCNA10 expression develops in opposite tonotopic gradients in the inner and outer hair cells. Kcna10TM45 homozygotes display only a mild elevation in pure tone hearing thresholds as measured by auditory brainstem response (ABR), while heterozygotes are normal. However, Kcna10TM45 homozygotes have absent vestibular evoked potentials (VsEPs) or elevated VsEP thresholds with prolonged peak latencies, indicating significant vestibular dysfunction despite the lack of any overt imbalance behaviors. Our results suggest that Kcna10 is expressed primarily in hair cells of the inner ear, with little evidence of expression in other organs. The Kcna10TM45 targeted allele may be a model of human nonsyndromic vestibulopathy.
PMCID: PMC3684051  PMID: 23528307
KCNA10; inner ear; vestibular dysfunction; Kcna10 knockout mouse
13.  Actin-Bundling Protein TRIOBP Forms Resilient Rootlets of Hair Cell Stereocilia That Are Essential for Hearing 
Cell  2010;141(5):786-798.
Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets, but distinct from bundles formed by espin, an actin cross-linker in stereocilia. We generated mutant Triobp mice (TriobpΔex8/Δex8) that are profoundly deaf. Stereocilia of TriobpΔex8/Δex8 mice develop normally, but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere.
PMCID: PMC2879707  PMID: 20510926
14.  DFNB79: reincarnation of a nonsyndromic deafness locus on chromosome 9q34.3 
Genetic analysis of inbred Pakistani family PKDF280, segregating prelingual severe to profound sensorineural hearing loss, provided evidence for a DFNB locus on human chromosome 9q34.3. Co-segregation of the deafness trait with marker D9SH159 was determined by a two-point linkage analysis (LOD score 9.43 at θ=0). Two additional large families, PKDF517 and PKDF741, co-segregate recessive deafness with markers linked to the same interval. Haplotype analyses of these three families refined the interval to 3.84 Mb defined by D9S1818 (centromeric) and D9SH6 (telomeric). This interval overlaps with the previously reported DFNB33 locus whose chromosomal map position has been recently revised and assigned to a new position on chromosome 10p11.23-q21.1. The nonsyndromic deafness locus on chromosome 9q segregating in family PKDF280 was designated DFNB79. We are currently screening the 113 candidate DFNB79 genes for mutations and have excluded CACNA1B, EDF1, PTGDS, EHMT1, QSOX2, NOTCH1, MIR126 and MIR602.
PMCID: PMC2795002  PMID: 19603065
hereditary deafness; DFNB79; DFNB33; Pakistan; chromosome 9q34.3
15.  Phenotypic variability of CLDN14 mutations causing DFNB29 hearing loss in the Pakistani population 
Journal of human genetics  2012;58(2):102-108.
Human hereditary deafness at the DFNB29 autosomal locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetra-membrane spanning that localizes to the apical tight junctions of organ of Corti hair cells and in many other tissues. Typically, the DFNB29 phenotype is characterized by pre-lingual, bi-lateral, sensorineural hearing loss. The goal of this study was to define the identity and frequency of CLDN14 mutations and associated inner ear phenotypes in a cohort of 800 Pakistani families segregating deafness. Hearing loss in 15 multi-generational families was found to co-segregate with CLDN14-linked STR markers. The sequence of the six exons and regions flanking the introns of CLDN14 in these 15 families revealed five likely pathogenic alleles. Two are novel missense substitutions (p.Ser87Ile and p.Ala94Val) while p.Arg81His, p.Val85Asp and p.Met133ArgfsX23 have been reported previously. Haplotype analyses indicate that p.Val85Asp and p.Met133ArgfsX23 are founder mutations. The p.Val85Asp accounts for approximately 67% of the mutant alleles of CLDN14 in our cohort. Combined with previously reported data, CLDN14 mutations were identified in 18 of 800 Pakistani families (2.25%; 95% CI, 1.4-3.5%). Hearing loss in the affected individuals homozygous for CLDN14 mutations varied from moderate to profound. This phenotypic variability may be due to environmental factors (e.g. drug and noise exposure) and/or genetic modifiers.
PMCID: PMC3596117  PMID: 23235333
CLDN14; claudin 14; DFNB29; mild hearing loss; profound deafness; Pakistan
16.  Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells 
PLoS Biology  2013;11(6):e1001583.
Backscatter scanning electron microscopy and conventional whole cell patch-clamp experiments reveal a two-step mechanism for the regeneration of tip links, the crucial element of mechanotransduction machinery in the hair cells of the inner ear.
Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca2+-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.
Author Summary
The inner ear detects sound when stereocilia, the mechanosensory projections on the apical surface of the hair cells, are deflected and tug on tiny extracellular tip links. These links interconnect stereocilia and convey forces to the mechanosensitive transduction channels. Current models postulate a static composition of the tip link with protocadherin 15 (PCDH15) at the link's bottom end and cadherin 23 (CDH23) at the upper end. Tip links are subjected to substantial sound-induced forces. Although hair cells can renew (regenerate) disrupted tip links and restore hearing, the molecular details of this process are unknown. Our study provides mechanistic insight into tip link regeneration. We used backscatter scanning electron microscopy to monitor the distribution of immuno-gold labeled molecular components of the tip links during their re-formation and a conventional whole-cell patch-clamp technique to follow the concomitant recovery of mechano-electrical transduction. According to our data, the mechanotransduction machinery is initially re-established by the formation of functional (mechanotransduction-mediating) links of a previously unknown composition, PCDH15–PCDH15. Transition to the PCDH15–CDH23 composition underlies final maturation of mechanotransduction. This two-step mechanism of tip link regeneration was unexpected. As tip links are continuously stressed by loud sounds and regenerated throughout an organism's life, we provide a plausible molecular mechanism for the life-long maintenance of mechanosensory function in nonregenerating cochlear hair cells.
PMCID: PMC3679001  PMID: 23776407
17.  Actin in hair cells and hearing loss 
Hearing Research  2011;288(1-2):89-99.
Hereditary deafness is genetically heterogeneous such that mutations of many different genes can cause hearing loss. This review focuses on the evidence and implications that several of these deafness genes encode actin-interacting proteins or actin itself. There is a growing appreciation of the contribution of the actin interactome in stereocilia development, maintenance, mechanotransduction and malfunction of the auditory system.
PMCID: PMC3403717  PMID: 22200607
18.  Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome 
Human genetics  2008;124(3):215-223.
Mutations of PCDH15, encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be comprised of 35 exons and encodes a variety of isoforms with 3 to 11 ectodomains (EC), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1 we identified homozygous mutant alleles (1 missense, 1 splice site, 3 nonsense and 2 deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F, which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15, mutations in the introns or regulatory elements of PCDH15, or an additional locus for type I USH at chromosome 10q21.1.
PMCID: PMC2716558  PMID: 18719945
DFNB23; Usher syndrome; protocadherin 15; PCDH15; deafness; retinitis pigmentosa
19.  Mutations in CIB2, a calcium and integrin binding protein, cause Usher syndrome type 1J and nonsyndromic deafness DFNB48 
Nature genetics  2012;44(11):1265-1271.
Sensorineural hearing loss is genetically heterogeneous. Here we report that mutations in CIB2, encoding a Ca2+- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). There is one mutation of CIB2 that is a prevalent cause of DFNB48 deafness in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In rodents, CIB2 is localized in the mechanosensory stereocilia of inner ear hair cells and in retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of Ca2+ binding, CIB2 significantly decreased the ATP-induced Ca2+ responses in heterologous cells, while DFNB48 mutations altered CIB2 effects on Ca2+ responses. Furthermore, in zebrafish and Drosophila, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We show that CIB2 is a new member of the vertebrate Usher interactome.
PMCID: PMC3501259  PMID: 23023331
20.  USH1H, a novel locus for type I Usher syndrome, maps to chromosome 15q22-23 
Clinical genetics  2008;75(1):86-91.
Usher syndrome (USH) is a hereditary disorder associated with sensorineural hearing impairment, progressive loss of vision attributable to retinitis pigmentosa and variable vestibular function. Three clinical types have been described with type I (USH1) being the most severe. To date six USH1 loci have been reported. We ascertained two large Pakistani consanguineous families segregating profound hearing loss, vestibular dysfunction, and retinitis pigmentosa, the defining features of USH1. In these families we excluded linkage of USH to the 11 known USH loci, and subsequently performed a genome-wide linkage screen. We found a novel USH1 locus designated USH1H that mapped to chromosome 15q22-23 in a 4.92 cM interval. This locus overlaps the non-syndromic deafness locus DFNB48 raising the possibility that the two disorders may be caused by allelic mutations.
PMCID: PMC2673543  PMID: 18505454
deafness; DFNB48; retinitis pigmentosa; Usher syndrome; USH1H; vestibular dysfunction; 15q22-23
21.  Expression of cadherin 23 isoforms is not conserved: implications for a mouse model of Usher syndrome type 1D 
Molecular Vision  2009;15:1843-1857.
We compared cadherin 23 (Cdh23) mRNA and protein variants in the inner ear and retina of wild-type and mutant mice and primates to better understand the pleiotropic effects of Cdh23 mutations, and specifically to understand the absence of retinal degeneration in Cdh23 mutant mice.
Semiquantitative real-time PCR was used to compare the level of expression of Cdh23 alternative transcripts in the inner ear and retina of wild-type and homozygous Cdh23v-6J (waltzer) mice. Antibodies generated against CDH23 isoforms were used in immunohistochemistry, immunohistology, electron microscopy, and western blot analyses of mouse and primate inner ear and retina to study the distribution of these isoforms in various cellular compartments.
Cdh23 mRNA alternative splice variants were temporally and spatially regulated in the inner ear and retina. In the mature mouse retina, CDH23 isoforms were broadly expressed in various cellular compartments of the photoreceptor layer. The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells. The longest CDH23 protein isoform, CDH23_V1, appeared by western blotting to be the only one affected by the Cdh23v-6J mutation; it was expressed in the wild-type mouse inner ear, but not in the mouse retina. However, CDH23_V1 was detected in western blot analyses of monkey and human retinas.
The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts. Many of these isoforms continue to be expressed in waltzer mice. The longest CDH23 isoform (CDH23_V1), however, is not expressed in mutant mice and is necessary for normal inner ear function. The longest isoform is expressed in the retinas of primates, but not detected in the mouse retina. This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.
PMCID: PMC2743805  PMID: 19756182
22.  TRPA1-Mediated Accumulation of Aminoglycosides in Mouse Cochlear Outer Hair Cells 
Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin–Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.
PMCID: PMC3214240  PMID: 21879401
gentamicin; ototoxicity; transient receptor potential A1 channel; organ of Corti; reactive oxygen species; 4-hydroxynonenal
23.  Double homozygous waltzer and Ames waltzer mice provide no evidence of retinal degeneration 
Molecular Vision  2008;14:2227-2236.
To determine whether cadherin 23 and protocadherin 15 can substitute for one another in the maintenance of the retina and other tissues in the mouse. Does homozygosity for both v and av mutant alleles (i.e., a double homozygous mouse) cause retinal degeneration or an obvious retinal histopathology?
We generated mice homozygous for both Cdh23v-6J and Pcdh15av-Jfb alleles. The retinal phenotypes of double heterozygous and double homozygous mutant mice were determined by light microscopy and electroretinography (ERG). Histology on 32 different tissues, scanning electron microscopy of organ of Corti hair cells as well as serum biochemical and hematological examinations were evaluated.
ERG waves of double heterozygous and double homozygous mice showed similar shape, growth of the amplitude with intensity, and implicit time for both rod and cone pathway mediated responses. Mice homozygous for both Cdh23v-6J and Pcdh15av-Jfb mutations showed no sign of retinitis pigmentosa or photoreceptor degeneration but, as expected, were deaf and had disorganized hair cell sensory bundles.
The simultaneous presence of homozygous mutant alleles of cadherin 23 and protocadherin 15 results only in deafness, not retinal degeneration or any other additional obvious phenotype of the major organ systems. We conclude that in the mouse cadherin 23 or protocadherin 15 appear not to compensate for one another to maintain the retina.
PMCID: PMC2593751  PMID: 19057657
25.  Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans 
Nature genetics  2008;40(11):1335-1340.
Many proteins necessary for sound transduction have been discovered through positional cloning of genes that cause deafness1–3. In this study, we report that mutations of LRTOMT are associated with profound non-syndromic hearing loss at the DFNB63 locus on human chromosome 11q13.3-q13.4. LRTOMT has two alternative reading frames and encodes two different proteins, LRTOMT1 and LRTOMT2, that are detected by Western blot analyses. LRTOMT2 is a putative methyltransferase. During evolution, novel transcripts can arise through partial or complete coalescence of genes4. We provide evidence that in the primate lineage LRTOMT evolved from the fusion of two neighboring ancestral genes, which exist as separate genes (Lrrc51and Tomt) in rodents.
PMCID: PMC3404732  PMID: 18953341

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