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Bacteria in humans play an important role in health and disease. Considerable emphasis has been placed in understanding the role of bacteria in host-microbiome interkingdom communication. Here we show that serotonin, responsible for mood in the brain and motility in the gut, can also act as a bacterial signaling molecule for pathogenic bacteria. Specifically, we found that serotonin acts as an interkingdom signaling molecule via quorum sensing and that it stimulates the production of bacterial virulence factors and increases biofilm formation in vitro and in vivo in a novel mouse infection model. This discovery points out at roles of serotonin both in bacteria and humans, and at phenotypic implications not only manifested in mood behavior but also in infection processes in the host. Thus, regulating serotonin concentrations in the gut may provide with paradigm shifting therapeutic approaches., Highlights • Serotonin can act as a bacterial quorum sensing molecule through the LasR quorum sensing pathway. • Serotonin stimulates quorum sensing-dependent virulence factor and biofilm production in Pseudomonas aeruginosa. • Serotonin exacerbates Pseudomonas aeruginosa infection in vivo. Bacteria play many roles in human health and disease, thus investigating host-microbiome communication is of great importance. To this end, our research demonstrates that serotonin, a neurotransmitter responsible for mood in the brain and motility in the gut, can act as a bacterial communication molecule for pathogenic bacteria, specifically Pseudomonas aeruginosa. Serotonin increases Pseudomonas virulence factor and biofilm production in vitro and enhances virulence in infected mice. These findings are beneficial in understanding the impact of the microbiome on human health and may open the door for novel treatments for human disease.

The unparalleled heterogeneity in genetic causes of hearing loss along with remarkable differences in prevalence of causative variants among ethnic groups makes single gene tests technically inefficient. Although hundreds of genes have been reported to be associated with nonsyndromic hearing loss (NSHL), GJB2, GJB6, SLC26A4, and mitochondrial (mt) MT-RNR1 and MTTS are the major contributors. In order to provide a faster, more comprehensive and cost effective assay, we constructed a DNA fluidic array, CapitalBioMiamiOtoArray, for the detection of sequence variants in five genes that are common in most populations of European descent. They consist of c.35delG, p.W44C, p.L90P, c.167delT (GJB2); 309kb deletion (GJB6); p.L236P, p.T416P (SLC26A4); and m.1555A>G, m.7444G>A (mtDNA). We have validated our hearing loss array by analyzing a total of 160 DNAs samples. Our results show 100% concordance between the fluidic array biochip-based approach and the established Sanger sequencing method, thus proving its robustness and reliability at a relatively low cost.

Phosphoribosylpyrophosphate synthetase 1(PRPS1) codes for PRS-I enzyme that catalyzes the first step of nucleotide synthesis.PRPS1gene mutations have been implicated in a number of human diseases. Recently, new mutations inPRPS1have been identified that have been associated with novel phenotypes like diabetes insipidus expanding the spectrum ofPRPS1-related diseases. The purpose of this review is to evaluate current literature onPRPS1-related syndromes and summarize potential therapies. The overexpression ofPRPS1results in PRS-I superactivity resulting in purine overproduction. Patients with PRS-I superactivity demonstrate uric acid overproduction, hypotonia, ataxia, neurodevelopment abnormalities, and postlingual hearing impairment. On the other hand, decreased activity leads to X-linked nonsyndromic sensorineural deafness (DFNX-2), Charcot-Marie-Tooth disease-5 (CMTX5), and Arts syndrome depending on the residual activity of PRS-I. Mild PRS-I deficiency (DFNX-2) results in non-syndromic progressive hearing loss whereas moderate PRS-I deficiency (CMTX5) and severe PRS-I deficiency (Arts syndrome) present with peripheral or optic neuropathy, prelingual progressive sensorineural hearing loss, and central nervous system impairment. Currently, purine replacement via S-adenosylmethionine (SAM) supplementation in patients with Arts syndrome appears to improve their condition. This suggests that SAM supplementation can alleviate symptoms ofPRPS1deficient patients and open new avenues of therapeutic intervention.

Hearing loss is the most common sensory deficit in humans. We show that a point mutation in DCDC2 (DCDC2a), a member of doublecortin domain-containing protein superfamily, causes non-syndromic recessive deafness DFNB66 in a Tunisian family. Using immunofluorescence on rat inner ear neuroepithelia, DCDC2a was found to localize to the kinocilia of sensory hair cells and the primary cilia of nonsensory supporting cells. DCDC2a fluorescence is distributed along the length of the kinocilium with increased density toward the tip. DCDC2a-GFP overexpression in non-polarized COS7 cells induces the formation of long microtubule-based cytosolic cables suggesting a role in microtubule formation and stabilization. Deafness mutant DCDC2a expression in hair cells and supporting cells causes cilium structural defects, such as cilium branching, and up to a 3-fold increase in length ratios. In zebrafish, the ortholog dcdc2b was found to be essential for hair cell development, survival and function. Our results reveal DCDC2a to be a deafness gene and a player in hair cell kinocilia and supporting cell primary cilia length regulation likely via its role in microtubule formation and stabilization.

The steroid hormone 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] plays an important role in maintaining a balance in calcium and bone metabolism. To study the effects of 1α,25-(OH)2D3 on osteoclast (OC) formation and bone resorption, OC differentiation was induced in bone marrow-derived mononuclear cells from Wistar rats with the addition of macrophage colony stimulating factor and receptor activator for nuclear factor-κB ligand in vitro. Cells were then treated with 1α,25-(OH)2D3 at 10−9, 10−8 or 10−7 mol/l. OCs were identified using tartrate-resistant acid phosphatase staining and activity was monitored in the absorption lacunae by scanning electron microscopy. Expression levels of functional proteins associated with bone absorption, namely carbonic anhydrase II, cathepsin K and matrix metalloproteinase-9 were evaluated by western blot analysis. The results showed that 1α,25-(OH)2D3 inhibited the formation and activation of OCs in a dose-dependent manner and downregulated the expression levels of bone absorption-associated proteins.

In a large consanguineous Turkish kindred with recessive nonsyndromic, prelingual, profound hearing loss, we identified in the gene FAM65B (MIM611410) a splice site mutation (c.102-1G>A) that perfectly cosegregates with the phenotype in the family. The mutation leads to exon skipping and deletion of 52-amino acid residues of a PX membrane localization domain. FAM65B is known to be involved in myotube formation and in regulation of cell adhesion, polarization, and migration. We show that wild-type Fam65b is expressed during embryonic and postnatal development stages in murine cochlea, and that the protein localizes to the plasma membranes of the stereocilia of inner and outer hair cells of the inner ear. The wild-type protein targets the plasma membrane, whereas the mutant protein accumulates in cytoplasmic inclusion bodies and does not reach the membrane. In zebrafish, knockdown of fam65b leads to significant reduction of numbers of saccular hair cells and neuromasts and to hearing loss. We conclude that FAM65B is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing.

Inherited deafness has been shown to have high genetic heterogeneity. For many decades, linkage analysis and candidate gene approaches have been the main tools to elucidate the genetics of hearing loss. However, this associated study design is costly, time-consuming, and unsuitable for small families. This is mainly due to the inadequate numbers of available affected individuals, locus heterogeneity, and assortative mating. Exome sequencing has now become technically feasible and a cost-effective method for detection of disease variants underlying Mendelian disorders due to the recent advances in next-generation sequencing (NGS) technologies. In the present study, we have combined both the Deafness Gene Mutation Detection Array and exome sequencing to identify deafness causative variants in a large Chinese composite family with deaf by deaf mating. The simultaneous screening of the 9 common deafness mutations using the allele-specific PCR based universal array, resulted in the identification of the 1555A>G in the mitochondrial DNA (mtDNA) 12S rRNA in affected individuals in one branch of the family. We then subjected the mutation-negative cases to exome sequencing and identified novel causative variants in the MYH14 and WFS1 genes. This report confirms the effective use of a NGS technique to detect pathogenic mutations in affected individuals who were not candidates for classical genetic studies.

Mutations in four members of the connexin gene family have been shown to underlie distinct genetic forms of deafness, including GJB2 [connexin 26 (Cx26)], GJB3 (Cx31), GJB6 (Cx30) and GJB1 (Cx32). We have found that alterations in a fifth member of this family, GJA1 (Cx43), appear to cause a common form of deafness in African Americans. We identified two different GJA1 mutations in four of 26 African American probands. Three were homozygous for a Leu-->Phe substitution in the absolutely conserved codon 11, whereas the other was homozygous for a Val-->Ala transversion at the highly conserved codon 24. Neither mutation was detected in DNA from 100 control subjects without deafness. Cx43 is expressed in the cochlea, as is demonstrated by PCR amplification from human fetal cochlear cDNA and by RT-PCR of mouse cochlear tissues. Immunohistochemical staining of mouse cochlear preparations showed immunostaining for Cx43 in non-sensory epithelial cells and in fibrocytes of the spiral ligament and the spiral limbus. To our knowledge this is the first alpha connexin gene to be associated with non-syndromic deafness. Cx43 must also play a critical role in the physiology of hearing, presumably by participating in the recycling of potassium to the cochlear endolymph.

Usher syndrome (USH) is characterised by hearing impairment and progressive pigmentary retinopathy. USH can be divided into three subtypes based on the severity and progression of the major clinical findings. These subtypes are genetically heterogeneous, with at least six loci for USH1, three for USH2 and one for USH3. In the present study, five unrelated consanguineous families with USH1 were analysed for linkage to markers flanking the six USH1 loci. Two of these families, one Pakistani and one Turkish, demonstrated linkage to the USH1D locus. In another family, haplotype segregation was consistent with linkage to USH1C. The remaining families were not linked to any of the six USH1 loci, providing support for the existence of at least one additional USH1 locus. Analysis of these two new USH1D families allowed us to narrow the USH1D candidate region to a 7.3-cM interval with a telomeric flanking marker at D10S1752. Comparison of the affected haplotypes in our Pakistani family with the original Pakistani USH1D family yielded no evidence for a founder effect. The identification of two additional affected families suggests that the USH1D may be a more common form of USH1 than originally suspected. The USH1D (CDH23) gene has recently been cloned. Mutation analysis has shown two different CDH23 mutations in the two Pakistani USH1D families studied, which confirmed our finding that there was no evidence for a founder effect by haplotype analysis. The interesting correlations between genotype and phenotype in CDH23 are also summarised.

Although more than 50% of recessive non-syndromic deafness is attributed to mutations in the connexin 26 (Cx26) gene, only a few reported families have shown dominant transmission of the trait. The W44C mutation was originally reported in two families from the same geographic region of France, which exhibited dominant non-syndromic hearing loss. In this report, we describe a third family with early-onset severe-to-profound non-syndromic hearing loss segregating with the W44C mutation. Our observation places W44C among recurrent mutations in the Cx26 gene and emphasizes the importance of screening for this as well as other Cx26 mutations in autosomal dominant families.

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E. Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14-15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18. The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Mutations in the GJB3 gene encoding connexin31 (Cx31) can cause a dominant non-syndromic form of hearing loss (DFNA2). To determine whether mutations at this locus can also cause recessive non-syndromic deafness, we screened 25 Chinese families with recessive deafness and identified in two families affected individuals who were compound heterozygotes for Cx31 mutations. The three affected individuals in the two families were born to non-consanguineous parents and had an early onset bilateral sensorineural hearing loss. In both families, differing SSCP patterns were observed in affected and unaffected individuals. Sequence analysis in both families demonstrated an in-frame 3 bp deletion (423-425delATT) in one allele, which leads to the loss of an isoleucine residue at codon 141, and a 423A-->G transversion in the other allele, which creates an Ile-->Val substitution at codon 141 (I141V). Neither of these two mutations was detected in DNA from 100 unrelated control subjects. The altered isoleucine residue lies within the third conserved alpha-helical transmembrane domain (M3), which is critical for the formation of the wall of the gap junction pore. Both the deletion of the isoleucine residue 141 and its substitution to valine in the two families could alter the structure of M3, and impair the function of the gap junction. The present data demonstrate that, like mutations in connexin26, mutations in Cx31 can lead to both recessive and dominant forms of non-syndromic deafness.

This work was supported by the Medical Research Council, by Defeating Deafness of the United Kingdom, by European Community grant CT96-1324, and by National Institutes of Health R01 grant DC02530. C.H. acknowledges support from the British Retinitis Pigmentosa Society and The Birmingham Eye Foundation.

This work was supported by the Medical Research Council (U.K.), Defeating Deafness—the Hearing Research Trust, and SENSE and by European Community grant CT96-1324. C.H. acknowledges support from the British Retinitis Pigmentosa Society and the Birmingham Eye Foundation.

The shaker-1 (Myo7a) mouse deafness locus is encoded by an unconventional myosin gene: myosin VIIA [Gibson, Walsh, Mburu, Varela, Brown, Antonio, Biesel, Steel and Brown (1995) Nature (London) 374, 62-64]. The myosin VIIA gene is expressed in hair cells in the cochlea, where it is thought to function in the development of the critical neuroepithelium where auditory transduction takes place. In order to understand better the function of myosin VIIA, we have determined the complete sequence of the mouse myosin VIIA cDNA and employed the wild-type sequence for mutational analysis of a number of shaker-1 alleles. Analysis of the mouse myosin VIIA tail sequence demonstrates a large internal repeat with regions of similarity to myosins IV, X and XII as well as members of the band 4.1 family. In addition, the myosin VIIA repeats are similar along their entire length to a tail domain from a plant kinesin. The mouse myosin VIIA tail also contains a putative Src homology 3 (SH3) domain. Along with three previously reported shaker-1 mutations, mutations for seven shaker-1 alleles in total have now been identified. The mutational changes have been analysed in terms of their predicted effect on both myosin motor head and tail domain function and the predictions related to the known phenotypes of the shaker-1 alleles. Five of the mutations lie in the motor head, and analysis of their likely effect on myosin head structure correlates well with the known severity of the shaker-1 alleles. Of the two mutations in the tail, one is a missense mutation within the kinesin and myosin IV, X and XII homology domains that substitutes a conserved amino acid and leads to a severe deafness phenotype. This and other data suggest that myosin VIIA may have properties of a myosin-motor-kinesin-tail hybrid and be involved in membrane turnover within the actin-rich environment of the apical hair cell surface.

Usher syndrome is recognized as the most frequent cause of hereditary deaf-blindness. Usher syndrome type I (USH1), the most severe form of the disease, is characterized by profound congenital sensorineural deafness, constant vestibular dysfunction, and retinitis pigmentosa of prepubertal onset. This form is genetically heterogeneous and five loci (USH1A-E) have been mapped thusfar. However, only the gene responsible for USH1 B (which accounts for approximately 75% of USH1 cases) has been characterized. It encodes a long-tailed unconventional myosin, myosin VIIA, with a predicted 2215 amino acid sequence. Primers covering the complete myosin VIIA coding sequence as well as the 3' non coding sequence were designed, allowing direct sequence analysis of each of the 48 coding exons and flanking splice sites in seven patients affected by USH1. Four novel mutations were thereby identified. The possibility should now be considered of a sequence-based prenatal diagnosis in some of the families affected by this very severe form of Usher syndrome.

Objectives:The Miami Otogenetic Program has provided a unique platform to carry out translational research on delivering genetic services to deafness patient care. Using target-enrichment/NGS, we will 1) determine the overall frequencies of different forms of genetic deafness, 2) identify new genes for ARSNHL and ADNSHL, and 3) create a Genomic Deafness Database (GDD) and Personalized Sequence Profile (PSP) for the clinical care of deaf patients.Methods:We collected a unique cohort of multiplex families derived from three unique sources from the U.S., China, and Turkey, suitable for determination of molecular epidemiology of hereditary deafness and for new gene identification using “target-enrichment,” methods and next generation sequencing (NGS). Our interdisciplinary and collaborative team will conduct outcomes evaluation of genetic service on deaf patient care in our diverse populations.Results:The infrastructure of our multidisciplinary otogenetics team is presented along with our use of testing algor...

To investigate 1α,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1α,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1α,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1α,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.

Background: Mitochondrial mutations have been shown to be responsible for syndromic and nonsyndromic hearing impairment. Aim: To assess the genotypic–phenotypic correlation of mitochondrial DNA mutations in three generations of a single family. Methods: A single family with maternally inherited diabetes and hearing loss was recruited. Genomic DNA was subject to polymerase chain reaction–restriction fragment length polymorphism analysis (ApaI) for A3243G mutation detection and confirmation with direct DNA sequencing. The degree of heteroplasmy for the A3243G mutation in blood DNA samples was quantified. In addition, we reviewed audiological data of A3243G-associated hearing loss cases from the literature to provide details of audiologic features. Results: Six of 11 family members were recruited. All affected members harbored the A3243G mutation. Four of six members had diabetes. Five of five affected members demonstrated hearing loss ranging from mild to severe. The degree of heteroplasmy ranged from 5.51% to 27.74%. Conclusions: Patients with a greater percentage of heteroplasmy have a trend toward more severe phenotypic presentations. Hearing loss is bilateral, sensorineural, and symmetric. The main audiogram shapes found were sloping. Additional studies are necessary to clarify the relationship between degree of heteroplasmy and phenotypic presentation.

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X 2 receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X 2 receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X 2 receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2 -null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X 2 function is required for life-long normal hearing and for protection from exposure to noise.

Item does not contain fulltext OBJECTIVE: The purpose of this review was to evaluate the current literature on phosphoribosylpyrophosphate synthetase 1 (PRPS1)-related diseases and their consequences on hearing function. DESIGN: A literature search of peer-reviewed, published journal articles was conducted in online bibliographic databases. Study sample: Three databases for medical research were included in this review. RESULTS: Mutations in PRPS1 are associated with a spectrum of non-syndromic to syndromic hearing loss. Hearing loss in male patients with PRPS1 mutations is bilateral, moderate to profound, and can be prelingual or postlingual, progressive or non-progressive. Audiogram shapes associated with PRPS1 deafness are usually residual and flat. Female carriers can have unilateral or bilateral hearing impairment. Gain of function mutations in PRPS1 cause a superactivity of the PRS-I protein whereas the loss-of-function mutations result in X-linked nonsyndromic sensorineural deafness type 2 (DFN2), or in syndromic deafness including Arts syndrome and X-linked Charcot-Marie-Tooth disease-5 (CMTX5). CONCLUSIONS: Lower residual activity in PRS-I leads to a more severe clinical manifestation. Clinical and molecular findings suggest that the four PRPS1 disorders discovered to date belong to the same disease spectrum. Dietary supplementation with S-adenosylmethionine (SAM) appeared to alleviate the symptoms of Arts syndrome patients, suggesting that SAM could compensate for PRS-I deficiency.

One hundred and thirty-four families or individuals with auditory-pigmentary syndromes such as Waardenburg syndrome (WS) or probable neurocristopathies were screened for mutations in the PAX3 and MITF genes. PAX3 mutations were found in 20/25 families with definite Type 1 WS and 1/2 with Type 3 WS, but in none of 23 with definite Type 2 WS or 36 with other neurocristopathies. The PAX3 mutations included substitutions of conserved amino acids in the paired domain or the homeodomain, splice-site mutations, nonsense mutations and frame-shifting insertions or deletions. No phenotype-genotype correlations were noted within WS1 families. With MITF, mutations likely to affect protein function were found in seven families, five of which had definite Type 2 WS. We conclude that Type 1 and Type 3 WS are allelic and are normally caused by loss of function mutations in PAX3; that Type 2 WS is heterogeneous, with about 20% of cases caused by mutations in MITF, and that individuals with auditory, pigmentary or neural crest syndromes which do not fit stringent definitions of Waardenburg syndrome are unlikely to have mutations in either the PAX3 or MITF genes. The molecular pathology of MITF/microphthalmia mutations appears to be different in humans and mice, with gene dosage having more significant effects in humans than in the mouse.

The Waardenburg syndrome (WS) consists of at least two distinct autosomal dominant hereditary disorders. WS Type I has been mapped to the distal part of chromosome 2q and the gene identified as PAX3. Other gene(s) are responsible for WS Type II. Mapping WS Type II requires accurate diagnosis within affected families. To establish diagnostic criteria for WS Type II, 81 individuals from 21 families with Type II WS were personally studied, and compared with 60 personally studied patients from 8 families with Type I and 253 cases of WS (Type I or II) from the literature. Sensorineural hearing loss (77%) and heterochromia iridum (47%) were the two most important diagnostic indicators for WS Type II. Both were more common in Type II than in Type I. Other clinical manifestations, such as white forelock and skin patches, were more frequent in Type I. We estimate the frequency of phenotypic traits and propose diagnostic criteria for WS Type II. In practice, a diagnosis of WS Type II can be made with confidence given a family history of congenital hearing loss and pigmentary disorders, where individuals have been accurately measured for ocular distances to exclude dystopia canthorum.

MYO3A, an unconventional myosin that localizes to the distal tips of stereocilia in the sensory epithelia of the vertebrate auditory and vestibular systems, consists of an N-terminal kinase domain, an actin-activated motor domain, and a class-specific tail domain. Previous work indicates that MYO3A tip-localization is regulated through autophosphorylation. MYO3A is thought to transport the actin regulatory protein, ESPIN1, to the stereocilia tips in inner ear hair cells and mutations in MYO3A lead to non-syndromic deafness in humans. We investigated the G488E mutation in MYO3A, a deafness associated mutation located near the conserved switch I region. The G488E mutation reduces the maximum actin-activated ATPase rate 2-fold and increases 10-fold the actin concentration required to reach one-half maximal ATPase. Interestingly, G488E MYO3A increases 2-fold the actin filament sliding velocity measured with the in vitro motility assay. We also investigated the impact of the G488E mutation on cellular localization by examining GFP tagged MYO3A dK constructs transfected into COS7 cells. The G488E mutation completely abolished MYO3A dK filopodia tip localization both in the presence and absence of ESPIN1. In addition, co-transfection of G488E MYO3A dK, ESPIN1 and wild-type (WT) MYO3A dK demonstrated that the presence of WT MYO3A does not rescue the tip localization of G488E. We hypothesize that the G488E mutation disrupts the duty ratio which prevents processive movement to the tips of parallel actin bundles. In support, chimeras of the MYO3A tail with other myosin motors (MYO1A, MYO19, MYO5A, and MYO10) demonstrate that only high duty-ratio motors are capable of filopodia tip localization. Our results demonstrate that intra-filopodia motility requires high duty ratio motor activity, which may be crucial for the formation, maintenance, and ultrastructure of parallel actin bundles.

This article is a review of the genes and genetic disorders that affect hearing in humans and a few selected mouse models of deafness. Genetics is playing an increasingly critical role in the practice of medicine. This is not only in part to the importance that genetic knowledge has on traditional genetic diseases but also in part to the fact that genetic knowledge provides an understanding of the fundamental biological process of most diseases. The proteins coded by the genes related to hearing loss (HL) are involved in many functions in the ear, such as cochlear fluid homeostasis, ionic channels, stereocilia morphology and function, synaptic transmission, gene regulation, and others. Mouse models play a crucial role in understanding of the pathogenesis associated with these genes. Different types of familial HL have been recognized for years; however, in the last two decades, there has been tremendous progress in the discovery of gene mutations that cause deafness. Most of the cases of genetic deafness recognized today are monogenic disorders that can be broadly classified by the mode of inheritance (i.e., autosomal dominant, autosomal recessive, X-linked, and mitochondrial inheritance) and by the presence of associated phenotypic features (i.e., syndromic; and nonsyndromic). In terms of nonsyndromic HL, the chromosomal locations are currently known for ∼ 125 loci (54 for dominant and 71 for recessive deafness), 64 genes have been identified (24 for dominant and 40 for recessive deafness), and there are many more loci for syndromic deafness and X-linked and mitochondrial DNA disorders (http://hereditaryhearingloss.org). Thus, today's clinician must understand the science of medical genetics as this knowledge can lead to more effective disease diagnosis, counseling, treatment, and prevention.

Item does not contain fulltext Hereditary hearing loss is characterized by a high degree of genetic heterogeneity. Here we present OTOGL mutations, a homozygous one base pair deletion (c.1430 delT) causing a frameshift (p.Val477Glufs( *)25) in a large consanguineous family and two compound heterozygous mutations, c.547C>T (p.Arg183( *)) and c.5238+5G>A, in a nonconsanguineous family with moderate nonsyndromic sensorineural hearing loss. OTOGL maps to the DFNB84 locus at 12q21.31 and encodes otogelin-like, which has structural similarities to the epithelial-secreted mucin protein family. We demonstrate that Otogl is expressed in the inner ear of vertebrates with a transcription level that is high in embryonic, lower in neonatal, and much lower in adult stages. Otogelin-like is localized to the acellular membranes of the cochlea and the vestibular system and to a variety of inner ear cells located underneath these membranes. Knocking down of otogl with morpholinos in zebrafish leads to sensorineural hearing loss and anatomical changes in the inner ear, supporting that otogelin-like is essential for normal inner ear function. We propose that OTOGL mutations affect the production and/or function of acellular structures of the inner ear, which ultimately leads to sensorineural hearing loss.

Waardenburg syndrome (WS), an autosomal dominant syndrome of hearing loss and pigmentary disturbances, comprises at least two separate conditions. WS type 1 is normally caused by mutations in PAX3 located at chromosome 2q35 and is distinguished clinically by minor facial malformations. We have now located a gene for WS type 2. Two families show linkage to a group of microsatellite markers located on chromosome 3p12-p14.1. D3S1261 gave a maximum lod score of 6.5 at zero recombination in one large Type 2 family. In a second, smaller family the adjacent marker D3S1210 gave a lod of 2.05 at zero recombination. Interestingly, the human homologue (MITF) of the mouse microphthalmia gene, a good candidate at the phenotypic level, has recently been mapped to 3p12.3-p14.4.

Direct evidence of the critical physiological role of connexins (Cxs) has come through the associations of several human diseases with pathogenic mutations in specific Cx genes. Currently, mutations in genes coding for five Cx proteins (Cx26, Cx30, Cx31, Cx32, and Cx43) have been shown to cause sensorineural hearing loss. Cx45 is another gap junction protein, coded by the GJA7 gene. To investigate the possible contribution of GJA7 mutations to deafness, we sequenced the GJA7 gene in 341 unrelated probands with nonsyndromic hearing loss from Turkey, South Africa, United Kingdom, United States, and China. Three nucleotide variants not affecting the amino acid sequence, c.213C > T, c.906C > T, and c.912G > T, and one missense change, c.889C > A (p.D297N), were found. None of the identified changes appeared to be pathogenic. Our data suggest that GJA7 alterations have no or low genetic relevance in nonsyndromic hearing loss in these populations.

The purpose of this study was to determine the prevalence of mutations in the GJB2 gene, the GJB6-D13S1830 deletion and the four common mitochondrial mutations (A1555G, A3243G, A7511C and A7445G) in a South African population.Using single-strand conformation polymorphism and direct sequencing for screening GJB2 mutation; Multiplex PCR Amplification for GJB6-D13S1830 deletion and Restriction Fragment-Length Polymorphism (PCR-RFLP) analysis for the four common mtDNA mutations. We screened 182 hearing impaired students to determine the frequency of these mutations in the population.None of the reported disease causing mutations in GJB2 nor any novel pathogenic mutations in the coding region were detected, in contrast to the findings among Caucasians. The GJB6-D13S1830 deletion and the mitochondrial mutations were not observed in this group.These results suggest that GJB2 may not be a significant deafness gene among sub-Saharan Africans, pointing to other unidentified genes as responsible for nonsyndromic hearing loss in these populations.

Objectives: The aim of the study is to assess clinical characteristics of individuals with nonsyndromic sensorineural hearing loss (NSSNHL) with genetic mutations in GJB2 and/or GJB6. We describe and compare one group with biallelic mutations against a group of heterozygote mutation carriers. Methods: A total of 350 patients between the ages of 3 months and 80 years referred to a tertiary care outpatient otology practice for NSSNHL were screened for genetic mutations. Direct sequencing of GJB2 and polymerase chain reaction analysis of GJB6 was performed and clinical data from history and physical, audiologic testing and radiographic studies were reviewed. Results: Thirty-two patients were found to have biallelic mutations (incidence of 9.1%). Twenty-five patients were found to have only one GJB2 mutation (incidence of 7.1%). Severe to profound hearing loss occurred in 85% of the homozygote group and 38% of the heterozygote group. Both groups similarly had a propensity toward bilateral, symmetric, nonprogressive hearing loss with rare inner ear malformations on radiologic imaging. Conclusions: These two patient populations have similar incidences in a cohort of patients evaluated for NSSNHL, which is higher than general population heterozygote carrier rates. Heterozygote mutation carriers had less hearing impairment, but most other factors demonstrated no differences. These results support the theory of an unidentified genetic factor contributing to hearing loss in some heterozygote carriers. Therefore, genetic counseling should consider the complexity of their genetic factors and the limitations of current screening. Laryngoscope, 2011

Genetic factors are among the main etiologies of severe to profound hearing loss and may play an important role in cochlear implantation (CI) outcomes. While genes for common forms of deafness have been cloned, efforts to correlate the functional outcome of CIs with a genetic form of deafness carried by the patient have been largely anecdotal to date. It has been suggested that the differences in auditory performance may be explained by differences in the number of surviving spiral ganglion cells, etiology of hearing loss, and other factors. Knowledge of the specific loci and mutations involved in patients who receive cochlear implants may elucidate other factors related to CI performance. In this review article, current knowledge of cochlear implants for hereditary hearing loss will be discussed with an emphasis on relevant clinical genotype-phenotype correlations.

The identities and frequencies of MYO15A mutations associated with hearing loss in different populations remained largely unknown. We screened the MYO15A gene for mutations in 104 unrelated multiplex and consanguineous Turkish families with autosomal recessive nonsyndromic sensorineural hearing loss using autozygosity mapping. The screening of MYO15A in 10 families mapped to the DFNB3 locus revealed five previously unreported mutations: p.Y289X (1 family), p.V1400M (1 family), p.S1481P (1 family), p.R1937TfsX10 (3 families), and p.S3335AfsX121 (2 families). Recurrent mutations were associated with conserved haplotypes suggesting the presence of founder effects. Severe to profound sensorineural hearing loss was observed in all subjects with homozygous mutations except for two members of a family who were homozygous for the p.Y289X mutation in the N-terminal extension domain and had considerable residual hearing. We estimate the prevalence of homozygous MYO15A mutations in autosomal recessive nonsyndromic deafness in Turkey as 0.062 (95% confidence interval is 0.020-0.105).

Usher syndrome type II (USH2) is an autosomal recessive disorder characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa (RP). To identify novel mutations and determine the frequency of USH2A mutations as a cause of USH2, we have carried out mutation screening of all 72 coding exons and exon–intron splice sites of the USH2A gene. A total of 20 USH2 American probands of European descent were analyzed using single strand conformational polymorphism (SSCP) and direct sequencing methods. Ten different USH2A mutations were identified in 55% of the probands, five of which were novel mutations. The detected mutations include three missense, three frameshifts and four nonsense mutations, with c.2299delG/p.E767fs mutation, accounting for 38.9% of the pathological alleles. Two cases were homozygotes, two cases were compound heterozygotes and one case had complex allele with three variants. In seven probands, only one USH2A mutation was detected and no pathological mutation was found in the remaining eight individuals. Altogether, our data support the fact that c.2299delG/p.E767fs is indeed the most common USH2A mutation found in USH2 patients of European Caucasian background. Thus, if screening for mutations in USH2A is considered, it is reasonable to screen for the c.2299delG mutation first.

Two reduced molybdenum(V) phosphates, (H2dien)4[CaMo12O24(OH)6(HPO4)8]·nH2O (1) and (H2dien)4[Ba2(H2O)4CaMo12O24(OH)6(HPO4)4(PO4)4]·nH2O (2) (dien = diethylenetriamine) have been prepared under hydrothermal conditions and characterized by elemental analyses, IR, UV–vis, cyclic voltammetry and single-crystal X-ray diffraction. [Mo6P4] units in both structures are linked by octahedrally coordinated Ca(II) cations into sandwich-shaped [Ca(Mo6P4)2] clusters. While the clusters in 1 are discrete, those in 2 are interconnected through BaO9 polyhedra into a three-dimensional framework structure. It was indicated that the compounds exhibit photocatalytic activity in the bleaching of rhodamine B.

We have characterized the spectrum of SLC26A4 mutations and clinical features in a population of mainland Chinese patients with nonsyndromic sensorineural hearing loss (SNHL) and enlarged vestibular aqueduct (EVA).Cross-sectional clinical genetic study.Tertiary care outpatient otolaryngology clinic.A total of 32 subjects identified with bilateral EVA using high-resolution CT were screened for mutations in SLC26A4 by denaturing high-performance liquid chromatography and direct sequencing methods.A total of 13 different mutations were identified in the SLC26A4 gene, five of which are novel. A total of 88 percent of the patients harbored biallelic mutations, 11 patients were homozygotes, and 17 were compound heterozygotes. Four patients were found to carry a single SLC26A4 mutation. The IVS7-2AG mutation was the most frequent, accounting for 60 percent of the mutant alleles. We have not found any correlations between the type of SLC26A4 mutations and the type, degree, and progression of hearing loss. There are significant proportions of patients with asymmetric (26%), progressive (32%), or fluctuating hearing loss (21%).Our data confirm the high prevalence of SLC26A4 mutations in Chinese patients with SNHL and EVA. We could not establish any relationship between genotype and phenotype. However, the high incidence of asymmetric, progressive, and fluctuating hearing loss found in the current study indicates that patients with those features should be routinely screened for SLC26A4 mutation in addition to diagnosis of EVA using CT or MRI.

Background Increasing evidence shows that whole genomes of eukaryotes are almost entirely transcribed into both protein coding genes and an enormous number of non-protein-coding RNAs (ncRNAs). Therefore, revealing the underlying regulatory mechanisms of transcripts becomes imperative. However, for a complete understanding of transcriptional regulatory mechanisms, we need to identify the regions in which they are found. We will call these transcriptional regulation regions, or TRRs, which can be considered functional regions containing a cluster of regulatory elements that cooperatively recruit transcriptional factors for binding and then regulating the expression of transcripts. Results We constructed a hierarchical stochastic language (HSL) model for the identification of core TRRs in yeast based on regulatory cooperation among TRR elements. The HSL model trained based on yeast achieved comparable accuracy in predicting TRRs in other species, e.g., fruit fly, human, and rice, thus demonstrating the conservation of TRRs across species. The HSL model was also used to identify the TRRs of genes, such as p53 or OsALYL1, as well as microRNAs. In addition, the ENCODE regions were examined by HSL, and TRRs were found to pervasively locate in the genomes. Conclusion Our findings indicate that 1) the HSL model can be used to accurately predict core TRRs of transcripts across species and 2) identified core TRRs by HSL are proper candidates for the further scrutiny of specific regulatory elements and mechanisms. Meanwhile, the regulatory activity taking place in the abundant numbers of ncRNAs might account for the ubiquitous presence of TRRs across the genome. In addition, we also found that the TRRs of protein coding genes and ncRNAs are similar in structure, with the latter being more conserved than the former.

To evaluate temporal bone CT findings in GJB2-related deafness (DFNB1) hearing loss and non-DFNB1 hearing loss children.Case-control series.Children with nonsyndromic hearing loss diagnosed as DFNB1 or non-DFNB1 after screening GJB2 allele variants and the large GJB6 deletion. Temporal bone CT images compared in a cohort of nine DFNB1 children with 10 non-DFNB1 children. Visual criteria and absolute measurements were compared against established normative values.Visual inspection failed to identify two patients with abnormalities identified by using absolute measurements. Only one of nine DFNB1 children had an ear anomaly versus seven of 10 non-DFNB1 (odds ratio 16.33; 95% CI, 1.35, 197.78; P = 0.050). The non-DFNB1 group had a mean vestibule width that was significantly larger, and a mean lateral semicircular canal island width and vestibular aqueduct that were significantly smaller than the DFNB1 group.Visual inspection of temporal bone CT images alone may not identify all anomalies and should be used with absolute CT measurements. Abnormal temporal bone CT findings are significantly less likely in children with DFNB1 compared with non-DFNB1 children despite similar age and degree of hearing loss.

A novel two-dimensional solid, [{Cu(en)2}4(H4W12O42)]·9H2O (en = ethylenediamine), has been prepared under mild hydrothermal conditions. Single crystal X-ray diffraction revealed that each paradodecatungstate [H4W12O42]8− cluster in the title compound is connected with its four adjacent clusters through eight {Cu(en)2}2+ bridging groups into a two-dimensional layered structure. Its cyclic voltammetric behavior in aqueous electrolyte demonstrated that its modified carbon paste electrode has a good stability. A probe reaction indicated that the catalytic activity of paradodecatungstates in epoxidation of maleic anhydride has been enhanced by assembling paradodecatungstate clusters into extended structures.

Remarkable progress has been made in the past decade in identifying genes involved with deafness in man and mouse. The identification of these genes and functional analysis of the proteins they encode are paving the way towards a better understanding of the physiology and pathophysiology of the auditory system. Given the complexity of auditory transduction and diversity of cochlear structures, it is not surprising that an estimate of at least 1 percent of human protein-coding genes are involved in perception of sound. Over 400 distinct syndromes of which hearing loss is a component have been reported (www.ncbi.nlm.nih.gov/omim). Approximately 113 loci for monogenic disorders for which hearing loss is the only manifestation and therefore is nonsyndromic, have been mapped to the human genome (http:webhost.ua.ac.be/hhh/). As of August 2007, there are approximately 46 genes identified from these loci. Here, we review some of the major advances in our knowledge of auditory function within an evolving understanding of the structure and regulation of the machinery of hearing.

Objective To evaluate the speech perception and language development with cochlear implants (CI) of DFNB1 children in comparison with non-DFNB1 deaf children. Study Design Retrospective case series. Setting Academic tertiary center. Results Thirty-one congenitally deafened children, screened for GJB2 allele variants, were followed for an average 32 months after CI. With the use of age-appropriate testing, 75% of DFNB1 and 53% of non-DFNB1 children achieved open set word recognition (speech perception category [SPC] level 6). Multivariate analysis showed that SPC was primarily dependent on duration of CI use, but not on the cause of hearing loss. In Reynell language tests, DFNB1 children showed more consistent and quicker gains than non-DFNB1 children. Conclusion Although children with CI with DFNB1 show faster gains in Reynell scores, duration of CI use appears to have a greater effect on speech perception than DFNB1 status. Significance Identification of DFNB1 children is useful in counseling of CI outcomes.