CYSRT1: An Antimicrobial Epidermal Protein that Can Interact with Late Cornified Envelope Proteins.

Late cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Constitutive CYSRT1 expression is present in fully differentiated epidermis and can be further induced in vivo by disruption of the skin barrier upon stratum corneum removal. Transcriptional regulation correlates to keratinocyte terminal differentiation but not to skin bacteria exposure. Similar to LCEs, CYSRT1 was found to have antibacterial activity against Pseudomonas aeruginosa. Comparative gene sequence analysis and protein amino acid alignment indicate that CYSRT1 is highly conserved among vertebrates and has putative antimicrobial activity. To summarize, we identified CYSRT1 in the outer skin layer, where it colocalizes with LCE proteins and contributes to the constitutive epidermal antimicrobial host defense repertoire.

Multimorbidity among persons aged 25-64 years: a population-based study of social determinants and all-cause mortality.

BACKGROUND: Despite increasing multimorbidity across the lifespan, little is known about the co-occurrence of conditions and risk factors among younger adults. This population-based study examines multimorbidity, social determinants and associated mortality among younger and middle-age adults. METHOD: Analysis was based on the Northern Ireland population aged 25-64 years enumerated in the 2011 Census (n = 878 345), with all-cause mortality follow-up to 2014 (8659 deaths). Logistic regression was used to examine social determinants and Cox proportional hazards models in the analysis of associated mortality. RESULTS: Prevalence of multimorbidity was 13.7% in females and 12.7% in males. There was a strong association between multimorbidity that included mental/cognitive illness and deprivation. Among those never married, multimorbid physical conditions were less likely [relative risk ratios (RRR) = 0.92: 95% confidence interval (CI) = 0.88, 0.95 for males; and RRR = 0.90: 0.87, 0.94 for females]. Rurality was associated with lower physical multimorbidity (RRR = 0.92: 0.89, 0.95) but higher mental/cognitive multimorbidity (RRR = 1.35: 1.12, 1.64) among females. All multimorbid categories were associated with elevated risk of mortality. CONCLUSION: The health and economic challenges created by multimorbidity should be addressed further 'upstream'. Future multimorbidity research should include younger adults to inform the development of preventative interventions and align health and social care services more closely with patients' needs.

The formulation of lozenge using black mulberries (Morus nigra L.) leaf extract as an α-glucosidase inhibitor.

BACKGROUND: Diabetes mellitus is a chronic metabolic disease, which possibly leads to kidney, brain, heart failure, and other organ complications, subsequently harming human health. These symptoms have been prevented using the leaf of black mulberry (BM), as a traditional medicine, because the phenolic compounds contained are able to decrease blood glucose concentration. Meanwhile, previous reports have shown that BM contains 1-deoxynojirimycin, with strong activity as an α-glucosidase inhibitor. The aim of this study, therefore, was to formulate and evaluate BM leaf extract in lozenge dosage form as an α-glucosidase inhibitor. MATERIALS AND METHODS: The leaves of BM were extracted using the maceration method, where ethanol (70%) served as a solvent, and the inhibitory activity of the sourced α-glucosidase enzyme was determined through in vitro study. Subsequently, the extract was formulated into lozenge dosage form and evaluated for physical stability and also the effect of α-glucosidase enzyme. RESULTS: The result showed an inhibitory activity of BM leaf extract against the enzyme α-glucosidase, with a half maximal inhibitory concentration (IC50) value of 357.6 µg/mL, whereas the lozenge formulation containing 43% of extract as well as 5% polyvinylpyrrolidone showed the best physical stability as compared to other formulas. However, the lozenge inhibits α-glucosidase enzyme with an IC50 value of 549.7 µg/mL. CONCLUSION: It was established that the lozenge of BM leaf extract possesses activity as an α-glucosidase inhibitor.

Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.

Deficiency of the cysteine protease inhibitor cystatin M/E (Cst6) in mice leads to disturbed epidermal cornification, impaired barrier function, and neonatal lethality. We report the rescue of the lethal skin phenotype of ichq (Cst6-deficient; Cst6-/-) mice by transgenic, epidermis-specific, reexpression of Cst6 under control of the human involucrin (INV) promoter. Rescued Tg(INV-Cst6)Cst6ichq/ichq mice survive the neonatal phase, but display severe eye pathology and alopecia after 4 mo. We observed keratitis and squamous metaplasia of the corneal epithelium, comparable to Cst6-/-Ctsl+/- mice, as we have reported in other studies. We found the INV promoter to be active in the hair follicle infundibulum; however, we did not observe Cst6 protein expression in the lower regions of the hair follicle in Tg(INV-Cst6)Cst6ichq/ichq mice. This result suggests that unrestricted activity of proteases is involved in disturbance of hair follicle biology, eventually leading to baldness. Using quenched activity-based probes, we identified mouse cathepsin B (CtsB), which is expressed in the lower regions of the hair follicle, as an additional target of mouse Cst6. These data suggest that Cst6 is necessary to control CtsB activity in hair follicle morphogenesis and highlight Cst6-controlled proteolytic pathways as targets for preventing hair loss.-Oortveld, M. A. W., van Vlijmen-Willems, I. M. J. J., Kersten, F. F. J., Cheng, T., Verdoes, M., van Erp, P. E. J., Verbeek, S., Reinheckel, T., Hendriks, W. J. A. J., Schalkwijk, J., Zeeuwen, P. L. J. M. Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.

Intercomparison of methods of coupling between convection and large-scale circulation: 2. Comparison over nonuniform surface conditions.

As part of an international intercomparison project, the weak temperature gradient (WTG) and damped gravity wave (DGW) methods are used to parameterize large-scale dynamics in a set of cloud-resolving models (CRMs) and single column models (SCMs). The WTG or DGW method is implemented using a configuration that couples a model to a reference state defined with profiles obtained from the same model in radiative-convective equilibrium. We investigated the sensitivity of each model to changes in SST, given a fixed reference state. We performed a systematic comparison of the WTG and DGW methods in different models, and a systematic comparison of the behavior of those models using the WTG method and the DGW method. The sensitivity to the SST depends on both the large-scale parameterization method and the choice of the cloud model. In general, SCMs display a wider range of behaviors than CRMs. All CRMs using either the WTG or DGW method show an increase of precipitation with SST, while SCMs show sensitivities which are not always monotonic. CRMs using either the WTG or DGW method show a similar relationship between mean precipitation rate and column-relative humidity, while SCMs exhibit a much wider range of behaviors. DGW simulations produce large-scale velocity profiles which are smoother and less top-heavy compared to those produced by the WTG simulations. These large-scale parameterization methods provide a useful tool to identify the impact of parameterization differences on model behavior in the presence of two-way feedback between convection and the large-scale circulation.

Prevalence and predictors of psychotropic medication use: results from the Northern Ireland Study of Health and Stress.

BACKGROUND: To identify the predictors of psychotropic medication use and to determine rates and patterns of use in Northern Ireland (NI) among the general population and various subgroups. METHOD: Analysis of data from the NI Study of Health and Stress, a representative household survey undertaken between 2004 and 2008 with 4340 individuals. Respondents were asked about prescribed psychotropic medication use in the previous 12 months along with a series of demographic questions and items regarding experience of traumatic life events. Mental health disorders were assessed using the World Health Organization's Composite International Diagnostic Interview. RESULTS: Females, individuals aged 50-64 years old, those who were previously married, and those who had experienced a traumatic lifetime event were more likely to have taken any psychotropic medication. Use of any psychotropic medication in the population in the previous 12 months was 14.9%. Use among individuals who met the criteria for a 12-month mental health disorder was 38.5%. Almost one in ten individuals (9.4%) had taken an antidepressant. CONCLUSIONS: Compared with other countries, NI has high proportions of individuals using psychotropic medication in both the general population and those who met the criteria for a 12-month mental disorder. However, these results still suggest possible under treatment of mental disorders in the country. In addition, rates of use in those with no disorder are relatively high. The predictors of medication use are similar to findings in other countries. Possible research and policy implications are discussed.

Malignant syphilis in an AIDS patient.

Malignant syphilis is an uncommon, but not unknown, ulcerative variation of secondary syphilis. The lesions typically begin as papules, which quickly evolve to pustules and then to ulcers with elevated edges and central necrosis. It is usually, but not mandatory, found in patients with some level of immunosuppression, such as HIV patients, when the TCD4(+) cell count is >200 cells/mm(3). Despite the anxiety the lesions cause, this form of the disease has a good prognosis. The general symptoms disappear right after the beginning of treatment, and lesions disappear over a variable period. This study reports the case of a 27-year-old man who has been HIV positive for 6 years, uses antiretroviral therapy incorrectly, has a TCD4(+) cell count of 340 cells/mm(3), a VDRL of 1:128 and itchy disseminated hyperchromic maculopapular lesions with rupioid crusts compatible with malignant syphilis.

Intercomparison of methods of coupling between convection and large-scale circulation: 1. Comparison over uniform surface conditions.

As part of an international intercomparison project, a set of single-column models (SCMs) and cloud-resolving models (CRMs) are run under the weak-temperature gradient (WTG) method and the damped gravity wave (DGW) method. For each model, the implementation of the WTG or DGW method involves a simulated column which is coupled to a reference state defined with profiles obtained from the same model in radiative-convective equilibrium. The simulated column has the same surface conditions as the reference state and is initialized with profiles from the reference state. We performed systematic comparison of the behavior of different models under a consistent implementation of the WTG method and the DGW method and systematic comparison of the WTG and DGW methods in models with different physics and numerics. CRMs and SCMs produce a variety of behaviors under both WTG and DGW methods. Some of the models reproduce the reference state while others sustain a large-scale circulation which results in either substantially lower or higher precipitation compared to the value of the reference state. CRMs show a fairly linear relationship between precipitation and circulation strength. SCMs display a wider range of behaviors than CRMs. Some SCMs under the WTG method produce zero precipitation. Within an individual SCM, a DGW simulation and a corresponding WTG simulation can produce different signed circulation. When initialized with a dry troposphere, DGW simulations always result in a precipitating equilibrium state. The greatest sensitivities to the initial moisture conditions occur for multiple stable equilibria in some WTG simulations, corresponding to either a dry equilibrium state when initialized as dry or a precipitating equilibrium state when initialized as moist. Multiple equilibria are seen in more WTG simulations for higher SST. In some models, the existence of multiple equilibria is sensitive to some parameters in the WTG calculations.

Anticancer potential of Syzygium aromaticum L. in MCF-7 human breast cancer cell lines.

BACKGROUND: The common treatment for cancer is unfavorable because it causes many detrimental side effects, and lately, there has been a growing resistance toward anticancer drugs, which worsens the future of cancer treatment. Therefore, the focus has now shifted toward natural products, such as spices and plants, among many others, to save the future of cancer treatment. Cloves (Syzygium aromaticum L.) are spices with the highest antioxidant content among natural products. Besides acting as an antioxidant, cloves also possess many other functions, such as anti-inflammatory, antibacterial, and antiseptic, which makes them an ideal natural source to be developed as an anticancer agent. OBJECTIVE: This study aims to evaluate the cytotoxic activity of cloves toward MCF-7 human breast cancer cell lines. MATERIALS AND METHODS: Different concentrations of water extract, ethanol extract, and essential oil of cloves were investigated for their anticancer potential in vitro through a brine shrimp lethality test (BSLT) and an MTT assay. RESULTS: In both BSLT and MTT assays, the essential oil showed the highest cytotoxic effect, followed by ethanol and water extract. The LD50 concentration of essential oil in the 24 hours BSLT was 37 µg/mL. Furthermore, the IC50 values in the 24 hours and 48 hours MTT assays of the essential oil were 36.43 µg/mL and 17.6 µg/mL, respectively. CONCLUSION: Cloves are natural products with excellent cytotoxicity toward MCF-7 cells; thus, they are promising sources for the development of anticancer agents.

Embarrassment when illness strikes a close relative: a World Mental Health Survey Consortium Multi-Site Study.

BACKGROUND: In this global study we sought to estimate the degree to which a family member might feel embarrassed when a close relative is suffering from an alcohol, drug, or mental health condition (ADMC) versus a general medical condition (GMC). To date, most studies have considered embarrassment and stigma in society and internalized by the afflicted individual but have not assessed family embarrassment in a large-scale study. METHOD: In 16 sites of the World Mental Health Surveys (WMHS), standardized assessments were completed including items on family embarrassment. Site matching was used to constrain local socially shared determinants of stigma-related feelings, enabling a conditional logistic regression model that estimates the embarrassment close relatives may hold in relation to family members affected by an ADMC, a GMC, or both conditions. RESULTS: There was a statistically robust association such that subgroups with an ADMC-affected relative were more likely to feel embarrassed compared to subgroups with a relative affected by a GMC (p<0.001), even with covariate adjustments for age and sex. CONCLUSIONS: . The pattern of evidence from this research is consistent with conceptual models for interventions that target individual- and family-level stigma-related feelings of embarrassment as possible obstacles to effective early intervention and treatment for an ADMC. Macro-level interventions are under way but micro-level interventions may also be required among family members, along with care for each person with an ADMC.

Cystatin M/E knockdown by lentiviral delivery of shRNA impairs epidermal morphogenesis of human skin equivalents.

The protease inhibitor cystatin M/E (CST6) regulates a biochemical pathway involved in stratum corneum homeostasis, and its deficiency in mice causes ichthyosis and neonatal lethality. Cystatin M/E deficiency has not been described in humans so far, and we did not detect disease-causing mutations in the CST6 gene in a large number of patients with autosomal recessive congenital ichthyosis, who were negative for mutations in known ichthyosis-associated genes. To investigate the phenotype of CST6 deficiency in human epidermis, we used lentiviral delivery of short hairpin RNAs that target CST6 in a 3D reconstructed skin model. Surprisingly, CST6 deficiency did not cause an ichthyosis-like phenotype, but prevented the development of a multilayered epidermis. From this study, we conclude that CST6 deficiency may be incompatible with normal human foetal development.

Lifetime prevalence of mental health disorders and delay in treatment following initial onset: evidence from the Northern Ireland Study of Health and Stress.

BACKGROUND: The current study provides the first epidemiological estimates of lifetime mental disorders across NI based on DSM-IV criteria. Risk factors, delays in treatment and the experience of conflict are also examined. METHOD: Nationally representative face-to-face household survey of 4340 individuals aged > or =18 years in NI using the composite international diagnostic interview. Analyses were implemented using SAS and STATA software. RESULTS: Lifetime prevalence of any disorder was 39.1% while projected lifetime risk was 48.6%. Individuals who experienced conflict were more likely to have had an anxiety, mood or impulse-control disorder. Treatment delays were substantial for anxiety and substance disorders. CONCLUSIONS: Results from this study show that mental disorders are highly prevalent in Northern Ireland. The elevated rates of post-traumatic stress disorder in relation to other countries and the association of living 'in a region of terror' disorders suggests that civil conflict has had an additional impact on mental health. Given substantial delays in treatment, further research is required to investigate the factors associated with failure and delay in treatment seeking.

CLRN1 mutations cause nonsyndromic retinitis pigmentosa.

OBJECTIVE: To describe the mutations in the CLRN1 gene in patients from 2 consanguineous Pakistani families diagnosed with autosomal recessive retinitis pigmentosa (arRP). DESIGN: Case-series study. PARTICIPANTS: Affected and unaffected individuals of 2 consanguineous Pakistani families and 90 unaffected controls from the same population. Informed consent was obtained from participants and the protocol was approved by a local institutional review board. METHODS: Patients of 2 consanguineous families were genotyped with single-nucleotide polymorphism microarrays for genome-wide linkage analysis. The search for potential candidate genes within the 8-Mb overlapping homozygous region in these families revealed the presence of CLRN1, a gene previously known to cause Usher's syndrome type III (USH3), which was analyzed by direct sequence analysis. The clinical diagnosis was based on the presence of night blindness, fundoscopic findings, and electroretinography (ERG) results. Additionally, pure tone audiometry was performed to rule out Usher's syndrome. MAIN OUTCOME MEASURES: Fundoscopy, single-nucleotide polymorphism microarray, DNA sequence analysis, ERG, and audiometry. RESULTS: Sequencing of CLRN1 revealed novel missense mutations (p.Pro31Leu and p.Leu154Trp) segregating in 2 families. Analysis of fundus photographs indicated attenuation of the retinal vessels, and bone spicule pigmentation in the periphery of the retina. The ERG responses were indicative of a rod-cone pattern of the disease. Audiometric assessment revealed no hearing impairment, thereby excluding Usher's syndrome. Subcellular localization studies demonstrated the retention of the mutant proteins in the endoplasmic reticulum, whereas the wild-type protein was mainly present at the cell membrane. CONCLUSIONS: The RP-associated mutations p.Pro31Leu and p.Leu154Trp may represent hypomorphic mutations, because the substituted amino acids located in the transmembrane domains remain polar, whereas more severe changes have been detected in patients with USH3. These data indicate that mutations in CLRN1 are associated not only with USH3, but also with nonsyndromic arRP.

Association of whirlin with Cav1.3 (alpha1D) channels in photoreceptors, defining a novel member of the usher protein network.

PURPOSE: Usher syndrome is the most common form of hereditary deaf-blindness. It is both clinically and genetically heterogeneous. The USH2D protein whirlin interacts via its PDZ domains with other Usher-associated proteins containing a C-terminal type I PDZ-binding motif. These proteins co-localize with whirlin at the region of the connecting cilium and at the synapse of photoreceptor cells. This study was undertaken to identify novel, Usher syndrome-associated, interacting partners of whirlin and thereby obtain more insights into the function of whirlin. METHODS: The database of ciliary proteins was searched for proteins that are present in both the retina and inner ear and contain a PDZ-binding motif. Interactions with whirlin were evaluated by yeast two-hybrid analyses and validated by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization in the retina with immunofluorescence and immunoelectron microscopy. RESULTS: The L-type calcium channel subunit Ca(v)1.3 (alpha(1D)) specifically interacts with whirlin. In adult photoreceptors, Ca(v)1.3 (alpha(1D)) and whirlin co-localize in the region of the connecting cilium and at the synapse. During murine embryonic development, the expression patterns of the Whrn and Cacna1d genes show significant overlap and include expression in the eye, the inner ear, and the central nervous system. CONCLUSIONS: The findings indicate that Ca(v)1.3 (alpha(1D)) is connected to the Usher protein network. This conclusion leads to the hypothesis that, in the retina, whirlin scaffolds Ca(v)1.3 (alpha(1D)) and therefore contributes to the organization of calcium channels in the photoreceptor cells, where both proteins may be involved in membrane fusions.

Usher syndrome and Leber congenital amaurosis are molecularly linked via a novel isoform of the centrosomal ninein-like protein.

Usher syndrome (USH) and Leber congenital amaurosis (LCA) are autosomal recessive disorders resulting in syndromic and non-syndromic forms of blindness. In order to gain insight into the pathogenic mechanisms underlying retinal degeneration, we searched for interacting proteins of USH2A isoform B (USH2A(isoB)) and the LCA5-encoded protein lebercilin. We identified a novel isoform of the centrosomal ninein-like protein, hereby named Nlp isoform B (Nlp(isoB)), as a common interactor. Although we identified the capacity of this protein to bind calcium with one of its three EF-hand domains, the interacton with USH2A(isoB) did not depend on this. Upon expression in ARPE-19 cells, recombinant Nlp(isoB), lebercilin and USH2A(isoB) were all found to co-localize at the centrosomes. Staining of retinal sections with specific antibodies against all three proteins revealed their co-localization at the basal bodies of the photoreceptor-connecting cilia. Based on this subcellular localization and the nature of their previously identified binding partners, we hypothesize that the pathogenic mechanisms for LCA and USH show significant overlap and involve defects in ciliogenesis, cilia maintenance and intraflagellar and/or microtubule-based transport. The direct association of Nlp(isoB) with USH2A(isoB) and lebercilin indicates that Nlp can be considered as a novel candidate gene for USH, LCA and allied retinal ciliopathies.

A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.

Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.

MPP1 links the Usher protein network and the Crumbs protein complex in the retina.

The highly ordered distribution of neurons is an essential feature of a functional mammalian retina. Disruptions in the apico-basal polarity complexes at the outer limiting membrane (OLM) of the retina are associated with retinal patterning defects in vertebrates. We have analyzed the binding repertoire of MPP5/Pals1, a key member of the apico-basal Crumbs polarity complex, that has functionally conserved counterparts in zebrafish (nagie oko) and Drosophila (Stardust). We show that MPP5 interacts with its MAGUK family member MPP1/p55 at the OLM. Mechanistically, this interaction involves heterodimerization of both MAGUK modules in a directional fashion. MPP1 expression in the retina throughout development resembles the expression of whirlin, a multi-PDZ scaffold protein and an important organizer in the Usher protein network. We demonstrate that both proteins interact strongly by both a classical PDZ domain-to-PDZ binding motif (PBM) mechanism, and a mechanism involving internal epitopes. MPP1 and whirlin colocalize in the retina at the OLM, at the outer synaptic layer and at the basal bodies and the ciliary axoneme. In view of the known roles of the Crumbs and Usher protein networks, our findings suggest a novel link of the core developmental processes of actin polymerization and establishment/maintenance of apico-basal cell polarity through MPP1. These processes, essential in neural development and patterning of the retina, may be disrupted in eye disorders that are associated with defects in these protein networks.

Mutations in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene cause autosomal recessive nonsyndromic hearing loss.

In two large Turkish consanguineous families, a locus for autosomal recessive nonsyndromic hearing loss (ARNSHL) was mapped to chromosome 6p21.3 by genome-wide linkage analysis in an interval overlapping with the loci DFNB53 (COL11A2), DFNB66, and DFNB67. Fine mapping excluded DFNB53 and subsequently homozygous mutations were identified in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene, also named tetraspan membrane protein of hair cell stereocilia (TMHS) gene, which was recently shown to be mutated in the "hurry scurry" mouse and in two DFNB67-linked families from Pakistan. In one family, we found a homozygous one-base pair deletion, c.649delG (p.Glu216ArgfsX26) and in the other family we identified a homozygous transition c.494C>T (p.Thr165Met). Further screening of index patients from 96 Turkish ARNSHL families and 90 Dutch ARNSHL patients identified one additional Turkish family carrying the c.649delG mutation. Haplotype analysis revealed that the c.649delG mutation was located on a common haplotype in both families. Mutation screening of the LHFPL5 homologs LHFPL3 and LHFPL4 did not reveal any disease causing mutation. Our findings indicate that LHFPL5 is essential for normal function of the human cochlea.

The DFNB31 gene product whirlin connects to the Usher protein network in the cochlea and retina by direct association with USH2A and VLGR1.

Mutations in the DFNB31 gene encoding the PDZ scaffold protein whirlin are causative for hearing loss in man and mouse. Whirlin is known to be essential for the elongation process of the stereocilia of sensory hair cells in the inner ear, though its complete spatial and temporal expression patterns remained elusive. Here, we demonstrate that, in embryonic development, the gene is not only expressed in the inner ear, but also in the developing brain and the retina. Various isoforms of whirlin are widely and differentially expressed, and we provide evidence that whirlin directly associates with USH2A isoform b and VLGR1b, two proteins that we previously reported to be part of the Usher protein interactome. These proteins co-localize with whirlin at the synaptic regions of both photoreceptor cells and outer hair cells in the cochlea. These findings indicate that whirlin is part of a macromolecular PDZ protein scaffold that functions in the organization of the pre- and/or postsynaptic side of photoreceptor and hair cell synapses. Whirlin might be involved in synaptic adhesion through interaction with USH2A and VLGR1b as well as in synaptic development as suggested by its spatial and temporal expression patterns. In addition, we demonstrate that whirlin, USH2A and Vlgr1b co-localize at the connecting cilium and the outer limiting membrane of photoreceptor cells and in spiral ganglion neurons of the inner ear. Our data show that whirlin is connected to the dynamic Usher protein interactome and indicate that whirlin has a pleiotropic function in both the retina and the inner ear.