Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.Usherin oireyhtymään (USH) kuuluu perinnöllinen kuuro-sokeus ja vaihteleva tasapainon häiriö. Oireyhtymä jaetaan kolmeen alatyyppiin kliinisten oireiden etenemisen ja vaikeusasteen mukaan. Oireyhtymän alatyyppi 3 (USH3) tunnistettiin alkujaan suomalaisissa potilaissa. Geeni, klariini, jonka mutatoituminen aiheuttaa USH3:n löydettiin Suomessa vuonna 2001 ja klariinista löytyi kaksi mutaatiota. Näistä toinen on suomalainen päämutaatio ja löytyy jokaiselta suomalaiselta USH3 potilaalta. Usherin oireyhtymän eri alatyyppejä aiheuttavat kuitenkin mutaatiot yhdeksässä geenissä, joiden tuottamien proteiinien uskotaan toimivan yhdessä niin silmän kuin korvan soluissa. Tämän väitöskirjan tekemisen aikana suomalaisilta potilailta löytyivät ensimmäiset USH1 ja USH2 geenien mutaatiot. Näistä neljä USH1 mutaatiota sekä yksi USH2 mutaatio on aikaisemmin tuntemattomia ja vain yksi Suomesta tunnistettu USH2 mutaatio on jo aikaisemmin raportoitu muualla. Suomalaisten USH -mutaatioiden kirjo onkin tyypillinen Suomen väestölle. Mutaatioiden kartoitus on tärkeää potilaiden diagnostiikassa koska mutatoituneen geenin tietäminen helpottaa mm. potilaiden taudin etenemisen ennusteessa ja hoitotoimenpiteiden päättämisessä. Tutkimus keskittyi klariini -geeniin ja sen geenivirheiden aiheuttamaan, suomalaiseen tautiperimään kuuluvaan USH3 alatyyppiin. Tutkimuksen aikana klariinista löydettiin kahdeksan uutta silmukointimuotoa. Tällöin geenin osat yhdistellään versioiksi, jotka mahdollisesti luetaan erilaisiksi proteiineiksi. Klariinin toiminnan säätelystä vastaava alue pystyttiin kartoittamaan ja sen toimintaa tutkimaan eri solutyypeissä. Lisäksi klariini -proteiinia tutkimalla nähtiin proteiinin kulkeutuvan solukalvolle ja mutatoituneiden proteiinien huomattiin jäävän solun sisäisiin kalvostoihin. Mutatoituessaan klariini muuttuu lisäksi usein epävakaaksi. Tiedämme, että klariini on kalvoproteiini ja klariinit muodostavat keskenään rykelmiä kalvoilla. Emme kuitenkaan tiedä mikä tehtävä klariinin mutatoituessa häiriintyy. Tiedämme vain sen että tuo tehtävä on elintärkeä verkkokalvolla että sisäkorvassa. Klariinia tutkittiin myös silmässä ja korvassa, joissa USH oireet esiintyvät. Hiirillä, joilla ei ole toimivaa klariinia, klariinin puutos aiheuttaa pelkkää kuuroutta muttei sokeutta. Seeprakaloissa klariinia tuotetaan korvan karvasoluissa sekä verkkokalvolla. Kun klariinin tuotto estetään seeprakalan kehityksen aikana, niille kehittyy kuulon/tasapainoaistin häiriöitä ja kalojen näkökyky heikentyy. Tutkimus on kuitenkin vasta alussa. USH3 -kalamalli alkaa tuottaa toimivaa klariinia parin päivän ikäisenä. Osalla kaloista kyky nähdä ja kuulla alkaa palautua. Kysymys siitä, voiko jo alkaneen USH3 -oireyhtymän pysäyttää tai jopa parantaa tuomalla normaalin klariini -geenin verkkokalvolle tai sisäkorvaan, on yhäkin vastaamatta. Onko klariinin tehtävä tärkeä kehityksen aikana, vai voiko sen korvata myöhemmin, vaikka silmä ja korva ovat kehittyneet ilman klariinia, ovat tärkeitä kysymyksiä geeniterapian onnistumisen kannalta

Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane

PurposeMutations of clarin 1 (CLRN1) cause Usher syndrome type 3 (USH3). To determine the effects of USH3 mutations on CLRN1 function, we examined the cellular distribution and stability of both normal and mutant CLRN1 in vitro. We also searched for novel disease-causing mutations in a cohort of 59 unrelated Canadian and Finnish USH patients.MethodsMutation screening was performed by DNA sequencing. For the functional studies, wild-type (WT) and mutant CLRN1 genes were expressed as hemagglutinin (HA) tagged fusion proteins by transient transfection of BHK-21 cells. Subcellular localization of CLRN1-HA was examined by confocal microscopy. The N-glycosylation status of CLRN1 was studied by using the N-glycosidase F (PNGase F) enzyme and western blotting. Cycloheximide treatment was used to assess the stability of CLRN1 protein.ResultsWe found three previously reported pathogenic mutations, p.A123D, p.N48K, and p.Y176X, and a novel sequence variant, p.L54P, from the studied USH patients. The WT HA-tagged CLRN1 was correctly trafficked to the plasma membrane, whereas mutant CLRN1-HA proteins were mislocalized and retained in the endoplasmic reticulum. PNGase F treatment of CLRN1-HA resulted in an electrophoretic mobility shift consistent with sugar residue cleavage in WT and in all CLRN1 mutants except in p.N48K mutated CLRN1, in which the mutation abolishes the glycosylation site. Inhibition of protein expression with cycloheximide indicated that WT CLRN1-HA remained stable. In contrast, the CLRN1 mutants showed reduced stability.ConclusionsWT CLRN1 is a glycoprotein localized to the plasma membrane in transfected BHK-21 cells. Mutant CLRN1 proteins are mislocalized. We suggest that part of the pathogenesis of USH3 may be associated with defective intracellular trafficking as well as decreased stability of mutant CLRN1 proteins

CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT–PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration

Cone Structure in Patients With Usher Syndrome Type III and Mutations in the Clarin 1 Gene

ObjectiveTo study macular structure and function in patients with Usher syndrome type III (USH3) caused by mutations in the Clarin 1 gene (CLRN1).MethodsHigh-resolution macular images were obtained by adaptive optics scanning laser ophthalmoscopy and spectral domain optical coherence tomography in 3 patients with USH3 and were compared with those of age-similar control subjects. Vision function measures included best-corrected visual acuity, kinetic and static perimetry, and full-field electroretinography. Coding regions of the CLRN1 gene were sequenced.ResultsCLRN1 mutations were present in all the patients; a 20-year-old man showed compound heterozygous mutations (p.N48K and p.S188X), and 2 unrelated women aged 25 and 32 years had homozygous mutations (p.N48K). Best-corrected visual acuity ranged from 20/16 to 20/40, with scotomas beginning at 3° eccentricity. The inner segment-outer segment junction or the inner segment ellipsoid band was disrupted within 1° to 4° of the fovea, and the foveal inner and outer segment layers were significantly thinner than normal. Cones near the fovea in patients 1 and 2 showed normal spacing, and the preserved region ended abruptly. Retinal pigment epithelial cells were visible in patient 3 where cones were lost.ConclusionsCones were observed centrally but not in regions with scotomas, and retinal pigment epithelial cells were visible in regions without cones in patients with CLRN1 mutations. High-resolution measures of retinal structure demonstrate patterns of cone loss associated with CLRN1 mutations.Clinical relevanceThese findings provide insight into the effect of CLRN1 mutations on macular cone structure, which has implications for the development of treatments for USH3.Trial registrationclinicaltrials.gov Identifier: NCT00254605

CLRN1 is nonessential in the mouse retina but is required for cochlear hair cell development.

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5-6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT-PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT-PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration