Auswahl der wissenschaftlichen Literatur zum Thema „USH1G“

Usher syndrome (USH) is an autosomal recessive disease characterized by hearing loss, retinitis pigmentosa (RP), and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous and is the most common cause underlying deafness and blindness of genetic origin. Clinically, USH is divided into three types. Usher type I (USH1) is the most severe form and is characterized by severe to profound congenital deafness, vestibular areflexia, and prepubertal onset of progressive RP. Type II (USH2) displays moderate to severe hearing loss, absence of vestibular dysfunction, and later onset of retinal degeneration. Type III (USH3) shows progressive postlingual hearing loss, variable onset of RP, and variable vestibular response. To date, five USH1 genes have been identified:MYO7A(USH1B),CDH23(USH1D),PCDH15(USH1F),USH1C(USH1C), andUSH1G(USH1G). Three genes are involved in USH2, namely,USH2A(USH2A),GPR98(USH2C), andDFNB31(USH2D). USH3 is rare except in certain populations, and the gene responsible for this type isUSH3A.

Abstract We studied the scientific literature and disease guidelines in order to summarize the clinical utility of genetic testing for Usher syndrome (USH). USH is mostly transmitted in an autosomal recessive manner and is caused by variations in the ADGRV1, CDH23, CIB2, CLRN1, HARS, MYO7A, PCDH15, PDZD7, USH1C, USH1G, USH2A, WHRN genes. Prevalence is estimated to be 1:30,000. Clinical diagnosis is based on audiogram, vestibular tests, visual acuity test, fundus examination, color test, optical coherence tomography and electroretinography. The genetic test is useful for confirming diagnosis, and for differential diagnosis, couple risk assessment and access to clinical trials.

Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf–blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.

Usher syndrome (USH) is the most common genetic condition responsible for combined loss of hearing and vision. Balance disorders and bilateral vestibular areflexia are also observed in some cases. The syndrome was first described by Albrecht von Graefe in 1858, but later named by Charles Usher, who presented a large number of cases with hearing loss and retinopathy in 1914. USH has been grouped into three main clinical types: 1, 2, and 3, which are caused by mutations in different genes and are further divided into different subtypes. To date, nine causative genes have been identified and confirmed as responsible for the syndrome when mutated: MYO7A, USH1C, CDH23, PCDH15, and USH1G (SANS) for Usher type 1; USH2A, ADGRV1, and WHRN for Usher type 2; CLRN1 for Usher type 3. USH is inherited in an autosomal recessive pattern. Digenic, bi-allelic, and polygenic forms have also been reported, in addition to dominant or nonsyndromic forms of genetic mutations. This narrative review reports the causative forms, diagnosis, prognosis, epidemiology, rehabilitation, research, and new treatments of USH.

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal.

Abstract Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.

La surdité est le déficit sensoriel le plus fréquent chez l'Homme et touche plus de 466 millions de personnes dans le monde. En France, un enfant sur 1000 naît avec une surdité sévère ou profonde. La surdité peut être associée à des troubles de l'équilibre. Face à l'absence de traitement curatif, la thérapie génique apparaît comme une alternative prometteuse pour le traitement des surdités et des troubles vestibulaires d’origine génétique. Les études de thérapie génique chez les modèles murins reposent notamment sur l’utilisation des virus adéno-associés (AAV) comme vecteurs de gènes. Dans le cadre de cette thèse, nous avons utilisé les AAV pour la thérapie génique des surdités et troubles vestibulaires et pour l’étude physiologique de la fonction des gènes. Plus précisément, nous nous sommes intéressés à trois protéines dont le dysfonctionnement entraine une atteinte neurosensorielle : l’OTOFERLINE, la SNAP-25 et SANS. Les résultats obtenus chez la souris montrent qu’il est possible de restaurer la structure et la fonction des cellules ciliées sensorielles de l'oreille interne, au niveau synaptique et stéréociliaire grâce au transfert in vivo de gènes thérapeutiques contenus dans un AAV. L’utilisation d’AAV exprimant Snap-25 a également permis de mettre en évidence le rôle de cette protéine dans la survie et l’exocytose de la cellule ciliée interne. Ainsi, nous avons restauré l'audition et l’équilibre dans différents modèles de synaptopathies auditives et vestibulaires. Ce projet ouvre la voie à de nouvelles approches thérapeutiques des formes génétiques de surdités et troubles vestibulaires chez l’Homme
Hearing loss is the most common sensory deficit, affecting more than 466 million people worldwide. In France, one in 1,000 children is born with severe to profound deafness, Hearing loss is often associated with balance impairments. Currently, there is no curative treatment available, however the possibility of gene therapy is a promising alternative treatment for deafness and vestibular disorders of genetic origin. Gene therapy studies in mouse models particularly rely on the use of adeno-associated viruses (AAVs) for gene delivery. In this thesis, we used AAVs for both gene therapy of hearing loss and vestibular disorders and to study physiological gene function. Specifically, we looked at three proteins whose dysfunction leads to neurosensory impairment: OTOFERLINE, SNAP-25 and SANS.Our results obtained in mice show that it is possible to restore the structure and function of inner ear sensory hair cells, at the synaptic and stereociliary levels, by in vivo transfer of therapeutic genes contained in an AAV. The use of AAV expressing Snap-25 demonstrated the role of this protein in both the survival and exocytosis of inner hair cells. Thus, we restored hearing and balance in different models of auditory and vestibular synaptopathies. This project opens up new perspectives for the treatment of genetic forms of deafness and vestibular disorders in humans

Usher syndrome is an autosomal recessive disease, displaying pathology of the auditory and visual systems. The three clinical phenotypes are linked to eleven different loci, and differ in the severity and age of onset of sensorineural hearing loss, vestibular areflexia and Retinitis Pigmentosa. The expression pattern of Ush1c which encodes a PDZ domain containing protein (Harmonin) has been established in the murine ear. This study examined the localisation of the protein in the murine eye, detecting Ush1c mRNA from E12.5 and localising the protein to the newborn photoreceptors from P2. In the developed retina, harmonin was localised to the photoreceptor outer segments. Further immunoreactivity was noted in unfixed tissue, showing harmonin colocalising with the USH1B and 1D proteins to the photoreceptor inner segments and outer plexiform layer. The USH1B and USH1D proteins are known to interact with and be involved in the correct localisation of harmonin in the ear. Yet in the eye, harmonin localisation in fixed tissue was maintained in the outer segments in the absence of either USH1 protein. To further examine the protein interactions of harmonin, a Yeast Two-Hybrid study was undertaken using a bovine retinal library. Six possible interacting proteins were identified and confirmed by yeast mating. Several of these positive interactors, although interacting with the whole harmonin protein, did not interact with any of its PDZ binding domains. One such protein was phosducin, a small acidic cytosolic protein. Its potential as a binding partner for harmonin in the retina was confirmed by a pull down assay and immunolocalisation studies. Phosducin is known to interact with proteins associated with glutamate receptors at the photoreceptor ribbon synapses. Its interaction with harmonin would suggest a role for harmonin in the outer plexiform layer and more specifically at the ribbon synapses of the retina.

Inherited Retinal Diseases (IRDs) represent a major cause of blindness worldwide. Adeno-associated viral (AAV) vector-based gene therapies represent the most promising treatments. We aimed to develop gene therapies for gyrate atrophy of the choroid and retina (GA) and Usher syndrome type 1B (USH1B) retinitis pigmentosa. GA is characterized by ornithine aminotransferase [OAT, coding sequence (CDS) ∽1.3 Kb] deficiency. We demonstrated in vitro expression and activity of 3XFlag-tagged human OAT (hOAT-3XFlag). AAV vector carrying the hOAT-3XFlag expression cassette improved the structural retinal defects in the Oat-/- mouse model of GA. Bi-allelic mutations in the Myosin7A gene (MYO7A) (CDS ∽6.7 Kb) cause USH1B, the most common combination of inherited congenital deafness and blindness. We demonstrated effective delivery and expression of MYO7A in mice and pigs using dual AAV vectors. During AAV manufacturing, we found a contaminant vector resulting from recombination between two homologous sequences in the AAV vector containing the 5’ half of hMYO7A. This was removed by changing one of the two sequences while maintaining the same MYO7A expression levels in vivo. We selected three therapeutic doses of dual AAV-hMYO7A that rescue retinal defects in shaker-1 mice, a mouse model of USH1B. These doses will be translated in patients with USH1B. In the same mouse model, we confirmed biological potency of dual AAV-hMYO7A that will be used in the clinical trial. Overall, these studies offer promising results, paving the way for a gene therapy of GA and for the clinical translation of dual AAV vectors in USH1B subjects.

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Usher syndrome is an autosomal recessive disease characterized by the association of retinitis pigmentosa and sensorineural hearing loss with or without vestibular dysfunction. Prevalence for this disease was estimated to be 3-4 per 100,000 individuals. Distinguished by clinical features, Usher syndrome can be divided in three types, where Usher syndrome type 1 is the most severe form. Causing disease mutations were reported in 10 genes, 6 of them associated to Usher syndrome type I. MYO7A gene is the most commonly mutated gene for this type, representing approximately 50% of the cases. MYO7A gene codes for myosin VIIa protein, previously described as a motor transport protein and participating in the establishment of cell-cell adhesions. This work aimed to evaluate the possibility of two novel MYO7A variants, identified in compound heterozygosity in a Usher syndrome type 1 Portuguese patient using a Targeted resequencing approach to 9 of the Usher syndrome associated genes, be responsible for the phenotype. Accordingly, the segregation analysis of these variants (c.3503+1delG and c.5561dupT) in the available patient relatives was performed, as well as the analysis of other five variants located between the two variant loci. Furthermore, 250 samples from normal individuals were analysed for the two novel variants and all of them presented the normal genotypes. Additionally, an in silico study was performed aiming to evaluate the evolutionary conservation of the two variant loci, revealing that the first is highly conserved among the mammalian analysed species while the second is highly conserved among the vertebrate analysed species. Since MYO7A c.3503+1delG variant is located at the donor splice site of MYO7A exon 27, alteration of splice site analysis was performed with in silico Splice site prediction and the complete absence of the normal donor splice site at MYO7A exon 27 was observed. Altogether, these results support the hypothesis that the two variants (c.3503+1delG and c.5561dupT) can be a compound heterozygous mutation responsible for Usher syndrome type I phenotype in the studied patient. In order to confirm the presence of the two variants in the MYO7A transcript nucleotide sequences, nasal epithelium samples from the patient and two normal individuals were studied and both wt and c.5561dupT alleles were expressed, while c.3503+1delG was only identified as the wt allele. However, it is predictable that a complex phenomenon be occurring at this splice site due to c.3503+1delG mutation. Finally, considering that it is x predictable that c.5561dupT mutation causes a MYO7A frameshift leading to a truncated myosin VIIa protein with a partially abnormal second MyTH4 domain and completely without the second FERM domain and the protein C-terminus, it seemed relevant to understand the effect of such mutation at the cellular and molecular level. Therefore, protein-protein interaction studies were performed with two constructions of C-terminal MyTH4-FERM (wt and mut), tagged with GFP. These studies suggested that the previously reported interaction of myosin VIIa with myrip was preserved in the presence of the MyTH4-FERM mut, and a gain of myosin VIIa function was achieved with the MyTH4-FERM mut, which seems to interact with munc13-4 protein, an interaction that was not seen with the MyTH4-FERM wt. Co-localization of both myosin VIIa with MyTH4-FERM mut and munc13-4 proteins was observed near vesicles structures when both were simultaneously expressed in HEK293a cells. This study reveals that MYO7A c.3503+1delG and c.5561dupT mutations are a compound heterozygous mutation causing USH1 in a Portuguese patient, and elucidate some functional myo7a protein alterations that may be important to understand the molecular mechanisms of this disease. However, further studies are required to clarify these implications at the cellular level.
O Síndrome de Usher é uma doença autossómica recessiva caracterizada pela associação de duas patologias, retinite pigmentosa e da perda auditiva sensoneural, às quais poderá estar também associada uma disfunção vestibular. Estima-se que esta doença afecta 3 a 4 indivíduos em cada 100 000. Distinguidos pelas características clínicas, o Síndrome de Usher pode ser dividido em três tipos, de entre os quais o Síndrome de Usher do tipo 1 é a forma mais severa. Até à data foram identificados 10 genes que possuem mutações causadoras desta doença, 6 dos quais foram associados ao Síndrome de Usher do tipo 1. O gene MYO7A é o gene onde foram encontradas mais mutações em doentes com este tipo de síndrome, representando cerca de 50% dos casos. O gene MYO7A codifica uma proteína designada de miosina VIIa, a qual já foi previamente descrita como uma proteína transportadora que também está envolvida no estabelecimento de adesões intercelulares. Este trabalho tem por objectivo avaliar a possibilidade de duas novas variantes do gene MYO7A serem responsáveis pelo fenótipo do Síndrome de Usher do tipo 1. Estas duas novas variantes foram identificadas em heterozigotia composta num doente português com Síndrome de Usher do tipo 1, através de uma abordagem de Targeted resequecing para 9 dos genes associados ao Síndrome de Usher. Assim sendo, foi feita a análise não só destas variantes (c.3503+1delG e c.5561dupT) mas também de outras cinco variantes localizadas entre entes loci, nos familiares em que foi possível. Além disso, as duas novas variantes foram analisadas em 250 amostras provenientes de indivíduos saudáveis, sendo que não foram encontradas em nenhuma destas amostras. Para além disso, com o objectivo de avaliar a conservação evolutiva destes loci foram realizados estudos in silico que demonstraram que ambos os loci são extremamente conservados entre as espécies de mamíferos analisadas. Uma vez que a variante c.3503+1delG do gene MYO7A está localizada no donor splice site do seu exão 27, foi feita uma previsão da alteração do local de splicing com um softweare online (splice site prediction), previsão esta que revelou uma completa destruição deste local de splicing na presença desta variante. Todos estes resultados sustentam a hipótese de que estas duas variantes (c.3503+1delG e c.5561dupT) possam constituir uma mutação heterozigótica composta responsável pelo fenótipo do Síndrome de Usher do tipo 1 no doente estudado. Com o intuito de perceber se estas duas novas variantes estariam alteradas na sequência de nucleótidos do transcrito do gene MYO7A, foram utilizadas uma amostra do epitélio nasal do doente e duas amostras do epitélio nasal de dois indivíduos normais, tendo xii sido verificada a expressão quer do alelo normal quer do alelo com a variante c.5561 na amostra do doente. No que diz respeito à variante c.3503+1delG, apenas foi possível identificar o alelo selvagem. Contudo, é espectável que na presença desta mutação possa estar a ocorrer um fenómeno complexo. Por último, considerando a probabilidade que a mutação c.5561dupT cause uma frameshift no transcrito do gene MYO7A, originando uma miosina VIIa truncada com um segundo domínio MyTH4 parcialmente anómalo que leva à destruição completa do segundo domínio FERM e do C-terminal da proteína, é relevante elucidar quais os efeitos desta mutação ao nível celular. Posto isto, foram realizados estudos de interacção proteína-proteína com duas construções dos domínios MyTH4-FERM do terminal carboxílico (selvagem e mutada) com uma tag de GFP. Estes estudos sugerem que a interacção já descrita entre a miosina VIIa e a myrip foi preservada na presença da mutação. Para além disso, foi identificado aquilo que parece ser um ganho de função por parte destes domínios do terminal carboxílico da miosina VII. Na presença da mutação, observou-se interacção com a munc13-4, o que não é detectado na presença destes domínios na sua forma selvagem. A ocorrência desta interacção e de um possível ganho de função na presença da mutação por parte da miosina VIIa é suportado pela observação da co-localização da muc13-4 e dos domínios do terminal carboxílico da miosina VIIa mutada perto de estruturas que se assemelham a vesículas, quando ambas são expressas nas células HEK293a. Este estudo revela que as mutações c.3503+1delG e c.5561dupT do gene MYO7A constituem uma mutação heterozigótica composta que é causa do fenótipo de Síndrome de Usher do tipo 1 num doente Português. Para além disso, elucida algumas alterações da miosina VIIa que podem ser importantes para elucidar os mecanismos moleculares desta doença. Contudo, mais estudos serão necessários para clarificar estas implicações ao nível celular

Abstract Hallgren estimated the frequency of Usher syndrome to be 3.0 in 100,000 in Scandinavia. According to Boughman et al., the prevalence in the United States is 4.4 in 100,000. The Usher syndromes constitute 2.5% of families with retinitis pigmentosa, and 24% to 54% of deafblind persons registered at the Helen Keller National Center for Deaf-Blind Youths and Adults have the syndrome. Thirty percent of deaf French Acadians in Louisiana have Usher syndrome, now identified as type 1C. There is genetic heterogeneity for USH1 in France. The gene in the families originating from the Poitou-Charentes region in western France (USH1A) maps to 14q32. The Acadian variety in Louisiana (USH1C) maps to 11p15.1, and the non-Acadian, non-14q variety (USH1B) maps to 11q13.5.