2299delG Mutation Research Articles

Expression of the human usherin c.2299delG mutation leads to early-onset auditory loss and stereocilia disorganization

Usher syndrome (USH) is the leading cause of combined deafness and blindness, with USH2A being the most prevalent form. The mechanisms responsible for this debilitating sensory impairment remain unclear. This study focuses on characterizing the auditory phenotype in a mouse model expressing the c.2290delG mutation in usherin equivalent to human frameshift mutation c.2299delG. Previously we described how this model reproduces patient’s retinal phenotypes. Here, we present the cochlear phenotype, showing that the mutant usherin, is expressed during early postnatal stages. The c.2290delG mutation results in a truncated protein that is mislocalized within the cell body of the hair cells. The knock-in model also exhibits congenital hearing loss that remains consistent throughout the animal’s lifespan. Structurally, the stereocilia bundles, particularly in regions associated with functional hearing loss, are disorganized. Our findings shed light on the role of usherin in maintaining structural support, specifically in longer inner hair cell stereocilia, during development, which is crucial for proper bundle organization and hair cell function. Overall, we present a genetic mouse model with cochlear defects associated with the c.2290delG mutation, providing insights into the etiology of hearing loss and offering potential avenues for the development of effective therapeutic treatments for USH2A patients.

Generation and Genetic Correction of USH2A c.2299delG Mutation in Patient-Derived Induced Pluripotent Stem Cells.

Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient’s healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient’s iPSCs also provide an unlimited resource for disease modeling and mechanistic studies.

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

Efficacy Outcome Measures for Clinical Trials of USH2A Caused by the Common c.2299delG Mutation

To determine the change in vision and retinal structure in patients with the common c.2299delG mutation in the USH2A gene in anticipation of clinical trials of therapy. Retrospective observational case series. Eighteen patients, homozygotes or compound heterozygotes with the c.2299delG mutation in USH2A, were studied with regard to visual acuity, kinetic perimetry, dark- and light-adapted static perimetry, optical coherence tomography (OCT), and autofluorescence (AF) imaging. Serial data were available for at least half of the patients, depending on the parameter analyzed. The kinetics of disease progression in this specific molecular form of USH2A differed between the measured parameters. Visual acuity could remain normal for decades. Kinetic and light-adapted static perimetry across the entire visual field had similar rates of decline that were slower than those of rod-based perimetry. Horizontal OCT scans through the macula showed that inner segment/outer segment line width had a similar rate of constriction as co-localized AF imaging and cone-based light-adapted sensitivity extent. The rate of constriction of rod-based sensitivity extent across this same region was twice as rapid as that of cones. In patients with the c.2299delG mutation in USH2A, rod photoreceptors are the cells that express disease early and more aggressively than cones. Rod-based vision measurements in central or extracentral-peripheral retinal regions warrant monitoring in order to complete a clinical trial in a timely manner.

USH2A Gene Editing Using the CRISPR System.

Usher syndrome (USH) is a rare autosomal recessive disease and the most common inherited form of combined visual and hearing impairment. Up to 13 genes are associated with this disorder, with USH2A being the most prevalent, due partially to the recurrence rate of the c.2299delG mutation. Excluding hearing aids or cochlear implants for hearing impairment, there are no medical solutions available to treat USH patients. The repair of specific mutations by gene editing is, therefore, an interesting strategy that can be explored using the CRISPR/Cas9 system. In this study, this method of gene editing is used to target the c.2299delG mutation on fibroblasts from an USH patient carrying the mutation in homozygosis. Successful in vitro mutation repair was demonstrated using locus-specific RNA-Cas9 ribonucleoproteins with subsequent homologous recombination repair induced by an engineered template supply. Effects on predicted off-target sites in the CRISPR-treated cells were discarded after a targeted deep-sequencing screen. The proven effectiveness and specificity of these correction tools, applied to the c.2299delG pathogenic variant of USH2A, indicates that the CRISPR system should be considered to further explore a potential treatment of USH.

A combination of two truncating mutations in USH2A causes more severe and progressive hearing impairment in Usher syndrome type IIa

ObjectivesUsher syndrome is an inherited disorder that is characterized by hearing impairment (HI), retinitis pigmentosa, and in some cases vestibular dysfunction. Usher syndrome type IIa is caused by mutations in USH2A. HI in these patients is highly heterogeneous and the present study evaluates the effects of different types of USH2A mutations on the audiometric phenotype. Data from two large centres of expertise on Usher Syndrome in the Netherlands and Sweden were combined in order to create a large combined sample of patients to identify possible genotype-phenotype correlations. DesignA retrospective study on HI in 110 patients (65 Dutch and 45 Swedish) genetically diagnosed with Usher syndrome type IIa. We used methods especially designed for characterizing and testing differences in audiological phenotype between patient subgroups. These methods included Age Related Typical Audiograms (ARTA) and a method to evaluate the difference in the degree of HI developed throughout life between subgroups. ResultsCross-sectional linear regression analysis of last-visit audiograms for the best hearing ear demonstrated a gradual decline of hearing over decades. The congenital level of HI was in the range of 16–33 dB at 0.25–0.5 kHz, and in the range of 51–60 dB at 1–8 kHz. The annual threshold deterioration was in the range of 0.4–0.5 dB/year at 0.25–2 kHz and in the range of 0.7–0.8 dB/year at 4–8 kHz. Patients with two truncating mutations, including homozygotes for the common c.2299delG mutation, developed significantly more severe HI throughout life than patients with one truncating mutation combined with one nontruncating mutation, and patients with two nontruncating mutations. ConclusionsThe results have direct implications for patient counselling in terms of prognosis of hearing and may serve as baseline measures for future (genetic) therapeutic interventions.

Análisis Molecular de las Mutaciones 2299delG y C759F en Individuos Colombianos con Retinitis Pigmentosa e Hipoacusia Neurosensorial

Determinar la presencia de las mutaciones 2299delG y C759F en 37 individuos colombianos no relacionados que presentan RP e hipoacusia neurosensorial. Materiales y métodos: análisis de secuencia directa del exón 13 del gen USH2A en todos los individuos seleccionados para el estudio. Resultados: la mutación 2299delG fue observada únicamente en individuos con Síndrome de Usher tipo II, mientras que la mutación C759F, no fue identificada en los individuos del estudio.

The effect of the common c.2299delG mutation in USH2A on RNA splicing

Recessive variants in the USH2A gene are an important cause of both Usher syndrome and nonsyndromic retinitis pigmentosa. A single base-pair deletion in exon 13 (c.2299delG, p.Glu767Serfs*21) is considered the most frequent mutation of USH2A. It is predicted to generate a premature termination codon and is presumed to lead to nonsense mediated decay. However the effect of this variant on RNA has not been formally investigated. It is not uncommon for exonic sequence alterations to cause aberrant splicing and the aim of the present report is to evaluate the effect of c.2299delG on USH2A transcripts. Nasal cells represent the simplest available tissue to study splicing defects in USH2A. Nasal brushing, RNA extraction from nasal epithelial cells and reverse transcription PCR were performed in five Usher syndrome patients who were homozygous for c.2299delG, two unaffected c.2299delG heterozygotes and seven control individuals. Primers to amplify between exons 12 and 15 and exons 10 and 14 were utilised. Significant variability was observed between different RT-PCR experiments. Importantly, in controls, PCR product of the expected size were amplified on all occasions (13/13 experiments); for patients this was true in only 4/14 experiments (Fisher exact test p = 0.0002). Bioinformatics tools predict the c.2299delG change to disrupt an exonic splicing enhancer and to create an exonic splicing silencer within exon 13. Here, we report an effect of the common c.2299delG mutation on splicing of exons 12 and 13 of USH2A. Future studies are expected to provide important insights into the contribution of this effect on the phenotype.

The USH2A c.2299delG mutation: dating its common origin in a Southern European population

Usher syndrome type II is the most common form of Usher syndrome. USH2A is the main responsible gene of the three known to be disease causing. It encodes two isoforms of the protein usherin. This protein is part of an interactome that has an essential role in the development and function of inner ear hair cells and photoreceptors. The gene contains 72 exons spanning over a region of 800 kb. Although numerous mutations have been described, the c.2299delG mutation is the most prevalent in several populations. Its ancestral origin was previously suggested after the identification of a common core haplotype restricted to 250 kb in the 5' region that encodes the short usherin isoform. By extending the haplotype analysis over the 800 kb region of the USH2A gene with a total of 14 intragenic single nucleotide polymorphisms, we have been able to define 10 different c.2299delG haplotypes, showing high variability but preserving the previously described core haplotype. An exhaustive c.2299delG/control haplotype study suggests that the major source of variability in the USH2A gene is recombination. Furthermore, we have evidenced twice the amount of recombination hotspots located in the 500 kb region that covers the 3' end of the gene, explaining the higher variability observed in this region when compared with the 250 kb of the 5' region. Our data confirm the common ancestral origin of the c.2299delG mutation.

Mutation analysis in the long isoform of USH2A in American patients with Usher Syndrome type II

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.

Mutational spectrum in Usher syndrome type II.

Usher syndrome type II is an autosomal recessive disorder characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa (RP). We carried out a mutation screening of the USH2A gene in 88 probands with Usher syndrome type II to determine the frequency of USH2A mutations as a cause for USH2. Six mutations, including 2299delG, 921-922insCAGC, R334W, N346H, R626X, and N357T were identified, with 2299delG mutation being the most frequent (16.5% of alleles), accounting for 77.5% of the pathologic alleles. Thirty-five percent (31/88) of the probands had a USH2A mutation. Nine of them carried two pathogenic mutations: six cases were homozygotes and three were compound heterozygotes. Twenty-two probands (25%) were found to carry only single USH2A mutations. One new missense mutation (N357T) occuring within the laminin N-terminal (type VI) domain of usherin was identified. Eight polymorphisms were found, five of which are novel. Our data support the view that the 2299delG is the most common mutation in USH2A.

A Common Ancestral Origin of the Frequent and Widespread 2299delG USH2A Mutation

Usher syndrome type IIa is an autosomal recessive disorder characterized by mild-to-severe hearing loss and progressive visual loss due to retinitis pigmentosa. The mutation that most commonly causes Usher syndrome type IIa is a 1-bp deletion, described as "2299delG," in the USH2A gene. The mutation has been identified in several patients from northern and southern Europe and from North America, and it has been found in single patients from South America, South Africa, and China. Various studies have reported a range of frequencies (.16-.44) among patients with Usher syndrome, depending on the geographic origin of the patients. The 2299delG mutation may be the one that most frequently causes retinitis pigmentosa in humans. Given the high frequencies and the wide geographic distribution of the mutation, it was of interest to determine whether the mutation resulted from an ancestral mutational event or represented a mutational hotspot in the USH2A gene. Haplotype analysis was performed on DNA samples from 116 unrelated patients with Usher syndrome type IIa; the patients were from 14 countries and represented 148 2299delG alleles. On the basis of six single-nucleotide polymorphisms within the USH2A gene, 12 core haplotypes were observed in a panel of normal chromosomes. However, in our analysis, only one core haplotype was found to be associated with the 2299delG mutation. The data indicate that the widespread geographic distribution of the 2299delG mutation is the result of an ancestral mutation that has spread throughout Europe and into the New World as a result of migration.

Identification of novel USH2A mutations: implications for the structure of USH2A protein.

Usher syndrome type II is an autosomal recessive disorder, characterised by stable hearing impairment from childhood and progressive retinitis pigmentosa from the late teens. Mutations in the USH2A gene, located on 1q41, were recently shown to be responsible for Usher syndrome type IIa. We have investigated the molecular pathology of Usher type II by screening the USH2A gene for mutations in 31 unrelated patients from Denmark and Norway. Besides the frequent 2299delG mutation, which accounted for 44% of the disease alleles, a heterogeneous spectrum of mutations was identified. Sixteen new, putative disease-causing mutations were detected, of which 12 were private and four were shared by unrelated patients. The disease-causing mutations were scattered throughout the gene and included six nonsense and seven missense mutations, two deletions and one small insertion. In addition, six non-pathogenic polymorphisms were identified. All missense mutations resulted in major amino acid side-chain alterations. Four missense mutations affected the N-terminal part of USH2A, whereas three missense mutations affected the laminin-type epidermal growth factor-like (LE) domain. The structural consequences of the mutations affecting the LE domain are discussed in relation to the three-dimensional structure of a LE-module of the mouse laminin gamma1 chain.