Identification of 13 novel USH2A mutations in Chinese retinitis pigmentosa and Usher syndrome patients by targeted next-generation sequencing.

BACKGROUND: The USH2A gene encodes usherin, a basement membrane protein that is involved in the development and homeostasis of the inner ear and retina. Mutations in USH2A are linked to Usher syndrome type II (USH II) and non-syndromic retinitis pigmentosa (RP). Molecular diagnosis can provide insight into the pathogenesis of these diseases, facilitate clinical diagnosis, and identify individuals who can most benefit from gene or cell replacement therapy. Here, we report 21 pathogenic mutations in the USH2A gene identified in 11 Chinese families by using the targeted next-generation sequencing (NGS) technology. METHODS: In all, 11 unrelated Chinese families were enrolled, and NGS was performed to identify mutations in the USH2A gene. Variant analysis, Sanger validation, and segregation tests were utilized to validate the disease-causing mutations in these families. RESULTS: We identified 21 pathogenic mutations, of which 13, including 5 associated with non-syndromic RP and 8 with USH II, have not been previously reported. The novel variants segregated with disease phenotype in the affected families and were absent from the control subjects. In general, visual impairment and retinopathy were consistent between the USH II and non-syndromic RP patients with USH2A mutations. CONCLUSIONS: These findings provide a basis for investigating genotype-phenotype relationships in Chinese USH II and RP patients and for clarifying the pathophysiology and molecular mechanisms of the diseases associated with USH2A mutations.

Full-field electroretinography, visual acuity and visual fields in Usher syndrome: a multicentre European study.

PURPOSE: Usher syndrome (USH) is a multisensory deficiency involving vision, hearing and the vestibular system. The purpose of this study is to report on the functional data (i.e. electroretinography, visual fields, visual acuity) of patients with retinitis pigmentosa (RP) due to Usher syndrome that were collected in a multicentre European study (TREATRUSH). METHODS: A total of 268 genetically confirmed USH patients underwent electrophysiological examinations in the context of multimodal ophthalmological examination in the study (75 USH1, 189 USH2 and four USH3). Full-field electroretinography (ERG) was performed according to ISCEV standards, visual field determination was carried out with either the Octopus or Goldmann perimeters and visual acuity was examined with either ETDRS or Snellen charts. The data were compared between USH subtypes (USH1/USH2/USH3) and correlated with age. RESULTS: Visual acuity decreases significantly with age for both USH1 and USH2 (p < 0.001), without a difference between the two cohorts. When corrected for age, the preserved kinetic visual field was significantly larger in USH2 than in USH1 (p = 0.04). Furthermore, the preserved kinetic visual field area showed a significant decrease with age (based on an exponential fit) in both USH1 and USH2 (p < 0.001). In USH1 patients, however, the visual field was already vastly reduced at an early age. The ERG results were abnormal in all patients. Detectable data for scotopic ERG were obtained from nine patients, and data of photopic ERG were obtained from 24 patients, without a difference between USH1 and USH2 subtypes. CONCLUSIONS: There are differences in the phenotypes of RP in USH subtypes, most visible in the progression of visual fields between USH1 and USH2. The perimetric reduction occurs earlier in USH1 than in USH2. In both subtypes, visual acuity decreases significantly with age and the ERG is not detectable already at early ages.

Comprehensive Molecular Screening in Chinese Usher Syndrome Patients.

Purpose: Usher syndrome (USH) refers to a group of autosomal recessive disorders causing deafness and blindness. The objectives of this study were to determine the mutation spectrum in a cohort of Chinese patients with USH and to describe the clinical features of the patients with mutations. Methods: A total of 119 probands who were clinically diagnosed with USH were recruited for genetic analysis. All probands underwent ophthalmic examinations. A combination of molecular screening methods, including targeted next-generation sequencing, Sanger-DNA sequencing, and multiplex ligation probe amplification assay, was used to detect mutations. Results: We found biallelic mutations in 92 probands (77.3%), monoallelic mutations in 5 patients (4.2%), and 1 hemizygous mutation in 1 patient (0.8%), resulting in an overall mutation detection rate of 78.2%. Overall, 132 distinct disease-causing mutations involving seven USH (ABHD12, CDH23, GPR98, MYO7A, PCDH15, USH1C, and USH2A) genes; 5 other retinal degeneration genes (CHM, CNGA1, EYS, PDE6B, and TULP1); and 1 nonsyndromic hearing loss gene (MYO15A) were identified, and 78 were novel. Mutations of MYOA7 were responsible for 60% of USH1 families, followed by PCDH15 (20%) and USH1C (10%). Mutations of USH2A accounted for 67.7% of USH2 families, and mutation c.8559-2A>G was the most frequent one, accounting for 19.1% of the identified USH2A alleles. Conclusions: Our results confirm that the mutation spectrum for each USH gene in Chinese patients differs from those of other populations. The formation of the mutation profile for the Chinese population will enable a precise genetic diagnosis for USH patients in the future.

Comprehensive molecular diagnosis of 67 Chinese Usher syndrome probands: high rate of ethnicity specific mutations in Chinese USH patients.

BACKGROUND: Usher syndrome (USH) is the most common disease causing combined deafness and blindness. It is predominantly an autosomal recessive genetic disorder with occasionally digenic cases. Molecular diagnosis of USH patients is important for disease management. Few studies have tried to find the genetic cause of USH in Chinese patients. This study was designed to determine the mutation spectrum of Chinese USH patients. METHODS: We applied next generation sequencing to characterize the mutation spectrum in 67 independent Chinese families with at least one member diagnosed with USH. Blood was collected at Peking Union Medical College Hospital. This cohort is one of the largest USH cohorts reported. We utilized customized panel and whole exome sequencing, variant analysis, Sanger validation and segregation tests to find disease causing mutations in these families. RESULTS: We identified biallelic disease causing mutations in known USH genes in 70 % (49) of our patients. As has been previously reported, MYO7A is the most frequently mutated gene in our USH type I patients while USH2A is the most mutated gene in our USH type II patients. In addition, we identify mutations in CLRN1, DFNB31, GPR98 and PCDH15 for the first time in Chinese USH patients. Together, mutations in CLRN1, DNFB31, GPR98 and PCDH15 account for 11.4 % of disease in our cohort. Interestingly, although the spectrum of disease genes is quite similar between our Chinese patient cohort and other patient cohorts from different (and primarily Caucasian) ethnic backgrounds, the mutations themselves are dramatically different. In particular, 76 % (52/68) of alleles found in this study have never been previously reported. Interestingly, we observed a strong enrichment for severe protein truncating mutations expected to have severe functional consequence on the protein in USH II patients compared to the reported mutation spectrum in RP patients, who often carry partial protein truncating mutations. CONCLUSIONS: Our study provides the first comprehensive genetic characterization of a large collection of Chinese USH patients. Up to 90 % of USH patients have disease caused by mutations in known USH disease genes. By combining NGS-based molecular diagnosis and patient clinical information, a more accurate diagnosis, prognosis and personalized treatment of USH patients can be achieved.

Novel USH2A mutations in Japanese Usher syndrome type 2 patients: marked differences in the mutation spectrum between the Japanese and other populations.

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A. In a recent mutation screening of USH2A in Japanese USH2 patients, we identified 11 novel mutations in 10 patients and found the possible frequent mutation c.8559-2A>G in 4 of 10 patients. To obtain a more precise mutation spectrum, we analyzed further nine Japanese patients in this study. We identified nine mutations, of which eight were novel. This result indicates that the mutation spectrum for USH2A among Japanese patients largely differs from Caucasian, Jewish and Palestinian patients. Meanwhile, we did not find the c.8559-2A>G in this study. Haplotype analysis of the c.8559-2G (mutated) alleles using 23 single nucleotide polymorphisms surrounding the mutation revealed an identical haplotype pattern of at least 635 kb in length, strongly suggesting that the mutation originated from a common ancestor. The fact that all patients carrying c.8559-2A>G came from western Japan suggests that the mutation is mainly distributed in that area; indeed, most of the patients involved in this study came from eastern Japan, which contributed to the absence of c.8559-2A>G.

Novel mutations of MYO7A and USH1G in Israeli Arab families with Usher syndrome type 1.

PURPOSE: This study investigated the genetic basis for Usher syndrome type 1 (USH1) in four consanguineous Israeli Arab families. METHODS: Haplotype analysis for all known USH1 loci was performed in each family. In families for which haplotype analysis was inconclusive, we performed genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array. For mutation analysis, specific primers were used to PCR amplify the coding exons of the MYO7A, USH1C, and USH1G genes including intron-exon boundaries. Mutation screening was performed with direct sequencing. RESULTS: A combination of haplotype analysis and genome-wide homozygosity mapping indicated linkage to the USH1B locus in two families, USH1C in one family and USH1G in another family. Sequence analysis of the relevant genes (MYO7A, USH1C, and USH1G) led to the identification of pathogenic mutations in all families. Two of the identified mutations are novel (c.1135-1147dup in MYO7A and c.206-207insC in USH1G). CONCLUSIONS: USH1 is a genetically heterogenous condition. Of the five USH1 genes identified to date, USH1C and USH1G are the rarest contributors to USH1 etiology worldwide. It is therefore interesting that two of the four Israeli Arab families reported here have mutations in these two genes. This finding further demonstrates the unique genetic structure of the Israeli population in general, and the Israeli Arab population in particular, which due to high rates of consanguinity segregates many rare autosomal recessive genetic conditions.

Deafblindness in French Canadians from Quebec: a predominant founder mutation in the USH1C gene provides the first genetic link with the Acadian population.

BACKGROUND: Usher syndrome type 1 (USH1) is the leading cause of deafblindness. In most populations, many private mutations are distributed across the five known USH1 genes. We investigated patients from the French Canadian population of Quebec (approximately 6 million people) that descends from about 8,500 French settlers who colonized the St Lawrence River valley between 1608 and 1759. We hypothesized that founder mutations in USH1 genes exist in this population. RESULTS: We have genetically characterized 15 patients from different regions of Quebec who were clinically diagnosed as USH1. Of these cases, 60% carried mutations of the USH1C gene, a genetic subtype that is rare outside the Acadian population. We have discovered a founder effect of the c.216G>A mutation, which has previously been designated the 'Acadian allele' because it accounts for virtually all Acadian USH1 cases. It represents 40% of disease alleles in Quebec, and a carrier of c.216G>A was identified in the general population. Mutations in other genes, except CDH23, are very rare. CONCLUSION: Based on our findings, approximately 0.5% of congenitally deaf children in Quebec are at risk of developing retinal degeneration due to homozygosity for c.216G>A. Although the Acadians and French Canadians from Quebec are descended from French ancestors, they have always been considered genetically distinct. The genetic conditions common in Quebec are generally not found in Acadians, or they are due to different mutations. Our results, however, show that carriers of the c.216G>A allele haplotype belonged to the early founders of both the Acadian and the Quebec population.

Novel USH2A mutations in Israeli patients with retinitis pigmentosa and Usher syndrome type 2.

OBJECTIVE: To identify USH2A mutations in Israeli patients with autosomal-recessive Usher syndrome type 2 (USH2) and retinitis pigmentosa (RP). METHODS: Patients from 95 families with RP and 4 with USH2 were clinically evaluated. USH2A exons 2-72 were scanned for mutations using single-strand conformation and sequencing analyses. The frequency of novel missense changes was determined in patients and controls using restriction endonucleases. RESULTS: The analysis revealed 3 USH2A mutations, 2 of which are novel, in 2 families with USH2 and a large family (MOL0051) with both USH2 and RP. Compound heterozygotes for 2 null mutations (Thr80fs and Arg737stop) in MOL0051 suffered from USH2 while compound heterozygotes for 1 of the null mutations and a novel missense mutation (Gly4674Arg) had nonsyndromic RP. CONCLUSIONS: Our results support the involvement of USH2A in nonsyndromic RP and we report here of a second, novel, missense mutation in this gene causing autosomal-recessive RP. CLINICAL RELEVANCE: Possible involvement of USH2A should be considered in the molecular genetic evaluation of patients with autosomal-recessive RP. Understanding the mechanism by which different USH2A mutations cause either USH2 or RP may assist in the development of novel therapeutic approaches.