USH2A sequencing in three affected members of a large family, referred for the recessive USH2 syndrome, identified a single pathogenic alteration in one of them and a different mutation in the two affected nieces. As the patients carried a common USH2A haplotype, they likely shared a mutation not found by standard sequencing techniques. Analysis of RNA from nasal cells in one affected individual identified an additional pseudoexon (PE) resulting from a deep intronic mutation. This was confirmed by minigene assay. This is the first example in Usher syndrome (USH) with a mutation causing activation of a PE. The finding of this alteration in eight other individuals of mixed European origin emphasizes the importance of including RNA analysis in a comprehensive diagnostic service. Finally, this mutation, which would not have been found by whole-exome sequencing, could offer, for the first time in USH, the possibility of therapeutic correction by antisense oligonucleotides (AONs).

The usherin gene (USH2A) has been screened for mutations causing Usher syndrome type II (USH2). Two protein isoforms have been identified: a short isoform of 1,546 amino acids and a more recently recognized isoform extending to 5,202 amino acids. We have screened the full length by genomic sequencing. We confirm that many mutations occur in the exons contributing solely to the longer form. USH2 is an autosomal recessive disorder and, in contrast to previous studies, both mutations were identified in 23 patients and a single mutation in 2 out of 33 patients. A total of 34 distinct mutated alleles were identified, including one complex allele with three variants and another with two. A total of 27 of these are novel, confirming that most mutations in usherin are private. Many of the mutations will lead to prematurely truncated protein but as there are a substantial number of missense variants, we have used in silico analysis to assess their pathogenicity. Evidence that they are disease-causing has been produced by protein alignments and three-dimensional (3D) structural predictions when possible. We have identified a previously unrecognized cysteine rich structural domain, containing 12 dicysteine repeats, and show that three missense mutations result in the loss of one of a pair of the defining cysteine-cysteine pairs.

No abstract

We have systematically analyzed the two known minor genes involved in Usher syndrome type 2, DFNB31 and GPR98, for mutations in a cohort of 31 patients not linked to USH2A. PDZD7, an Usher syndrome type 2 (USH2) related gene, was analyzed when indicated. We found that mutations in GPR98 contribute significantly to USH2. We report 17 mutations in 10 individuals, doubling the number of GPR98 mutations reported to date. In contrast to mutations in usherin, the mutational spectrum of GPR98 predominantly results in a truncated protein product. This is true even when the mutation affects splicing, and we have incorporated a splicing reporter minigene assay to show this, where appropriate. Only two mutations were found which we believe to be genuine missense changes. Discrepancy in the mutational spectrum between GPR98 and USH2A is discussed. Only two patients were found with mutations in DFNB31, showing that mutations of this gene contribute to only a very small extent to USH2. Close examination of the clinical details, where available, for patients in whom no mutation was found in USH2A, GPR98, or DFNB31, showed that most of them had atypical features. In effect, these three genes account for the vast majority of USH2 patients and their analysis provide a robust pathway for routine molecular diagnosis.