Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes.

Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch-point mapping strategy was also used to investigate further a putative branch-point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes.

Sequence contexts that determine the pathogenicity of base substitutions at position +3 of donor splice-sites.

Variations at position +3 of 5' splice-sites (5'ss) are reported to induce aberrant splicing in some cases but not in others suggesting that the overall nucleotidic environment can dictate the extent to which 5'ss are correctly selected. Functional studies of three variations identified in donor splice-sites of USH2A and PCDH15 genes sustain this assumption. To gain insights into this question, we compared the nucleotidic context of U2-dependent 5'ss naturally deviated (+3G,+3C, or+3T) from the+3A consensus with 5'ss for which a +3 variation (A>G, A>C, or A>T) was shown to induce aberrant splicing. Statistical differences were found between the two datasets, highlighting the role of one peculiar position in each context (+3G/+4A; +3C/-1G; and +3T/-1G). We provided experimental support to the biostatistical results through the analysis of a series of artificial mutants in reporter minigenes. Moreover, different 5' end-mutated U1 snRNA expression plasmids were used to investigate the importance of the position +3 and of the two identified compensatory positions -1 and +4 in the recognition of 5'ss by the U1 snRNP. Overall, our findings establish general properties useful to molecular geneticists to identify nucleotide substitutions at position +3 that are more likely to alter splicing.

UMD-USHbases: a comprehensive set of databases to record and analyse pathogenic mutations and unclassified variants in seven Usher syndrome causing genes.

Using the Universal Mutation Database (UMD) software, we have constructed "UMD-USHbases", a set of relational databases of nucleotide variations for seven genes involved in Usher syndrome (MYO7A, CDH23, PCDH15, USH1C, USH1G, USH3A and USH2A). Mutations in the Usher syndrome type I causing genes are also recorded in non-syndromic hearing loss cases and mutations in USH2A in non-syndromic retinitis pigmentosa. Usher syndrome provides a particular challenge for molecular diagnostics because of the clinical and molecular heterogeneity. As many mutations are missense changes, and all the genes also contain apparently non-pathogenic polymorphisms, well-curated databases are crucial for accurate interpretation of pathogenicity. Tools are provided to assess the pathogenicity of mutations, including conservation of amino acids and analysis of splice-sites. Reference amino acid alignments are provided. Apparently non-pathogenic variants in patients with Usher syndrome, at both the nucleotide and amino acid level, are included. The UMD-USHbases currently contain more than 2,830 entries including disease causing mutations, unclassified variants or non-pathogenic polymorphisms identified in over 938 patients. In addition to data collected from 89 publications, 15 novel mutations identified in our laboratory are recorded in MYO7A (6), CDH23 (8), or PCDH15 (1) genes. Information is given on the relative involvement of the seven genes, the number and distribution of variants in each gene. UMD-USHbases give access to a software package that provides specific routines and optimized multicriteria research and sorting tools. These databases should assist clinicians and geneticists seeking information about mutations responsible for Usher syndrome.