Abstract
Splice site mutations contribute to a significant portion of the genetic causes for mendelian disorders including deafness. By next-generation sequencing of 4 multiplex, autosomal dominant families and 2 simplex, autosomal recessive families with hereditary deafness, we identified a variety of candidate pathogenic variants in noncanonical splice sites of known deafness genes, which include c.1616+3A > T and c.580G > A in EYA4, c.322-57_322-8del in PAX3, c.991-15_991-13del in DFNA5, c.6087-3T > G in PTPRQ and c.164+5G > A in USH1G. All six variants were predicted to affect the RNA splicing by at least one of the computational tools Human Splicing Finder, NNSPLICE and NetGene2. Phenotypic segregation of the variants was confirmed in all families and is consistent with previously reported genotype-phenotype correlations of the corresponding genes. Minigene analysis showed that those splicing site variants likely have various negative impact including exon-skipping (c.1616+3A > T and c.580G > A in EYA4, c.991-15_991-13del in DFNA5), intron retention (c.322-57_322-8del in PAX3), exon skipping and intron retention (c.6087-3T > G in PTPRQ) and shortening of exon (c.164+5G > A in USH1G). Our study showed that the cryptic, noncanonical splice site mutations may play an important role in the molecular etiology of hereditary deafness, whose diagnosis can be facilitated by modified filtering criteria for the next-generation sequencing data, functional verification, as well as segregation, bioinformatics, and genotype-phenotype correlation analysis.
Highlights
RNA splicing refers to the process of removing introns from the initial transcript, transcribed from its DNA template, and connecting exons to form a continuous RNA molecule
Targeted or whole-exome nextgeneration sequencing (NGS) has been increasingly employed for mutation screening of heterogeneous diseases such as deafness
The routine sequencing data analysis usually focus on non-synonymous variants in exon and canonical splice site (CSS) variant only, while the potential noncanonical splice site (NCSS) variants are often ignored
Summary
RNA splicing refers to the process of removing introns from the initial transcript (preRNA), transcribed from its DNA template, and connecting exons to form a continuous RNA molecule. Sequences near the splicing sites of preRNA are conserved, which include the GT bases at the 5′ donor site of the intron, the AG bases at the 3′ acceptor site, the branch point composed of the polypyrimidine trace and splicing regulatory sequences such as exonic splicing enhancer (ESE), exonic splicing silencer (ESS), intronic splicing enhancer (ISE) and intronic splicing silencer (ISS) (Glisovic et al, 2008; Anna and Monika, 2018) The existence of these conserved sequences ensures the accurate RNA splicing, while mutations in these sequences may lead to structural alteration of the protein products and a variety of genetic disorders (Wang et al, 2012). Intronic or exonic mutations may create a new splice site, resulting in partial intron retention, exon shortening or formation of pseudo exons (VazDrago et al, 2017)
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