Detection and Functional Verification of Noncanonical Splice Site Mutations in Hereditary Deafness

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.

Detailed Clinical Features of PTPRQ-Associated Hearing Loss Identified in a Large Japanese Hearing Loss Cohort

“…Among the 17 variants identified in this study, 15 (88.2%) were loss of function variants, with the majority of variants (16/25, 64%) previously reported as also being loss of function variants [23,[28][29][30][31][32][33][34][35]. PTPRQ variants have been reported as causative for autosomal recessive and autosomal dominant HL [2].…”

“…His high frequency hearing then deteriorated from moderate to profound over 20 years, with his HL changing from flat-type to high-frequency steeply sloping HL. In previous studies, the hearing level in the patients with autosomal recessive inheritance ranged from moderate to profound [7,[28][29][30][31][32][33][34][35]. Most patients had progressive, high-frequency HL regardless of autosomal recessive or autosomal dominant inheritance [7,23,28,[37][38][39][40][41][42][43].…”

Hereditable variants of classical protein tyrosine phosphatase genes: Will they prove innocent or guilty?

“…Knockout mouse studies demonstrated that PTPRQ is an essential component of the stereocilia hair-bundle shaft connectors since deficiency results in hearing loss ( Goodyear et al, 2003 ). In 2010 the formal proof was published that inactivating mutations in the gene are also responsible for deafness in humans ( Schraders et al, 2010 ; Shahin et al, 2010 ) and many more have followed since ( Gao et al, 2015 ; Sang et al, 2015 ; Eisenberger et al, 2018 ; Wu et al, 2018 ; Ozieblo et al, 2019 ; Sang et al, 2019 ; Safka Brozkova et al, 2020 ; Chen et al, 2021a ; Mahmood et al, 2021 ; Yang et al, 2021 ). For a more detailed description of the genetic data we refer to excellent recent reviews ( Kremer, 2019 ; Richardson and Petit, 2019 ).…”