Abstract
Aims: The aim of this study was the genetic diagnosis by next generation sequencing (NGS) of a patient diagnosed with Usher syndrome type 2 and the functional evaluation of the identified genetic variants to establish a phenotype–genotype correlation. Methods: Whole exome sequencing (WES) analysis identified two heterozygous intronic variants in CDH23, a gene responsible of Usher syndrome type 1. Evaluation of the putative splicing effects was performed in vivo, in whole blood samples, and in vitro, by transfection of midigene constructs in HEK293T cells. Results: Two intronic variants were identified in intron 45 of CDH23—one novel, c.6050-15G>A, and the other, c.6050-9G>A, already reported as a noncanonical splice site (NCSS) mutation—with partial functional characterization. In vivo and in vitro analyses showed aberrant transcripts by the addition of 13 and 7 nucleotides to exon 46, respectively. Transcript degradation by nonsense mediated decay (NMD) in blood cells could only be prevented by cycloheximide treatment. Midigene constructs showed that the two variants contributed to exon skipping and generated aberrantly spliced transcripts. Conclusions: A combination of in vivo and in vitro assays provided a comprehensive view of the physiological effects of NCSS variants, which in this case led to a clinical reassignment of the proband as affected with atypical USH1 syndrome.
Highlights
Usher syndrome (USH) is a group of inherited autosomal recessive disorders characterized by partial or total progressive hearing and vision loss
The comparison of the audiometry tests recorded at 5 years old (1995) and 2019 showed stable congenital severe hearing loss (Figure 1C)
The initial clinical diagnosis was Usher syndrome type II, and genetic diagnosis of the patient and family was required for confirmation
Summary
Usher syndrome (USH) is a group of inherited autosomal recessive disorders characterized by partial or total progressive hearing and vision loss. All Usher disease forms are clinically and genetically heterogeneous and constitute the most common cause of combined deafness and blindness [1]. Sensorineural hearing impairment is caused by abnormalities of the inner ear, and vision loss occurs as photoreceptor cells in the retina deteriorate progressively, leading to retinitis pigmentosa (RP) [2]. Genes 2019, 10, 732 form, showing extensive genetic and allelic heterogeneity, and is characterized by severe to profound congenital deafness, variable vestibular areflexia, and adolescent onset retinitis pigmentosa [4]. The major gene associated with USH1 is MYO7A, which accounts for up to 70%
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