An innovative strategy for the molecular diagnosis of Usher syndrome identifies causal biallelic mutations in 93% of European patients

Abstract

Usher syndrome (USH), the most prevalent cause of hereditary deafness-blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1-3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.

Figure 1

Schematic representation of the large rearrangements identified at USH loci. Novel rearrangements are represented by red (deletion) or green (duplication) left-right arrows, and previously reported rearrangements are highlighted in gray. For each rearrangement, the corresponding number of USH patients from our cohort is indicated between parentheses.

Figure 2

Schematic representation of the proteins encoded by USH1 genes, annotated with the novel variants identified by TES. For each protein, the longest isoform is shown, and the novel pathogenic sequence variants are indicated. Abbreviations: IQ, isoleucine-glutamine motifs; MyTH4, myosin tail homology 4 domain; FERM, band 4.1-ezrin-radixin-moesin domain; SH3, src homology 3 domain; PDZ, PSD95-discs large-ZO1 domain; CC, coiled coil domain; PST, proline–serine–threonine–rich region; EC, extracellular cadherin domain; TM, transmembrane domain; Ank, ankyrin domain; SAM, sterile alpha motif domain.

Figure 3

Schematic representation of the proteins encoded by USH2 genes and PDZD7, annotated with the novel variants identified by TES. For each protein, the longest isoform is shown, and the novel pathogenic sequence variants are indicated. LamG/TspN/PTX, N-terminal thrombospondin/pentaxin/laminin G-like domain; Lam Nter, laminin N-terminal domain; Lam EGF-like, laminin-type EGF-like domain; LamG-like, laminin G-like domain; FNIII, fibronectin type III domain; TM, transmembrane domain; Calx, Ca²⁺-binding calcium exchanger β; EAR, Epilepsy Associated Repeats; PDZ, PSD95-discs large-ZO1 domain; GPS, G-protein-coupled proteolysis site.

Figure 4

Prevalence and European distribution of the mutations of USH1, USH2, and USH3 genes identified in this study. For each participating country, the pie chart is equally divided in 11 sectors, representing each of the different USH1 (blue), USH2 (pink, and dark pink for the USH2 modifier PDZD7), and USH3 (green) genes. In each sector, the colored area indicates the proportion of the USH1 patients, or the proportion of the USH2 and USH3 patients, carrying mutations in the corresponding gene. The inset illustrates the proportion of three prevalent USH2A mutations relative to the total number of USH2A mutations identified, in France, Germany, Italy, and Slovenia.